DNA Sequencing Protocols

CHAIVER

1

DNA Sequencing Hugh G. Grifin

and Annette M. Grifin

1. Introduction Methods to determine the sequenceof DNA were developed in the late 1970s (1,2) and have revolutionized the science of molecular genetics. The DNA sequences of many different genes from diverse sources have been determined, and the information is stored in international databanks such as EMBL, GenBank, and DDBJ. Many scientists now accept that sequence analysis will provide an increasingly useful approach to the characterization of biological systems. Projects are already underway to map and sequencethe entire genome of organisms such as Escherichia coli, Saccharomyces cerevisiae, Caenorhabditis elegans, and Homo sapiens. In the recent past, large-scale sequencing projects such as these were often dismissed as prohibitively expensive and of little short-term benefit, while DNA sequencing itself was seen as a repetitive and unintellectual pursuit. However, this view is now changing and most scientists recognize the importance of DNA sequence data and perceive DNA sequencing as a valuable and often indispensable aspect of their work. Recent technological advances, especially in the area of automated sequencing, have removed much of the drudgery that used to be associated with the technique, and modern innovative computer software has greatly simplified the analysis and manipulation of sequence data. Large-scale sequencing From Methods m Molecular Biology, Vol. 23. DNA Sequencmg Protocols Edited by. H. and A. Gnffm Copyright Q1993 Humana Press Inc., Totowa,

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projects, such as the Human Genome Project, produce the DNA sequences of many unknown genes. Such data provide an impetus for molecular biologists to apply the techniques of reverse genetics to produce probes and antibodies that can be used to identify the gene product, its cellular location, and its time of appearancein the developing cell (3). A function can be assigned by mutant analysis or by comparison of the deduced amino acid sequence with proteins of known function. Therefore, DNA sequencing can act as a catalyst to stimulate future research into many diverse areas of science. The two original methods of DNA sequencingdescribed in 1977 (1,2) differ considerably in principle. The enzymatic (or dideoxy chain termination) method of Sanger (I) involves the synthesis of a DNA strand from a single-stranded template by a DNA polymerase. The Maxam and Gilbert (or chemical degradation) method (2) involves chemical degradation of the original DNA. Both methods producepopulations of radioactively labeled polynucleotides that begin from a fixed point and terminate at points dependent on the location of a particular base in the original DNA strand. The polynucleotides are separated by polyacrylamide gel electrophoresis, and the order of nucleotides in the original DNA can be read directly from an autoradiograph of the gel (4). Although both techniques are still used today, there have been many changes and improvements to the original methods. While the chemical degradation method is still in use, the enzymatic chain termination method is by far the most popular and widely used technique for sequence determination. This process has been automated by utilizing fluorescent labeling instead of radioactive labeling (Chapters 33-37), and the concepts of polymerase chain reaction (PCR) technology have been harnessed to enable the sequencing reaction to be “cycled” (Chapters 21, 26, and 34). Other recent innovations include multiplexing (Chapter 28), sequencing by chemiluminescence rather than radioactivity (Chapter 29), solid phasesequencing (Chapter 25), and the use of robotic work stations to automate sample preparation and sequencing reactions (Chapter 38). 2. Maxam and Gilbert Method In the original Maxam and Gilbert method (2) a fragment of DNA is radiolabeled at one end and then partially cleaved in four different chemical reactions, each of which is specific for a particular base or

DNA Sequencing

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type of base. This results in four populations of labeled polynucleotides. Each radiolabeled molecule extends from a fixed point (the radiolabeled end) to the site of chemical cleavage, which is determined by the DNA sequence of the original fragment. Since the cleavage is only partial, each population consists of a mixture of molecules, the lengths of which are determined by the base composition of the original DNA fragment. The four reactions are electrophoresed in adjacent lanes through a polyacrylamide gel. The DNA sequence can then be determined directly from an autoradiograph of the gel. The original method has been improved over the years (5). Additional chemical cleavage reactions have been devised (6), new end-labeling techniques developed (7,8), and shorter, simplified protocols have been produced (Chapter 32). The main advantage of chemical degradation sequencing is that sequence is obtained from the original DNA molecule and not from an enzymatic copy. It is therefore possible to analyze DNA modifications such as methylation, and to study protein/DNA interactions. Chemical sequencing also enables the determination of the DNA sequence of synthetic oligonucleotides. However, the Sanger method is both quicker and easier to perform and must remain the method of choice for most sequencing applications. 3, Sanger Method The Sanger (or chain termination) method (I) involves the synthesis of a DNA strand from a single-stranded template by a DNA polymerase. The method depends on the fact that dideoxynucleotides (ddNTPs) are incorporated into the growing strand in the same way as the conventional deoxynucleotides (dNTPs). However, ddNTPs differ from dNTPs becausethey lack the 3’-OH group necessaryfor chain elongation. When a ddNTP is incorporated into the new strand, the absence of the hydroxyl group prevents formation of a phosphodiester bond with the succeeding dNTP and chain elongation terminates at that position. By using the correct ratio of the four conventional dNTPs and one of the four ddNTPs in a reaction with DNA polymerase, a population of polynucleotide chains of varying lengths is produced. Synthesis is initiated at the position where an oligonucleotide primer anneals to the template, and each chain is terminated at a specific base (either A, C, G, or T depending on which ddNTP was used). By using the four different ddNTPs in four separate reactions the com-

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plete sequence information can be obtained. One of the dNTPs is usually radioactively labeled so that the information gained by electrophoresing the four reactions in adjacent tracks of a polyacrylamide gel can be visualized on an autoradiograph. The original method used the Klenow fragment of DNA polymerase I to synthesize the new strands in the sequencing reactions, and this enzyme is still used today (Chapter 12). Other enzymes such as Sequenase (Chapter 14), T7 polymerase (Chapter 13), and Taq polymerase (Chapter 15) are also widely used. Each enzyme has its own particular properties and qualities, and the choice of polymerase will depend on the type of template and the sequencing strategy employed. 4. Templates

for DNA Sequencing

The polymerase reaction requires single-stranded template. This is usually achieved by utilizing Ml3 phage that can produce large amounts of just one strand of DNA as part of its normal replicative cycle. Double stranded (replicative form) Ml3 can also be isolated, and this is used to clone the DNA fragment to be sequenced. The qualrty of DNA sequencedata achieved using Ml3 template is extremely good and many researchers prefer to subclone to Ml3 prior to sequencing. Sequencing reactions can be performed directly on plasmid DNA, the double stranded molecule being denatured prior to sequencing. Recent innovations in DNA purification techniques and the availability of improved polymerases have greatly enhanced the quality of data produced by plasmid sequencing methods (Chapters 14, 18, and 19). Sequence determination can also be performed directly on cosmid clones (Chapter 21), lambda clones (Chapter 20), and on PCR products (Chapters23-25). In particular, the adventof cycle sequencing(Chapter 26) has vastly increased the range of templates that can be used. 5. Sequencing

Strategies

A lot of sequencing performed is confirmatory sequencing to check the orientation or the structure of newly constructed plasmids, or to determine the sequence of mutants. This type of sequencing can be easily achieved by subcloning a restriction fragment into Ml3 and sequencing using the universal primer. Alternatively, a custom-designed oligonucleotide primer can be synthesized and sequencing performed without the need for any subcloning.

DNA Sequencing

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The determination of long tracts of unknown sequence, however, requires careful planning and the utilization of one of a variety of strategies including: the shotgun approach, directed sequencing strategies, and the gene walking technique. A random, or shotgun, approach involves subcloning random fragments of the target DNA to an appropriate vector such as Ml3 (Chapter 7). Sequencesfrom these recombinants are determined at random until the individual readings can be assembled into a contiguous sequence. This is achieved using a sequence assembly computer program (9,lO). The disadvantage of this method is the redundancy in the sequence data obtained, each section of DNA being sequenced several times over. However, the strategy benefits from making no prior assumptions about the DNA to be sequenced, such as base composition or the presence of certain restriction sites. Directed strategies usually involve the construction of a nested set of deletions of the fragment to be sequenced. Progressive deletions of the fragment are generatedwith a nuclease,eachdeletion being approximately 200-300 bp. Following deletion the fragments are recloned into Ml3 or a plasmid vector adjacent to the universal primer site. The subclones are then sequenced in order of size, with the sequence of each clone overlapping slightly with the one before. In this way, a large tract of contiguous sequenceis determined on one gel. The disadvantage is the labor and time involved in constructing the deletions. Several methods are available for deletion construction including the use of exonuclease III (Chapter 8), T4 DNA polymerase (Chapter 9), and DNase I (Chapter 10). It is essential to sequence both strands of the DNA and this usually entails generating two sets of deletions. Perhaps the simplest method of sequencing is the gene walking technique (Chapter 11). This involves the initial sequencing of approximately 200-400 bp of the end of a cloned fragment using the universal primer (the sequence of the other end can be achieved with the reverse primer). This sequence information is then used to design a new oligonucleotide primer, which will provide the sequence of the next 200-400 bp, and so on across the entire length of the insert. This method is the least labor intensive because no deletion construction or generation of random clones is necessary, and template DNA can be made in the one batch since the template is the same for all sequencing reactions. However, the delay involved in synthesizing a new oli-

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gonucleotide primer before the next reaction can be performed may considerably prolong the time taken to sequence a long tract of DNA. The cost of oligonucleotide synthesis may also be prohibitive. 6. Automation

in DNA Sequencing

One of the major advances in sequencing technology in recent years is the development of automated DNA sequencers. These are based on the chain termination method and utilize fluorescent rather than radioactive labels. The fluorescent dyes can be attached to the sequencing primer, to the dNTPs, or to the terminators, and are incorporated into the DNA chain during the strand synthesis reaction mediated by a DNA polymerase (e.g., Klenow fragment of DNA polymerase I, Sequenase, or Taq DNA polymerase). During the electrophoresis of the newly generated DNA fragments on a polyacrylamide gel a laser beam excites the fluorescent dyes. The emitted fluorescence is collected by detectors and the information analyzed by computer. The data are automatically converted to nucleotide sequence. Several such instruments are now commercially available and are becoming increasingly popular (11; Chapters 33-37). Other aspects of the sequencing procedure that are being automated include template preparation and purification, and the sequencing reactions themselves. Robotic workstations are currently being developed to perform these tasks (Chapter 38). 7. Cycle Sequencing Cycle sequencing is a new and innovative approach to dideoxy sequencing. Its advantages over conventional sequencing techniques are that the reactions are simpler to set up, less template is required, the quality and purity of template are not as critical, and virtually any single- or double-stranded DNA can be sequenced (including lambda, cosmid, plasmid, phagemid, M13, and PCR product). In this method, a single primer is used to linearly amplify a region of template DNA using Taq polymerase in the presence of a mixture of dNTPs and a ddNTP. Either radioactive or fluorescent labels can be used, making cycle sequencing technology as relevant to automated processes as it is to manual methods (Chapters 26, 34-36). As in conventional dideoxy sequencing methods, cycle sequencing involves the generation of a new DNA strand from a single-stranded

DNA Sequencing template, synthesis commencing at the site of an annealed primer, and terminating on the incorporation of a ddNTP. The difference is that the reaction occurs not just once but 20-30 times under the control of a thermal cycler (or PCR machine). This results in more and better sequence data from less template. The process of denaturing a doublestranded molecule is eliminated, with denaturation occurring automatically in the thermal cycler. The development of cycle sequencing techniques has made a major contribution to DNA sequencing methodology, improving the reliability and efficiency of DNA sequence determination and eliminating time-consuming steps. 8. Aim of This Book The purpose of this book is to provide detailed practical procedures for a number of DNA sequencing techniques. Although protocols for DNA sequencing methods are available elsewhere, there was a need for a book that comprehensively covered the vanguard techniques now being applied in this rapidly evolving field. Each contribution is written so that a competent scientist who is unfamiliar with the method can carry out the technique successfully at the first attempt by simply following the detailed practical procedures that have been described by each author. Even the simplest techniques occasionally go wrong, and for this reason a “Notes” section has been included in most chapters. These notes will indicate any major problems or faults that can occur, their sources, and how they can be identified and overcome. Since the purpose of this book is to describe practical procedures and not to go into great depth regarding theory, a comprehensive reference section is included in most chapters, enabling the reader to refer to other publications for more detailed theoretical discussions on the various techniques. References 1. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing wrth chaintermmator inhibitors. Proc. Natl. Acad. Sci USA 74, 5463-5467. 2. Maxam, A. M and Gilbert, W (1977) A new method for sequencing DNA Proc. Natl. Acad. Scl USA 74,560-564.

3. Barrell, B. (1991) DNA sequencmg: present limitations and prospects for the future. FASEB J 5,40-45 4. Sambrook, J., Frrtsch, E F., and Maniatrs, T. (1989) Molecular Cloning. ,4 Laboratory Manual 2d ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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5 Maxam, A. M. and Gilbert, W. (1980) Sequencing end-labeled DNA with basespecific chemical cleavages. Meth. Enzym. 65,499-560. 6. Ambrose, B J. B. and Pless, R. C. (1987) DNA sequencing. Chemical methods. Meth. Enzymol. 152,522-538. 7. Volckaert, G. (1987) A systematic approach to chemical DNA sequencing by subcloning in pGV451 and derrved vectors. Meth Enzym. 155,23 l-250 8 Eckert, R. L. (1987) New vectors for rapid sequencing of DNA fragments by chemical degradation. Gene 51,247-254. 9. Dolz, R. (1993) Fragment assembly programs, in DNA sequencmg: Computer Analysis ofSequence Data, (Griffin, A. M. and Grrffm, H. G., eds.), Humana Press, Totowa, NJ. (Ch. 2). 10 Staden, R. (1992) Managing sequencing projects, m DNA sequencing. Computer Analysis of Sequence Data, (Griffin, A. M. and Griffin, H. G., eds.), Humana Press, Totowa, NJ. (Ch 17). 11 Hunkapiller, T , Karser, R J., Kopp, B F , and Hood, L. (1991) Large-scale and automated DNA sequence determination. Science 254,59-67

&IAE’TER

Ml3 Cloning Their Contribution

2

Khicles to DNA Sequencing

Joachim

Messing

1. Introduction For studies in molecular biology, DNA purification has been essential, in particular for DNA sequencing, probing, and mutagenesis. The amplification of DNA in E. coli by cloning vehicles derived from M13mp or pUC made expensive physical separation techniques like ultracentrifugation unnecessary.Although today the polymerase chain reaction is a valuable alternative for the amplification of small DNA pieces (I), it cannot substitute for the construction of libraries of DNA fragments. Therefore, E. coli has served not only as a vehicle to amplify DNA, but also to separate many DNA molecules of similar length and the two DNA strands simultaneously. For this purpose, a bacteriophage like M 13 can be used. The various viral cis- and trunsacting functions are critical not only for strand separation, but also to separate the single-stranded DNA from the E. coli cell by an active transport mechanism through the intact cell wall. Although it may have been somewhat surprising to some how many changes in its DNA sequence the phage tolerated, manipulations of this amplification and transport system have been extended today even to the viral coat proteins for the production of epitope libraries (2). Much of the work is now more than a decade old, but experience has confirmed the usefulness of some simple biological paradigms. TechFrom Methods m Molecular Biology, Vol 23’ DNA Sequencmg Protocols Edlted by. H and A Gnffm Copyright 01993 Humana Press Inc , Totowa,

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niques that were new and limiting fifteen years ago included automated oligonucleotide synthesis and the use of thermostable enzymes, which add a critical dimension to molecular biology today. Neither necessarily replaces the previous techniques, but they create greater flexibility, enormously accelerate scientific investigations, and even make certain analyses possible for the first time. However, DNA sequencing of larger contigs (several overlapping sequences that can be linked) have benefited from economizing on the synthesis of new oligonucleotides (3,4). Even in the absence of automated oligonucleotide synthesis in the earlier years, the concept of a universal primer could be developed by alternative techniques. 2. Development

of DNA Sequencing A Discussion

Techniques:

In 1974, at one of the first meetings on the use of restriction endonucleases in molecular biology some of these ideas became clear, Work on the chemical synthesis of a tRNA gene was presented, and the initial work on sequencing phage $X174 using restriction fragments as primers for the plus-minus method was discussed. At that time, oligonucieotide synthesis required a major effort and could not easily be generally applied. Restriction fragments offered an alternative. They could be used as primers for DNA synthesis in vitro and for marker-rescue experiments to link genetic and physical maps of viruses like SV40, both forerunners for DNA sequencing and sitedirected mutagenesis. There were several reasons to use @X174 as the first model in developing DNA-sequencing techniques and determining the sequence of an entire autonomous genome. First, it was one of the smallest DNA viruses; it is even smaller than M13. Second, the mature virus consists of single-stranded DNA, eliminating the need to separate the two strands of DNA for template preparation; this is even more critical if one wishes to use double-stranded restriction fragments as primers. Third, a restriction map was superimposed on the genetic map by marker-rescue experiments (5). The latter feature still serves today as a precondition for other genomes. Restriction sites were critical as signposts along the thousands of nucleotides and provided the means to dissect the double-stranded replicative form or RF of @X174 in small, but ordered pieces that permitted the DNA sequencing effort

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Cloning Vehicles

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to proceed in a walking manner along the genome. Today the restriction map can be replaced by any DNA sequence (e.g., STS), since the synthesis of oligonucleotides is so rapid that researchers can use the DNA sequence that was just read from a sequencing gel to design and produce an oligonucleotide to extend the sequencing gel further in the 5’ direction. Therefore, the use of oligonucleotides instead of restriction fragments in such a primer walking method would have enormously accelerated the @X174 project. At the Cleveland Conference on Macromolecules in 1981, after a talk that I had given, the replacement of shotgun sequencing by such a method was suggestedby a colleague, who, as a pioneer in DNA synthesis and its automation, saw a perfect match of this emerging technology with DNA sequencing. Another expert in the chemical synthesis of DNA, Michael Smith, had more interest in the applications than improving the method, and had switched from the diester method to the phosphoramidite method (6), as well as conceiving the idea of using oligonucleotides in marker rescue (7). These innovative researchers pioneered marker rescue and established the physical and genetic map of $X174 (5). It is clear that today’s protein engineering had its roots right there with the right people at the right time, because they also recognized that oligonucleotides could eliminate the need for strand separation for DNA sequencing (8), and developed this method until it reached greater maturity (4,9). Even double-stranded DNA sequencing with universal primers became easier with the development of the pUC plasmids (IO). Despite all the advantages of choosing @X174 as a model system, Sanger’s group nearly picked a different single-stranded DNA phage, fd. In principle, E. coli has two different types of single-stranded DNA phage, represented by $X174 and fd. The first is packaged into an icosahedral head; it kills and lyses the host cell, but does not require F pili, which are receptor sites on the surface of the cell wall encoded by F factors. Its host range is restricted to E. coli C. Phage like fd can only infect male-specific E. cd, producing pili at their surface that are packaged in a filamentous coat and discharged from the cell without lysis; infected cells can continue to divide. These differences are critical, but there was another reason for choosing fd originally, The major coat protein encoded by gene VIII of the phage, a very small but very abundant protein, had been sequenced by protein sequen-

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cing methods. Therefore it seemed obvious, particularly to those who had pioneered protein sequencing, to use protein sequenceto check the DNA sequence. The protein sequence allowed the design and synthesis of an oligonucleotide that would prime in vitro DNA synthesis within the coat protein gene. Furthermore, the derived DNA sequence had to match the protein sequence. Of course, the codon redundancy of many amino acids made it difficult to design a unique primer, and it might have been not too surprising that the approach did not lead to the correct DNA sequence (11). It turned out later that this was not caused by the choice of codons, but to a mistake in the protein sequence instead, Still, the paradigm of reverse genetics again has its roots right there. 3. Replication Systems and Ml3 In 1974, a research group at the Max Planck Institute of Biochemistry in Munich became interested in viruses, initially in E. coli more than in mammalian cells. The “Abteilung” was organized in subgroups, and one subgroup was interested in developing in vitro rephcation systems using both bacteriophage and small plasmids. This had a strong biochemical emphasis, and the researchers rapidly began to learn about single-stranded DNA replication. Eleven years earlier, Hofschneider had isolated a similar filamentous phage from the Munich sewers that he named after a series of phage with the initial M (12). Number 13 was the one that was studied most. Looking for a different research topic than plasmids or DNA replication, the possibility of combining Ml3 phage production with the in vitro DNA synthesis-based method of DNA sequencing was seen. Although this might have been obvious to those familiar with phage replication, innovative methods were needed for adaptation to DNA cloning techniques. The walking method for sequencing $X174 was the strategy used at that time, and some thought that it would be difficult to clone large fragments into Ml3 (although the author’s record was around 40 kb) and that a walking method might therefore have a limited use. Logically, the only alternative to the walking method was the use of shotgun clomng and a universal primer. The replicative form of Ml3 could be used to clone DNA fragments of a size slightly larger than necessary for single sequencing reactions, and a universal sequence near the cloning site would be used as a primer.

Ml3 Cloning Vehicles This would shift the work from preparing primers to preparing templates, which still remains more economical (13). If they were numerous, cloning was much faster than any biochemical technique, and with these thoughts in mind, a plan took shape to construct in vitro recombinants of phage Ml3 without using existing methods. One might recall that in vitro recombinants were usually based on drugresistance markers. This led to the development of plasmid vectors with unique cloning sites that were scattered ail over the plasmid genome (14). 4. Transposons Mutagenesis Both Zinder’s and Schaller’s laboratory had in mind, and actually later used, transposons to develop f 1 and fd transducing phage (15,16). However, one could predict that such a course of experiments, although useful for plasmid cloning vehicles, would be less useful for M13. It seemed plausible, and such an experiment could demonstrate that, in contrast to $X174, filamentous phage can accommodate additional DNA by extending the filamentous coat; infected cells can be treated like plasmid-containing cells. To some degree this had already been proved since one group had already described mutants of more than unit length (17). Another advantage of transposon mutagenesis was that insertion mutants would be naturally selected. This was one of the biggest obstacles from the beginning. Although plasmids and bacteriophage h were natural transducing elements, filamentous phage had never been shown to have this property, and it was not obvious whether insertion mutants would be viable. This was difficult because it was already known that amber mutants of most viral genes not only cause abortive infection, but also lead to killing of the host, something that does not happen when these mutations are suppressed. Therefore, insertion mutants that can be used as plasmids would kill the cell. It was thus predictable that insertion mutants had to be restricted to noncoding regions. Therefore, a decision was made to use a restriction enzyme that recognized at least two different sites in the intergenic region of the RF. Rather than asking whether the intergenic region contains a target site for transposons, the restriction map showed that it was possible to get at least two different insertion mutants within the intergenic region. The only difficulty in such an experiment was

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to find conditions where the restriction enzyme would cut RF only once at any of the possible target sites, so that a population of unit length RF could be ligated to the appropriate marker DNA fragment. However, there was another reason not to use drug-resistance markers. Infected cells still divide, but very slowly. Therefore, selection takes much longer than with plasmids, but it makes it very easy to distinguish infected from noninfected cells on a bacterial lawn. A single infection grown on a bacterial lawn forms a turbid plaque. If bacterial cells are then transfected by the calcium chloride technique of Mandel and Higa (18), a transformed cell can be recognized as a plaque. Hence, no selection technique is necessary, but the ability to distinguish between wild-type M 13 and M 13 insertion mutants would remain a problem. Although it was quite plausible to think of the histochemical screen used for bacteriophage hplac by Malamy et al. (19), the la& gene would have been a large insertion. However, it turned out that, rather than using entire genes as markers, one could clone only the portion encoding the amino-terminal and the repressible control region, and provide the rest in tram by the host of the phage. This became clear when Landy et al. (20) wrote on the purification of an 800-bp Hind11 fragment from hplac capable of a-complementation in a cell-free transcription-translation system. An informal sequence of this fragment showed that it was 789-bp long and included the first 146 codons of the ZacZ gene, but it was still necessary to assemble many components and purify several restriction endonucleases. Work began after some strains and purified Zac repressor were traded; this allowed the purification of the 789-bp Hind11 fragment out of about fifty other restriction fragments by simply filtering the DNA/lac repressor complex through a nitrocellulose filter. After adding IPTG, it was possible to recover the DNA in solution. Using DNA-binding proteins for purifying and cloning promoter regions is now a well established technique, but ligating restriction fragments via blunt ends was not established at the time when this experiment was ready. As described elsewhere (21), only two transformants were obtained-one of them was saved and named M 13mp 1. Electron microscopy proved that added DNA was packaged as a filamentous phage and produced as single-stranded DNA (22,23). Now the path took a more formal shape. Not only would the histochemical screenwork by detecting a blue among colorless plaques, but

Ml3 Cloning Vehicles 1 2 3 4 5 6 ATG ACC ATG ATT AC-

7 8 TCA CTG (+ GGmAT TC CT TA AG (-

15 9 10 WlJmpl GCC GTC (+ or Viral strand) or viral strand) or complementary strand)

12 3 4 5 6 7 8 9 10 M13mp2 A CTG GCC GTC (+ or Viral ATG ACC ATG ATT ACE AAT TC EcoRI

strand)

Fig. 1. Creation of an EcoRI sue by chemical mutagenesis. By screening the nucleotides of the first ten codons of the 1ucZ gene, we found that the sequence GGATTC could erther be converted into an EcoRI site GAATTC or a BamHI sue GGATCC by a single base change. Since there already was a BumHI site in gene III, but no EcoRI site m M13mp1, and I had an ample supply of EcoRI enzyme purified myself, we decided to select the GAATTC site that also changed codon GAT for asparttc acid to AAT for asparagine (24).

it could be reversed. One uncertainty was how to introduce new restriction sites in the right region, Such a site had to be unique for M 13mp 1

and positioned not somewhere in the viral genome, but in the ZacZ region, so that insertion mutants would not give rise to a-complementation. Inspection of the sequence showed that there were not many sequences in the amino-terminal region that could be converted in a single step into a unique restriction site. Attempts to use EcoRI linkers that became available at the time to “marker rescue” them did not succeed. Without somebody to synthesize oligonucleotides that were more homologous to the Zac region, it was hopeless. As an alternative, a chemical mutagen was tried. It was known that methylated G could mispair with uracil or thymine, therefore, by methylating the single-stranded Ml3 DNA with nitrosomethylurea, a mutation could be introduced into the minus strand and the subsequent RF molecules (Fig. 1). Unfortunately, there was no good genetic selection for this procedure. It would require brute-force methods of enriching EcoRI-sensitive RF from a transformed phage library by gel electrophoresis of linear versus circular molecules. Still, it was hard to believe when this author isolated RF that was not only sensitive to EcoRI, but had exactly the predicted base change in codon 5 of the 1acZ gene (24).

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The same mutagenesis led to two more EcoRI mutants and a mutant RF that was resistant to BumHI, a site within gene III. At that time, there was still much concern that many mutations might not be tolerated because of changes in the protein sequence or the secondary structure of RNA. Therefore, changes in the lac DNA should probably occur at a higher frequency than in the viral DNA. Furthermore, the aminoterminus of the 1acZ gene appeared to be more flexible since it was demonstrated that fusion proteins retained P-galactosidase function. On the other hand, this author did not recognize the tremendous selection power for suppressor mutations. In other words, any mutation that was introduced could potentially be compensated for by another mutation somewhere else. The primary mutant might give a low titre, but because of the growth advantage a suppressor mutant would rapidly take over. An example of such a case is M 13mp 1, Later, Dotto and Zinder (25) showed that insertion mutants at the mpl Hue111 site gave a low titre phenotype: Since M13mpl gave a normal titer, they searched for a suppressor mutation. Codon 40 in gene II of M 13mp 1 was indeed changed. 5. The Need for a Universal

Primer

Researchers continue to use chemical mutagens to get rid of restriction sites. The reason for this becomes clear when they return to the use of Ml 3 in DNA sequencing. The EcoRI site in M13mp2 allowed cloning by screening for colorless plaques. Now one could readily purify these plaques and prepare a template for sequencing the inserted DNA. Still, as with the $X174 project, a restriction fragment from the adjacent Zuc DNA needed to be purified as a primer, and such a fragment was subcloned into a plasmid for convenience (26). Such a primer fragment still needed to be denatured since it was doublestranded, and had to be cut off after the sequencing reaction to produce a shift of the 3’ end in the sequencing gel. Although such a protocol could still be improved on, a more serious obstacle arose suddenly from the concern over the biological containment of Ml 3 recombinant DNA. The NIH Recombinant Advisory Committee (RAC) thought that the conjugation proficient E. coli host strains could lead to the spread of Ml3 infection and pose a risk in using Ml3 as a cloning vehicle. On the other hand, using one of the truD or tru1 mutants reduces conjugation by a factor of 106, but they

Ml3 Cloning Vehicles

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still were infectious to M13. Since this F factor carrying the traD mutation was wild-type lac, a histochemical screen with the mp vectors would not be possible, and one would have to return to drug-resistanttype M 13 vectors. The scientific reasoning of RAC is hard to understand. First, nobody argued against Agrobacterium tumefaciens as a plant transformation vector, although it was conjugation proficient and could easily spread in the environment. Second, F pili were never made under stress or anaerobic conditions, something that was already known as “phenocopies.” Conjugation in the human gut was in any case nearly zero. Third, Ml3 infection per se reduces conjugation by a factor of 106. It was hard to argue with so many well known scientists at that time, so a new seriesof E. coli strains (JM series) all carrying the Ml5 deletion on the F’ traD36 episome was constructed. Since it was a concern of NIH, it was necessary to summarize the status of all the strains for potential users in the NIH Bulletin (27). Although DNA sequencing of eukaryotic origin by Ml3 cloning was now possible, preparation of the primer from the plasmid was still cumbersome. It was clear that an oligonucleotide to replace the restriction fragment as a universal primer was needed. Inquiries regarding the synthesis of a universal primer by a commercial supplier revealed that the cost of such an attempt made it out of the question A further attempt and timely support produced success, not only with a universal primer but also with another application of oligonucleotides (3). In 1978 this author made another interesting observation, namely that in-frame insertions of linkers in the EcoRI site could still give a positive color reaction, One of these isolates, called M13mp5, could be used to clone both EcoRI and Hind111 fragments at the same site and with the same primer for sequencing (27). The utility of creating cloning sites on top of each other was based on the universal primer concept, but in turn caused the development of multiple cloning sites (MCS) or polylinkers that are now found in all cloning vehicles and provide many additional uses (Fig. 2). Therefore, work began on the synthesis of an oligonucleotide that could be inserted into the EcoRI site and generate restriction sites recognized by six basepair cutters like BamHI, AccI, SmaI, or Hind11 useful for cloning either bluntended fragments or fragments with sticky ends produced by four base cutters like Sau3A, TaqI, and HpaII (Fig. 3) (consistent with a DNA

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A

Kanr

ECORI-BamHI-SalI-PstI-SalI-BamHI-EcoRI AccI AccI HincII HincII

B

EcoRI-SmaI-BamHI-SalI-PstI-Hind111 XmaI AccI HincII

Imp8 1

HindIII-PstI-SalI-BamHI-SmaI-EcoRI AccI HincII

(mpg 1 XmaI

--->3 ’ ~xxxxxxxxxxxxxxxxxxx~ <---exoII1 GAATTCCCGGG CTTAAGGGCC<-- exoVII----> BarnHI 5

I--

GCCAAGCTT ACGEGGTTCGAA PstI

Fig. 2. Symmetric (a) and asymmetric (b) polylinkers. Two other “tricks” were used m the construction of polylmkers. One type of polylmker was symmetrical where all sites except the central one occurred twice. By cloning a drug-resistance marker into the central site, the polylinker could be used in a linker scanning method of coding regions (10) and (34). The other type of polylinker was a pair, where two vectors contain an array of sites only once, but each of them m the opposite onentation Cloned DNA no longer could be cloned out with a single enzyme as in the first type, but DNA could be cloned by using two different sites at the same time. This had the advantage that the orientation of a cloned fragment could be determined. By using a vector pair, both orientations can be obtained with the same pair of restriction cuts and therefore each strand of a restriction fragment could become the viral strand of Ml3 and available as a template for sequencing Furthermore, by using two restriction enzymes that produce 3’ and 5’ overhangs, one can either use It for cloning oligonucleotlde hbrarles or to generate umdlrectional deletions with exonuclease III

shotgun sequencing approach). This also required a renewed chemical mutagenesis to eliminate the AccI and the HincII sites naturally occurring in M13. All single mutations were combined by marker rescue to give rise to M13mp7 (3). This was just the system, but did shotgun sequencing succeed?

Ml3 Cloning Vehicles

Unique

vector

sequence

19

Compatible

target

G'GATC C C CTAG'G

N'GATC N N CTAG'N MYbOT

GT'CG AC CA GC'TG

NT’CG AN

sites

NA GC'TN NC'CG

GN

NG GC'CN GTC'GAC CAG'CTG HincII

NNN'NNN NNN'NNN Restrlction

enzymes

AALL, HaeIII,

like

etc.;

Ba.UlorExoIIIIV; sheared

and repaired

DNA

Fig. 3. Sticky and blunt-end cloning of small fragments mto unique cloning sites. By destgning a unique sequence for the M13mp vectors that were recogmzed by restriction enzymes that could cut a hexanucleotide sequence in various ways by etther producing sticky ends of 4 or 2 bases, or blunt ends, the variety of DNA fragments that could be cloned next to the umversal primers were endless Note that the sequence GTCGAC was recognized by SufI, AccI, and H&II, each producing different ends. In our sequencing project wtth cauliflower mosatc virus we generated small DNA fragments for shotgun DNA sequencing by cleaving CAMV with EcoRI*, Mb&, HpaII, TagI, HincII, HueIII, and A/u1 (28) Later we used DNaseI (35), sonication (30), and a combination of exonuclease III and VII to generate blunt ends (36)

Initially, lack of funds and proper laboratory facilities made a demonstration impossible. Financial support finally arrived, however, and a dedicated research team began producing the sequence of cauliflower mosaic virus on the side (8031 bp). This was accomplished in a record time of three months, and the results were finally published a year later (28).

20

Messing

6. Conclusion During the same time, several researchers reported that they could not finish their work on sequencing the mitochondrial DNA if they could not use a host for Ml3 approved by the NIH guidelines. This author gave them not only the JM strains, but also the newly developed M13mp7 for blunt-end cloning to facilitate their work. Although this author’s work was not published, having been rejected by PNAS as trivial at the time, in the end, I recognized that there was no sense in competing but that the scientific community should be used as a laboratory at large, and this indeed became reality (29). Becoming overwhelmed by requests for strains and protocols, the newly developing reagent companies were turned to for help. Their educational and service role helped immensely to disseminate the knowledge needed to train students and investigators in academia and industry in M 13 cloning, sequencing, and site-directed mutagenesis (30). Along the way, the first Apple-based software on shotgun sequencing was developed (31,32), in addition to a textbook for an undergraduate course (33). A good overview of M13mp, pUC vectors, and helper phage has also appeared (21). As with all methods of wider scope, modifications and refinements have been produced in many laboratories as many chapters in this book show. However, some principles have not changed: whenever there is a way to use a cell or parts of one as a machine, scientists seem to get ahead faster and cheaper, and that is what biotechnology is all about. Acknowledgment Most of this author’s work on Ml3 was supported by the Deutsche Forschungs Gemeinschaft, grant no. 5901-9-0386, and the Departments of Agriculture and Energy, grant no. AC0243 lER10901. References 1. Saiki, R. K., Gelfand, D. H., Stoffel, S , Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Ehrlich, H A. (1988) Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239,487- 491 2. Scott, J. and Smith, G. (1990) Searchmg for peptIde ligands with an epitope library. Science 249,386 -390. 3. Messing, J., Crea, R., and Seeburg, P. H. (1981) A system for shotgun DNA sequencing. Nucl. Acids Res. 9, 309-321. 4. Norrander, J., Kempe, T., and Messing, J (1983) Improved Ml3 vectors using oligonucleotide-directed mutagenesis. Gene 26, 101-106.

Ml3 Cloning Vehicles

21

5. Edgell, M. H., Hutchison, C. A., III, and Sclair, M. (1972) Specific endonuclease R fragments of bacteriophage $X174 deoxyribonucleic acid. J. Viral. 9,574-582 6 Beaucage, S. L. and Caruthers, M H. (1981) Deoxynucleoside phosphoramidites-a new class of key Intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett. 22, 1859-l 862. 7. Hutchison, C. A., III, Phtlhps, S., Edgell, M. H., Gillam, S., Jahnke, P., and Smith, M (1978) Mutagenesis at a specific position in a DNA sequence. J. Biol. Chem. 253,655 l-6560. 8. Smith, M , Leung, D W , Gillam, S., Astell, C. R , Montgomery, D. L., and Hall, B D (1979) Sequence of the gene for iso-1-cytochrome C in Saccharomyces cerevisiae. Cell 16, 753-761. 9 Zoller, M and Smith, M. (1982) Oligonucleotide-directed mutagenesis using M13-derived vectors. an efficient and general procedure for the production of point mutations in any fragment of DNA. Nucl. Acids Res. 10,6487-6500 IO. Vieira, J. and Messing, J (1982) The pUC plasmtds, an M 13mp7 derived system for msertion mutagenesis and sequencing with synthetic universal prtmers Gene 19,259-268. 11 Sanger, F , Donelson, J E., Coulson, A. R., Kossel, H., and Fischer, H (1973) Use of DNA polymerase I primed by a synthetic ohgonucleotide to determine a nucleotide sequence in phage f 1 DNA. Proc. Nat1 Acad. Ser. USA 70,1209-l 2 13 12. Hofschneider, P H (1963) Untersuchungen uber “kleine” E coli K12 Bacteriophagen M12, M13, und M20. Z. Naturforschg. Mb, 203-205. 13. Davison, A. J. (1991) Experience in shotgun sequencing a 134 kilobase pair DNA molecule DNA Sequence 1,389-394 14 Bolivar, F , Rodriguez, R. L., Greene, P. J., Betlach, M. V., Heynecker, H L , Boyer, H. W , Crosa, J. W , and Flakow, S (1977) Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2,95-l 13 15. Herrmann, R., Neugebauer, K., Zentgraf, H., and Schaller, H. (1978) Transposition of a DNA sequence determining kanamycin resistance into the singlestranded genome of bacteriophage fd MOE Gen. Genet. 159, 17 1. 16 Vovis, G F. and Ohsumi, M. (1978) The filamentous phages as transducing particles, m The Single-Stranded DNA Phages (Denhardt, D. T , Dressler, D., and Ray, D S , eds ), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 445-448 17 Salivar, W. O., Henry, T. J., and Pratt, D. (1967) Purtfication and properties of diploid particles of coliphage M13. Virology, 32,41-5 1 18. Mandel, M. and Higa, A (1970) Calcium-dependent bacteriophage DNA mfection. J. Mol Blol., 53, 159-162. 19 Malamy, M H., Fiandt, M , and Szybalski, W. (1972) Electron microscopy of polar insertions in the lac operon of Escherichia co11 Mol. Gen Genet 119, 207-222 20 Landy, A , Olchowski, E , and Ross, W. (1974) Isolation of a functional lac regulatory region. Mol Gen Gene& 133,273-28 1. 2 1. Messing, J. (1991) Clonmg m Ml 3 phage or how to use biology at its best Gene 100,3-12

22

Messing

22 Messing, J , Gronenborn, B., Muller-Hill, B., and Hofschnetder, P H. (1977) Frlamentous cohphage Ml3 as a cloning vehtcle insertion of a HrndII fragment of the fat regulatory region m the Ml3 rephcattve form in vitro Pruc Natl. Acad. SCL USA 74,3642-3646. 23. Messing, J. and Gronenborn, B (1978) The filamentous phage Ml3 as carrter DNA for operon fusions in vitro, m The Single-Stranded DNA Phages (Denhardt, D T., Dressler, D., and Ray, D. S., eds.), Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 449-453. 24 Gronenborn, B. and Messing, J. (1978) Methylatron of single-stranded DNA m vttro mtroduces new restrtctrons endonuclease cleavage sites. Nature 272, 375-377 25 Dotto, G. P. and Zmder, N D (1984) Reduction of the mmimal sequence for mmatton of DNA synthesis by qualitative and quantttatrve changes of an mltrator protein Nature 311,279-280 26. Heidecker, G., Messmg, J , and Gronenborn, B. (1980) A versatile prrmer for DNA sequencing m the M13mp2 clonmg system Gene 10,69-73. 27 Messing, J (1979) A multrpurpose cloning system based on the single-stranded DNA bacteriophage M13. Recombinant DNA Technical Bulletin, NIH Publtcanon No. 79-99,2, No. 2,43-48 28 Gardner, R. C., Howarth, A J., Hahn, P 0, Brown-Leudi, M , Shepherd, R J., and Messmg, J. (198 1) The complete nucleotide sequence of an mfectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencmg. Nucl Acrds Res. 9,2871-2888 29 Holden, C (1991) Briefings Science 254,28 30. Messing, J (1983) New Ml3 vectors for clonmg. Methods Enzymol. 101,20-78 31 Larson, R. and Messing, J. (1982) Apple II software for Ml3 shotgun DNA sequencmg Nucl Acids Res 10, 39-49 32 Larson, R. and Messing, J. (1983) Apple II computer software for DNA and protean sequence data DNA 2,3 l-35. 33 Hackett, P. H., Fuchs, J A , and Messing, J (1984) An introduction to recombmant DNA techniques Basic Experiments in Gene Manzpulution BenjaminCummings, Menlo Park, CA 34 Messing, J., Vieira, J , and Gardner, R (1982) Codon insertion mutagenesis to study functional domains of P-lactamase In vrtro mutagenesis Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp 52 35 Messmg, J. and Seeburg, P H (1981) A strategy for hrgh speed DNA sequencing, in Developmental Biology Using Purified Genes (Brown, D. and Fox, F , eds ), ICN-UCLA Symposia on Molecular and Cellular Biology, vol 23 Academic, NY, pp 659-669. 36 Yanisch-Perron, C , Vieira, J , and Messing, J (1985) Improved Ml3 phage cloning vectors and host strains, nucleottde sequences of the M 13mp and pUC vectors Gene 33,103-l 19

hAP!l’ER

Cloning

3

into Ml3

Qingzhong

Yu

1. Introduction The bacteriophage M 13 has been developed into a cloning vector system for obtaining single-stranded DNA template required for the dideoxy chain termination method of sequencing DNA (1,2). General aspects of bacteriophage Ml3 as a cloning vector system are reviewed in Chapter 2, and the preparation of foreign DNA fragments for Ml3 cloning is described in Chapter 7. In this chapter the preparation of Ml3 vectors and the ligation of foreign DNA fragments (inserts) into Ml3 vectors are described. 2. Materials 2.1. Preparation

of Replicative

Form

(RF) Ml3

DNA

1. L-Broth: Bacto-tryptone l%, bacto-yeast extract O.S%,NaCl 1%. 2. 2X YT: Bacto-tryptone 1.6%, bacto-yeast extract 1%, NaClO.5%. 3. M9 minimal medium: Na,HFQ, . 7H20 12.8 g, IU-12P043 g, NaC10.5 g, NH&l 1.0 g, 20% glucose 20 mL, Hz0 to 1 L. 4. BacteriophageM13: A single blue plaque from a freshly transformed plate. 5. E. coli JM 103 or JM 109: A colony grown on an M9 minimal agar plate. 6. Solution 1: 50 mII4 Glucose, 25 mM Tris-HCI, pH 8.0, 10 rniI4 EDTA, pH 8.0, autoclaved and stored at 4°C. From. Methods m Molecular Brology, Vol 23. DNA Sequencmg Protocols E&ted by- H. and A Gnffm Copyright 01993 Humana Press Inc., Totowa,

23

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24

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7. Solution 2: 0.2M NaOH, 1% SDS, freshly mix together equal volumes of 0.4M NaOH and 2% SDS stocks before use. 8. Solution 3: 60 mL 5M Potassium acetate, 11.5 mL glacial acetic acid, 28.5 mL H20, stored at 4OC. 9. TE: 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, pH 8.0. 10. Phenol-chloroform: Mix well equal volumes of TE (pH 8.0)-saturated phenol (nucleic actd grade) and chloroform (AR grade), stored at 4°C m a dark glass bottle. 11. 3M NaAc, pH 5.2, stored at 4°C. 12. Ethanol: Absolute alcohol, stored at -20°C. 13. RNase: 1 mg/mL DNase-free pancreatic RNase A. 2.2. Preparation of Ml3 Vectors 1. Restriction enzymes. 2. 10X RE buffers. 3. Calf intestinal alkaline phosphatase (CIP). 4. 10X CIP dephosphorylation buffer. 5. Proteinase K 10 mg/mL. 6. 10% SDS. ’ 7. 0.5M EDTA, pH 8.0. 8. 3M NaAc, pH 7.0, stored at 4°C. 9. 10X TBE: Tns base108 g, Boric acid 55 g, EDTA 92H20 9.5 g, HZ0 to 1 L. 10. 0.8% Agarose gel in 1X TBE. 2.3. Ligation of Inserts into Ml3 Vectors 1. T4 DNA ligase. 2. 10X Ligation buffer. 3. 10 mM ATP, stored at -20°C. 4. Ml3 vectors (prepared as described in the methods section). 5. Inserts (having termini compatible with the vectors).

All enzymes and buffers are stored at -20°C. Other solutions can be stored at room temperature except when indicated otherwise. 3. Methods

A number of M 13 vectors have been constructed (.2,3) and are commercially available from several companies. Therefore, it would be more convenient to purchase the Ml3 vectors than to prepare them oneself. Sometimes, however, you may need a Ml3 vector with a special cloning site to fit your cloning strategy. Thus the preparation

of Ml3 vectors is described below.

Cloning

25

into Ml3

3.1. Mini

Preparation

of

RF Ml3 DNA

1. Inoculate 5 mL of L-Broth in a 20-mL sterile culture tube (e.g., universal) with one bacterial colony (e.g., JM103 or JM109) from an M9 minimal agar plate. Incubate at 37OCin an orbital shaking incubator overnight. 2. Add 50 l,tL of the bacteria culture to 2 mL of L-Broth in a 5-mL culture tube (e.g., bijoux). Inoculate this culture with Ml3 bacteriophage by touching a single blue plaque from a transformatton plate with a sterile toothpick and washing its end m the culture. Incubate the infected culture at 37°C for 4-5 h m an orbital shaking incubator. 3. Transfer 1.0-l .5 mL of the culture to a microfuge tube and centrifuge at 12,OOOgfor 2 min at room temperaturein a microfuge. Remove supernatant to a fresh tube, being careful not to disturb the pellet. If desired, the single strand Ml3 (single-stranded) DNA can be prepared from the supernatant. 4. Remove any remaining supernatant by aspiration from the tube containing the bacterial pellet. Resuspend the pellet by ptpeting it or vigorous vortexing in 100 pL of solution 1 and leave at room temperature for 5-10 mm. 5. Add 200 cls,of freshly prepared solution 2. Close the tube and mix the contents by mvertmg the tube rapidly five times. Do not vortex. Store the tube on ice for 5 mm. 6. Add 150 pL of ice-cold solution 3. Vortex the tube gently m an inverted position for 10 s. Store on ice for 5 min. 7. Centrifuge at 12,OOOgfor 5 min and transfer the supernatant to a fresh tube. 8. Add an equal vol of phenol:chloroform, mix by vortexing for 20-30 s. Spin as in step 7 and transfer the aqueous phase (top layer) to a fresh tube. 9. Add 2 vol of ethanol, mix by vortexing, and stand for 5 min at room temperature. 10. Spin as m step 7 and remove the supernatant by gentle asptratton. 11, Wash the pellet with 1 mL of 70% ethanol. Spin for 2 mm m the same orientation of the pellet. Remove the supernatant as m step 10. Vacuum dry for 3-5 mm or air-dry for 10 min. 12. Dissolve the pellet m 20 pL of TE (pH 8.0) contammg RNase (20 pg/ mL) to remove RNA. Vortex briefly. The double-stranded RF Ml 3 DNA is now ready for analysis by digestion with restriction enzymes.

3.2. Preparation

of

Ml3 Vectors

3.2.1 Digestion with a Single Restriction Enzyme (RE) 1. Digest RF Ml3 DNA with a single RE in the followmg reaction: RF Ml3 DNA 10 $L (400 ng) Restriction enzyme 3-5-fold excess 10X appropriate buffer 2 pL make up to 20 pL total vol H2O

26

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Incubate for 2 h. Remove 2 pL of the reaction and analyze the extent of digestion by electrophoresis through 0.8% agarose gel, using undigested Ml3 DNA as a marker. If digestion is mcomplete, add more RE and continue the incubation. 2. When digestion is complete, extract the Ml3 DNA with phenol:chloroform and precipitate DNA with 0.1 vol of 3M NaAc, pH 5.2, and 2.5 vol of ethanol for 1 h at -20°C or overnight if convenient. 3. Recover the DNA by centrifugation at 12,OOOgfor 10 min in a microfuge. Wash the pellet with 70% ethanol and vacuum dry. Dissolve the pellet in 20 p.L of TE, pH 8.0. Now it is ready to use for ligation with inserts if dephosphorylatton is not required. 3.2.2. Dephosphorylation

of Ml3

Vectors

4. The single RE-dtgested Ml3 vector can be dephosphorylated by treatment with calf intestinal alkaline phosphatase (CIP) to reduce the background of nonrecombinant molecules formed by circularization of the vector during ligation. To 20 pL of linearized Ml3 vector obtained in step 3, add: 10X CIP buffer 5 pL CIP 1 U/100 pmoles for protruding 5’ termmt 1 U/2 pmoles for blunt or recessed termim make up to 50 pL total vol H20 5. Incubate for 30 min at 37OCfor protruding 5’ termini vectors. For blunt or recessedterminus vectors, incubate for 15 min at 37”C, then add the same amount of CIP and continue incubation for a further of 45 min at 55°C. 6. At the end of the incubation period, add the following to the reaction: 10% SDS 2.5 pL (final concentration 0.5 %) 0.5 pL (final concentration 5 n-&f) 0.5M EDTA (pH 8.0) 10 mg/mL proteinase K 0.5 pL (final concentration 100 Clg/mL) Incubate for 30 min at 56OCto remove the CIP. 7. Cool the reaction to room temperature, and extract with phenol:chloroform twice. Add 0.1 vol of 3M NaAc, pH 7.0, mix well and add 2.5 vol of ethanol. Mix and store at 20°C for 1 h or overnight. Recover the DNA as in step 3. The final pellet is dissolved in 20 pL of TE, pH 8.0. Now it is ready to use for ligation with inserts. 3.2.3.

Digestion

with

Two Restriction

Enzymes

8. If a different RE digestion is required for generating a Ml3 vector with incompatible termini (forced dtrectional cloning vector), the DNA recovered in step 3 above is dissolved in 10 pL of TE (pH 8.0) and steps l-3 are repeated with the second RE. To check the extent of the digestion by the second RE, set up a separate RE digestion reaction with the sec-

Cloning into Ml3

27

ond RE in the same ratio of RF Ml 3 DNA and the enzyme. Analyze the digestion by electrophoresis as m step 1. 9. If two different enzymes require the same buffer condition, the RF Ml3 DNA can be digested simultaneously with both REs. To momtor the extent of the digestion, set up two reaction mixtures with the two REs separately as controls. After the RF Ml3 DNA has been completely digested, repeat steps 2-3. The final pellet is dissolved in 20 pL of TE, pH 8.0. It is ready to use for ligation with inserts. 10. For vectors digested with two different REs it is not necessary to dephosphorylate to reduce the background. But, to avoid religation of the small fragment generated by RE digestion of the polycloning sites into the M 13 vectors, it can be removed by electrophoresis through agarose gel or polyacrylamide gel (refer to Chapter 10 in Volume 2 of this series). 3.3. Ligation of Inserts into Ml3 Vectors 1. Set up ligation reaction m the followmg order: Ml 3 vector 1 r-1L(20 w) 10 mM ATP lc1L 10X ligation buffer 1 pL inserts l-4 pL (3-5-fold molar excess) T4 DNA ligase 5 U for blunt termini 1 U for cohesive termmi make up to 10 pL total vol H2O Incubate at 14OC overnight. Then store at -2OOC or use directly for transformation. 2. Set up two control reactions contammg the same components except that in the place of the mserts, be sure that one control contains H,O, and the other contains an appropriate amount of a test DNA that has been successfully cloned mto the Ml3 vector on a previous occasion. Incubate under the same conditions as m step 1. 3. After ligation, analyze 1 pL of each ligation reaction, using the same amounts of the Ml3 vector and inserts without ligase as control, by electrophoresis through 0.8% agarose gel to check that the ligation has been successful. 4. Transform 5 w of the remaining ligation sample mto competent bacteria of the appropriate strain of E. coli, e.g., JM103 or JM109. 4. Notes 1 Bacteria carrying an F’ episome (e.g., JM103 or JM109) can grow m M9 muumal medium, but much more slowly than m L-Broth and do not survive prolonged storage at 4°C. It is, therefore, better to streak a master culture of the bacteria on the M9 minimal agar plate every month

28

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The master culture bacteria should be stored at -70°C in L-Broth containing 15% glycerol. 2. Bacteriophage Ml3 can drffuse a considerable distance through the top agar on the transformation plate. Therefore, it is important to pick up a single blue plaque that is well-separated from its neighbors to avoid crosscontamination with other plaques. And it is also better to pick up a single blue plaque from a freshly transformed plate to get a good yreld of RF Ml3 DNA. 3. Since RF Ml3 DNA remains Inside infected bacteria and Ml3 phage progeny containing single-stranded M 13 DNA is released to the medium, it is important to remove any remaining supernatant from infected bacterial pellet in step 4 of the mmipreparation of RF Ml3 DNA to mmimaze single-stranded Ml3 DNA contammation. Even so, sometimes the mmiprepared RF Ml3 DNA is still contaminated with singlestranded Ml3 DNA, which may confuse the pattern of DNA fragments obtained by RE digestion. However, it can be distinguished from the RF Ml 3 DNA by analyzing RE-digested and undigested RF Ml 3 DNA by electrophoresis through the same agarose gel. Because the singlestranded M 13 DNA cannot be digested by REs commonly used in M 13 cloning, Its migration through the gel does not change after RE drgestion, whereas the digested RF Ml3 DNA will change its migration through the gel. 4. When recovermg Ml 3 DNA after centrifugation of precipitated samples, sometimes the Ml3 DNA pellet is not visible. Therefore, it is necessary to carefully remove the supernatant and leave a little bit (20-30 l.tL) behind in the tube to minimize the loss of the DNA. Do likewise when washing with 70% ethanol, then vacuum dry. 5. To remove CIP which may affect subsequent ligation of Ml3 vectors with inserts after dephosphorylation, an alternative method is to mactivate the CIP by heating at 70°C for 10 min in the presence of 5 mM of EDTA, pH 8.0, and then extract with phenol:chloroform. Because EDTA precipitates from solution at acid pH if its concentration exceed 5-10 mM, the 3M NaAc, pH 7.0, is used instead of the commonly used 3M NaAc, pH 5.2, for precipitation of dephosphorylated Ml 3 DNA. 6. The efficiency of ligation of blunt termini is somewhat lower than for ligation of cohesive termini. To improve the efficrency of the ligation of blunt termmi, higher concentration of inserts and T4 DNA hgase is required. The extent of ligation of Ml3 vectors with inserts can be checked by electrophoresis through 0.8% agarose gel, using unhgated Ml3 vectors and inserts as control. Normally, the ligated Ml3 vectors with inserts will mrgrate slowly through the gel because of the increased

Cloning into Ml3

29

molecular weight, sometimes glvmg fuzzy or smeared bands which mdlcates the extent of the ligation.

Acknowledgment The author wishes to thank D. Cavanagh for advice in the preparation of the manuscript.

References 1 Messmg, J. (1983) New Ml3 vectors for cloning Meth Enzymol. 101,20-79 2. Sanger,F., Nicklen, S , and Coulson, A. R. (1977) DNA sequencmg with chamterminating mhlbltors Proc. Nat1 Acad. Sci. USA 74, 5463-5467 3 Yamsch-Perron, C , Vlelra, J., and Messing, J (1985) Improved M 13 phage cloning vectors and host strains: nucleotlde sequences of the M13mp18 and pUC19 vectors. Gene 33,103-l 19.

&IAFTER

Transfection

4

of E. coli with

Ml3 DNA

Fiona M. lbmley

1. Introduction In recent years several techniques have been described for the introduction of DNA molecules into strains of E. coli by transformation or transfection. These are based on the findings of Mandel and Higa (I) who demonstrated that incubation of cells with naked bacteriophage h DNA in cold calcium chloride resulted in uptake of virus and that a transient heat-shock of the mixture greatly enhancedthe efficiency of transfection. Subsequently, this method was used to introduce a variety of circular and linear DNAs into strains of E. coli, and many variations in the technique have been described that are aimed at increasing the yield of transformants or transfectants. The central requirements for success are the presence of multivalent cations, an incubation temperature close to O”C, and a carefully controlled heatshock at 42OC,but a number of other compounds and procedures have been found to increase efficiency in some or all strains of E. coli that have been tested. The mechanisms involved in DNA transformation of cells are not fully understood and even with the most efficient methods that are available the proportion of cells that become “competent” for transformation is limited to around 10% of the total population. The procedures for Ml3 transfection of E. coli cells are simple, requiring the establishment of a log phase bacterial culture, the prepaFrom Methods m Molecular Bfology, Vol. 23 DNA Sequenong Protocols Edlted by Ii. and A Gnffm Copyright 01993 Humana Press Inc , Totowa,

31

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Tomley

ration of competent cells and the introduction of DNA into these cells, Fresh cells can be prepared for each transfection or, alternatively, frozen aliquots of competent cells can be stored at -70°C and thawed as required. This chapter includes two alternative procedures for preparing competent cells, one based on the original Calcium method and a second based on that of Hanahan (2). 2. Materials 1. An E, co11 strain smtable for propagating bacteriophage Ml 3 vectors. Recommended strains include JMlOl, JM103, JM107, JM109, TGl, and TG2 (see Note 1). 2. Bacterial growth media: Sterilize by autoclaving in suitable ahquots a. L-broth: 1% tryptone, 0.5% yeast extract, 200 mM NaCl. b. SOB-broth: 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, pH 7.0. Just before use, add Mg2+ to 20 mM from a sterile ftltered stock of 1M MgC12, 1M MgSO+ c. H-agar: 1% tryptone, 140 rniV NaCl, 1.2% bacto-agar. To pour plates, melt agar by boiling or microwavmg, cool to around 50°C and pour mto Petri-dishes on a level surface (15-20 ml/plate). Once agar has set, dry plates by storing inverted at 37°C for several hours before use. d. H-top agar: 1% tryptone, 140 mM NaCl, 0.8% bacto-agar. Melt agar as above and hold at 48OC m a waterbath until needed. 3. Sterile, detergent-free 1-L conical flasks fitted with porous tops or cotton-wool bungs and an orbital incubator capable of shaking these vigorously (around 250 rpm) at 37°C. 4. 50-mL polypropylene tubes (e.g., disposable Falcon 2070 or reusable Oakridge, sterile and detergent-free), and a centrifuge, preferably refrigerated, capable of spinning these tubes at 4,000g. 5. Transformation buffers (see Note 2): a. 100 mM Calcium chloride: Store 1M CaC12 at -20°C m 1.5-mL aliquots. When needed, thaw, dilute to 15 mL, filter, and chill on ice. b. TFB: 10 mM K-MES, 100 mM RbCl or KCl, 45 mMMnC12. 4H20, 10 mM CaC12. 2H20, 3 mM hexamminecobalt chloride. Make up a 1M stock of MES, adJust the pH to 6.3 with 5M KOH, and store at -20°C m IO-mL ahquots. To make up 1 L of TFB, use one 10 mL aliquot of IM K-MES, add all the other salts as solids, filter, and store m 15mL aliquots at 4OC, where tt is stable for over a year. It is important that the final pH of the buffer is 6.15 f 0.1. c. FSB: 10 mM potassium acetate, 100 rniV KCl, 45 mM MnC12 . 4H20, 10 mM CaCl, . 2H20, 3 rruV hexammmecobalt chloride, 10% glyc-

Transfection

of E. coli Cells

33

erol. Make up a 1M stock of potassium acetate, adjust the pH to 7.2 with 2M acetic acid, and store at -20°C in lo-mL ahquots. To make up 1 L of FSB, use one lo-mL aliquot of 1M potassium acetate, add the other salts as solids, glycerol to lo%, adjust the pH to 6.4 with 0.W HCl (N.B. if you add too much, do not attempt to readjust the pH with base: discard the batch and start again), filter, and store in 15mL aliquots at 4°C. During storage, the pH of this buffer drifts down to 6.1-6.2 and then stabilizes. d. DMSO/DTT (see Note 3): Make up 1M potassium acetate as in step 5c, above, and store in 100~& aliquots. Take a fresh bottle of highest grade DMSO, divide into IO-mL aliquots and store m sterile, tightly capped tubes at -7OOC. To make up 10 mL of DMSO/DTI’, use one lOO+L aliquot of 1M potassium acetate, 9 mL DMSO, 1.53 g of DTT, sterilize through a filter that will withstand organic solvents (e.g., Millex SR, millipore), and store in 300-w aliquots at -20°C. 6. Fresh cells/X-gal/IPTG: Make this up just before you need it, calculating the volumes required for your total number of samples. Per sample, take 200 pL of fresh E. coli cells and gently add 40 pL of X-gal (20 mg/ mL m dimethylformamide) and 40 p.L of IPTG (24 mg/mL in water). 7. Sterile, disposable 5-mL tubes (e.g., Falcon 2054).

3. Methods All the methods relate to the preparation of competent cells from a 50 mL log-phase culture. 3.1. Preparation

of Log Phase Cells

1. Pick a single bacterial colony from a fresh plate, or take 500 pL of a fresh overnight culture, and transfer into 50 mL of L-broth (for Calcium method) or SOB-broth (for Hanahan method) in a conical flask. Set up duplicates, one for preparing competent cells and the other for fresh plating cells that are required in step 3 of Section 3.4. 2. Incubate flasks at 37OCwith vigorous shaking until the cell concentration reaches around 5 x 107/mL. For most E. coli strains this means an Abe,,of between 0.3 and 0.4, and will take around 3 h incubation from a single colony, around 2 h from an overnight culture. 3. Transfer the contents of one flask to a sterile, precooled, 50-mL tube and store on ice for 10 mm to cool the cells. 4. Pellet cells by spinning at 4000g for 10 min and carefully pour off the broth, mverting the tube to drain away the last traces. Choose either method .2 or .3 for making competent cells, with all steps carried out aseptically.

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3.2. Fresh or Frozen Competent Using Calcium Chloride

Cells

1. Suspend cells in 10 mL of cold O.lM CaCl*, store on ice for 5 min, then pellet as in step 4 of Section 3.1. 2. Suspend cells gently in 2.5 mL of cold O.lM CaClz and incubate on ice until ready for use. At this point cells can be dispensed m small aliquots into cooled sterile 1.5-mL tubes, snap-frozen in liquid nitrogen, and stored at -70°C until needed, when they should be thawed rapidly then immediately stored on ice.

3.3. Fresh or Frozen Competent Using Hanahan Method

Cells

1. Suspend cells in 10 mL of cold TFB (for fresh) or FSB (for freezing), store on ice for 10 mm then pellet as m step 4 of Sectton 3.1, 2. Suspend cells gently in 4 mL of cold TFB or FSB and store on me. 3. Add 140 pL of DMSO/DTT (for fresh) or DMSO alone (for freezing), mix immediately by gentle swirling, and store cells on ice for 15 min. 4. Add a further 140 pL of DMSO/DTT or DMSO, and mix immediately. Fresh cells should be stored on ice for at least 15 min then dispensed m small aliquots mto cooled 5-mL tubes. For freezing cells, proceed with step 5, below. 5. Dispense cells in small aliquots into cooled 5-mL tubes, snap-freeze m liquid nitrogen, and store at -70°C until needed. Thaw ahquots rapidly, then store cells on ice for 15 mm prior to use.

3.4. Introduction of Ml3 DNA to Competent and Generation of Progeny Plaques

Cells

1. Incubate tubes of competent cells m an rcewater bath. For most purposes 50-pL aliquots of cells will generate sufficient plaques, but more may be used if very high numbers are required (see Notes 4 and 5). 2. Carefully add 5 pL of an Ml3 ligation containing approximately 40400 ng total DNA to each ahquot and swirl gently (see Note 6). Leave m the icewater bath for 30-45 min. In addition to the ligations, two transformation controls should be included, one containing 5-10 pg of ds circular Ml3 DNA, and one containmg no DNA. 3. Meanwhile, melt sufficient H-top agar (3 ml/plate, plus a few mL excess) and keep It m a waterbath at 48OC. Just before moving on to step 4, below, take the second flask of cells (from step 1 of Section 3.1.) and make up sufficrent fresh cells/X-gal/IPTG mix (280 pL/plate). 4. Transfer the tubes of competent cells/ligations to a rack in a waterbath, preheated to 42°C and leave for exactly 90 s without shaking (see Note

Transfection

of E. coli Cells

7). Transfer rapidly back into the icewater bath and make up to 200 pL with SOB-broth (minus MgZ2+). If you think you are going to get too many plaques, then split the samples at this point, e.g., leave a 180 pL in the first tube and transfer 20 pL to a second. 5. Add 280 pL of fresh cells/X-gal/IPTG mix to each sample then, working one sample at a time, add 3 mL of H-top agar, mrx quickly, and pour onto a dry H-agar plate. Be careful not to mtroduce air bubbles. 6. Allow plates to dry thoroughly then incubate overnight at 37°C (see Note 8). Plaques formed by wild-type Ml 3 will be blue, and those formed by recombinants will be white (see Note 4).

4. Notes 1. Host bacteria can be stored for short periods at 4°C on mmtmal (M9) agar, which maintains the F’episome, but should be replated frequently from a master stock stored at -7OOC in L-broth plus 15% glycerol. 2. For making up and storing all transformation buffers: use only high quality pure water, e.g., Milli-Q or equivalent; use sterile, detergent-free glassware or plasticware; 9 sterilize solutrons by filtration through 0.45pm pore filters, e g., Nalgene or Acrodisc. 3. In the original Hanahan method, DMSO and DTT solutions were added sequentially. However, the efficiency of transformation is just as high rf they are added together (3). 4. The number of plaques that are obtained from each transfection varies enormously and is dependent on many factors, such as the amount of DNA added, the ratio of insert to vector in the ligation reaction, the efficiency of ligation, and the efficiency of transformatron. As a guide, closed circular Ml3 DNA should give over lo7 plaques&g using the Hanahan method thus the control transfected with 10 pg of ds Ml3 DNA should yield over 100 plaques. Vector DNA that has been linearized then religated to an excess of Insert with compatible ends will give anything from 102-lo4 less plaques per pg, depending on the religation efficiency, thus, each test transfection of 40-400 ng may give between 40 and 40,000 plaques. For the Calcium method, the numbers of plaques are around 2-lo-fold lower. Freshly prepared Calcium-treated cells can be stored for up to 48 h in CaCl, at 4OC and the efficiency of transfection increases up to six-fold over the first 24 h then declines to the original level (4). 5. The efficiency of transformation using frozen competent cells is reduced compared to freshly prepared cells but, unless very high numbers of plaques are required, frozen cells are adequate and very convenient, l

l

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6. The volume of ligated DNA added to the cells should not exceed 10% of the total volume. 7. The length of time for heat-shock is calibrated for Falcon 2054 tubes and for others the heat-up time may be different. 8. It is important that agar plates are thoroughly dried before top agar 1s poured onto them.

References 1 Mandel M. and Higa A (1970) Calcium-dependent bacteriophage DNA mfection J. Mel Biol. 53, 109-118. 2 Hanahan D. (1983) Studies on transformation of Escher&ia coli with plasmids J Mol. Blol 166,557-580. 3 Sambrook J , Fritsch E. F , and Maniatrs T (1989) ‘Preparatron and Transformation of competent E coli,’ m Molecular Cloning, A Laboratory Manual, 2nd ed , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 1 74-1.84 4 Dagert M and Ehrlich S D (1979) Prolonged incubation m calcium chloride improves the competence of Escherichia colr cells Gene 6,23-28

Ml3 Phage Growth and Single-Strand DNA Preparation

1. Introduction Ml3 phages do not lyse their host, but are released from infected cells as the cells continue to grow and divide. Cells infected with Ml3 have a longer replication cycle than uninfected cells, and as the infection proceeds, areas of slower-growing cells can be visualized as turbid plaques on lawns of E. coli (1). Well-separated plaques contain cells infected with phages derived from a single transformation event and these can be picked and regrown to provide pure stocks of recombinant phage particles and DNA. During infection of its host cell, the single-stranded DNA (ssDNA) of the phage is converted and amplified into double stranded replicative forms (RF) that are intermediates in the production of progeny ssDNA. These ssDNA molecules are packaged into a protein coat and extruded out of the cell and, since there is no size constraint on packaging, recombinants containing foreign DNA are readily produced. From liquid culture, packaged particles are readily recoverable from the broth and the ssDNA can be rapidly extracted (2,3). The method is straightforward, and because Ml3 phages replicate rapidly, both phage growth and DNA purification can be carried out in one day provided that a fresh overnight culture of host bacteria is available. From* Methods m Molecular Slology, Vol. 23: DNA Sequencrng Protocols E&ted by. H. and A. Griffin Copynght 01993 Humana Press Inc., Totowa,

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2. Materials 1. An E. coli strain suitable for propagating bacteriophage Ml3 vectors Recommended strains mclude JMlOl, JM103, JM107, JM109, TGI, and TG2. 2. 2X TY broth: 1% tryptone, 1% yeast, 100 mM NaCl. Sterthze by autoclaving in 50-mL aliquots. 3. Stertle culture tubes for growmg up 1.5 mL cultures (e.g., disposable lo-mL Falcon tubes or glass/plastic universal bottles) and an orbital incubator for shaking the tubes vigorously at 37OC. 4. A mrcrofuge and 1S-mL mrcrofuge tubes. 5. Solutions a. PEG/NaCl: 20% PEG, 2.5M NaCl. Make up 100 mL at a trme and sterilize by ftltration. Store at 4°C. b. TE: 10 mA4Tris-HCl, 1 mIt4 EDTA (pH 8.0). For convenience, store 1M Tris-HCl, 100 mM EDTA, pH 8.0, m I-mL aliquots at -20°C. When needed, thaw, dilute to 100 mL, check the pH, filter, and store at room temperature. c. Phenol: Use high-grade redistilled phenol stored at -2OOC in aliquots of 100 -500 mL. To equtlibrate, warm to room temperature, then melt at 65°C and add hydroxyquinoline to 0.1%. To the melted phenol, add an equal volume of 0.5M Trts-HCl, pH 8.0, mtx for a few minutes to allow phases to separate, and take off the upper layer (buffer). Do repeated extractions with O.lM Trts-HCl, pH 8.0, unttl the pH of the phenolic phase gets up to around 7.8. Extract once wrth TE, remove the aqueous phase, then store the phenol at 4°C in a dark bottle under a layer of fresh TE (around 20 mL for 100 mL phenol). Fresh phenol should be prepared once a month. d. Chloroform: Use high-grade chloroform that has not been exposed to the air for long periods of time. Mix 24.1 with isoamyl alcohol and store m a tightly capped bottle at room temperature. e. 3M sodium acetate, pH 5.2: Dissolve solid sodmm acetate in water, adjust the pH to 5.2 with glacial acetic actd, dtspense into aliquots, autoclave, and store at room temperature. f. Ethanol: For convenience, store in tightly capped bottles at -20°C.

3. Method 1. Pick a single colony from a freshly streaked plate of a suitable E. coli host strain into 10 mL of 2X TY m a conical flask and grow with shaking at 37°C overmght. 2. Dilute the overnight culture 1:OOin 2X TY to give sufftctent fresh culture for 1.5 ml/plaque. Ahquot 1.5-mL vol into sterile tubes.

Liquid

Culture and SSDNA Preparation

39

3. Carefully touch a sterile toothpick into a well-separated single plaque and wash the end of the toothpick in a 1.5-mL aliquot of fresh cells (see Notes 1 and 2). This IS sufficient to transfer Infected cells from the plaque to the new culture. Incubate the 1.5-mL cultures with vigorous shaking at 37°C for 4.5-5.5 h (see Notes 3 and 4). 4. Transfer cultures to 1.5~mL microfuge tubes and spin for 5 min in a microfuge. 5. Transfer supernatants to clean tubes making sure the pellet is undisturbed, add 200 uL of PEG/NaCl to each, vortex well, and leave at room temperature for at least 15 min. 6. Spin for 5 min in microfuge to pellet the phage particles. Remove the PEG-containing supernatant with a pipet, respin the tubes for a few seconds and carefully remove all remaming traces of supernatant from around the phage pellet using a drawn-out Pasteur pipet (see Note 5). The phage pellet should be visible at the bottom of the tube. 7. Add 100 pL of TE to each phage pellet and vortex vigorously to ensure that they are properly resuspended, then add 50 pL of phenol, vortex well, and leave for a few minutes. Vortex again and spin for 2 mm to separate the phases. 8. Carefully remove the upper aqueous layer into a fresh 1.5~mL tube, being careful to leave behind all the precipitated material at the interface. 9. Add 50 pL of chloroform, vortex and spin for 1 min to separatethe phases (see Note 6). 10. Carefully remove the upper aqueous layer into a fresh 1.5-mL tube, add 0.1 vol of 3M sodium acetate and 2.5 vol of absolute ethanol and precipitate the DNA. 11. Spin in microfuge for 5 min and remove the ethanol by aspiration, being careful not to disturb the DNA pellet that is often barely visible at this stage. Add 200 l,tL of 70% ethanol, spin for 2 min, remove the ethanol very carefully, and dry the pellet either under vacuum for a few minutes or by leaving the open tubes on the bench until dried by evaporation, 12. Dissolve the pellet in 30 pL of TE. At this stage, a few microliters (2-5 pL) can be removed and run on a minigel to check the quality and yield of DNA (see Note 7). The remainder of the DNA should be stored at -20°C until required.

4. Notes 1. If plaques have been picked and regrown on plates as colonies (e.g., because of the need to screen inserts by hybridization), cultures for ssDNA preps are prepared by touching the toothpicks onto the colomes and washing in the 1 S-mL fresh cells.

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2. Once plaques have been obtamed, it is best to grow phages and purify the DNA as quickly as possible as there is an increase m plaque contamination and a deterioration m quahty of DNA obtamed tf plaques are stored at 4°C. 3. Do not grow up 1.5mL cultures for extended perrods of time as this increases the possibtlity of selecting for mutants and also increases cell lysis and htgher levels of contaminating host chromosomal DNA. 4. Do not grow at temperatures above 37°C because although the host cells ~111grow the ytelds of Ml3 phages drop rapidly with increasing temperature. 5. Make sure all the PEG is removed as this causes high backgrounds m sequencing reactions. 6. The chloroform extraction can be omitted, but the highest quality templates are obtamed tf rt 1sincluded. 7 A yield of ssDNA of between 5 and 10 pg/mL ts normal. If the final pellet 1ssuspended in 30 pL of TE, approx 4-8 pL IS ample for obtaming high-quality sequence using standard dtdeoxy methods.

References 1 Marvm, D. A. and Hohn, B. (1969). Filamentous bacterial viruses. Bacteriol Rev. 33, 172-209. 2 Bankier, A. and Barrell, B. G. (1983) “Shotgun DNA Sequencing.” In Techniques in the Life Sciences (Biochemistry), ~0185, Techniques in Nuclerc Acid Biochemistry (Flavell, R A , ed ), pp l-34, Elsevier, Amsterdam 3 Sambrook, J., Fntsch, E. F., and Maniatis, T. (1989) “Small-scale preparation of single-stranded Bacteriophage Ml3 DNA.” In Molecular Closmg, A Laboratory Manual. 2nd Ed., Cold Sprmg Harbor Laboratory Press, Cold Sprmg Harbor, NY, pp 4-29,4-30.

&4PTER

6

Ml3 Phage Growth and DNA Purification Using 96 Well Microtiter Trays Alan l! Bankier 1. Introduction The growth and purification of M 13 DNA from small volume (1 SmL) cultures is a rapid and easily performed procedure (1). The samples can be processed in microcentrifuges in disposable polypropylene tubes and yield sufficient, pure single-stranded DNA (4 M) for five or more sequencing experiments. Even when several cultures are to be grown and purified simultaneously, up to 100 can be processed to completion in a day. The handling of this number of samples is tedious, however, and much time is spent opening and closing tubes and transferring tubes in and out of microcentrifuges. One hundred small volume phenol extractions and ethanol precipitations tasks even the more dedicated sequencer. When contemplating larger sequencing projects, the thought of processing the several thousand recombinants needed is indeed daunting. Shotgun sequencingof 100kb requires over 3000 random templates (2,3) and each template is rarely sequencedmore than just the once. Clearly, any further simplification of the procedure would be of great benefit. If reduced yields can be tolerated, one solution lies in the use of microtiter trays (4,5). Phage can be grown, in small volume, in the ninety-six wells of a microtiter tray and, by modification of conventional purification methods, they can also be processedin the trays. Using microtiter From: Methods m Molecular Wology, Vol. 23’ DNA Sequencrng Protocols EdIted by H and A Gnffm Copynght 01993 Humana Press inc., Totowa,

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trays removes most of the tedious handling needed when tubes are

used and makes the process even quicker. Several microtiter trays can be handled at once, making the preparation of several hundred recombinants fairly trivial. Each culture provides sufficient DNA for 2-3 conventional sequencing experiments. A small residue can be kept in a library for regrowth, should this become necessary. The procedure is summarized in Fig. 1.

2. Materials 1. Autoclaved 2X YT broth: 1% bactotryptone, 1% yeastextract, 0.5% NaCl. 2. Sterile polystyrene 96-well microtiter trays (e.g., Cornmg cell wells) There appears to be little advantage to using either tissue culture treated or nontreated plates. 3. Rigid plastic “sandwich” boxes (with lids) approx dimensions 12 x 18 x 6 cm. These are used simply as a convement holder for microttter trays, providmg sufficient air supply for good phage growth but maintaming an isolated environment. The trays can be held m position within the boxes using blocks of foam sponge. 4. Sterile, uncoated, wooden toothpicks. 5. A benchtop centrifuge capable of spinning microtiter trays at speeds up to 4000 rpm (such as the I.E.C. Centra 4X). If this speed cannot be attained, the centrifugation times will need to be extended. 6. PEG/NaCI: 20 % polyethylene glycol (mol wt 6000-8000), 2.5M sodium chloride. 7. T.E.: 10 mM Tris-HCl, pH 8.0,O.l mM Na,EDTA. 8. T.E./SDS: 1% sodmm dodecyl sulfate in T.E. 9. Ethanol/NaOAc: 95% ethanol and 3M sodium acetate, pH 5.0, equrlibrated usmg acetic acid, combined in the ratio 25: 1. 10. Self adhesive microtiter plate sealers (e.g., Cornmg 3095).

3. Method 1. Prepare an overnight culture of suitable host strain (e.g., TGl or JM109) by toothpickmg a single colony into 10 mL of 2X YT. Grow the culture at 37°C m an orbital shaker at 300-350 rpm for 18 h. 2. Make a dilutton of the stationary phase, overnight culture 1: 100 m 2X YT (25 mL for each plate of 96 phage cultures) and pipet 250 pL into each well of the microtiter tray(s). 3. Transfer a single recombinant phage into each well by carefully touchmg the center of the plaque with a sterile toothpick and flushing the toothpick pomt by rotating it m the diluted cell culture in the well. Take care to touch the plaque only lightly and to avoid touchmg any neighbor-

Preparing

Ml3

DNA in Microtiter

Toothplck

Plaques

1 Grow

I

I

6 Hours

Pellet

1

Cells I Supernatant

1 Transfer PEG

43

Trays

Preclpltate

Phage

I Phaae In SDS

Denature

1

I

1 Ethanol

Preclpltate

DNA 1

I Redissolve Fig. 1 A

DNA In T.E.

summary of template growth and purification m mrcrotiter trays.

mg plaques. As a guide, rt should only just be apparent which plaques have been lifted (see Notes 1-3). 4. Secure the tray, uncovered, inside a plasttc box fitted with a lid. Place the box instde an orbrtal shaker and grow the culture for 6 h at 37OCat a speed of 300 rpm(see Notes 4-7). 5. Remove the culture tray from the box and centrifuge it for 10 min at 4000 rpm. Using a multichannel pipet, very carefully remove 200 pL of the supernatant and transfer it to a clean microtiter tray (see Note 8). 6. Repeat the centrrfugatton step and very carefully transfer 150 pL of supernatant to a clean tray. 7. Add 30 pL of PEG/NaCI to each well, cover the tray with a plate sealer to isolate each well, and mtx tt thoroughly (a multttube vortexer is very convenient) leave it at room temperature for 15 min (see Note 9). 8. Centrifuge the tray for 10 min at 4000 rpm and discard the supernatant. This IS most easily done by inverting the tray sharply (catching the supernatant in disinfectant) and leaving the inverted tray on tissue to drain for 5 min with occasional gentle tapping.

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9. Add 50 pL of T.E./SDS to each well and seal the tray completely with a plate sealer. Ensure complete dissolution of the phage pellet by shaking the tray in a multitube vortexer for 2-3 mm. Put the tray lid on over the sealer, and place the tray m an incubator at 80°C for 20 mm (see Note 10). 10. Remove the tray and allow tt to cool to room temperature. Add 125 pL of ethanol/NaOAc to each well, cover the tray with a plate sealer, and mtx It thoroughly. Leave the DNA to precipitate at room temperature for 15 mm (see Note 11). 11. Centrifuge the tray for 10 mmutes at 4000 rpm and discard the supernatant by invertmg the tray sharply. Add 200 pL of 95% ethanol to each well to wash the pellet and immediately discard the supernatant as before. Leave the tray inverted on tissue to dram for 5 mm and dry the pellets under vacuum for 5 mm. 12. Redissolve the DNA in 20 pL of T.E., seal the tray with a plate sealer, and store it at -20°C. 4. Notes 1, Always dispose of phage and cell contaminated materials by soaking m dtstnfectant/autoclavtng.Bad technique can contammate stocksand can even result in the growing of hundreds of individual cultures of the same phage 2. Recombinant phage can be stored m T.E. at 4°C for many days or even weeks and 1s preferable to storage on agar plates. Plaques can be toothpicked into 15 pL of T.E. m individual wells of a 96-well mrcrottter tray, sealed with a plate sealer and stored in a refrigerator. When DNA is to be prepared from these recombinants, tt ts only necessary to add 250 pL of diluted host cells and continue wtth described procedure. 3. Optimal results are only obtained using the healthiest of cells that are growing vtgorously. The overnight culture should be started using a single colony from a minimal media plate that was streaked from frozen stock host cells no more than about one week prevtously. 4. When using an orbital shaker havmg a bed designed for tubes, the sandwich boxes can be secured m posmon by glumg cylindrtcal feet of appropriate size and spacing to the bottom of the boxes The feet will then slot mto the tube holes holding the box m place. Tubes and trays can then be shaken stmultaneously and without changing shaker format. 5. Ml3 phage growth is very sensitive to both temperature and rate of shakmg. Ensure that the temperature is not permuted to rise even margmally above 37°C and that the shaker 1srestricted to between 30&350 rpm. 6. The frequency of poor sequence quality increases wtth prolonged phage growth times. Do not exceed the recommended period of 6 h.

Preparing

Ml3 DNA in Microtiter

Trays

7. When insufficient time exists for complete processing of the samples, it is best to set up the phage cultures late m the day. The tray can be placed m the shaker, at room temperature, connected to the voltage supply through a timer set to turn it on in the early hours of the morning. The following day, 6 h after the timer is set to start, the tray of cultures can be processed to completion. 8. The most difficult aspect of this procedure is transfer of the phage supernatant without disturbing the cell pellet, and it is this difficulty that necessitates a second cell centrifugation step. It takes a steady hand and a keen eye to handle a multichannel pipet accurately and consistently without stirring up cells. One solution is to fabricate a pipet holder that constrains the height of the pipet and permits a microtiter tray to be stepped in columns beneath it. The most consistent results, however, have been obtained using a robotic workstation, as described in Chapter 38. 9. It IS very important to completely redissolve the PEG precipitated phage. Incomplete dissolution results in reduced yields and poorer quality. Using a flat bed multitube vortexer at maximum speed for 2-3 min is the easiest way to do this. 10. At the elevated temperatures used during the SDS step, microtiter tray plate sealers tend to warp and peel off. Putting the tray lid on durmg this incubation helps to keep the sealer m place and minimizes evaporation loss. 11. Ethanol tends to dissolve the adhesive used on plate sealers. Once mixed, do not be tempted to repeat the shaking.

References 1, Messing, J and Bankier, A. T. (1989) The use of single-strandedDNA phage in DNA sequencing,in Nucleic Acids Sequencing. A Practical Approach (Howe, C. J. and Ward, E. S., eds ), IRL Press, Oxford, pp. l-36 2. Bankrer, A. T., Beck, S., Bohm, R., Brown, C. M., Cerny, R., Chee, M. S., Hutchison, C. A., III, Kouzarides, T., Martrgnetti, J. A., Preddie, E., Satchwell, S. C., Tomlinson, P., Weston, K. M., and Barrell, B. G. (1991) The DNA sequence of the human cytomegalovirus genome. DNA Sequence 2, l-12. 3 Davrson, A. J (1991) Experience m shotgun sequencmg a 134 kilobase pair DNA molecule. DNA Sequence 1,389-394. 4. Eperon, I. C (1986) Rapid preparation of bacteriophage DNA for sequence analysis in sets of 96 clones, using filtration. Anal. Biochem. 56,406-412. 5 Smith, V., Brown, C. M , Bankrer, A. T., and Barrell, B. G. (1990) Semiautomated preparation of DNA templates for large-scale sequencing projects. DNA Sequence 1,73-78

&AE’TER

Generation

7

of kdom Fragments by Sonication Alan

CCBankier

1. Introduction Except for all but the shortest of sequencing projects, the resolution limits of polyacrylamide gels (being somewhat less than 1 kb) prevents determining the entire sequencein one run. There are several ways to overcome this by subcloning or by primer walking. Each has its advantages and disadvantages.These alternatives should be considered whenever a new project is undertaken.Frequently, the best approach would in fact use more than one method. A high proportion of large-scale sequencing projects use random cloning and sequencing(shotgun sequencing) to produce in excess of 95% of the data, and then employ more directed means to complete it (I-3). The shotgun method (4) benefits from making no prior assumptions about the DNA to be sequenced,such as base composition or the presence of certain restriction enzyme sites. The DNA to be sequenced is randomly broken down to clonable sizes, optionally size selected, and subcloned. Sequences from these recombinants are determined at random until the individual readings can be joined together by virtue of overlaps. There are several ways of producing random breaks either enzymatically or physically. Enzymic methods such as DNAse 1 (5) require calibration for each enzyme preparation, to accurately assessits activFrom Methods m Molecular Biology, Vol. 23: DNA Sequencrng Protocols Edited by: H. and A. Griffm Copyrlght 81993 Humana Press Inc., Totowa,

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ity, and for each DNA solution since the degree of enzyme action is specific to the length of the DNA and its concentration. Physical methods such as sonication (6,7), are insensitive to concentration and, to a high degree, even length. Mechanical shearing has a greater effect on larger molecules; the shorter the length, the more difficult it is to shear. The effect of this is that a wide range of initial lengths (from hundreds of bases to hundreds of kilobases) will be reduced to a similar size range when the limits of a particular device is approached. The size range generated will be solely dependant on the power of the sonicator or the force used in the physical process. When using a sonicator this is very easy to control. The method described here uses a Heat Systems Ultrasonics (Farmingdale, NY) W-385 sonicator fitted with a cup horn. The benefits of using a cup horn are that samples in a few microliters can be sheared and no potential for crosscontamination exists. Several samples can even be processed simultaneously. Any sonicator can be used all that is needed is a simple time course of sonication, at a given power setting, to determine the minimum time needed to give the desired size range. Any unwanted DNA can be used for thus time course. 2. Materials 1. 10X Ligase buffer: 500 mM Tris-HCI, pH 7.5, 100 mM magnesium chlorrde, 100 mA4dithiothreitol, 10 mM ATP, 250 clg/mL bovine serum albumen. 2. T4 DNA ligase. 3. A Heat Systems Ultrasonics W-385 somcator fitted with a cup horn probe, or any other precalibrated somcator. 4. Chasemix: 0.5 mMdATP, 0.5 mMdCTP,0.5 n-uV!dGTP, 0.5 mMdl’“TP. 5. Klenow fragment DNA polymerase. 6. T4 DNA polymerase. 7. 20% P.E.G.: 20% polyethylene glycol (mol wt 6000-8000), 1M NaCl. 8. A flat bed agarose gel apparatus. This need only be a minigel system of dimensions 10 x 10 cm or even smaller. 9. Low melting temperature agarose. 10. 10X T.B.E.: 108 g Tris base, 55 g boric acid, 9.3 g Na,EDTA dissolved and made up to 1 L m deionized water. 11. T.B.E. dye mix: 0.1 g xylene cyan01 F.F., 0.1 g bromophenol blue, 20% sucrose in T.B.E. buffer. 12. 3M sodium acetate, pH 5.0 equilibrated using acetic acid. 13 T.E. buffer 10 mM Tris-HCl, pH 8.0,O.l mM Na,EDTA.

Generating Fragments

by Sonication

49

3. Method 1. Self-ligate 5-10 pg of the DNA to be sequenced by mixing m a microcentrifuge tube: 5-10 pg of the linear DNA, 3 pL 10X ligase buffer, 10 U T4 DNA hgase (use 100 U for blunt-ended fragments), make up the volume to 30 pL with sterile distilled water, and incubate at 15°C for 2 h (see Notes 1 and 2). 2. Position the tube m the cup horn filled with tee-cold water so that the bottom of the tube is just above but not touching the probe. Sonicate the sample on full power for 20 s. Using a W-385, this produces a peak in size around 1 kb (see Note 3). 3. End-repair the somcated fragments by adding to the tube 4 pL of chase mix, 10 U of Klenow fragment DNA polymerase, and 10 U of T4 DNA polymerase. Incubate the reaction for 30 mm at 37OC and heat-kill the enzymes by heating to 70°C for 10 mm. Add distilled or deionized water to 50 pL and ethanol precipitate the DNA. 4. Redissolve the fragments in 100 l.tL of T.E. and selectively precipitate the fragments longer than 400 bp by adding 66 pL of 20% P.E.G.IlM sodium chloride and leaving on ice 10 min. Centrifuge the tube for 10 mm in a microcentrifuge and remove all of the supernatant. Briefly respin the tube and remove any residual P.E.G. Redissolve the DNA in 50 pL of T.E. This should provide sufficient material for 50 blunt hgation/cloning experiments that, under ideal conditions, would yield several thousand recombmants (see Notes 4 and 5).

4. Notes 1. As little as 1 pg of DNA can be used to prepare sonicated fragments, but smce the overall efficiency of the process is low, it IS not recommended. 2. The mechanism of physical shearing tends to break molecules around their center. If the starting material is a linear molecule, the subfragments produced, therefore, will not have a random distribution. By simply self-ligating the linear molecule, a truly random fragment generation is produced. 3. If the only available sonicator is one fitted with a direct immersion probe, it is very important to clean it thoroughly to avoid crosscontamination. The required sonication time is also likely to be much shorter, as short as a few seconds. 4. The method described here uses P.E.G. precipitation for stze selection. Alternatives to this include size selection from agarose or no selection at all. For agarose selection, load the entire sonicated, end-repaired sample onto a l-cm slot on a low gelling temperature agarose minigel. Run the sample m 2-3 cm and cut out the size range needed. The fragments can be purified by any of the many described procedures (8).

50

Bankier

5. Fragments purified from low gelling temperature agarose frequently fail to hgate. This is assumed to be caused by mhlbitors not removed during the phenol extraction/ethanol preclpltatlon. The assumption would seem to be substantiated by the observation that the problem IS associated with particular batches of agarose. Similar inhibition by agarose contaminants would occasionally appear to prevent adequate end repair. If either of these 1s suspected, the only recourse is to try altemative batches of agarose or alternative means of fragment purification.

References 1. Chee, M S., Bankier, A. T , Beck, S., Bohm, R., Brown, C. M., Cerny, R , Horsnell, T , Hutchison, C. A., III, Kouzarides, T , Martignetti, J A., Predche, E , Satchwell, S C , Tomhnson, P , Weston, K M , and Barrell, B. G (1990) Analysis of the protein-coding content of the sequence of human cytomegalovlrus strain AD169, m Current Topics in Mzcrobzology and Immunology vol. 154, (McDougall, J. K., ed.), Springer-Verlag, Berlin, Heidelberg, pp. 125-169. 2. Baer, R., Bankier, A. T , Biggm, M D., Deininger, P. L., Farrell, P. J., Gibson, T. J., Hatfull, G , Hudson, G. S., Satchwell, S. C , Segum, C , Tuffnell, P S , and Barrell, B. G. (1984) DNA sequence and expression of the B95-8 EpsteinBarr virus genome. Nature 310,207-211 3. Davison, A. J. and Scott, J E (1986) The complete DNA sequence of vancellazoster virus J. Gen. Virol 67, 1759-l 816 4. Messing, J. and Bankier, A T (1989) The use of single-stranded DNA phage in DNA sequencing, in Nucleic Acids Sequencing. A Practical Approach (Howe, C. J. and Ward, E. S., eds.), IRL Press, Oxford, pp. l-36. 5. Anderson, S. (1981) Shotgun DNA sequencing usmg cloned DNAse l-generated fragments. Nucleic Acrds Res. 9,3015 6 Deininger, P. L. (1983) Random subcloning of sonicated DNA: application to shotgun DNA sequence analysis. Anal. Biochem. 129,216. 7 Bankier, A.T., Weston, K. M. and Barrel], B.G. (1987) Random clonmg and sequencing by the Ml3ldideoxynucleotide chain termrnatlon method, in Methods in Enzymology vol. 155, (R. Wu, ed.), Academic Press, London, pp. 51-93. 8. Sambrook, J , Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: a Laboratory Manual 2nd ed Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y

CHAPTER8

Generation of a Nested Set of Deletions Using Exonuclease III George

Murphy

1. Introduction Exonuclease III (Exo III) will digest double-stranded DNA in a 3’ to 5’ direction if the DNA is blunt-ended or possessesa 5’ overhang. It will not digest if there is a 3’ overhang of three or more bases, or if the 3’ end has had thiophosphate-containing bases incorporated into it. In order to generate a set of insert deletions using Exo III it is necessary to cut the polylinker twice with different restriction enzymes so that the cut end nearest the primer site possesses a 3’ overhang, or has thiophosphate residues at the 3’ end, and the end of the polylinker attached to the insert possessesa 5’ overhang or blunt end. If this can be achieved, digestion with Exo III will result in progressive deletion of the 3’ end of the insert, leaving a single-stranded 5’ overhang that can be removed by treatment with mung bean nuclease (I) or exonuclease VII (Exo VII). The blunt ends thus formed are ligated, a suitable host transformed, and colonies are picked at random and screened for insert size (2), following which a suitable range of inserts is then sequenced. One of the advantages of using directed deletions is the accuracy with which insert sizes can be measured by restriction enzyme digestion. Size selection is an essential part of this technique since there is a very broad distribution in size of insert obtained at each timepoint, From Methods m Molecular Wology, Vol 23 DNA Sequencing Protocols Edited by H and A Gnffln Copynght 01993 Humana Press Inc , Totowa,

51

NJ

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even if a clearly defined band is observed when the rate of deletion is being measured. A rapid screening procedure employing the isolation of crude plasmid from colony streaks, or more easily from small cultures grown up in microtiter wells, is outlined here. The number of clones that can be screened in a working day using this method is limited only by the running of the gels, as the isolation of crude plasmid takes about 30 min. Selected transformants can then be miniprepped and a more accurate estimation made of their insert size by restriction enzyme digest.

2. Materials 2.1. Exonuclease

Deletions

2.1.1. Linearizing the Insert 1. 10X TA buffer: 330 mM Trrs-acetate (pH 7.9), 660 mM potassium acetate, 100 mA4 magnesium acetate, 40 mM spermrdme, and 5 mM DTT NaCl can be added to TA buffer for those enzymes requumg a higher salt concentration. 2. Phenol/chloroform: Equal volumes of phenol (equilibrated wrth TE) and chloroform:isoamyl alcohol (25: 1 v/v). 3. Chloroform. 4. Sepharose-CL-6B: Equilibrate the Sepharose (Pharmacia, Upssala, Sweden) in TO.lE and adjust to a packed gel: buffer ratio of 2:l. 5. 10X Exo III buffer: 500 mM Tris-HCl, pH 8.0, 50 mM MgC12, and 10 mh4 DTT. 6. TE: 10 mM Tris-HCl (pH S.O), 1 mM EDTA. 7. TO.lE: 10 mM Tris-HCl (pH 8,0), 0.1 mM EDTA. 2.1.2. Protecting the Primer Site with Thiophosphates 1, 10X TM: 100 miJ4 Tris-HCI, pH 8.0, 50 mM MgCl*. 2. Thio-dNTPs: 20 mM in each thiophosphate dATP, dCTP, dGTP, or dTTP (Amersham, Arlington Heights, IL), pH 8.0. 3. Klenow fragment of DNA polymerase (BRL, Richmond, LA) 4. Phenol/chloroform. 5. Chloroform. 6. Sepharose-CL-6B. 2.1.3. Digestion with Exo III 1. Exo III (BRL). 2. Stop buffer: 10 mM Tris-HCl, pH 8.0, 10 mM EDTA. 3. 10 Exo VII buffer: 500 mM potassium phosphate pH 7.9,90 mM EDTA and 10 mM DTT.

Exonuclease

Deletions

53

2.1.4. Blunting the DNA 2.1.4.1. BLUNTING WITH MUNG BEAN NUCLEASE 1. Mung bean nuclease buffer: 30 mkf sodium acetate,50 miJ4NaCl, 1 rnk! ZnCl*, 1 mM cysteine, 0.001% (v/v) triton, and 5% (v/v) glycerol. 2. Mung bean nuclease (BRL). 3. Phenol/chloroform. 4. Chloroform. 5. Sodium acetate: 3M sodium acetate, pH 5.0. 6. 96% Ethanol. 7. TE. 2.1.4.2. BLUNTING WITH Exo VII EXONUCLEASE 1, Exo VII buffer. 2. Exo VII (BRL).

3. Sodium acetate. 4. Ethanol. 5. Sepharose-CL-6B. 6. TE. 7. 10X TA buffer. 8. T4 DNA polymerase (BRL). 9. dNTPs: Solution 2.5 mM m each of dATP, dCTP, dGTP, and dTTP.

2.1.5. Ligation 1. 10X Ligation buffer: 500 rnkf Tris-HCl, pH 7.5, 100 mJ4 MgC&, 100 mM DTT, 10 mM spermidine, 10 mkf ATP, and 1 mg/mL BSA. 2. T4 DNA ligase (BRL). 2.2. Template Screening 1. LB: 1% bacto tryptone, 0.5% bacto yeast extract, and 1% NaCl (all w/v). 2. Protoplasting buffer: 20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 50 rnk! NaCl, 20% w/v sucrose, 100 clg/mL RNase A, and 100 crg/mL lysozyme. 3. Lysis buffer: 2.5 mM EDTA, 2% SDS, 10% w/v sucrose, and 0.04% w/v bromophenol blue. 4. Gel buffer: 1X TBE, 0.05% SDS (w/v). 5. Gel stain: 0.5 pg/mL ethidium bromide in water. 3. Methods 3.1. Exonuclease Deletions To prepare the DNA for Exo III treatment the sample is first digested with two restriction enzymes, one leaving a blunt end or a 5’ overhang at the end of the insert to be deleted, the other protecting the

54

Murphy

vector primer site from deletion by leaving a 3’ overhang of at least three bases. As an alternative, the digestion is performed in two steps. In the first step the site to be used to protect the priming site is 3’ endfilled with thiophosphates. In the second step the insert is prepared for deletion by digesting off its 3’ thiophosphate protection with an enzyme leaving a blunt end or a 5’ overhang (see Notes 1 and 2). 3.1.1. Linearizing the Insert 1. Mix 15 pg DNA, water, 8 pL 10X TA buffer, and 40 U of both restriction enzymes m a total volume Notes 3 and 4).

of 80 pL. Digest for 2 h at 37°C (see

2. Add 50 pL phenol/chloroform and vortex for 1 min before centrrfuganon at 10,OOOgfor 4 mm. Repeat the extraction with 50 pL chloroform. 3. Spin-dtalyze the sample on acolumn prepared from 500 pL of Sepharose slurry (see Chapter 19 and Note 5). Add 14 pL 10X Exo III buffer, adjust the volume to 140 pL, and use 70 p.L for digestion with Exo III, storing the remainder of the sample at -20°C. 3.1.2. Protecting

the Primer Site with Thiophosphate

1. Perform steps l-3 from Section 3.1.1. above as far as the spin-dialysis, but using in step 1 40 U of the restriction enzyme distal to the site to be used to expose the insert (i.e., the restriction site closest to the primer site of the vector).

2. Add 10 pL 10X TM and 2 pL of thlophosphate-dNTPs, followed by 15 U Klenow fragment of DNA polymerase Incubate 30 mm at room temperature.

I in a total volume

of 100 pL

3. Heat at 65°C for 10 min then cool to room temperature. 4. Add 10 pL of 1OX TA, 40 U of an enzyme that will produce a blunt end or a 5’ overhang

at the end of the insert, releasing

the thiophosphate

labeled site, and water to 120 pL. Incubate for 1 h at 37°C. 5. Extract with phenol/chloroform and chloroform as above then spm-dialyze the sample on a column prepared from 600 & Sepharose slurry (see Chapter 19). Contmue as m 3.1.1. above.

3.1.3. Digestion with Exo III The prepared sample is then treated with Exo III to progressively delete through the insert and samples removed at appropriate times, including a zero-time sample. 1. Premcubate the sample for 2 mm at 37°C then add 125 U Exo III Mix rapidly and immediately

remove 10 pL to (1) 10 pL stop buffer if using

Exonuclease

55

Deletions

mung bean nuclease to generate blunt ends, or (ii) to 1.5 pL 10X Exo VII buffer and 4 pL water if usmg Exo VII. Place on ice. 2. Remove a 10 l.tLsample as above every minute for 6 mm. Heat the samples for 10 mm at 65°C then cool to room temperature (see Notes 6,7, and 8). 3.1.4. Blunting

the DNA

Two methods may be usedto generateblunt ends for ligation. The use of mung bean nucleaseis more rapid than the method with Exo VII, but has the disadvantage of causing occasional loss of the primer site due to “nibbling-back” or destruction of DNA caused by digestion from nicks or through an excessive ratio of enzyme to substrate. Use of Exo VII has the additional advantage that up to five times more colo-

nies are obtained per Itg of DNA than in the nuclease S 1 method. 3.1.4.1. BLUNTING WITH MUNG BEAN NUCLEASE 1. Add 100 pL mung bean buffer containing 100 U/mL mung bean nuclease to each sample. Incubate at 37OC for 30 min. 2. Extract with 100 pL phenol/chloroform, followed by 50 pL chloroform, centrtfugmg each time at 10,OOOgfor 4 mm. 3. Add 12 cls,sodium acetate and 400 pL ethanol to each sample and leave at -70°C for 1 h before centrifugation at 10,OOOgfor 10 mm. Rinse with cold ethanol, dry the pellet and dissolve m 20 pL TE.

3.1.4.2. BLUNTING WITH Exo VII NUCLEASE AND T4 DNA POLYMERASE 1. Add 1.5 U Exo VII to each tube and incubate for 1 h at 37°C then heat at 65°C for 15 mm. 2. Add 10 pL water and spin-dialyze the sample on a spin-column made from 250 pL Sepharose slurry (see Chapter 19) to remove phosphate that will otherwise interfere with ligation, add 3 pL sodium acetate and 100 pL ethanol and leave at -70°C for 1 h before centrifugation at 10,OOOgfor 10 min. Rinse with cold ethanol, dry the pellet and dissolve in 10 6 TE. 3. Add 2 pL 10X TA buffer, 1 pL dNTPs, 5 U of T4 DNA polymerase and water to 20 pL. Incubate at 25’C for 30 min. Heat at 65°C for 10 min then cool to room temperature. 3.1.5. Measurement of the Rate of Deletion and DNA Ligation

In order to ascertain the rate of deletion and to determine which DNA samples should be ligated and used for transformation, part of the sample is run out on an agarose minigel. Large inserts in small

56

Murphy

vectors may be separated directly, but it is often more accurate to cut out the remaining insert with a suitable restriction enzyme with a site on the polylinker distal to the end subjected to deletion. 1. Take 8 pL of the blunted sample and add 1 pL 10X TA and 10 U of an polylinker enzyme cleaving at the undeleted end of the insert or Pvu II, which cuts m the flanking lac regions of many vectors. Digest for 1 h at 37°C and then separate on a suitable agarose gel. Ligate suitable timepoints (see Note 9). 2. Add 3 pL of the remaining sample to 3 pL 10X ligation buffer, 3 U T4 DNA ligase and water to 30 pL 3. Incubate at 15°C overnight. Use 3 pL of the ligation mixture to transform 200 pL of competent cells and plate out all the sample 3.2. Template Screening Following deletion and transformation, colonies are picked at random from each timepoint and screened for insert size. A rough approximation of size is obtained through sizing intact plasmid using protoplast lysis. This is followed by making minipreps and screening them by restriction digest. 3.2.1. Screening by Streaking Colonies 1. Prck twelve colonies from each timepoint, usmg sterile toothpicks. Streak each colony onto a fresh LB plate prepared with a suitable antibiotic and marked on the bottom into twelve sectors. Grow overnight at 37°C. 2. Scrape off about 0.5-l cm of each streak with a pipet tip, picking up as little agar as possible, and transfer to a 0.5~mL microcentrifuge tube containing 10 pL of protoplasting buffer, vortexing vigorously to disperse the sample. Leave the tip m the sample for loading onto the gel. Incubate for 15-30 min at room temperature (see Note 10). 3. At the same time prepare Insert size markers, combmmg streaks of cells transformed either with the vector or with vector contammg the fulllength insert. 4. Load the samples onto a 0.7% agarose gel, into wells preloaded with 5 pL of lysis buffer. Use a supercoiled DNA ladder (BRL) as a size marker (see Notes 11 and 12). 5. Leave the gel for 5 mm after loading to ensure lysis of the cells, then run the gel at 50 V for 15 mm before mcreasmg to 100 V and runnmg until the dye band runs off the bottom of the gel. 6. Stain the gel and photograph the DNA bands.

Exonuclease

57

Deletions

3.2.2. Screening Using Microtiter Plates 1. Add 100 pL LB plus ampicillm to each required microtiter well and maculate with a single colony. Incubate at 37°C overnight, shaking at about 150 rpm. To prevent evaporation of the medium it is best to place autoclave tape over the wells. 2. Remove 10 pL of the medium to a second microtiter plate containing 5 pL of protoplasting buffer in each well. Incubate and run on an agarose gel as above.

4. Notes 1. A potential problem with this strategy, particularly with long inserts, IS the need to find two restriction enzyme sites in the region between the insert and the primer site that will satisfy the above conditions and are also not present in the insert. The more sites there are in the polylmker, the easier this process becomes. For this reason vectors such as Bluescript (Stratagene) are ideal for this approach. If inserts can be cloned mto the central sites of the Bluescript polylinker then it may be possible to delete the insert from either end, with the additional advantage that three priming sites exist at each end of the polylinker. 2. By Judicious selection of enzyme sites close to the insert and using thiophosphates to block exonuclease activity it may be possible to delete through an insert m several stagesif enough sites are left on the primer side of the polylinker to provide a second lmearization satisfying the criteria for Exo III digestion. 3. It is essential that the initial digestions with restriction enzyme to open up the insert for deletion go to completion. The presence of nonlmearized plasmrd will eventually result in the recovery of large numbers of fulllength inserts during template screening. 4. The amount of DNA to be digested depends on the length of insert that it is desired to remove. The values suggested in the methods are sufficient to provide about 1 J.QDNA per time-point over 6 mm of treatment, with a deletion rate of around 200 bp/min, that should provide enough deletion to sequence an insert of around 1.5 kb. The suggested amounts and volumes should be increased proportionately for longer inserts. There is a broadening of the size-range of the insert in samples taken at later time-points. 5. It is unnecessary to use caesium gradient-purified DNA for this technique, although use of such DNA has the advantage that enough material is available for several trials to determine the rate of deletion. In this laboratory, deletions are mainly performed on miniprep DNA to

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Murphy

avoid the time-consuming process of caesium gradient banding. The use of spm-columns to clean up linearized DNA provides excellent substrates for dtgestion. However, the digestion rates of miniprep plasmtd DNA tend to be more variable than when using more purified material. If using mmiprep DNA the plasmid from two overnight lo-mL cultures are pooled m a volume of 100 pL. This provides enough material for stx trials of the digestion rate if 30 & is used per lmearization. 6. The activity of Exo III falls off with length of incubation and so if a longer insert 1sbeing treated later, samples should be taken at longer intervals than the imtial samples. For example, if digesting an insert of 5 kb the amount of DNA, exonuclease and the reaction volumes should be tripled, and samples 7-12 removed at 2 mm intervals, with samples 13-l 8 being removed every 5 mm. 7. Substantial variation m the rate of digestion is often observed, because of factors such as quality of the Exo III and purity of the DNA substrate. If insufficient or excessive digestion rates are observed, the digestion may be repeated using the retained portion of the linearized plasmid and altering the amount of enzyme used. 8. Since Exo III is moderately temperature sensitive, the rate can also be controlled by altering the digestion temperature, a particularly useful parameter if only a few nucleottdes need to be removed (e.g., in studies on promoter location). If, under the conditions used, 200 bp are removed every mmute at 37”C, then about 120 bp will be removed at 30°C and 50 bp at 20°C. 9. When the rate of Exo III deletion is measured by cutting out the insert before gel separation, any change in mobility of the vector band indicates substantial digestion through the primer site. If this is observed the samples should not be ligated, as the primer sites will have been lost. 10. If the initial screening for insert size 1s done by streaking out the transformant, some practice is required in Judging the amount of cells to take. After vortexmg, the solution should be turbid rather than dense. Better results are obtained when too little, rather than too many, cells are used. The mimmum sample which can still be seen on the gel would be about half of the cells from a large colony on the original plate. 11. When screening the transformants by protoplast lysis tt is important not to use a submerged gel. Buffer should be added to the gel tank until tt is a few mm below the gel surface. Failure to do thts will result in the DNA being sucked out of the well by changes in surface tension as the SDS in the lysis buffer mixes wtth the gel buffer. 12. After a first round of screenmg by protoplast lysts, it is preferable to screen again by making muuprep DNA and measuring the insert size

Exonuclease Deletions

59

more accurately by restriction enzyme digest. If a restriction enzyme site on the end of the polylinker has been used to protect the primer site, it will clearly be removed during the linearization process. The Pvu II sites flanking the polylinker in pUC and Bluescript plasmids provide useful sites for digestion m such circumstances, or other rare sites in the vector close to the insert may be used.

References 1. Henikoff, S. (1984) Umdirectional digestlon with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28,351-359 2 Wen-Qin, X and Potts, M (1989) Quick screening of plasmid deletion clones carrymg Inserts of desired sizes for DNA sequencing. Gene Anal Tech. 6,17-20

&IA.FTER

9

Sequential Deletions of Single-Stranded DNA “Cyclone Sequencing”

George Murphy 1. Introduction A major problem of the directed deletion of double-stranded DNA using Exonuclease III (Chapter 8) is the necessity to find two enzyme sites to open up the insert for digestion and to protect the primer site. The “Cyclone Sequencing” technique of Dale, McClare, and Houchins (1) overcomes this problem as it is totally independent of the restriction sites in the insert. In this procedure single-stranded Ml3 is hybridized to an oligomer that spans the Hind111(even-numbered M 13 phage) or EcoRI site (odd-numbered phage) of the polylinker, generating a region of double-stranded DNA. This region is digested with the appropriate enzyme, linearizing the phage, and then the 3’ to 5’ exonuclease activity of T4 DNA polymerase is used to digest through the insert. The 3’ end is then homopolymer tailed using terminal transferase and the original oligomer used to create the double-stranded region, which has a 3’ end complementary to the homopolymer tail, is reannealed across the restriction site and the homopolymer tail. After ligation the DNA is used to transform competent cells and the resulting plaques picked at random for screening. The procedure has the great advantage that, as the restriction site used to linearize the DNA is regenerated in this procedure, the insert may be deleted in several stages. From Methods In Molecular B!ology, Vol. 23. DNA Sequencmg Protocols Edlted by Ii and A Griffin Copynght 01993 Humana Press Inc , Totowa, NJ

61

62

Murphy

T4 DNA polymerase does not digest single-stranded DNA at an even rate, so that the size-dispersion of templates is considerable.It is therefore essential to screen templates for size of insert before sequencing, rather than pick plaques at random from each time point. The large size of the Ml3 vectors, particularly if the insert is short, makes direct selection of templates by insert size difficult. Aliquots of randomly selected clones may be separatedovernight on agarosegels, but accurate sizing of small inserts may not be possible. A more sensitive, though time-consuming, alternative is to estimate insert size by hybridizing to single-stranded DNA complimentary to the insert followed by nuclease S1 digestion (2). This can be performed either on purified template or directly on the phagecontaining supernatant after the cells have been removed. The problem of insert size-selection when using large Ml3 vectors is eliminated if phagemid vectors are used. These can be propagated as plasmids, but single-stranded DNA can be generatedthrough the use of helper phages. 2. Materials

2.1. DNA Preparation 2.1.1.

Single-Stranded

Ml3

DNA

1. Minimal medium: Minimal medium Ingredients are autoclaved separately then mixed aseptically. MIX together 887 mL water, 10 mL 20% (w/v) glucose, 1 mL 1M MgS04, 1 mL O.lM CaCl*, 1 mL 1M thiamine HCl, and 100 mL M9 salts (7 g Na2HP0,, 3 g KH,PO,, 1 g NH&I, and 0.5 g NaCl in a total vol of 100 mL). 2. YT: 8 g bacto tryptone, 5 g yeast extract, and 5 g NaCl in a vol of 1 L. 3. PEG: 20% (w/v) PEG (PEG 8000, Sigma, St. LOUIS,MO) in 2.5MNaCl 4. TE: 10 m&I Tris-HC1 pH 8.0, 1 mM EDTA. 5. Phenol/chloroform: Equal volumes of phenol, equlhbrated with TnsHCl, pH 8.0, and chloroform:lsoamyl alcohol, (25: 1 v/v). 6. Chloroform. 7. Sodium acetate: 3M sodium acetate, pH 5.0. 8. 96% (v/v) ethanol. 9. Bacterial host strain: JMlOl, JM109, or XLl-Blue (Stratagene). 2.1.2. Plasmid

Rescue

1. Helper phage: VCSM13 helper phage (Stratagene), selected on kana-

mycin (20 pg/mL). 2. YT. 3. PEG. 4. Phenol/chloroform.

Cyclone Sequencing

63

2.2. Restriction Enzyme and T4 Polymerase Digestion 1. Annealing buffer: 100 mM Tris-HCl, pH 7.4, 100 mM MgCl,. 2. Primers: 20 pg/mL m deionized water, RD29 if digesting with HindIII, or RD20 if using EcoRI. 3. Hind111 or EcoRI (BRL). 4. Polymerase buffer: 330 mM Tris acetate, pH 7.9, 660 mM potassium acetate, 100 mM magnesium acetate, 5 mM DTT, and 1 mg/mL BSA. 5. T4 DNA polymerase (BRL).

2.3. Homopolymer

Tailing

and Ligation

1. Nucleotides: 0.2 mM dATP or dGTP. 2. Terminal transferase (BRL). 3. ATP: 10 mM ATP in Tris-HCl pH 8.0. 4. T4 DNA ligase (BRL). 2.4. Template

Screening

2.4.1. Screenmg by Size Selection on Agarose Gels 1. Loading buffer: 0.25% bromophenol blue in 50% glycerol, 100 mM EDTA and 1X TBE. 2. 10X TBE: 162 g Tris, 27.5 g boric acid, and 9.5 g EDTAn. 3. SDS solution: 1% (w/v) SDS, 0.2M EDTA, pH 8.0. 4. Ethidium bromide: Stain gels m water containing 0.5 g/mL ethidmm bromide. 2.4.2. Screening Using N&ease Sl Digestion 1. Nuclease Sl buffer: 30 mM sodium acetate, pH 4.5,4 mM ZnS04, and 250 mM NaCI. 2. Nuclease Sl (BRL). 3. SDS solution. 4. Loading buffer. 5. 10X TBE.

3.1. Preparation

3. Methods of Single-Stranded

Ml3 DNA

1. Grow an overmght culture of a suitable host m 10 mL munmal medium. If using XLI-Blue, grow on YT contammg 12.5 pg/mL tetracyclrne. Add 5 mL of the overnight culture to 45 mL YT prewarmed to 37°C and grow with vigorous shaking at 37°C for 1 h. Add one well-isolated plaque from a transformation of host cells with vector plus msert DNA and grow for 6 h.

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2. Spin down the cells at 10,OOOgfor 5 min and decant the supernatant to a fresh tube without carrying over cells. 3. Add 5 mL of PEG, mix and leave for 30 mm before centrifugation for 10 min at 10,OOOg.Decant most of the supernatant, spin again for 30 s and remove all the remaining lrqurd with a drawn-out Pasteur. 4. Suspend the phage pellet in 1 mL TE and add 500 pL phenol/chloroform. Vortex for 1 min, then centrifuge at 10,OOOgfor 4 mm and remove the supernatant to a fresh tube. Repeat the phenol/chloroform extraction 5-6 times until no precipitated material is observed at the interface. A final extraction is carried out with 500 pL chloroform. Vortex for 20 s and centrifuge as above. 5. Transfer the supernatant to 100 pL sodium acetate and 3 mL ethanol and leave at -70°C for 1 h before centrifugation for 10 min. Rinse with cold ethanol, centrifuge, and dry the pellet before dissolving in 0.5 mL TE. 3.2. Isolation of Single-Stranded DNA by Plasmid Rescue 1. Use a fresh overnight culture of host cells and fresh helper phage of known titer. Inoculate 45 mL of YT medium m a 250-mL flask with 5 mL of overnight culture of bacterial host cells and grow with vrgorous shaking to an AS6aof 0.3, equivalent to about 2.5 x lo8 cells. 2. Add helper phage to a multiplicity of infection of 20: 1 phage:cells and continue shakmg for 8 h. Heat the cells to 65OCfor 10 mm and centrifuge at 10,OOOgfor 15 min. At this stage the supernatant can be stored at 4OC overnight. 3. Add 5 mL PEG, mix, and leave for 30 min before centrifuging at 10,OOOg for 20 min. Decant off the supematant, centrifuge briefly, and remove the remnants of liquid with an asptrator. 4. Dissolve the phage pellet m 2 mL TE and add 2 mL phenol/chloroform. Vortex for 1 min, centrifuge at 10,OOOgfor 5 min and transfer the supernatant to a fresh tube. Repeat the phenol/chloroform extraction until there is no prectpitate at the interface, usually 5-6 times. 5. Perform a final extraction just with chloroform, add 0.2 mL sodium acetate and 6 mL ethanol and leave at -20 “C for 1 h. Centrifuge, rinse with ethanol, dry, and dissolve in 0.5 mL TE. 3.3. Preparation of DNA for Sequencing DNA for sequencing can be prepared by either of the above methods, scaling the incubation volume down to 1.5 tnL, and adjusting all other volumes accordingly. Inoculate the medium with one well-isolated plaque or colony and resuspend the final purified DNA in 30 ltL TE.

Cyclone Sequencing 3.4. Restriction

65

Enzyme

and T4 Polymerase Digestion T4 DNA polymerase has a slow 3’ to 5’ exonuclease activity, but lacks any 5’ exonuclease function. Single-stranded DNA is linearized by hybridizing an oligonucleotide primer to the 3’ end of the polylinker, generating a double-stranded Hind111 or EcoRI site. This site is then cleaved with the appropriate restriction enzyme and the insert progressively deleted with T4 DNA polymerase. 1. Combme smgle-stranded DNA (4 pg), 8 pL 10X annealing buffer, 2 pL of RD20 (for an EcoRI digest), or 4 pL of RD29 (for a Hind111 digest) m a volume of 60 pL. Heat to 65°C for 10 mm and cool slowly to room temperature over 1 h (see Notes l-3). 2. Add 40 U of restrrction enzyme and increase the volume to 80 pL. If using HindIII, incubate at 37°C for at least 3 h, preferably overmght, before inacttvatmg the enzyme by heating at 85°C for 10 mm. With EcoRI, incubate for 1 h at 42°C and mactlvate by heating to 85’C for 10 mm. 3. Check for complete digestion by separating 10 pL of the sample on a 0.7% agarose gel, alongside uncut DNA as a control (see Note 4). 4. Cool to room temperature and add 10 pL 10X T4 DNA polymerase buffer, 8 U T4 DNA polymerase, and water to 100 pL. Remove 25 pL samples every 15 mm and heat to 65’C for 10 mm to macttvate the polymerase (see Note 5).

3.5. Homopolymer

Tailing

and Ligation

Following insert deletion the DNA is then recircularized by adding on a homopolymer tail to the 3’ end with terminal transferase and rehybridizing with the primer used to linearize the DNA. This primer has a 3’ end complementary to the homopolymer tail and anneals to this as well as the original restriction enzyme site. The annealed DNA is then ligated and used to transform competent cells. 1. To each 25 JJLdigest add 2 J.ILof 0.2 mM dGTP (for RD20 annealed samples) or dATP (for RD29 annealed samples), 5 U terminal transferase, and water to 30 pL. Incubate at 37°C for 20 mm and heat at 65°C for 10 mm before cooling (see Note 6). 2. Remove 10 pL as nonannealed control and add 1 pL of RD20 or RD29 (20 pg/mL) to the remainder. Anneal as for the restrtctton enzyme digest (Section 3.4.) then cool to room temperature. 3. Add 3 cls, ATP, 2 U T4 DNA ligase, and water to 30 pL. Incubate at room temperature for at least 90 min.

66

Murphy

4. Dilute 2 + of the control to 15 @, and use 1 pL of the control or from the ligated sample to transform 200 ~JLof competent cells.

3.6. Screening Transfbrmants for Size of Insert A nested set of deletions for sequencing is identified by screening for insert size. Screening by separation of crude phage supernatant on agarose gels is rapid but provides only a rough indication of size, while the use of nuclease S 1 digestion is slower but provides a much more accurate estimate. 3.6.1. Screening of Phage-Containing Supernatant by Agarose Gel 1. Add 15 pL of the phage-containing cell supernatant to 1 pL 1% SDS and 5 + loading buffer. 2. Electrophorese overnight on a 20-cm 0.7% agarose gel in TBE buffer at 50V. 3. Visualize the phage by stammg with ethldlum bromide. 3.6.2. Screening of the Phage-Containing Cell Supernatant by Nuclease Sl Digestion 1. Combme 50 @ of the phage suspension with an equal volume of solution from a transfection using a full-length insert m the opposite onentation and add 8 pL SDS/EDTA. 2. Incubate at 65°C for 1 h, cool to room temperature, and extract with 50 cls,each of phenol/chloroform, followed by 50 $ of chloroform, then ethanol precipitate. 3. Dissolve the DNA m 10 pL of Nuclease Sl buffer containing 200 U/ mL of Nuclease Sl and incubate for 30 min at 37°C 4. Add 5 $ loading buffer and separate on an agarose gel.

4. Notes 1. For even-numbered Ml 3 vectors the annealmg 1sperformed with the Hind111site oligomer RD 29, whde for odd-numbered vectors the EcoRI site primer RD 20 ISused DigestIon with EcoRI 1svmually complete after one hour, but dtgestlon with Hind111may require overnight incubation 2. Since the restrictton site used to linearize the DNA is recreated durmg the ligation reactton, it can be reused If a further round of deletmg 1s needed because no clones are recovered m which the insert IS completely deleted

67

Cyclone Sequencing

3. Because the procedure works with as Irttle as 2 pg of DNA for one reaction set, template from normal 1S mL sequencing-type cultures of M 13 DNA may be used. 4. It IS essential to check for complete digestion by separating a small sample of the digest on a 0.7% agarose gel, because even traces of uncut DNA will produce a high background of full-length insert in the templates. Thts background can be removed by separating the digested DNA on a 0.7% agarose gel and recovering the DNA before exonuclease treatment. 5. T4 DNA polymerase will remove about 30 bases/mmunder the conditions given here and the mean digestion rate remains linear over an hour. There is a wide variation of digestion rates between batches of T4 polymerase, however, and it may be advisable to estimate the rate of digestion using larger initial amounts of DNA and separating an aliquot of the digest on an overnight gel or measuring the length of insert remaining. 6. It is important to keep the length of homopolymer tail to as short a length as is consistent with efficient annealing to the oligomer. Long tails, particularly G tails with RD20, may create problems when they are sequenced. T7 DNA polymerase has a better capacity to read through G-C tatls than Klenow.

References 1. Dale, R. M. K., McClure, B. A., and Houchins, J. P. (1985) A rapid singlestranded strategy for producing a sequential seriesof overlapping clones for use in DNA sequencing,Plasmid 13,3 l-40. 2. Oshima,R. G. (1988) Rapididentification of Ml3 phagedeletionsfor sequence analysis.Biotechniques 6,s IO-5 11.

Subcloning

for DNA Sequencing

Gary M. Studnicka, Shau-Ping Lei, Hun-Chi Lin, and Gary Wilcox

1. Introduction Chemical (I) and enzymatic (2) methods for determining DNA sequences have revolutionized the techniques of molecular genetics and subsequently our understanding of the gene. The use of singlestranded Ml3 bacteriophage (3-5), in conjunction with Sanger’s dideoxy chain-termination sequencing procedure (2), has greatly increased the rate at which genes can be analyzed. One prerequisite for an efficient sequencingstrategy is a set of subcloneswhose endpoints areevenly distributed along the entire sequence. Shotgun subcloning methods (6,7) require rigorous fractionation of the DNA fragments before cloning, to insure appropriately sized subclones in the final library. Since clones are then chosen at random from the library, shotgun methods are very inefficient at completing the last few small sequencegapsthat remain near the end of the project. Various nonrandom subcloning methods have also beendeveloped,utilizing partial restriction digests(8), BAL-3 1 digestion (9), exonuclease III digestion (l&13), and DNase I (14). The method described here (15) is an improvement of Hong’s procedure (14). It requires two unique restriction sites, separated by only a short distance within the polylinker region of the vector, as shown in Fig. 1. Its overall objective is to create a deletion that begins at the first site, removes the second site along with a large piece of the cloned From Methods in Molecular Biology, Vol. 23 DNA Sequencrng Protocols Edlted by H and A Grifhn Copynght 01993 Humana Press Inc , Totowa, NJ

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et al.

polylmker redrictlon altas

re&rictmn

sites

Fig. 1 Diagram of subcloning strategy The large ctrcle represents the complete cloning vector m crrcular double-stranded form (elements are not drawn to scale) Its sequencing primer region is shown as a small grey arc at the upper left, and the cloned inserted gene fragment (to be sequenced) is shown as a long black arc at the right. The vector’s polylinker region contains many adjacent restriction sites, two of which are utilized in this subcloning procedure. Cleavage at the first site provrdes the fixed endpoint for the deletion, and digestion with DNase I provides the random endpoint for the deletion. Cleavage at the second site ensures that nondeleted vectors and other aberrant forms are destroyed. Additional restriction sites are present m the polylinker regions on either side of the cloned insert

insert, and terminates at some random point withm the insert. Partial digestion with DNase I is used to linearize the circular double-stranded vector and create a random endpoint for the deletion. The first restriction digest createsthe fixed endpoint for the deletion, so that the excised fragment can separate from the rest of the vector. The two endpoints of the linearized and deleted vector are then made blunt using Klenow fragment, and the vector is recircularized with T4 DNA ligase. The second restriction digest destroys the circularity (and thus the transfection efficiency) of most aberrant molecules that escaped linearization and deletion, or that contained a mislocated random endpoint (e.g., in the vector genes or in the primer region), or were ligated as

Subcloning

for DNA Sequencing

71

concatemers. After transfection, plaques that contain the desired subclones in high yield are harvested. 2. Materials 1. TYE broth: 15 g tryptone, 10 g yeastextract,5 g NaCI, fill to 1 L with water. 2. Double-stranded RF (“replicative form”) DNA of the inserted vector (e.g., M13mplO or M13mpl l), prepared as described (16) from 0.5 L overnight cultures in TYE broth. The DNA is purified by the alkaline method (26), and then further purified by cesium chloride-ethidmm bromide equilibrium gradient centrifugation. 3. DNase I buffer (10X): 200 mM Tris-HCl, pH 7.5, 10 mM MnCl,, 1 mg/ mL bovme serum albumin, as described (I 7). 4. TE buffer: 10 n&f Tris-HCl, pH 7.60.1 mM EDTA. 5. Universal buffer (1X): 50 mM Tris-HCl, pH 7.8,5 mM MgCI,, 25 mM NaCI, 10 n&f (NH,),S04. 6. PEG solution: 13% polyethylene glycol8000, 1.6M NaCl, asdescribed (18). 7. H buffer: 6.6 rnZt4Tris-HCl, pH 7.4, 6.6 mM MgCl,. 8. Klenow fragment (DNA polymerase I large fragment). 9. Two different restriction endonucleases that cut uniquely, only within the polylinker of the vector, on the same side of the cloned insert. 10. Two other restriction enzymes (with unique sites) that can excise the subcloned fragment to determine its size. 11. Agarose gel apparatus, 1 and 1.5% agarose gels, tracking dye. 12. T4 DNA ligase buffer: 50 mM Tris-HCI, pH 7.5, 10 rru!4 MgCl*, 5 rnJt4 dithiothreitol, 0.2 mM ATP. 13. Competent E. coli cells, obtained by treating a culture of rapidly growmg log-phase cells with cold calcium chloride, as described (3). 14. DNase I (Worthington), prepared as described (141, and stored at -20°C.

3. Method 1. Dilute 10X DNase I buffer IO-fold immediately before use. Suspend 12.5 pg circular double-stranded vector DNA in 240 pL of 1X DNase I buffer. Aliquot this into five 1.5~mL Eppendorf tubes (48 pL each). Serially dilute DNase I to concentrations of 0.5,0.25,0.125,0.062, and 0.03 1 ng/pL. Add 2 pL of each diluent to each tube to give final DNase I concentrations of 20, 10, 5, 2.5, and 1.25 pg/pL. Incubate at room temperature for 5 mm. 2. Add an equal volume of phenol saturated with TE buffer to stop the reaction. Remove the aqueous layer and extract with an equal volume of chloroform. Mix 5 pL (‘/lo vol) of each sample with 1 pL of tracking dye, and run on 1% agarose gel to determine optimal digestion conditions.

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3. Select samples that contam sufficient smgle-cut linearized vectors, but that do not have the prominent smear typically associated with multiple DNase I digestions. Three distmct bands will appear m each track of the gel: Open circular forms run slow, closed circular forms run in the middle, and single-cut linear forms run fast. Small overly digested fragments will appear as a smear running faster than the lmear forms (see Note 1). Combme contents of appropriate tubes and precipitate the DNA with ethanol. Resuspend the DNA pellet in 200 pL of universal buffer. Digest with the first restriction endonuclease for 2 h at 37°C. 4. Heat to 68°C for 10 mm. Add 200 pL PEG solution and place mixture on ice for 1.5 h. This step preferentially precipitates large DNA molecules, leaving small pieces (includmg the deleted fragments) m solution. 5 Centrifuge at 4°C for 10 mm. Resuspend DNA m 100 pL TE buffer. Repeated PEG precipitation normally is not required. Extract with equal volumes of phenol. Extract with 50:50 phenol plus chloroform. Extract with chloroform. Ethanol precipitate. Resuspend the DNA pellet m 20 pL of H buffer. Add 5 U of Klenow fragment (DNA polymerase I large fragment) and incubate at room temperature for 15 mm. 6. Ethanol precipitate. Resuspend the DNA pellet m 200 Ils, of T4 DNA ligase buffer, add 40 U of T4 DNA ligase, and incubate overrught at 4OC. Blunt-end ligatton proceeds most efficiently at low temperature overnight. 7. Heat to 68°C for 15 min to inactivate enzyme acttvity. Add 100 pL universal buffer (1X) contammg an appropriate amount of NaCl for the second restriction enzyme (if it requires some), since the T4 DNA IIgase buffer contains no NaCl. Save one half of the sample as a control. Digest the other half with second enzyme for 2 h at 37’C. 8. Transfect 5 pL of digested sample mto 0.2 mL of competent E. co11 strain JM103 and plate (see Note 2) as described (3) 9. About loo-150 plaques per plate should be expected (see Note 3). Grow isolated plaques m 5 mL of TYE broth supplemented with JM 103 bacteria for 8 h or overnight, as described (3). 10. Harvest bacteriophages and prepare minilysate RF DNA, asdescribed (16) 11. Double digest with appropriate endonucleases (one at each end of the insert) to excise the subcloned fragment and determme its size on a 1.5% agarose gel. 12. Sequence according to Sanger (2).

4. Notes 1 In selecting DNase I digestion conditions, it is important to avoid samples with multiple hits (Indicated by a fast-runnmg smear on the gel), but the mclusion of samples with significant amounts of circular forms does

Subcloning

for DNA Sequencing

not matter. The restrictton endonuclease digestion at the second site destroys the transfection efficiency of circular forms, and thus eliminates unwanted background. 2. It takes less than two days from DNase I digestion to plating, and there is no bias toward subclones with small deletions. 3. Starting with 12.5 pg of DNA, this improved method produced approximately 10,000 plaques, and thus increased the transfectant yield by 60fold over that of the original procedure (14).

References 1. Maxam, A. M and Gilbert, W. (1977) A new method for sequencing DNA Proc Natl. Acad Sci. USA 74,560-564.

2. Sanger, F., Nicklen, S., and Coulson, A. R (1977) DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Set. USA 74, 5463-5467. 3 Messing, J., Crea, R., and Seeburg, P. H. (1981) A system for shotgun DNA sequencing. Nucl. Acids Res. 9,309-321.

4. Vieira, J. andMessing,J. (1982) The pUC plasmids,an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal prtmers. Gene 19,259-268 5 Messmg, J. and Vieira, J (1982) A new pair of Ml3 vectors for selecting either DNA strand of double-digest restriction fragments Gene 19,269-276. 6. Anderson, S (1981) Shotgun DNA sequencing using cloned DNase I-generated fragments. Nucl Aads Res 9,3015-3027. 7 Deininger, P. L (1983) Approaches to rapid DNA sequence analysis. Anal. Biochem 129,216-223 8 Lamperti, E. D. and Villa-Komaroff, L. (1990) Generation of deletion subclones for sequencing by partial digestion with restriction endonucleases. Anal. Biochem. 185, 187-193. 9. Poncz, M., Solowiejczyk, D., Ballantine, M., Schwartz, E , and Surrey, S. (1982) “Nonrandom” DNA sequence analysis in bacteriophage Ml3 by the dideoxy chain-termination method. Proc. Natl. Acad See. USA 79, 4298-4302 10. Guo, L. H. and Wu, R. (1982) New rapid methods for DNA sequencing based on exonuclease III digestion followed by repair synthesis Nucl. Acids Res 10,

2065-2084. 11. Henikoff, S. (1984) Umdirectional dtgestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 351-359. 12 Henikoff, S (1990) Ordered deletions for DNA sequencing and m vitro mutagenesis by polymerase extension and exonuclease III gapping of circular templates Nucl. Acids Res. 18,2961-2966 13 Gronostajski, R. M and Sadowski, P. D (1985) Determmatton of DNA sequences essential for FLP mediated recombination by a novel method J Biol. Chem. 260, 12,32O-12,327. 14. Hong, G. F (1982) A systematic DNA sequencing strategy .I Mol. Biol. 158, 539-549

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15 Lm, H.-C., Lei, S.-P., and Wilcox, G. (1985) An improved DNA sequencmg strategy Anal. Biochem. 147, 114-119. 16. Ish-Horowitz, D., and Burke, J. F. (1981) Rapid and efficient cosmtd clonmg Nuci Acids Res. 9,2989-2998. 17. Rigby, P W J., Dieckmann, M , Rhodes, C , and Berg, P. (1977) Labelmg deoxyribonucletc acid to high specific activity by nick translation with DNA polymerase I. J Mol. Biof. 113,237-251 18 Lis, J T. (1980) Fractionatton of DNA fragments by polyethylene glycol induced precrpitation. Meth. Enzymol 65, 347-353

&APTER

Sequencing Ulrike

11

Using Custom Designed Oligonucleotides

Gerischer

and Peter Diirre

1. Introduction The dideoxy chain termination DNA sequencing procedure introduced in 1977 (I) has the advantage of being fast, simple to perform, and very accurate. Therefore, it became the method of choice to obtain several hundred bases of sequence information per reaction. The procedure is based on the enzymatic elongation and radioactive labeling of oligonucleotides that are complementary to the beginning of the single-stranded DNA template. Chain extension competes with the infrequent but specific termination by incorporation of a dideoxyribonucleotide. The products of four nucleotide-specific reactions can be separated on a polyacrylamide gel. An autoradiogram of such a gel finally provides the sequence information. Detailed descriptions of the various modifications of this method have been presented in preceding chapters of this volume. The synthetic oligonucleotide primer required for the synthesis of the labeled strands can easily be synthesized in automatic synthesizers or by hand, the latter of which is more laborious. Various so-called “universal sequencing” and “reverse sequencing” primers, complementary to the beginning of the polylinker region in Ml3 Zac cloning phages and plasmids, are commercially available. Specifically designed oligonucleotides can also be prepared upon request by several molecular biological companies, From: Methods m Molecular Biology, Vol 23. DNA Sequencmg Protocols E&ted by: H and A. Gnfftn Copyright 01993 Humana Press inc., Totowa,

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In principle, there are three different ways of obtaming DNA sequenceinformation by the dideoxynucleotide method. The first strategy involves sequencing of randomly cloned fragments by using only commercially available primers complementary to regions close to the insertion site. This methodology requires a high number of recombinant clones, many time- and money-consuming sequencing reactions, and a computer for putting the data together into a whole. Alternatively, defined subclones can be constructed, characterized, and finally sequenced. This approach is more straightforward, but also takes a lot of work and time. The second procedure makes use of a set of different deletions in the original DNA fragment, originating close to the commercially available primer. These nested deletions extend various lengths along the target DNA. Thus, the longer the deletions are the more new target DNA is brought into sequencing range (see also Chapters 8,9, and 10). Again, this procedure requires additional recombinant DNA work on the original clone and the preparation of many DNA templates. The third approach is based on start of sequencing with primers that are complementary to a known sequence. This can either be the vector or any other sequence adjacent to the DNA to be analyzed. Sequencing proceeds by successive synthesis of new primers at the edges of the newly obtained sequence in such a way that their 3’ ends are pointing off into the unknown target DNA. This method is quick and straightforward, but usually requires the availability of a DNA synthesizer. 2. Materials Synthesis of oligonucleotides may be performed by hand (24) or can be done by several molecular biological companies. The latter possibility certainly is easierbut also time-consuming and relatively expensive. The most convenient way to obtain specific primers is the use of an automatic DNA synthesizer. If such a machine is available an oligonucleotide (17mer) can be prepared within approximately 2.5 h. When using an automatic DNA synthesizer chemicals such as solvents, protected deoxyribonucleoside-3’-0-cyanoethyl phosphoramidites, and special reagents are normally provided by the manufacturer (see Note 1). Cheaper offers from different companies are usually acceptable, provided the quality of these chemicals is high. Special attention should be paid to contamination with water. Water repre-

Custom Primer-Directed

Sequencing

77

sents the main problem in efficient oligonucleotide synthesis, and should therefore be kept away from all reagents and solutions. Since solvents are hazardous, care should be taken to perform the synthesis in a well-ventilated area. Detailed manual methods and the necessary equipment and material have been described in a previous volume of this series (2,3). For postsynthetic procedures such as deprotection, cleavage of the oligonucleotide from the support, and removal of impurities the following materials are required: 1. 25% ammonia m water (solution should be of highest quality). 2. Sephadex G-25 columns. It is recommended to work with prepacked columns that give a defined elution profile. 3. Sterile water or TE buffer (10 mM Tris-HCI, pH 7.5, 1 mM EDTA).

3. Design of Primers Working with custom primers, new oligonucleotides

are designed

according to the results of the first sequencing reactions with primers complementary to the beginning of the polylinker region in all M 13 lac cloning phages and plasmids. Thus, determined sequences serve to plan the next primer and so on. As a rule for primer design the formula : Primer length = 18 + 1 extra nucleotide for each 2% off of 50% G + C has been reported (5). This is because oligonucleotides with too high an A + T-content might not prime satisfactorily with special template DNA strands. However, working with an A + T-rich organism (Clostrz’dium acetobutylicum) we routinely used 17mer primers (6) with G + C-contents between 2 and 9 nucleotides and never experienced any problems. New primer and known sequence should well overlap to ensure that the starting nucleotides of the new part can be read and to have an internal control of each sequence start. As a rule of thumb the 3’

end of the primer should be located about 30-40 nucleotides from the end of the already known sequence. Before synthesizing a primer its sequenceshould be compared carefully to the whole known sequence of template DNA and vector (preferably by means of a computer program). This will identify regions where undesired hybridizations could take place. Of special importance in this respect are the last ten 3’-terminal bases. More than 80% homology in this region might cause major background

problems,

Gerischer and Diirre particularly in G + C-rich templates (5). If undesired homology indeed can be found, a new primer should be designed (see Note 2). In general, it is not possible to predict the functionality of a new primer. Even oligonucleotides that are designed according to all rules might fail. In such a case synthesis of a different primer is recommended. 4. Synthesis

of Primers

Detailed procedures for manual oligonucleotide synthesis have been described in a previous volume of this series (2,3). Using an automatic DNA synthesizer the instructions of the respective manufacturer should be followed exactly. Therefore, only the principle of a standard method (using phosphoramidites) and the postsynthetic procedures will be described here. To avoid undesired reactions during synthesis, the hydroxyl group at position 5 of the monomers is protected by a 4,4’-dimethoxytrityl group, the hydroxyl group at the phosphorus atom by a P-cyanoethyl moiety, and primary ammes either by benzoyl groups (N6 in case of deoxyadenosine, N4 in case of deoxycytidine) or by the isobutyryl group in position N2 of deoxyguanosine. Thymidine is usually not protected (3). A fully protected dA monomer is shown in Fig. 1. The first nucleotide is coupled via its 3’-OH group and a spacer to a solid support such as silica, glass, or plastic beads. Then its S-dimethoxytrityl group is split off and the next monomer is added using the activator tetrazole. After a wash to remove this substance and unreacted nucleotides, unreacted Y-OH groups are acetylated (capping). Oxidation of the phosphite triester bridge renders the molecule ready for the next round of elongation (3). After completion of the synthesis the oligonucleotide is separated from the support by a short centrifugation (2000 g, 1 min) to remove residual solvent and incubation in 25% ammoma (15 h at 55°C). Care should be taken to completely submerge the support using an appropriate tube, Residual air bubbles can be removed by short centrifugation (2000 g, 1 min). The support is then taken out of the solution and centrifuged in a tube to obtain the residual liquid (2000 g, 1 mm). Both solutions are pooled and represent a crude mixture of a variety of oligonucleotides and some ammonium salts in ammonium hydroxide. The concentration of impurities is higher the longer the sequence of the oligonucleotide is. Removal of ammonia and other impurities

Custom Primer-Directed

Sequencing

79

/\ 0 P-OCHzCHzCN

Fig 1. Protected dA monomer using the phosphoramldlte

method (3).

is achieved by size exclusion chromatography on Sephadex G-25. The use of prepacked columns is recommended. Equilibration and elution is done with sterile water or TE buffer (seeNote 3). For sequencing reactions it is usually unnecessary to further purify the primer (which could be done by HPLC or gel electrophoresis). However, if the sequence of the oligonucleotide allows hairpin formation by selfcomplementation, denaturing conditions might be necessary in the purification procedure. 6. Sequencing

Reaction

Automatic DNA synthesizers such as Gene Assembler Plus (Pharmacia LKB GmbH, Freiburg, Germany) have a coupling efficiency of 98% under optimal conditions. Thus, one usually obtains a highly concentrated primer solution after removal from the solid support. Many sequencing protocols recommend the use of 10 ng primer per sequencing reaction, Using a 0.2~pm01 capacity support column in

80

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the above mentioned machine we routinely obtain approx 600 ~18oligonucleotide (in 1.5 mL buffer). The concentration can be determined by measuring the absorbance at 260 nm (1 corresponds to 3 1 p.g/mL). This solution is diluted loo-fold and 2 w are used for sequencing in the annealing reaction. We also routinely used 1 p.L of the undiluted endproduct and the results were as good as with less primer. Sequencing was performed as described in Chapter 13 of this volume. A complete cycle of designing and synthesizing a primer, performing the sequencing reaction, and analysis of the DNA sequence can be done in 48 h. Thus, every 2 d approx 400 bases of new sequence information become available. If subclones, or similar clones, and the necessary sequencing equipment are at hand, this number could easily be increased. This might be desirable in large sequencing projects. Advantages of this method are that no additional steps such as subcloning (including recombinant DNA work, transformation, and plasmid characterization), medium preparation for cultivation of clones, and template isolation from a large number of clones are necessary. A possible disadvantage might be the costs for primer synthesis. Using an automatic sequencer, the cost comes to approx $40-50 per 17mer.

6. Notes 1. Synthesis reagents for an automatic DNA synthesizer were found to be stable for at least 3-4 wk under laboratory conditions. 2. We strongly recommend to check the template sequence carefully after every elongation. Ambiguous regions can thus be resequenced at once which might help to avold later unnecessary primer synthesis. Direct computer analysis will also enable the identlflcatlon of matching stretches if sequencing was started from both ends of the template. If only the end of a newly obtained sequence 1s read and used for new primer design this could finally lead to unnecessary sequencmg of large parts of the vector. 3. If there are any doubts concerning the quality of the synthesis, the o11gonucleotlde can be examined m a sequencing gel (20% polyacrylamide, 8M urea) by comparison with an oligonucleotide of good quality or with a commercially available oligonucleotide marker. Visualization can be performed by UV-shadowing (by simply putting the gel on a fluorescent thin layer chromatography plate and dluminatmg It with UV light at 254 nm [7]) or by autoradrography after kinasmg the oligo-

Custom Primer-Directed

Sequencing

81

nucleotide with [‘y- 32P]ATP. Attention: Polynucleotide kinase shows unfavorable reaction kmettcs with a C at the S-end. The back reaction under these conditions is much stronger than the forward reaction. 4. Oligonucleotides synthesized as primers might also serve additional functions. They might be used in primer extenston expertments to determine transcription start points or for sequencmg a large number of mutants in a defined region within a short period of time. They can also be used as probes to detect specific DNA fragments by Southern hybrtdization.

Acknowledgments Work reported from this laboratory has been supported by grants

from the Bundesminister fiir Forschung und Technologie and the Deutsche Forschungsgemeinschaft. References 1. Sanger,F., Nlcklen, S., and Coulson, A. R. (1977) DNA sequencmg with chamterminatmg mhrbitors. Proc. Natl. Acad. Scr USA 74, 5463-5467 2. O’Callaghan, D. M and Donnelly, W. J. (1988) Ohgonucleotrde synthesis using the manual phosphotriester method, in Methods in Molecular Biology, vol 4. New Nucleic Acids Techniques (Walker, J M , ed >, Humana, Clifton, NJ, pp 165-192. 3. White, H. A. (1988) Manual ohgonucleotide synthesis using the phosphoramidite method, in Methods in Molecular Biology, vol. 4: New Nucleic Acids Techniques (Walker, J. M., ed ), Humana, Clifton, NJ, pp 193-213. 4. Gait, M. J. (1984) Oligonucleotide Synthesis-A Practical Approach. IRL Press, Oxford. 5. Barnes, W. M. (1987) Sequencing DNA with drdeoxyrrbonucleotides as cham terminators: Hints and strategies for big projects. Methods Enzymol. 152,538556. 6. Gerischer, U. and Durre, P. (1990) Cloning, sequencmg, and molecular analysis of the acetoacetate decarboxylase gene region from Clostridium acetobutylicum. J. Bacterial. 172,6907-69 18. 7. Thurston, S J and Saffer, J. D. (1989) Ultraviolet shadowing nucleic acids on nylon membranes. Anal Biochem. 178,41-42.

&IAPTER

12

Dideoxy Sequencing Reactions Using Klenow Fragment DNA Polymerase 1 Alan II Bankier 1. Introduction Historically, DNA sequencing by primed synthesis and chain terminators has been performed using the Klenow fragment of DNA polymerase 1 (I). This enzyme was derived from E. coli. DNA Pol I which, in addition to polymerase activity, has both 3’-5’ and S-3’ exonuclease activities. As described in Chapter 1, the principle of dideoxy sequencing is that four base specific sequence reactions are performed. Each reaction generates a large range of product lengths with a single 5’ end common to all fragments, and 3’ ends that terminate at every possible occurrence of a specific nucleotide. The four sets of products are fractionated through a polyacrylamide gel and the position of a specific base can be deduced by virtue of the comparison of each fragment’s length (2). The native enzyme would be no good for this procedure since the 5’-3’ exonuclease would destroy the unique 5’ end, and hence the reference point. The Klenow fragment is a proteolytic derivative of Pol I lacking this 5’-3’ exonuclease, but retaining both the polymerase action and the 3’-5’ exonuclease action, This latter enzymic action serves a useful function in terms of “proofreading” the polymerase incorporation From Methods m Molecular Biology, Vol 23’ DNA Sequencing Protocols Edlted by H and A Grlffln Copynght 01993 Humana Press Inc , Totowa,

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and thereby improving fidelity. However, some problems encountered with Klenow DNA sequencing can be attributed to the presence of this action. Klenow polymerase is far from ideal for use as a sequencing enzyme. It is fairly simple to describe the properties of the perfect enzyme. The ideal qualities of a sequencing enzyme are: 1. It should be easily preparedm large quantities. 2. It needs to be stable upon storagefor considerableperiods. 3. The enzyme should acceptall deoxy and dideoxy nucleotides and analogues as substrateswith equal affinities and high fidelity. 4. The polymerase action should be highly processive over nucleotide extensionsto 1 kb andbeyond,eventhrough regionsof secondarystructure within the template.

5. This activity should remain high, even in suboptimal conditions. 6. It should be inexpensive.

Klenow enzyme does not fully match any of these criteria. So why is it still used? The answer is that none of the other commercially available enzymes, described for use in DNA sequencing, fully match these requirements either, although some are much better than others. The Klenow enzyme is fairly well understood, and many of its failings can be compensated for by an experienced user. It comes closer than some of its rivals in meeting certain aspects of the ideal description. The use of Klenow, at least for the moment, is on the decline, in favor of the use of T7 polymerase and its derivatives, such as Sequenase (Chapter 14), and of the thermally stable enzymes, such as 7izq polymerase (Chapter 15). Sequenaseproduces a much more even band intensity pattern and, under ideal conditions, can give visually stunning results. The thermally stable enzymes that can be used in amplification or cycling procedures are particularly useful for fluorescence based automated systems where increased yields of reaction products, and hence signals, is of high importance. The simplicity of the procedures used for sequencing with Klenow, lend themselves very well to the manual handling of many samples simultaneously (3,4). This makes Klenow an acceptable alternative for large scale, radioactive sequencing. The methods described here assume that sequencing is being performed on single-stranded DNA derived from M 13 cloning vehicles

Sequencing

Using Klenow Polymerase

85

(51, although simple modifications can broaden this applicability. The template quantities assume a concentration of DNA around 0.125 pg/ pL. This is typical of the yield to be expected from a conventional prepa-

ration of a 15mL culture, dissolved in less than 50 pL. Making this assumption, it is not normally necessary to assay the concentration

any more accurately. The procedure for microtiter tray preparation, described in Chapter 6, also yields template DNA of around this con-

centration, although in smaller volume. These methods also describe reagent volumes for a single sequence reaction. This is seldom the case, but actual reagent volumes can simply be scaled. Very often, because the method employs microtrter trays, the most convenient numbers of samples handled simultaneously are 8, 12, and 24, or multiples thereof. These numbers are also convenient in terms of how many samples can be loaded onto standard sized polyacrylamide gels. In these radioactive sequencing reactions, any a-labeled nucleotide

triphosphate can be used although the reagents described here are specifically for dATP. Currently used radiolabel markers are 32P (I), 35S (6), or 33P (7). The first method detailed here is the conventional technique whereby chain extension/termination and incorporation of the labeled nucleotide occur in the same step. The second method separates the

labeling step from termination. This latter approach is reported to give readings over a longer length but is sensitive to template DNA concentration and has shorter incubation times, making the processing of lots of samples difficult. 2. Materials All materials and chemicals used in molecular biology techniques should be considered to be a potential health hazard and need to be handled with this in mind. Where appropriate, some materials have been singled out as being particularly hazardous. 2.1. Materials Common to Methods 1 and 2 1. Nonsterile, untreated 96-well microtiter trays such as Falcon 3911 or preferably heat resistant polycarbonate microtiter trays (e.g., Techne, Duxford, England, Hi-Temp 96, cat. no. FMW 11). 2. Synthetic oligonucleotide primer at a concentration of 0.5 pmol/pL in TE. This molecule should be between 15 and 30 nucleotides in length.

Bankier

86

3.

4. 5. 6. 7. 8.

9.

10. 11.

12.

13.

It should also hybridize specifically m the 5’ region of the template DNA that is to be sequenced. For templates derived from the Ml 3 series of vectors, or vectors having related polylmker cloning regions, a single “universal” primer (5’ d(GTAAAACGACGGCCAGT)) can be used. The template DNA to be sequenced, at a concentratton of around 0.05 pmol/pL. Thts corresponds to a concentration of around 0.125 pg/pL of Ml3 DNA, the approximate yield from a standard 1.5 mL preparation being around 5 pg. TM buffer: 100 mM Trts-HCl, pH 8.5, 50 mM MgC12 . TE buffer: 10 mM Tris-HCl, pH 8.0,O. 1 mM Na*EDTA. Distilled or deionized water. Microtiter tray plate sealers (e.g., Costar cat. no. 3095). Deoxynucleotide triphosphates, dATP, dCTP, dGTP, and d?Tp dissolved m TE to 50 mM and diluttons of these stocks to give 0.5 mM working solutions. These solutions can be stored at -20°C for years although it is best to freeze them m ahquots to prevent repeated freeze/thaw cycles. Dideoxynucleotide trtphosphates, ddATP, ddCTP, ddGTP, and ddTTP dissolved m TE to 10 mM workmg solutions These solutions can be stored at -20°C for years although it is best to freeze them m aliquots to prevent repeated freeze/thaw cycles. O.lM dithiothreitol (Cleland’s reagent). Store at -2OOC. 35S, 32P, or 33P alpha labeled dATP at ~400 Ci/mmol, 10 mCt/mL. These materials are a potential health hazard and should be handled and disposed of appropriately. The 35S analog has a much lower energy emission (35S 0.16 MeV 33P, 0.248 MeV, and 32P, 1.7 MeV) and can be used without radiation shields. Klenow fragment DNA Polymerase I at a concentration of around 5 U/ pL or higher. Enzyme at a lower concentration adds a proportionally higher amount of glycerol to the reactions, which can affect electrophoresis unless an ethanol precipitation is performed. Formamide dye mix: 100 mL deionized formamide, 0.1 g xylene cyan01 F.F., 0.1 g bromophenol blue, 2 mL 0.5MNa2EDTA. This solution seems stable at ambient temperatures for several months, but it is recommended that aliquots be stored frozen. Formamide is hazardous and may cause irritation to skm and eyes. 2.2. Materials

Specific

to Method

1

1. Four nucleottde-speciftc dideoxy nucleotide mixes (ddNTP/dNTPs) as follows: ddATP mix: 250 w dCTP, 250 l.uW dGTP, 250 l.uW dTTP, 10 w ddATP.

Sequencing

Using Klenow

Polymerase

87

ddCTP mrx: 12.5 w dCTP, 250 @4 dGTP, 250 w dTTP, 80 pIU ddCTP. ddGTP mix: 250 pAI dCTP, 12.5pA4dGTP, 250 @4 dlTP, 160 pA4 ddGTP. ddTTP mix: 250 w dCTP, 250 w dGTP, 12.5 @4 dTTP, 500 ruz/l ddTTP. These solutions are prepared using the workmg nucleotxde solutrons and can be kept for years at -20°C. 2. Chase mix: 0.5 rn/WdATP, 0.5 mMdCTP, 0.5 mMdGTP, 0.5 mJ4dTTP. 2.3. Materials Specific to Method 2 1. Labeling mrx: 7.5 @I dCTP, 7.5 rJM dGTP, 7.5 @4 dTTP. 2. Four nucleotide-specific termmatron mixes (ddNTP/dNTPs) as follows: A mix: 25 p/U dATP, 250 w dCTP, 250 piU dGTP, 250 @4 dTTP, 300 /.uI4ddATP. C mix: 250 @4 dATP, 25 p/U dCTP, 250 w dGTP, 250 @4 dTTP, 100 @4 ddCTP. G mix: 250 w dATP, 250 w dCTP, 25 w dGTP, 250 w dTTP, 150 w ddGTP. T mix: 250 p/t4dATP, 250 @I dCTP, 250 w dGTP, 25 @4 dTTP, 500 /.&I ddTTP.

3. Methods Two methods of sequencing using Klenow polymerase are described here. The first is a single-step labeling and termination procedure, the second employs an initial labeling step, followed by a termination step. 3.1. Single-Step Labeling and Termination 1. Prepare a drlutron of the primer in sequence reaction buffer TM as follows: 1 pL primer (0.5 pmol/pL), 1 pL TM, 6 pL distilled water. Aliquot 2 pL of this primer dilution onto the sides of four adjacent wells of a disposable mrcrotrter tray (see Notes 1-3). 2. Dispense 2 pL of the template DNA into each of the same four wells, on the opposite side from the primer. Cover the wells to form an arrtight seal using a nonporous food wrap, such as Saranwrap@, or using a self-adhesive plate sealer. 3. Centrifuge the tray briefly (accelerate rapidly to 1000 g and rmmedtately stop) to combine and mix the droplets, and place the microtiter tray in an incubator at 55°C for at least 30 min (see Notes 4 and 5). 4. Centrifuge the tray to recover any condensation and add 2 pL of each specific dideoxy nucleotrde mix (ddNTP/dNTPs) to the side of one of

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Bankier

the four wells (assign each well in the order the polyacrylamide gel will be loaded (seeNote 6). 5. Prepare a label/enzyme mix, on ice, as follows: 6 pL drstilled water, 1 pL O.lM dithiothreitol, 0.5 pL radiolabeled dATP (10 mCi/mL, - 400 Gil mmol), 0.5 pL Klenow DNA Polymerase (5 U/pL). Mix the reagents by gently pipeting up and down(see Note 7). 6. Dispense 2 pL of the label/enzyme mix onto the side of each well, centrifuge the tray briefly, and place it at 37OCfor 15 min (see Notes 8 and 10). 7. Add 2 pL of nucleotide chase solution to each of the wells, centrifuge the tray briefly, and place it at 37°C for 15 minutes (see Note 9). 8. Inactivate the enzyme by adding 2 pL of formamide stop solution to each of the wells and centrifuge the tray briefly (see Notes 12 and 13). 3.2. Two-Step

Labeling

and

Termination

1. Usmg a single well of a microtiter tray or a microcentnfuge tube, add and mix: 1 pL primer (0.5 pmol/pL), 1 pL TM, 8 pL template DNA (= 1 pg), Cover the well to form an airtight seal or cap the tube and place it in an incubator at 55°C for at least 30 min (see Notes 1, 2,4, 5). 2. Toward the end of the annealing period, dispense 2.5 pL of the four termination mixes into four adjacent wells of a microtiter tray. 3. Centrifuge the annealing container to recover any condensation and add: 2 pL labeling mix. 1 pL O.lM dithiothreitol (Cleland’s reagent). 0.5 pL radiolabeled dATP (10 mCi/mL, = 400 Ci/mmol). 0.5 pL Klenow DNA Polymerase (5 U&L). 2 pL distilled water. Mix the reagents by gently pipeting m and out and leave at room temperature for 5 min (see Note 7) 4. Aliquot 3.5 pL of the labeling/extenston reaction into the four termmation wells and centrifuge the tray briefly to mix the reagents. Place the tray at 37°C for 5 mm (see Notes 10 and 11). 5. Inactivate the enzyme by adding 2 pL of formamrde stop solution to each of the wells and centrifuge the tray briefly (see Notes 12 and 13).

4. Notes 1. Unlike many recommended buffers for DNA sequencing,the TM described here does not contam sodium chloride. Some sequence dependent, template secondary structures, which inhibit polymertzatton, are stabilized m salt soluttons. Removal of the sodium chloride improves the results on these templates without adversely affecting routine sequencmg with Klenow polymerase.

Sequencing

Using Klenow Polymerase

89

2. Microtiter trays are recommended as the reaction vessel; they are ideally suited to the small volumes employed, they are disposable, many samples can be handled at once, no caps need to be opened or closed, and individual contamers do not need to be labeled for identification. 3. In the two-step labeling/extension method, all of the reagent volumes have been standardized at 2 pL. This means that a single microliter pipet can be used for all additions. It also becomes possible to use a repetittve dispenser to greatly speed up multiple additions. The most convenient of these repetitive dispensers is a simple ratchet fittmg for a Hamilton gastight microliter syringe. When used with an LTlOO syringe fitted with a luer, disposable tip adapter, 50 x 2 pL additions can be made from a single charging of a disposable tip. 4. Annealing of the template DNA to primer can be carried out at a wide range of temperatures and incubation times. Using a single temperature of 55°C gives a good compromise between mcubatton time and ease of handling. The precise temperature is not crittcal. 5. Once annealed, the template/primer can be stored for several weeks at -2OOCwithout noticeable deterioration. This can be convenient for largescale sequencing (using robotics in particular) or when lack of time prevents continuing with the procedure. 6. The ratio of the dideoxy:deoxy nucleotide within a particular mix mfluences the rate of termination within that reaction. At low terminator concentration, chain extension will proceed farther. The mixes described here should be regarded as a starting point from where fine-tunmg can begin. By altering this ratio within the working nucleotide terminator mix, a peak of distribution of bands within the desired range can be easily obtained. 7. Klenow fragment DNA polymerase should be kept at a constant cold temperature whenever possible (especially when diluted). When removing an aliquot from the stock, it is best done rapidly within the confines of the freezer. 8. Sequence extensions usmg Klenow can be carried out at ambient temperature rather than at 37OCwithout noticeable deterioration of the end result. 9. It is possible to omit the chase step, as an extreme measure m minimizing the handling of large numbers of samples, by mcreasmg the extension time to around 30 min. Under these conditions, be sensitive to the signs of incomplete extension (termination in all four lanes at positions Immediately before “A” bands, the concentration limited, radiolabeled nucleotide). Usually it is best to assume the chase IS necessary rather than generate a large number of useless reaction products. 10. Microtiter tray thermal cyclers are very convenient for DNA sequenc-

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Bankier

ing. The annealing, extension, and denaturation can all be carried out without transferring the tray between drfferent locations. 11. The two-step labeling/termmation procedure becomesdtfficult when many samples are being processedstmultaneously. The short incubation periods and the many ptpetings involved make it difficult to standardize the reaction conditions. One way to overcome thts problem is to predispense the termination mixes prior to startmg. Also, carefully aliquot the labeling mixture as droplets on the side of the wells immediately

after mixing.

Ter-

mination reactions can then be started at the appropriate time by driving the droplets to the bottom of the well by brief centrifugatton. I2 If the sequence reaction samples are to be stored for any period of time before loading on a polyacrylamide gel, it is best to freeze the samples without

adding formamrde.

Normally

no degradation

1s apparent upon

storage in formamide but, occastonally, a degradation smear can appear after only a short period. 13. Despite the many tmprovements in the field of DNA sequencing, there are still many areas where things can go wrong. Since many of the reagents store well at -20°C it is highly recommended that a complete control kit is kept m the freezer. Problem solvmg IS simply a question of substituting reagents into this control system until the source of the problem is pinpointed. By far the most common problems are associated with template purity and enzyme quality.

References 1. Sanger, F., Nicklen, S , and Coulson, A. R. (1977) DNA sequencing with chainterminating inhibitors Proc Nat1 Acad. Sci USA 74, 5463-5467. 2 Bankier, A. T. and Barrell, B. G (1983) Shotgun DNA sequencing, rn Techniques in Life Sciences, Nucleic Acid Biochemistry vol. B5, (Flavell, R. A , ed.), Elsevier Scientific Publishers, Ireland, pp. l-34. 3. Bankier, A T , Weston, K. M , and Barrell, B G (1987) Random cloning and sequencing by the M13/dideoxynucleotide chain termination method, in Methods in Enzymology vol. 155, (Wu, R., ed.), Academic, London, pp. 51-93 4. Bankrer, A. T. and Barrell, B. G. (1989) Sequencing single-stranded DNA using the chain termination method, in Nuclerc Acids Sequencrng. A Practical Approach (Howe, C. J. and Ward, E. S., eds.), IRL Press, Oxford, pp. 37-78 5. Messing, J. and Bankier, A T (1989) The use of single-stranded DNA phage in DNA sequencing, in Nucleic Acids Sequencing. A Practical Approach (Howe, C. J. and Ward, E. S., eds.), IRL Press, Oxford, pp l-36 6 Biggin, M. D , Gibson, T. J., and Hong, G. F. (1983) Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination Proc. Natl. Acad. Sci. USA 80,3963-3965.

7. Zagursky, R J., Conway, P S , and Kashdan, M A. (1991) Use of 33P for Sanger DNA Sequencing Biotechniques 1,37-38

&A.E’TER

13

Dideoxy Sequencing Reactions Using T7 Polymerase Peter Diirre and Ulrike

Geriscker

1. Introduction The chain termination DNA sequencing procedure introduced by Sanger and coworkers in 1977 (I) meant a revolutionary achievement in fast, simple, and accurate deciphering of DNA fragments. Long stretches of this nucleic acid could from then on be sequenced within a relatively short time. The method makes use of the enzymatic elongation of specific oligodeoxyribonucleotide primers that are complementary to a specific sequence of the DNA template. Four different reaction mixes each contain all four conventional deoxyribonucleotide triphosphates (dNTPs) plus a small amount of one dideoxyribonucleotide triphosphate (ddNTP), an analog that leads to chain termination since the lacking 3’-hydroxyl group prevents formation of a phosphodiester bond with a succeeding nucleotide. The four assays each contain a different dideoxynucleotide triphosphate. The chain extension reaction thus competes with the infrequent but specific termination by a ddNTP. This leads to a mixture of DNA fragments of varying lengths that all possess the same 5’-end owing to the common primer. Since one of the dNTPs contains a radioactive isotope (either 32P or 35S) these fragments become radioactively labeled and can be detected by autoradiography after separation by gel electrophoresis. The corresponding sequence is derived from the From. Methods m Molecular S/ology, Vol 23 DNA Sequencmg Protocols Edlted by. H and A Grlffm CopyrIght 01993 Humana Press Inc , Totowa,

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band pattern of the four different reaction mixtures. Single-stranded phage and denatured double-stranded plasmid DNA can serve as a template, thus opening the possibility to use a large variety of vectors for cloning. Primers that are complementary to various regions of the multicloning sites of these vectors are commercially available from a number of molecular biological companies or can easily be synthesized in the lab (compare Chapter 11). The sequencing reaction is catalyzed by DNA polymerases such as Klenow fragment of Escherichia coli DNA polymerase I, reverse transcriptase (from Avian Myeloblastosis Virus [AMV] or MoloneyMurine Leukemia Virus [M-MLV]), a number of thermostable polymerases (from Thermus aquaticus [Tuq], Thermus thermophilus [Tth], Thermococcuslitoralis [Vent,], Bacillus stearothennophilus pst]), and T7 DNA polymerase. Originally, the Klenow fragment was used for dideoxy sequencing. However, this enzyme has a low processivity, meaning that it will randomly dissociate from the template and thus give rise to fragments that were not terminated by incorporation of a dideoxyribonucleotide. Thus, only relatively short sequences can be determined reliably in one run. Furthermore, chain elongation proceeds at a relatively low rate, secondary structures of the template create a problem, special nucleotides used in case of band compressions are not equally well incorporated, and the enzyme is very sensitive to contaminants in template preparations. Reverse transcriptase can better cope with problems caused by secondary structures in the template DNA, but thermostable polymerases usually are the method of choice in this case since even stable secondary structures are precluded at temperatures of 70-75°C. The bacteriophage T7 DNA polymerase complex consists of two polypeptides, the gene 5 product encoded by the phage and thioredoxin encoded by the E. coli host (2,3). This enzyme is now widely used for dideoxy sequencing because of its high processivity, high rate of polymerization, and tolerance for nucleotide analogs. Since the enzyme has been reported to possess a high 3’-5’ exonuclease activity (4,5) two different ways have been used to render it suitable for sequencing. One approach was to modify the enzyme either chemically by oxidation (6,7,Jor genetically to remove the exonuclease activity. The resulting products are known under the trade name “Sequenase” and

DNA Sequencing

Using T7 Polymerase

their use is described in Chapter 14. The other approach made use of a specific FPLC-purification of cloned T7 DNA polymerase to obtain a protein suitably low in exonuclease activity. Such a preparation proved to be similarly advantageous or even superior to modified T7 polymerase (8). This chapter describes the dideoxy sequencing procedure using unmodified T7 DNA polymerase. 2. Materials 2.1. Primers Various primers for a number of commonly used cloning vectors are commercially available (e.g., from Amersham [Arlington Heights, IL], GIBCO BRL [Gaithersburg, MD], International Biotechnologies Inc. [New Haven, CT], New England Biolabs [Beverly, MA], Pharmacia LKB [Piscataway, NJ], Sigma [St. Louis, MO], Stratagene [La Jolla, CA], United States Biochemical Corporation [Cleveland, OH]). Alternatively, appropriate primers can be synthesized by automatic machines or by hand (compare Chapter 11). The concentration of primer in the annealing reaction should be approx 0.11 w (corresponding to 0.67 pg/mL for a 17mer). However, even much higher concentrations have been used successfully (compare Chapter 11). 2.2. Template Preparation (Double-Stranded Plasmid DNA) 1. STEbuffer: 100mM NaCl, 10mM Tr~s-HCl,pH 7.5,l mM EDTA, pH 7.5. 2. Lysozyme solution: 50 mM glucose, 50 rnk! Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0. This solution should be autoclaved or filter sterilized and stored at 4°C. Prior to use 5 mg/mL crystalline lysozyme are added and completely dissolved. 3. SDS solution: Sodium dodecyl sulfate (l%, w/v) m NaOH (0.2M). Prepare fresh before use. Store at room temperature. 4. Sodrum acetate solution: 3M sodium acetate, pH 4.8. This solution IS prepared by dissolving 246.1 g of waterfree CH,COONa m 500 mL HzO, adjusting the pH to 4.8 with glacial acetic acid, and then adjusting the volume to 1 L. Store at room temperature. 5. Isopropanol: 100% (v/v). 6. Phenol/chloroform solution: Melt crystalline phenol at approx 50°C. Equilibrate the liquid with 0.2M Tris-HCI, pH 7.8. Only dlstllled phenol should be used. Do not use higher concentrated buffers for equili-

94

Diirre

and Gerischer

bration, smce that will affect phase separatton. MIX chloroform and lsoamyl alcohol m a ratio of 24: 1. Mix this new solution with the equiltbrated phenol in a ratio of 1:l to obtain the final reagent. Store m a brown glass bottle (if possible, under a nitrogen atmosphere) at 4°C. 7. Ethanol: 96 and 70% (v/v). 8. TE buffer: 10 mM Tris-HCl, pH 7.5, 1 r&f EDTA, pH 7.5. 9. RNase solutron: 10 mM Trrs-HCl, pH 7.5, 15 n&I NaCl, RNase (2 mg/ mL). Dissolve enzyme completely and keep the solutton at 100°C for 15 min to inactivate possible DNase contaminants. Let cool to room temperature, divide in small ahquots, and store at -20°C. 10 Chloroform solution, MIX chloroform and isoamyl alcohol tn a mtto of 24: 1. 2.3. Sequencing Reactions 2.3.1. Primer Annealing 1. Template: single- or double-stranded DNA, 0.25 pg/pL. 2. NaOH solutton: 2M NaOH. 3. Sodmm acetate solution: 3M sodmm acetate, pH 4.5. For preparatton see Section 2.2., step 4. 4. Ethanol: 96 and 70% (v/v). 5. Primer solution: 0.8 @4 in TE buffer or H20. 6. Annealing buffer: 280 mMTris-HCI, pH 7.5,100 m/t4MgCl,, 350 mM NaCl. 7. DTE solutton: 0.3M dithioerythrttol. 2.3.2. Labeling Reaction 1, T7 DNA polymerase, FPLC-purified, suitable for sequencing (Pharmacta LKB). Be careful to keep the enzyme constantly at -20°C. For takmg an ahquot the polymerase should be removed only briefly from the freezer. The aliquot IS then diluted wrth drlutron buffer and used for sequencing. The dilutton can be kept on Ice until the sequencing reactions are set up. 2. Drlutron buffer: 20 mM Trts-HCI, pH 7.5, 5 nut4 drthroerythrttol, 50 pg/mL bovine serum albumin. 3. Radioactive nucleotide: [a-32P]dATP or [a-35S]dATPaS, 370 MBq (10 mCl)/mL and 370 463 MBq (10 -12.5 mCi)/mL, respectively. Both nucleotides are mcorporated equally well mto the growing obgodeoxyribonucleotlde chain by T7 DNA polymerase. Using 35S,sharper bands are obtained in the autoradrogram and, as a result of the avoidance of scattering, longer sequencescan be deciphered in one run. The shelf life of this isotope IS longer, and the radiation dose of the personnel IS reduced (9). Advantages of 32Pare that shorter exposure times are required and that the sequencing gel does not need to be dried before autoradiography.

DNA Sequencing

Using T7 Polymerase Table 1 of Termination

Composition A-Mix

Component stock solution, mM dATP (1) dCTP (1) dGTP (1) dTTP (1) ddATP (0.5) ddCTP (0.5) ddGTP (0.5) ddTTP (0.5) Sequencing buffer MgC12 (50) Tris-HCl, pH 7.5 (200) NaCl(250) H2O

Mixes

C-Mix

G-Mix

T-MIX

PL

w

PL

PM

PL

w

w

PM

15 15 15 15 3

150 150 150 150 15

15 15 15 15

150 150 150 150

15 15 15 15

150 150 150 150

15 15 15 15

150 150 150 150

-

3 -

20

3

20 10 40 50

17

-

15

-

15 3 20

20 10 40 50

17

-

10 40 50 17

15 10 40 50

17

4. Labeling mtx: 1.375 p.M dCTP, 1.375 @I dGTP, 1.375 @I dTTP, 333.5 mM NaCl. This mix can be stored m alrquots at -20°C.

5. DTE solutron: 0.3M dithioerythrrtol. 2.3.3. Termination

Reaction 1, Termmation mixes: dATP, dGTP, dCTP, dTTP, ddATP, ddCTP, ddGTP, ddTTP, concentratronssee Table 1 (see Note 1). 2. Sequencing buffer: Composition see Table 1. 3. Stop solution: 95% (v/v) formamide, analytical grade, 20 mM EDTA, pH 7.5, 0.05% (w/v) xylene cyanol, 0.05% (w/v) bromophenol blue.

This solution should be stored at -20°C (if possible, under a nitrogen atmosphere). T7 DNA polymerase is also available in a kit, together with most of the required solutions (Pharmacia LIB). The composition of these solutions was not disclosed by the manufacturer. Sequencing using the reagents described above yields results that are practically identical to those obtained by the premade sequencing kit (Fig. 1). However, use of this kit saves the time for preparation of the solutions and, furthermore, provides so-called “read long sequencing mixes” that allow sequencing of up to 1000 nucleotides per run if a high resolution sequencing equipment is available.

96

Diirre

and Gerischer

Fig. 1, T7 DNA sequencing using different solutions and reagent mixtures. (A) Results from sequencing using solutions and chemicals described in this chapter. (B) Results using a commercially available sequencing kit from Pharmacia LKB. A part of the DNA sequence of Closrridium acefoburylicum, a strictly anaerobic, sporeforming bacterium, is shown. A 6% sequencing gel has been used. G, A, T, C, products from sequencing reactions.

2.4. Gel Electmphomsis The respectivematerials and methods aredetailed in Chapters16 and 17. 2.5. Autoradiography 1. Acetic acid: 10% (v/v). 2. X-ray film: Kodak (Rochester, NY) or Amersham. 3. Light-protected box for washing and autoradiography

of the gel.

3. Method 3.1. PrimQrs Primer design and synthesis are detailed in Chapter 11.

DNA Sequencing

Using T7 Polymerase 3.2. Template

97

Preparation

Isolation procedures of single-stranded phage DNA and doublestranded plasmid DNA suitable for sequencing are described in other chapters of this volume. In case of plasmid DNA we found a modification of the method of Birnboim and Doly (10) especially well suited. This procedure is detailed below. 1. Centrtfuge 5 mL of bacterial culture grown overnight at 6,000g for 5 min at 4°C. 2 Discard supematantand suspendthe sedimenttn 150pL lysozymesolution. 3. Transfer the preparation to a sterilized microcentrifuge cup and mcubate for at least 5 mm at room temperature. 4. Add 300 pL SDS solution and mix gently by hand for a few seconds 5. Immedtately add 225 uL sodium acetate solution. 6. Mix gently by hand for a few seconds and Incubate for 15 mm at 0°C 7. Centrifuge for 5 min at 4°C m a microcentrtfuge at maximal speed. Use either a model with coolmg system or place centrifuge in the coldroom 8. Transfer supernatant to a new microcentrifuge cup using a microliter prpet with sterilized tip. Be very careful to take only the supernatant 9. Add 600 pL isopropanol to precipitate the DNA and vortex a few seconds, 10 Incubate for 5 mm at room temperature. 11. Centrifuge for 5 mm at room temperature in a microcentrifuge at maximal speed. 12. Remove supernatant completely and rinse sediment with 1 mL cold ethanol (70%). 13. Let the sediment au dry (3-5 min) and then suspend it m 100 pL TE buffer. 14. Add 1 pL RNase solution and incubate for 15 min at room temperature. 15. Add 260 pL H,O and 40 pL sodium acetate solution. 16. Add 1 vol of phenol/chloroform solution. Attention: Wear gloves to avoid any harm to your fingers if spilling this solution 17. Vortex a few seconds and centrifuge for 5 mm at room temperature m a microcentrifuge at maximal speed. 18. Transfer upper phase to a new microcentrifuge cup and add 1 vol chloroform solution. 19. Mix gently by mvertmg the closed cup several times. 20. Centrifuge a few seconds for phase separation and transfer upper phase to a new microcentrifuge cup 21. Add 2 vols of cold ethanol (96%) to precipitate the DNA and vortex a few seconds. 22. Incubate for 15 mm at -70°C.

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23. Centrifuge for 10 mm at 4OC in a microcentrifuge at maximal speed. 24. Rinse sediment with 1 mL cold ethanol (70%) and mix briefly. 25. Centrifuge for 10 min at 4°C m a microcentrifuge at maximal speed and discard supernatant. 26. Dry the sediment under vacuum (3-5 mm) and then suspend it in 20 pL TE buffer. This procedure usually yields 10 -20 pg plasmid DNA from a 5mL culture. 3.3. Sequencing Reactions The procedure described below represents a modification method of Tabor and Richardson (6).

of the

3.3.1. Primer Annealing Whereas single-stranded phage DNA can be used directly as a template, double-stranded plasmid DNA must be denatured first. 1. Pipet 8 pL double-stranded plasmid DNA (contau-nng 2 pg nucleic acid) mto a sterilized microcentrifuge cup. 2. Add 2 pL NaOH and vortex the cup briefly. 3. Centrifuge the cup for a few seconds m a microcentrifuge at maximal speed to concentrate the complete solution at the bottom of the cup. 4. Incubate the cup for 10 min at room temperature. 5. Add 7 pL sterilized HZ0 and 3 pL of sodium acetate solution. 6. Add 60 pL of cold (-20°C) ethanol (96%), mix well and incubate the cup for 20 mm at -70°C. 7. Centrifuge for 10 min at room temperature m a microcentrifuge at maximal speed and discard supernatant. 8. Add 300 pL of cold ethanol (70%) and mix briefly. 9. Centrifuge for 10 mm at room temperature in a microcentrifuge at maximal speed. 10. Remove the supernatant carefully and dry the sediment under vacuum (3-5 mm). 11. Suspend the pellet in 10 pL sterihzed H20. 12. Add 2 pL annealing buffer, 2 pL of primer solution, and 1 pL of DTE solution. 13. Incubate for 20 mm at 37°C. 14 Keep the cup at room temperature for at least 10 mm. If the sequencing reaction is not carried out subsequently, the solution should be stored at -20°C until needed. For single-stranded DNA the following procedure can be used:

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1. Pipet 10 pL template (contaming 2 pg DNA) into a sterthzed microcentrifuge cup. 2. Add 2 Ils, of primer solution, 2 pL of annealing buffer, and 1 pL of DTE solution, 3. Vortex the cup briefly. 4. Centrifuge the cup for a few seconds m a microcentrifuge at maximal speed to concentrate the complete solution at the bottom of the cup. 5. Incubate the cup for 10 mm at 60°C. 6. Keep the cup at room temperature for at least 10 mm. If the sequencing reaction is not carried out subsequently, the solution should be stored at -20°C until needed. 3.3.2. Labeling Reaction Since T7 DNA polymerase exhibits a high processivity and a high rate of polymerization the proper sequencing reaction is divided into two parts. In the first stage (labeling reaction) low concentrations of the four deoxyribonucleotides and a relatively low temperature result in the formation of short oligodeoxyribonucleotide chains (approx 2030 bases). Since a radioactive nucleotide is present in the reaction mixture, all fragments become uniformly labeled, which is important for equal band intensities in the autoradiography step. In the second stage (termination reaction) the four standard reactions mixtures are set up that contain high concentrations of the four deoxyribonucleotides and a single dideoxyribonucleotide each. Higher temperature

and nonlimiting dNTP concentration then allow a high rate of polymerization that is only terminated by incorporation of a ddNTP. 1. Dilute T7 DNA polymerase with dilution buffer to a concentration of 1.5 u&L. 2. Add 0.8 -1 l.tL radioactive dATP (370 p.Bq-10 pCi) to the microcentrifuge cup contammg annealed primer/template. 3. Add 3 ~.ILlabeling mix. 4. Add 1 pL DTE solutton. 5. Add 2 pL diluted T7 DNA polymerase. 6. Mix by pipeting several times up and down in the cup with a mlcroltter pipet and a sterilized tip. 7. Incubate for exactly 5 min at room temperature and then proceed directly with the termination reactton. 3.3.3. Termination Reaction 1. Prepare the 4 termmation mixes. This should be done in advance (e.g., during the incubation period of the annealing reaction).

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2. Set up appropriate vials for the termination reaction (microcentrifuge cups or a microtiter plate) and mark them carefully with “A,” “C,” “G,” and “T.” 3. Pipet 2.5 pL of the respective termmation mix into the correspondmg cup or well. 4. Warm mixtures to 37°C in a water bath (takes approx 1 mm). 5. Pipet 4.5 pL of the labeling mixture into each cup or well. Use a fresh tip for each transfer. 6. Mix briefly by pipetlng several times up and down with the rmcrohter pipet. 7. Incubate the reaction mixture for exactly 5 mm at 37°C. Since 4 reactions have to be processed,use defined time intervals (e.g., 30-60 s) for pipetmg to ensure that every mixture was incubated the same amount of time. 8. Add 5 pL of stop solution to each cup or well. 9. Mix briefly by pipettng several times up and down with the microliter pipet. 10. Denature template and oligodeoxynucleotides by heating the cups or microtiter plate for 3 mm at 80°C. Il. Put vials on ice. 12. Load 1.8-2 pL of each mixture onto a sequencing gel. The remammg material of the termination reactions can be stored at -20°C and used for further sequencing runs if necessary. Be sure to denature the mixtures agam before loading onto a gel. 13. In order to read as many bases as possible, 2 samples from each reaction mixture should be run on the same gel, with a period of electrophoresis (approx 2-3 h, until the first colored marker band reaches the end of the gel) between the two loadmgs. 3.4. Gel Ebctmphoresis The respective methods are detailed in Chapters 16 and 17. 3.5. Autoradiography 1. After fmlshmg the electrophoresis as detailed m the above-mentioned chapters remove the glass plates from the sequencing equipment. 2. Remove one of the plates so that the gel still sticks to one glass plate. 3. Put gel on plate into a horizontal box and cover it with 2 L acetic acid (10%) (see Notes 2 and 3). 4. Incubate for 30 -40 min at room temperature with occasional agitation to remove the urea from the gel. 5. Dry gel on plate at 80°C for 90 mm. Liquid should be wiped off the plate before putting it mto the oven. Alternatively, the gel can be transferred to Whatman filter paper and dried on an ordinary gel dryer. If 32P-labeled dATP is used, drying is optional. Instead, the gel can be

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covered with a plastic wrap. Suitable X-ray film is then put on top of it and exposed at -20 or -70°C for approx 4 h. 6. Cover the gel directly with a suitable X-ray film and expose it overnight (35S-labeling) in a light-protected box at room temperature. 7. Develop the film according to the manufacturer’s instructions and determme the sequence (see Note 4).

4. Notes 1. To avoid band compressions by G-G or G-C band pairing nucleotide analogs such as 7-deaza-dGTP, 7-deaza-dITP, or dITP can be used instead of dGTP. Complete premade mixes with c7dGTP and c7dITP are available from Pharmacia LKB. 2. To remove urea from sequencing gels with a final acrylamide concentration of more than 12%, the acetic acid wash solution should contam 2% (w/v) glycerol. Otherwise, the gel might crack upon drying. 3. The acetic acid wash solution might be reused for not more than four times. This will help to reduce the amount of radioactive waste. 4. Starting with prepared template DNA and primers one round of sequencmg including the autoradiography can be performed m 24 h (calculating overnight as exposure time). 5. Using this procedure we found that even PCR-products could be directly sequenced after only one phenol treatment, one chloroform extractton, and one ethanol precipitation as described in Section 3.2. 6. If multiple sequencing reactions with the same double-stranded plasmid are planned, it is recommended to use a large amount of plasmid in the denaturation reaction. This will save time for future sequencing reactions. The denatured DNA after being dissolved in 10 pL sterilized HZ0 can be stored at -20°C.

Acknowledgments We thank R. Fischer, K. Herwig, and U. Sauer for helpful discussions and preparation of Fig. 1. Work reported from this laboratory has been supported by grants from the Bundesminister fiir Forschung und Technologle and the Deutsche Forschungsgemeinschaft. References 1. Sanger, F., Nlcklen, S., and Coulson, A R (1977) DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463-5467. 2 Modrich, P and Richardson, C. C. (1975) Bacteriophage T7 deoxyribonucleic acid replication in vitro. Bacteriophage T7 DNA polymerase. An enzyme composed of phage- and host-specified subunits J Blol Chem. 250,55 15-5522

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3 Mark, D F. and Rtchardson, C C (1976) Escherichia colr thioredoxin A subumt of bactertophage T7 DNA polymerase Proc Nut1 Acad. Sci. USA 73, 780-784 4 Hori, K , Mark, D. F., and Richardson, C. C. (1979) Deoxyribonucleic actd polymerase of bacterrophage T7. Characterizatton of the exonuclease activrties of the gene 5 protein and the reconstituted polymerase. J Blol Chem 254, 11,598-l 1,604 5 Adler, S. and Modrtch, P (1979) T7-induced DNA polymerase. Charactertzation of associated exonuclease acttvtttes and resolutton mto btologtcally actrve subunits. J Blol Chem 254, 11,605-l 1,614 6. Tabor, S. and Rtchardson, C. C. (1987) DNA sequence analysis with a modrfied bacteriophage T7 DNA polymerase. Proc Nat1 Acad Sci USA 84,47674771. 7 Tabor, S and Rrchardson, C C (1987) Selectrve oxidation of the exonuclease domain of bactertophage T7 DNA polymerase. J Biol Chem 262, 15,33015,333. 8. Krtstensen, T., Voss, H , Schwager, C , Stegemann, J., Sproat, B., and Ansorge, W. (1988) T7 DNA polymerase in automated dtdeoxy sequencing. Nucl. Aczds Res 16,348 l-3496. 9 Van Helden, P. D (1988) Use of 35S nucleottdes for DNA sequencmg, m Methods in Molecular Biology, vol. 4. New Nucleic Acids Techniques (Walker, J. M., ed ), Humana, Chfton, NJ, pp. 81-88. 10 Birnborm, H. C. and Daly, J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl. Acids Res 7, 15 13-1523

&WI’ER

14

Dideoxy Sequencing Reactions Using Sequenase Version 2.0 Hugh G. Griffin

and Annette M. Grij”fZn

1. Introduction The dideoxy chain-termination method of DNA sequence analysis involves the synthesis of a DNA strand by enzymatic extension from a specific primer using a DNA polymerase (I). Several different enzymes are available for this purpose each having different qualities and properties. The use of the Klenow fragment of DNA polymerase I, DNA polymerase, and T7 DNA polymerase in DNA sequence analysis is described elsewhere in this volume. Bacteriophage T7 polymerase is an enzyme which has both a DNAdependent DNA polymerase activity and a 3’-5’ exonuclease activity, and can be used for DNA sequence analysis. However, the nucleotide analogs incorporated into the DNA strand by the polymerase activity are efficiently removed again by the exonuclease activity (2). The 3’-5’ exonuclease activity associated with T7 DNA polymerase also means that this enzyme does not undergo strand-displacement synthesis (2). In addition, the exonuclease activity has the effect of lowering processivity (the average number of nucleotides polymerized on a particular primer-template prior to dissociating), and giving an apparent polymerase activity much lower than it otherwise might be (2). From Methods m Molecular Bology, Vol 23 DNA Sequencing Protocols E&ted by Ii and A Grlffm Copynght 01993 Humana Press Inc , Totowa,

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Some years ago a chemically modified version of T7 DNA polymerase was produced that lacked most of the exonuclease activity but retained all of the polymerase activity (3,4). This chemically modified enzyme was called Sequenase. Later, a genetically engineered version of T7 DNA polymerase was produced which had a total absence of 3’-5’ exonuclease activity, was extremely stable and had a higher specific activity than the chemically modified enzyme (2,.5,6). This form of the enzyme was named Sequenase Version 2.0. Sequenase and Sequenase Version 2.0 have proven themselves to be very useful for dideoxy sequencing. They have properties markedly different from those of T7 DNA polymerase and other enzymes used for DNA sequencing. These properties include low (Sequenase) or no (Sequenase Version 2.0) 3’-5’ exonuclease activity, high processivity, high rate of polymerization, and efficient incorporation of nucleotide analogs (dITP, a-thio-dATP, 7-deaza-dGTP, dideoxy NTP’s, etc.). Both versions of the enzyme have higher processivity; a single molecule will incorporate thousands of nucleotides before dissociating from the template than the Klenow fragment of E. coli DNA polymerase I, whose processivity is only about lo-50 nucleotides (7). The rate of polymerization by Sequenaseis about 300 nucleotides per second compared with rates of 30-45 nucleotides per second for the Klenow fragment (7). The Sequenase enzymes can be used directly in the sequencing protocols available for other DNA polymerases. However, a special two-step protocol has been developed and appears to give better and more reliable results with Sequenaseand SequenaseVersion 2.0 (2,7). Template DNA is purified by standard techniques (described elsewhere in this volume) and is annealed to the oligonucleotide primer. The first step of the two-step reaction is the labeling step and is performed by mixing annealed template-primer DNA, dNTP’s including labeled nucleotide, and Sequenase. In this step, extension occurs for a limited number of bases utilizing most of the labeled nucleotide. In the second, or termination step, the reaction mixture is split into four aliquots. Each aliquot is added to a mixture of additional dNTP’s and a single dideoxynucleotide. Polymerization will only continue until a dideoxynucleotide is incorporated into the growing chain. This incorporation occurs randomly, providing the necessary range of sequence-dependent terminations. The entire two-step reac-

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tion can be performed in about ten minutes. The reactions are stopped by the addition of EDTA and formamide, heat denatured, and electrophoresed on an acrylamide gel. The unique characteristics of Sequenase Version 2.0 (high processivity, absence of 3’-5’ exonuclease activity, and efficient use of nucleotide analogs) produce radioactive bands of more uniform intensity and less background radioactivity than those obtained usmg other polymerases (2,6,7), and make it the enzyme of choice when sequencing long tracts of DNA. 2. Materials 1. Sequence Version 2.0 enzyme. 2. Enzyme dllutton buffer: 10 nnI4 Trrs-HCI, pH 7.5, 5 mM drthtothrettol, 0.5 mg/mL bovme serum albumm. 3. Sequenase buffer 5X concentrate: 200 nM Trrs-HCI pH 7.5, 100 mM MgC12, 250 rnk! NaCl 4 Primer DNA 0.5 pmol/pL. Universal primer available commercrally from many sources or use custom-designed primer. 5. Dithiothrertol O.lM. 6. Labeling mix 5X concentrate: 7.5 pkf dGTP, 7.5 pk! dCTP, 7.5 @! dTTP (see Note 1). 7 ddG termmatron mix* 80 w dGTP, 80 j&I dATP, 80 J..&IdCTP, 80 pkI dTTP, 8 @4 ddGTP, 50 n-&I NaCl. 8. ddA termination mix: 80 pA4dGTP, 80 pk! dATP, 80 @4 dCTP, 80 pk! dTTP, 8 @I4ddATP, 50 mM NaCl. 9. ddT termmatton mix: 80 pkI dGTP, 80 pkI dATP, 80 pkf dCTP, 80 @4 dTTP, 8 J&I ddTTP, 50 miI4 NaCl. 10. ddC termination mix: 80 pA4dGTP, 80 pk! dATP, 80 pi14dCTP, 80 w dTTP, 8 pkf ddCTP, 50 mM NaCl. 11. Labeled dATP: [a-35S]-dATP (see Note 2). Specific activity should be 1000-1500 Ci/mmol. 12. Stop solutron: 95% formamrde, 20 mikf EDTA, 0.05% bromophenol blue, 0.05% xylene cyanol.

All solutions should be stored at -20°C and kept on ice when thawed for use. Sequenase enzyme should be stored at -20°C (not m a frost free

freezer). Dilute the enzyme in ice-cold dilution buffer when required. Sequenase Version 2.0 is available from United States Biochemical Corporation (USB), PO Box 22400, Cleveland, Ohio, 44122 USA, or from USB c/o Cambridge Bioscience, 25 Signet Court, Stourbrrdge Common Business Centre, Swann’s Road, Cambridge CB5 8LA,

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England. USB offers a sequencing kit that includes all the buffers and nucleotide mixes needed for sequencing with Sequenase, the universal primer, control Ml3 DNA, and Sequenase Version 2.0 enzyme. Alternatively, the individual buffers and nucleotide mixes can be purchased separately. Radioactive safety procedures should be adhered to when handling solutions containing radioactively labeled nucleotides. Radioactive waste should be disposed of properly. 3. Methods 3.1. Annealing Reaction 1. In a small centrifuge tube (seeNotes 3 and 4) setup the following reactton: Primer (0.5 pmol/pL) 1 pL Sequenase buffer (5X) 2 pL 1 pg Ml3 or 5 pg plasmid (see Note 5) DNA Sterile drsttlled water to 10 pL. 2. Place in a 65OC waterbath for 2 mm. Allow to cool slowly to 30°C over a period of about 30 mm (see Note 6). Place on ice (see Note 7). 3.2. Labeling Reaction (see Note 1) 1. Dilute the labeling mrx concentrate 1:5 (1 + 4) wtth water (see Notes 5 and 12). 2. Dilute the Sequenase enzyme 1:8 in ice-cold enzyme dilution buffer. 3. To the annealed primer-template add further soluttons as follows: Primer-template lo& Dithiothreitol (O.lM) lI.IL Diluted labeling mix 2w [a-35S] dATP (10 @4, 10 pCi/pL) 0.5 pL Diluted Sequenase 2 clL* 4. Mix and incubate for 2 min at room temperature (see Note 8). 3.3. Termination Reactions (see Note 1) 1. Label four tubes G, A, T and C (see Notes 3 and 4). 2. Add 2.5 pL of ddGTP termmatton mtx to tube G. Add 2.5 pL of ddATP termmatton mtx to tube A. Add 2.5 pL of ddTTP termination mix to tube T. Add 2.5 pL of ddCTP termination mtx to tube C. 3. Prewarm the four tubes by mcubatmg at 37°C for 1 mm (see Note 9). 4. Add 3.5 cts,of the labeling reaction to each of the four tubes, mtxmg after the addition.

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5. Incubate at 37OCfor 5 min. 6. Add 4 pL of stop solution to each tube. 7. Heat the tubes at 80°C for 2 min immedtately prior to loading on a gel (see Notes 10-12).

4. Notes 1. If compresston artifacts are a problem m the sequencing gel (often seen with G-C rich templates) try running dITP reactions alongside the dGTP reactions. Compressions occur when the newly synthesized DNA strand does not remain fully denatured during electrophoresis and can usually be eliminated using dITP. The reactions are performed as normal using dITP labeling mix and dITP termination mixes. The dITP labeling mix (5X concentrate) contains: 15 piJ4 dITP, 7.5 pJ4 dCTP, and 7.5 @4 dTTP. The dITP termination mixes are as follows: ddG termination mix (for dITP): 160 ~JVdITP, 80 pit4 dATP, 80 luV dCTP, 80 luV dl’TP, 1.6 pA4 ddGTP, 50 mM NaCI. ddA termination mix (for dITP): 80 l.uV dITP, 80 pJ4 dATP, 80 l,uV dCTP, 80 @4 dTTP, 8 l&V ddATP, 50 mM NaCI. ddT termination mix (for dITP): 80 lU4 dITP, 80 l.&f dATP, 80 @k! dCTP, 80 pil4 dTTP, 8 @f ddTTP, 50 mM NaCl. ddC termination mix (for dITP): 80 l.Ul4dITP, 80 @4 dATP, 80 l.04 dCTP, 80 l.uV dTTP, 8 @4 ddCTP, 50 mM NaCI. 2. Either [a-32P]-dATP or [a-35S]-dATPcan be used although [a-35S]-dATP has the advantagesof htgher resolution, operator safety,and longer half-life. 3. Multi-well microtiter plates with “V-bottom wells are ideal for performing sequencing reactions. The annealing and labeling reactions with Sequenase are probably more conveniently performed in small microcentrifuge tubes and the microtiter plates used for the terminatton reactions. 4. When using multi-well microtiter plates, ensure that samples do not evaporate by using a lid or, if necessary, plastic film. Sticky tape placed over the wells is also useful for this purpose, but take care that samples do not splash when removing it. 5. To read sequence very close to the primer (within 30 bases) it may be necessary to use more template DNA (about 2 pg M13) and to dilute the labeling mix 1:15 rather than 1:5. 6. Allowing the template-primer annealing mixture to cool from 65OC to room temperature is best achteved by incubation in a small beaker of 65°C water that is allowed to stand at room temperature for about 30 min. 7. It is recommended that the annealed template-primer is stored on ice and used within 4 h.

Griffin

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8. Do not perform the labeling reaction at a temperature above 20°C or for longer than 5 mm. To do so may result in pause sites close to the primer. 9. Do not perform the termmations at temperatures below 37°C or for longer than 5 min. To do so may cause pause sites above 100 bases from the primer. Performing termination reactions at higher temperatures (up to 50°C) may improve results in some cases 10. When using 35S,the sequencing reactions can be stored at -20°C for up to 1 wk before electrophoresls. Denature by heating at 80°C for 2 mm immediately prior to loading. 11. Incubation steps, especially the denaturatlon at 80°C for 2 min, are best performed in a water bath rather than an incubator. This is because samples may not equilibrate to the desired temperature in an incubator m the relatively short mcubatlon times used m this protocol. 12. Sequence 300 -400 bases from the primer should be readily obtained following the normal procedure. There are two ways to extend the sequence further. One is by increasing the nucleotlde concentration m the labeling reactlon. To achieve this, use the labeling mix undiluted and double the amount of radioactive Isotope. The other method 1sto change the ratio of deoxynucleotldes m the termination step. This is readily achieved usmg the Sequence Extendmg Mix marketed by USB. Increasing the deoxy concentration with respect to the dideoxy concentration ~111extend the sequence further from the primer. Note that the quality of gel electrophoresis will limit the amount of sequence

information

obtainable.

References 1 Sanger, F , Nicklen, S., and Coulson, A R. (1977) DNA sequencmg with chaintermmatlon inhibitors. Proc. Natl. Acad Sci USA 74, 5463-5467. 2. Fuller, C. W. (1988) Sequenase Version 2.0. CJSBEdltorlal Comments 15, l-4 3. Tabor, S. and Richardson, C. C (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad Scl USA 84,4767477 1 4 Tabor, S and Richardson, C. C (1987) Selective oxldatlon of the exonuclease domain of bacteriophage T7 DNA polymerase J Blol. Chem. 262, 15,33015,333. 5. Tabor, S. and Richardson, C. C. (1989) Effect of manganese ions on the incorporation of dideoxynucleotldes by bacteriophage T7 DNA polymerase and Escherirhia coli DNA polymerase I. Proc. Nat1 Acad. SCL USA 86, 40764080 6 labor, S. and Richardson, C C (1989) Selective mactlvatlon of the exonuclease activity of bacteriophage T7 DNA polymerase by rn vitro mutagenesis J Biol Chem 264,6447-6458. 7 Fuller, C. W (1988) DNA Sequencing. USB Editorial Comments 14, 1-7.

Dideoxy Sequencing Reactions Using Taq Polymerase Fabrizio

Arigoni

and P. Alexandre

Kaminski

1. Introduction Tuq DNA polymerase is a highly stable polymerase isolated from the thermophilic organism Thermus aquaticus. Because of its unique

properties this enzyme has been extensively used for amplification of DNA fragments by the polymerase chain reaction (PCR). In addition, the thermostability of Tuq DNA Polymerase has proven to be extremely useful for sequencing single-stranded templates by the dideoxy chain termination reaction (l-3). The advantage of sequencing with Tuq DNA polymerase is that the reaction can be extended at high temperature, thus eliminating most band compression encountered when sequencing G + C-rich templates or hairpin regions. This method is particularly useful when sequencing DNA from organisms with a high G + C content where ambiguities are frequently found on the gels (see Fig. 1). Currently several companies have developed sequencing kits using Tuq DNA polymerase. We have tried the TAQenceTM (U.S. Biochemicals, Cleveland, OH) and the Tuq multiwellTM (Amersham, Arlington Heights, IL) kits, both of which gave comparably good results. However these kits are relatively expensive and similar results can be obtained by preparing the different solutions with little loss of time. Here we describe a protocol From Methods in Molecular B/ology, Vol 23 DNA Sequencmg Protocols Edlted by H and A Gnffm Copynght 01993 Humana Press Inc , Totowa, NJ

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Fig. 1. Autoradiography of polyacrylamide gel comparing sequence of a G + C rich DNA template with Tuq DNA polymerase using either dGTP (I) or 7-deazadGTP (II) in the reaction mixes. All reactions were performed as described in the text using an M 13mp 19 derived single-stranded templates labeled by incorporation of [(x-~~S] dATP. The reactions were analyzed on a 6% polyacrylamide-urea gel using 0.5X TBE buffer (see Chapter 16). The brackets indicate the area of compression caused by secondary structure formed by the template DNA that are resolved by the 7-deaza-dGTP substitution.

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based on the chain termination reaction as described by Stambaugh and Blakesley (4) that involves two major steps. The first step is the so-called extension reaction, or labeling reaction, that includes annealing of a synthetic primer to the template DNA and synthesis of the complementary strand including radioactively labeled dATP. This step is carried out at relatively low temperature (45°C) to avoid separation of the primer and the template DNA. The second step, the termination reaction, is carried out at 70°C in four different tubes, each containing a mixture of nucleotides and one of the four dideoxynucleotides (ddNTP). The dideoxynucleotides are nucleotide analogs that lack the 3’-OH group necessary for chain elongation. Once a ddNTP has been incorporated the reaction will be terminated. Since the ddNTPs are incorporated randomly, the reaction will give rise to DNA fragments from several to hundreds of nucleotides in length. The reaction is then stopped with a solution containing EDTA and formamide, and separated on a polyacrylamide-urea electrophoresis gel. Despite the high temperature at which the reaction is carried out, some clones are reluctant to denaturation, and band compression will still form during electrophoresis. This artifact can be eliminated by substituting dGTP with 7-deaza-dGTP, a dGTP analog that forms weaker bonds with dCTP, thus weakening the secondary structure formed (5). 2. Materials 1. 10X Reaction buffer: 700 rnIt4 Tris-HCl , pH 8.8, 20 mM MgC12, 1% Triton X-100. 2. Taq DNA polymerase (Cetus or Beckmann) 5 U&L. 3. 5X Labeling mix: 12 @V dCTP, 12 @4 dGTP, 12 pII4 dTTP. 4. [a-35S] dATP > 600 Ci/mmol 5. Termination mixes: Prepare the 4 termination mixes named TMA, TMC, TMG, TMT at the following concentration in Hz0 shown in Table 1. If 7-deaza-dGTP is to be used, replace dGTP by 7-deaza-dGTP at the same concentration in all termination mixes as well as in the labeling mrx (see Notes 4 and 8). 6. Stop solution: 95% formamide, 20 rmt4 EDTA, 0.05% bromophenol blue, 0.05% xylene cyan01 FF. 7. Single-stranded DNA (l-2 pg) resuspended in TloEo 1 buffer (10 nuI4 Tris-HCl, 0.1 n&I EDTA, pH 8.0). For preparation of template DNA (see Chapters 5 and 6). 8. Prtmer: 0.5 pmol; usually 15-20mer.

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Table 1 Four Termmatlon Mixes TMA ddATP ddCTP ddGTP ddTTP dATP dCTP dGTP dTTP

100 25 250 250 250

pM

w ~.IM @4 j.M

TMC

TMG

TMT

100 pkl -

3o@f 250 w 250 @cl 25 w 250 /.IM

l(@cIM 250 pA4 250 pM 250 @I 25 w

250 25 250 250

@f w /.&I /.M

3. Method 1, To anneal the template and primer, combme the followmg in a 1.5mL Eppendorf tube (see Notes 3 and 7): 9.5 pL smgle-stranded template DNA (l-2 pg), 1.6 pL 10X reaction buffer, 1 pL primer (0.5 pmol). Close the tube, place it at 70°C m a heating block filled with water, and allow it to cool slowly by placing the heating block at room temperature until the temperature reaches about 30°C. Centrifuge the tube briefly to collect the droplets formed on top and store on ice. The labelmg reaction should be performed on the sample in the following hour. 2. To carry out the labelmg reactton, add the followmg (see Notes l-3): 2 pL 1X labeling mix, 1 ltL [a-35S] dATP, 1 pL Taq DNA polymerase (dilute m 1X reaction buffer to 2.5 U&L). Incubate at 45°C m a water bath for 5 mm. 3. For the termination reaction, prepare four different mtcrocentrifuge tubes labeled A, C, G, and T, each contammg 3.5 & of the correspondmg termination reaction (3.5 @LTMA in the tube labeled A, 3.5 pL TMC in the tube labeled C, etc.). If several clones are sequenced at the same time it is advtsable to prepare the tubes contammg the termmatton mtxes m advance and to store them on ice, capped to prevent evaporation. Add 3.5-w aliquots of the labeling reaction to each of the 4 tubes and place them immediately in a waterbath at 70°C for 5 min. Remove the reaction tubes from the water bath, let them cool to room temperature, add 3.5 pL Stop solution, and store on ice until ready to deposit on gel. Heat the samples 5 min at 70°C and load 2-3 pL on a high resolution polyacrylamtde gel (see Notes 4-6). The reaction mixture can be frozen and kept at -2O’C for several days. Samples should always be heated 5 mm at 70°C before loading.

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4. Notes 1. When performing sequencing reaction, be sure to respect the same order as described in the protocol. 2. Use only 7-deaza-dGTPtermmation nuxes with 7-deaza-dGTPlabeling mtx. 3. Reactions can be performed in microtiter plates, which are particularly useful when several samples are to be analyzed. However, great care should be taken to avold excessive evaporation. 4. dNTP and ddNTP concentration were optimized to sequence DNA from organisms with high G + C content (65%) and may have to be adjusted for other organisms. This is best done by altering the ddNTP concentratron m the different termination mixes (Le., if the A lane is too faint in the bottom of the gel, increase the ddATP concentration 2-3 folds in the TMA mix). 5. The amount of sequence Information obtained by this protocol ranges from several nucleotldes to 350 -400 nucleottdes, and is mostly limited by gel electrophoresls. 6. Some compression may also appear when sequencing with 7-deazadGTP but generally at different places than those observed with dGTP. Therefore, we recommend running both reactions m parallel when sequencing a G + C rich hairpin region. 7. S-end-labeled primers can also be used for sequencing, m this case omit the [a-35S] dATP m the labeling reaction. 8. From our experience, 7-deaza-dGTP appears to be lessstable than dGTP, which can result in fuzzy bands on the gel. Therefore, labeling and termmation mixes with 7-deaza-dGTP should be freshly prepared or aliquoted when stored at -2OOC. 9. The annealing step can be replaced by heating at 45°C for 10 min after denaturing at 70°C for 2 min and subsequent cooling on ice, however, we observed that when using nonpurifed synthetic oligonucleotide as primer, heating at 70°C as described in this protocol gives higher quality results. This is particularly true when the oligonuceotide is relatively G + C rich.

Acknowledgments The authors wish to thank J.-P. Aubert and C. Elmerich in whose laboratory this work was carried out. We also would like to thank C. Vieille for providing technical data and D. Maze1 for critical reading of the manuscript.

References 1. Sanger,F., Nlcklen, S.,andCoulson,A. R. (1977) DNA sequencingwith chain terminating inhibitors Proc. Natl. Acad. Sci. USA 74, 5463-5467.

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and Kaminski

2 Innis, M. A , Myambo, K. B., Gelfand, D. H , and Brown, M A D (1988) DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA Proc. Natl. Acad. Sci. USA f&9436-9440

3. Brow, M. A. D. (1990) Sequencing wtth Tuq DNA polymerase, in PCR Protocols. A Guide to Methods and Applications (Innis, M , Gelfand, D., Snmski, J., and White, T , eds.), Academic, NY, pp 189-196. 4. Stambaugh, K. and Blakesley, R. (1988) Extended DNA Sequencmg with Klenow Fragment. The Kilobase Sequencingm System Focus 10:2,29-3 1 5. Barr, P. J., Thayer, R. M., Laybourn, P , NaJarion, R C., Seela, F , and Tolan, D. R. (1986) 7-deaza-2’-deoxyguanosine-S-triphosphate: enhanced resolutton in Ml3 dideoxy sequencing. Biotechnrques 4,428-432

&TARTER

Pouring and Buffer-Gradient Paul

16

Linear Sequencing

Gels

Littlebury

1. Introduction The products of sequencing reactions are separated on thin, low percentage (usually 6%) polyacrylamide gels. Normally, these gels are 40-50 cm in length, 20 cm wide, and between 0.3 and 0.4 mm thick. Longer gels (to enable more sequence to be read from a single run) up to 100 cm in length can be poured, as can wider gels (to enable more clones to be loaded onto a single gel). However, these larger gels are more difficult to handle after electrophoresis, that IS, during fixing, drying, and autoradiography. The methods given in this chapter deal only with the “standard” size sequencing gels, but the principles are the same for larger gels, except that more gel mix will be needed, and different sizes of combs, spacers, and glass plates may be needed. Sequence gels are poured between two plates of glass, separated by two thin spacers, ensuring that a constant thickness of gel is maintained (changes in the thickness of the gel will alter the migration rate of the samples). The gel is also loaded and run when it is still in this mold, with the glass plates providing support for the fragile gel. A consequence of this is that one of the plates has a notch cut from it, several centimeters deep, and is almost the width of the plate. Thus plate will subsequently be referred to as the “notched” plate. This From Methods m Molecular B!ology, Vol. 23 DNA Sequennng Protocols Edited by H and A Grtfm Copyright 01993 Humana Press Inc , Totowa, NJ

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enables contact between the gel and running buffer at the anode. Sample wells are also formed at this end by inserting a comb, made from material of the same thickness as the spacers, into the gel immediately after pouring. Both the combs and the spacers can be bought commercially, or they can be made using modeling card (PlastikardTM) that is cut according to requirements. A typical comb for a 20-cm wide gel would have 50 teeth (2 mm wide; 3 mm deep), to enable 12 clones to be loaded per gel. Instructions will be given in this chapter on pouring both linear and buffer gradient gels. Linear gels are the simplest to pour, but usually only 200-250 bases of sequence can be read per clone. Buffer gradient gels (I) increase the amount of sequence that can be read in comparison to a linear gel, but pouring one correctly takes a little practice. Buffer gradient gels work by slowing the migration rate of DNA molecules in the final 30% of the gel, thus reducing the spacing between adjacent bands, allowing more sequence to be read from one gel. This effect is achieved by increasing the ionic concentration of the gel mix in the final 30% of the gel’s length. Buffer gradient gels are, therefore, poured using two gel mixes of differing ionic concentration, the mix with the higher ionic concentration being poured first, followed by the mix with the lower ionic concentration. Care must be taken when pouring the gel to ensure that the gradient forms properly, in order to avoid distortion of the resultant sequence bands. 2. Materials 1. 10X TBE: 108 g trizma base, 55 g boric acid , 9.3 g EDTA (di-sodium salt). Make up to 1 L with deronized water. Drscard when a precipitate forms. 2. 40% Acrylamide (19: 1): 380 g acrylamrde, 20 g NJ!‘-methylene bisacrylamide. Make up to 1 L with deionized water, add 20 g Amberlrte MB 1 resin and stir gently for 10 min. Falter and store at 4°C (see Note 1). 3. 0.5X TBE Gel MIX: 75 mL 40% Acrylamrde (19:1), 25 mL 10X TBE, 230 g urea (ultrapure grade). Make up to 500 mL with deionized water. Filter and store at 4OC for up to 1 mo. 4. 5X TBE Gel MIX: 30 mL 40% Acrylamide (19:1), 100 mL 10X TBE, 92 g urea (ultrapure grade). Make up to 200 mL with deiomzed water. Filter and store at 4OC for up to 1 mo. 5. 25% Ammomum persulphate: 25% (w/v) m deionized water, and should only be kept for about 1 wk at 4°C.

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6. TEMED: N,N,N’,N’-Tetramethyl-1,2-diaminoethane. 7. Dimethyl dichlorosilane solution: For silanizing glass plates. 8. Glass plates: 50 x 20 cm. 9. Spacers and combs: Spacers: 50 x 0.5 cm x 0.35 mm thick PlastikardTM. Combs: 5 x 15 cm x 0.35 mm PlastikardTM, 10. Waterproof tape: [The above items are available from most molecular biology suppliers.] 11. 4X “Bulldog” or “Foldback” clips.

3. Methods 3.1. Preparation of Sequencing

Plates

1. Wash one pair of plates in warm water, and then rinse them with distilled water. If the plates are cleaned after each use, there ~111be no need to use detergents on them. Allow the plates to air dry. 2. Working in a fume hood, silanize one surface of each plate (see Note 2). To do this, spread approx 2 mL of stlanizmg solutton over the selected surface using kimwipes, taking care to ensure that the enttre surface of the glass plate is covered. The plates are then left to dry in the fume hood for 5-10 min. 3. Wipe each plate with a small quantity of ethanol. Ensure that the plates are well polished, and free of dust that may otherwise encourage the formation of air bubbles when the gel is poured (see Note 3). 4. Lay one spacer along each of the long edges of one plate (usually the unnotched one). Lay the second plate directly onto the first plate so that the two silanized sides form the inner surfaces of the mold assembly. 5. Clamp the plates together on one side using Foldback clips, and seal along the other side and the bottom of the plates with gel sealmg tape. Transfer the clips to the sealed side, and then seal the remaining side. If desired, a second layer of tape could be used along the bottom and the corners of the plates to further reduce the risk of leaks.

3.2. Pouring

a Linear

Sequencing

Gel

1. Allow 50 mL of 0.5X TBE gel mix to warm to room temperature. 2. Add to this gel mix 100 pL of 25% ammonium persulphate and 100 pL of TEMED. Mix by swtrlmg. 3. Take the gel mix up into a 50-mL syringe (without a needle), and inject the mix between the plates, maintaining a steady flow. The rate of flow of gel mix into the mold can be controlled by altering the angle at which the plates are held.

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4. Once the gel mold has been filled to the top, insert the comb just past the teeth. Clamp the edges of the plates at the top and bottom, to ensure that a uniform gel thickness is maintained. Leave the gel to polymerize for 30 min. 3.3. Pouring a Buffer Gradient Gel 1. Allow sufficient gel mix to warm to room temperature. For a 50-cm long buffer gradient gel: 50 mL 0.5X TBE gel mix, 10 mL 5X TBE gel mix, 2. Add 100 pL each of TEMED and 25% ammonium persulphate to the 0.5X TBE gel mix, and 20 pL of each to the 5X TBE gel mix (see Note 4). 3. Using a 25-mL Pasteur pipet, take up 7 mL of 0.5X TBE gel mix, followed by 7 mL of 5X TBE gel mtx (see Note 5). The two gel mixes should be mixed together at the interface by introducing a few air bubbles into the pipet. 4. Pour this mix into the mold either down one stde or down the center, 5. As soon as the ptpet IS empty, lower the mold to the hortzontal to stop the flow of gel mix. Take the remaining 0.5X TBE gel mix into a 50mL syringe, and fill the gel mould to the top, washing the gradient mtx to the bottom. Insert the comb, and clamp the plates, leavmg the gel to polymerize for 30 mm. 4. Notes 1. Acrylamide is a potent neurotoxm, the effects of which are cumulative Gloves and a mask should always be worn when weighing out solids, and when handling solutions containing acrylamtde, and gels should be poured in plasttc trays to contain sptllages and leaks. Polymerized acrylamide is considered to be nontoxic, although gloves should still be worn, as there may be residual unpolymerized acrylamtde present. 2. Many workers silamze only one of the glass plates, the theory being that the gel will stick to the nonsdanized plate. In practice, the gel seems to stick to either plate with equal frequency, and silanizmg both plates seems to aid the pouring of the gels. 3. Bubbles can be removed m one of two ways, should they occur. The gel assembly can be raised vertically and the glass lightly tapped to dislodge the bubble, which should then rise to the top of the gel. Alternatively, a piece of used X-Ray film cut to form a long strip can be used to push the bubble to the side, where it will not affect the running of the samples. 4. The rate at which the gel polymerizes can be altered by changing the amount of TEMED added to the gel mrx. It 1soften helpful, when first pourmg buffer gradient gels, if the amount of TEMED is reduced by

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10-20 pL. This ~111gave the inexperienced worker a little more time to pour the gel, and remove any bubbles that may have occurred during pouring. 5. When using buffer gradient gels, it is possible to change the gradient simply by altering the relative ionic concentratton of the two mixes. For example, if more 5X TBE mix is added during step 3., the gradient would become steeper.

Reference 1. Biggin, M. D., Gibson,T J., and Hong, G. F. (1983) Buffer gradientgels and35S label asanaid to raprdDNA sequencedetermination Proc. Natl. Acad. Sci USA 80,3963-3965

&LWl?ER

17

Electrophoresis of Sequence Reaction

Samples

Alan l! Bankier

1. Introduction The underlying principle of both main DNA sequencing methods by Sanger (I) and Maxam and Gilbert (21, is the ability to fractionate and resolve long, single-stranded DNAmolecules that differ in length by only one nucleotide. Denaturing polyacrylamide gels have been reported to give interpretable separation of molecules up to 0.6 kb in length, on l-m long gels (3). More practical systems for extended readings employ shorter buffer gradient gels (see Chapter 16), multiple loadings, giving run lengths of around 3 and 6 h, and reduced acrylamide concentration down to as low as 3.5%. Even using these techniques, a more reasonable estimate of the limit to accurate sequence that can be obtained on a routine basis, is 450-500 nucleotides per template. The redundant nature of shotgun procedures, where gel running tends to be the rate limiting step, make it more efficient to accept a reading of up to 350 nucleotides from each single run on a buffer gradient gel or a wedge gel (4-6). Following electrophoresis, the gel is subjected to autoradiography, where the position of each band can be visualized by virtue of the radioactive nucleotide incorporated during polymerization. Samples visualized by chemiluminescence (see Chapter 29), currently use similar exposure to film for detection. Fluorescence-based autoFrom. Methods m Molecular Biology, Vol 23. DNA Sequencmg Protocols Edited by H and A Gnffm Copynght 01993 Humana Press Inc , Totowa, NJ

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mated procedures (see Chapters 34-37) use direct in-gel detection without the need for gel handling and autoradiography. Direct autoradiography of wet gels, covered with Saranwrap@, is possible with 32P-labeled sequence reactions, but a considerable improvement in resolution is observed when the gel is dried. Particles from radioactive emissions in the lower part of the gel radiate outward in all directions and the resulting exposure on the film, positioned on the upper surface of the gel, appears broad and diffuse. If the gel is dried before autoradiography, this radiant “spread” is dramatically reduced. When 35S label is used, drying is essential because of the lower energy of emission. Drying is most conveniently carried out on a heated, vacuum gel drier, and takes less than one hour. Having negotiated all of the pitfalls of subcloning, risked one’s health handling hazardous materials, fiddled with almost nonexistent volumes of reagents, and successfully manipulated a very fragile gel that often seems bent on self-destruction to produce an autoradiograph, perhaps the hardest part remains. Reading sequencing films accurately is a skilled process. Although some broad guidelines can be learned from a chapter like this, there is no substitute for practical experience gained from comparing deduced sequences with known sequences and reconciling any differences. Several film scanners are available, with software intended to interpret sequencing data, notably the Amersham Autoreader (Amersham, Arlington Heights, IL). The accuracy of these devices is continually improving, but they are totally dependant upon being “fed” good quality input. At the end of it all, it can be very difficult to accept that the film may be truly unreadable.

2. Materials 1. A gel electrophoresis apparatus. This need not be too complicated, since satisfactory results can be obtained using very cheap and simple systems. 2. A high voltage power supply capable of at least 2000 V (d.c. output). For a 20 x 50 cm x 0.3-mm thick gel, the running conditions are around 1500 V at 30 mA. 3. 10X TBE: 108 g Tris base, 55 g boric acid, 9.3 g NazEDTA dissolved and made up to 1 L in deionized water. 4 Formamide dye mix: 100 mL deionized formamide, 0.1 g xylene cyan01 FF, 0.1 g bromophenol blue, 2 mL 0.5M Na,EDTA. There seems to be little or no deterioration of results using dye mix that has been stored at ambient temperatures for several months, but it is recommended that

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aliquots be stored frozen. Formamide is hazardous and may cause irritation to skm and eyes. 5. Narrow or flattened point, disposable pipet tips for gel loading. A small volume Hamilton syringe with a fine gage needle can also be used. Experrenced users frequently prefer to use drawn-out capillaries fitted to a mouth pipet, but this practice should be avoided on safety grounds. 6. Whatman 3MM paper, cut just larger than the size of the gel. 7. Saranwrap@ or equivalent, nonporous food wrap. 8. Slab gel drier, Savant SGD4050 or BioRad SE1 125B. 9. A high vacuum pump. Oil pumps require considerable maintenance and are quickly damaged by acetic acid if the gels are fixed before drying. Teflon diaphragm pumps are cheaper and are low maintenance (e.g., Vacuubrand GMBH). 10. Fuji RX or equivalent, X-ray film. Some films are considerably faster (and more expensive) but may not be suitable for automatic processors. 11. Light-tight film cassettesfor autoradiography. 12. Chemicals for film processing.

3. Methods The processdescribed in this chapter can be broadly categorized under three headings: gel running, autoradiography, and film reading. 3.1. Gel Running 1. Remove any sealing tape from the bottom of the polymerized gel, remove the well former from the top of the gel and wash the well(s) under runnmg deionized water (see Note 1). 2. If using a “shark’s tooth” comb, carefully insert it until the teeth just enter the gel surface evenly across the full width of the gel. 3. Assemble the gel in the electrophoresis apparatus with the well recess of the glass plate facing the upper buffer chamber and fill the upper and lower chambers with 1X TBE. 4. Thoroughly flush the top of the well with TBE, usmg a Pasteur pipet or syringe, to remove any unpolymerized acrylamide. It is not absolutely necessary to prerun the gel, but if desired, put the cover on the apparatus and connect it to a high voltage power supply and run it at around 30 V/cm for 30 mm (see Note 2). 5. If the sampleshave been stored at -20°C allow them to thaw and add 2 pL of formamide dye mix to each. Immediately before loading, denature the samples and place them on ice. Samplesprepared in tubes should be denatured for no longer than 3 mm in a boiling waterbath. If microtiter trays were used, place the trays, uncovered, in an incubator at 80°C for 20 mm.

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6. Isolate the gel running apparatus from the power supply and flush the wells at the top of the gel to remove the urea that will have diffused out of the gel. 7. Using a narrow polypropylene tip fitted to a small volume pipet, load around 2 p,L of sample into each well. The same tip can be used repeatedly by rinsing the tip once in the bottom buffer chamber. If difficulty is encountered with air pockets trapping some residual sample in the tip, it is easier to discard it into the radioactive waste and to use a clean tip (see Notes 3 and 4). 8. When all the samples are loaded, put the cover on the apparatus and connect it to a high voltage power supply. Electrophorese the gel at around 30 V/cm, until the bromophenol blue reaches or has migrated off the bottom of the gel (see Notes 5 and 6).

3.2. Gel Drying

and Autoradiography

1. When the run is complete, disconnect the electrophoresis unit from the power supply, discard the top buffer, and dispose of the radioactive bottom buffer m the recommended manner. Remove the gel/glass assembly from the unit and remove any remainmg sealing tape. 2. With the notched plate uppermost, pry the two glass plates apart by inserting a thin spatula between them and carefully twisting or levering it. Should the gel prefer to stick to the notched plate, simply remove the spatula, turn over the whole gel/glass assembly, and pry off the backplate. If the gel perststs in sticking equally to both plates, the whole assembly is best submerged in water or 10% acetic acid where the gel is free to float off the upper glass plate when pried open. The gel can be held in position during subsequent handlmg, using a piece of firm plastic netting (see Note 7). 3. Submerge the gel in around 1 L of 10% acetic acid m a seed tray for lo-15 min to dialyze out the urea. If the tray does not have a ridged base, gently agitate the solution periodically. Remove the gel, still on the glass plate, and dram off as much liquid as possible (see Note 8). 4. Transfer the gel to 3MM paper by placing a sheet smoothly and evenly on top of the gel by “rolling” the curved paper across from one end, or outward from the center. Gently ensure complete direct paper/gel contact over the whole gel and then peel the paper back and away from the glass. 5. Cover the gel with a layer of Saranwrap@ and cut off any excess 3MM paper or plastic wrap. Trim the top and sides of the gel to fit Into a slab gel drier and dry the gel for 15 mm under vacuum at 80°C. 6. Remove the plastic wrap from the dry gel and place it m a light-tight X-ray film cassette m direct contact with X-ray film. Exposure times will vary

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dependingupon whtch label hasbeenused.If the entire samplehasbeen loaded and the label was 35S,exposuretimes between 18-36 h should be adequate.When using 32P,the ideal exposurecan be as short as 1 h (seeNote 9). 7. Processthe X-ray film according to the supplier’s recommendations. 3.3. Film Reading In principle, the interpretation of a single sequence from four tracks on an autoradiograph should be simple. Because the individual molecules are separatedaccording to size, all that is required is that the positions of successive bands from bottom to top are noted. If the next band IS in the A track, then the next base in the sequencemust be an A. Several different problems may disrupt this idealized situation. It may not be clear which band comes next, particularly if the bands in question are in the outer tracks. Two or more bands may appear to occupy the same vertical position, or spurious bands may even prevent an absolute assignment at a particular position. Some positions expected within an even “ladder” of bands may appear to be vacant. Superimposed upon this will be the effects of poor template quality, less than perfect sequence reactions, and gel running and autoradiography problems. Probably the most important lesson to learn when reading sequence films is when not to read. Most mistakes are made by trying to read sequencesof inferior quality or by reading too far up the gel into regions of poor resolution. Eventually sequence-reading mistakes will have to be reconciled with other, more accurate readings, and this can be very time consuming. Very often, some weeks or months later, when the film is read again, the first impression is one of disbelief that it could have been read at all. Guidelines on reading sequence films range from the general, to a description of the very specific artifacts expected from particular cloning or sequencing strategies. Because of the broad nature of the chapters in these volumes, only the more relevant points will be listed and then in a general way. Many of the artifacts can be attributed to specific reagents and can be verified using a control system. 3.3.1. General Tips on Reading Sequences

1. Since many of the anomalousmobility problems associatedwith gels are predominantly related to high G/C sequences,it makes sense to

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have these two tracks adjacent to each other. The most commonly used track orders are ACGT and TCGA. 2. Before starting, make a general assessment of the quality of the data and decide whether or not it is worth reading. This overall inspection would consider the sharpness of the bands, the presence of background smear, and whether extra bands are present. 3. Ensure an optimum exposure. Too short an exposure can conceal the presence of weak bands, particularly when Klenow is used. Too long an exposure will increasethe background smear and accentuateartifact bands. If 32Pis used, over-exposure wtll dramatically reduce resolution, 4. Mark out or mask the four lanes to avoid confusion with other tracks, and mark the end-point. This may be the point where the quality or resolution falls below the required standard, or where a short insert meets the vector. 5. Reading the spaces between bands can be as important as reading the bands themselves. It should always be considered whether or not there is “space” for the band m question. Where there is ambigutty regarding the exact order of bands, it can often be deduced from the comparative spacing of consecutive bands within a track m the immediate vicmity. One should have some idea of how many bands to expect in a particular gap. 6. If consistent difficulty is encountered in interpreting the correct register of bands, a sharkstooth comb may prove to be beneficial. When these are used, there is often a small overlap between adjacent tracks, making order interpretation simpler. 7. Do not stop reading in “mid-sequence.” Once started, read the entire sequence from bottom to top. It is quite common, after a break, to recommence reading from the wrong point, particularly when the sequence is of a repetitive nature. 8. Speckles and streaks on autoradiographs usually arise from particles or small bubbles in the gel and can generally be avoided. Ensure that the glass plates are scrupulously clean before pouring the gel, and avoid using tissues that drop particles to wipe down their surfaces. 9. Patches of fuzziness can be attributed to regions of the gel not being m direct, intimate contact with the film, or with differential polymerization of the gel matrix. These patches can also be caused by regions of overheating in the gel by running it too fast. 3.3.2. Common Problems and Artifacts in Sequences 1. A high background smear is generally specific to the template DNA. Further purification of the template, using phenol or chloroform extractions and ethanol precipitation, is not always successful.

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2, Variations in band intensity are an intrinsic property of most enzymes, particularly Klenow. These are generally base sequence specific and occur in a predictable manner. Sequenase used m the presence of manganese produces a very even band intensity. 3. “Pile-ups,” where bands appear in the same position in all four tracks, result from nonspecific terminations, Typical causesare secondary structure within the template that the polymerase has difficulty passing through (different polymerases and different batches of the same polymerase can cope with this to varying degrees), and renaturation of a double stranded template causing a block to polymerization. This is quite common in sequencing PCR products. Both of these problems can often be reduced by destabilizing the structure, for example, by increasing the temperature of the extension reaction or reducing the salt concentration in the reaction. Failing this the polymerase has to be changed, since the most likely cause will then be poor quality enzyme. 4. Faint extra bands, or “shadow” bands, in other tracks can have one of several causes. If the shadow bands appear only in adjacent tracks, a sample may have spilled over between wells. Also random distribution of faint bands can indrcate a second background sequence. This may be caused by a contaminating plaque inadvertently picked off the agar plate, a lower abundance template resulting from a deletion during phage growth, or nonspecific priming. Finally, some faint artifact bands are polymerase-specific and appear at base sequence-dependent posttrons. 5. A generally clean, but very weak, sequence is caused by low template or primer concentratron, or by inefficient priming resulting from too high an annealing temperature. 6. Compressions, where band spacings are reduced or nonexistent at a specific point in a sequence, followed by a region of abnormally large spacing, result from secondary structure during gel electrophorests. 7. An uneven distribution of band intensity is indicative of an incorrect dideoxy:deoxy nucleotide ratio. A peak of intensity at a shorter length implies too high a dideoxy concentration, and a high intensity at longer lengths too low a dideoxy nucleotide concentration. When using a twostep labeling procedure, as recommended for Sequenase,a similar intensity problem is caused by variations in template concentration such as low template concentrations producing an increase m intensity of the longer molecules. 8. No signal at all m a single track is usually caused by accidentally missing the addition of a single ingredient. No signal in a set of four tracks, on the other hand, would indicate that the primer or buffer was omitted from the annealing, no template was recovered from the DNA prepara-

Bankier tion, the primer 1sInappropriate, or a deletton has occurred durmg growth of the recombinant that encompassed the priming site.

4. Notes 1. It is important to rinse the wells immediately after removing the slot former, as any unpolymerized acrylamide may subsequently polymerize and leave an uneven gel surface. Flushing the wells lust before loading removes any urea that may have dialyzed out of the gel, making it easier to load the samples as a tight band. 2. The presence of “smiling,” where samples toward the edge of the gel migrate slower than those in the center, is a consequence of variations of heat loss (and hence temperature) across the width of the gel. This can be mmimized m two sample ways: First, by not loading samples in the outermost 2 cm on each side of the gel, because this 1swhere the temperature difference is greatest, and second, by placing an alummmm sheet on the outer surface of the glass plate, because this evens out the temperature across the gel. More sophisticated and expensive methods of temperature control are available, but except for particular situations, such as in automated systems, it is unlikely they will be needed. 3. If possible, tt is better to load the sample around l-2 mm from the bottom of the well, giving a sharp sample band, rather than to let it trickle down from the top. 4. The extended denaturation period for mtcrotiter trays is intended to reduce the volume so that the entire sample can be loaded. The duration can be altered to adjust this volume, For example, fan assisted ovens will require a much shorter time. Only a fraction of samples denatured in tubes can be loaded since their volume is not reduced considerably. Do not attempt to overload the wells. As a guide, the sample height should be less than the well width, but ideally much less. 5. The conditions described in this method refer to running the gel at a constant voltage. In practice, though, it 1sbest to run denaturmg gels at constant power. This IS because the gel temperature is the most important factor to control. An internal gel temperature of 60-70°C should be attamed. For 20 x 50 cm x 0.3 mm thick gels a constant power of 35-40 watts is sufftcient. In caseswhere compresstons still exist this may need to be even higher to increase the gel temperature. 6. The precise time at which to stop electrophoresis will of course depend upon the length of the primer and the distancefrom the primer that sequence is to be read, as well as the concentration of acrylamtde in the gel. On 6% gels the Ml 3 cloning sitescorrespond approximately to the position of the bromophenol blue marker dye when the universal primer is used.

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7. When opening the glass plates, do not lever against the “ears” of the notched plate. These tend to be fairly fragile and easily broken. 8. There is no need to fix gels before they are dried. Without fixing they will take 45-60 min to dry on a vacuum gel drier at 80°C. If no drying facilities are available, it is possible to air dry them overnight. 9. The introduction of thin gels and 35S label was designed to improve resolution. Using smgle-sided film can improve the resolution of autoradiographs even more, although care must be taken to ensure that the emulsion side faces the dried gel and the Saranwrap@ is removed.

References 1 Sanger, F , Nicklen, S , and Coulson, A R. (1977) DNA sequencmg wtth chamterminating mhrbitors. Proc. Natl. Acad. Sci. USA ,74,5463-5467. 2. Maxam, A. M. and Gilbert, W. (1977) A New Method for Sequencing DNA Proc. Natl. Acad. Sci USA 74,560-564.

3 Ansorge, W. and Barker, R. (1984) System for DNA sequencing with resolution of up to 600 base paus. J Biochem. Blophys. Meth. 9,33-47. 4. Bankier, A. T. and Barrel], B. G. (1983) Shotgun DNA sequencing, m Techniques in Life Sciences, Nucleic Acid Biochemistry vol. B5 (Flavell, R. A , ed.), Elsevier Screntific Publishers, Ireland, pp. l-34. 5 Biggin, M. D., Gibson, T. J., and Hong, G. F. (1983) Buffer gradient gels and 35S label as an aid to raptd DNA sequence determination Proc. Natl. Acad. Sci. USA 80,3963-3965.

6. Bankier, A. T and Barrell, B. G. (1989) Sequencing smgle-stranded DNA using the chain termmation method, m Nucleic Acids Sequencing: A Practical Approach (Howe, C J and Ward, E. S., eds.), IRL Press, Oxford, pp. 37-38.

Plasmid

Sequencing

Hugh G. Grij’j%t and Annette M. Griffin

1. Introduction The dideoxy chain-termination method of DNA sequence analysis involves the synthesis of a DNA strand from a single-stranded template (I). The enzymatic synthesis is initiated at the site where an oligonucleotide primer anneals to the template. The most consistently satisfactory sequencing results are probably obtained by using bacteriophage M 13 (2,3) or phagemid (4) single-stranded DNA as the template. However, plasmid DNA that has been denaturedcan also serve as satisfactory template DNA. It is particularly important when preparing plasmid DNA for sequencing to give the utmost care and attention to the DNA isolation and purification techniques. Most problems that occur with plasmid sequencing are related to poor quality template. CsCl-ethidium bromide gradient preparations of plasmid DNA (5) can be used as template for sequencing but do not necessarily provide better results than mini-prep DNA. Many plasmid mini-prep methods are available for preparing template for sequencing. Denaturation is usually achieved with the use of alkali or by boiling (in the presenceof primer). Several excellent methods are presentedelsewhere in this book. This chapter presents a mini-prep method that involves an alkaline lysis procedure (6) followed by DNA purification by GENECLEAN@ -a commercial kit for the purification of DNA. The From. Methods In Molecular &logy, Vol. 23 DNA Sequencing Protocols E&ted by H and A Griffin Copynght 01993 Humana Press Inc , Totowa, NJ

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GENECLEAN@ kit is based on the principle that DNA will bind to a silica matrix, whereas DNA contaminants will not bind and can be removed by repeated washing of the silica-DNA matrix (7). The DNA is then denatured by alkali and sequenced using Sequenase Version 2.0. Although few people would claim that sequencing of plasmid DNA consistently provides as good results as sequencing M 13 or phagemid templates, with careful attention to template preparation very satisfactory results can be achieved. Sequencing plasmid DNA has the advantage of eliminating the need to subclone fragments into M13, and also enables both strands to be sequenced from the same plasmid by the use of reverse primers.

2. Materials 1. GET buffer: 50 mM glucose, 10 mM EDTA, 25 n&f Tris-HCl pH 8 0. This solution should be autoclaved for 15 mm at 10 lb/m2 (68.9 kPa) and stored at 4°C. 2. Lysozyme buffer: 20 mg/mL lysozyme m GET buffer. Make up fresh. 3 5M NaOH. 4. 10% sodium dodecyl sulfate (sodium lauryl sulfate, SDS). 5.2 n-&f EDTA. 6. Alkaline SDS: 200 mM NaOH, 1% SDS. Make up fresh as follows: 200 pL 5M NaOH, 500 pL 10% SDS, 4.3 mL water. 7. 3M sodium acetate, pH 4.8. AdJust pH to 4.8 with glacial acetic acid. 8. Phenol-chloroform: This is prepared by mixing equal amounts of phenol and chloroform. If desired, the mixture can be equilibrated by extracting several times with 0.1M Tris-HCl, pH 7.6, although this is not essential 9. 100% ethanol. 10. 70% ethanol. 11. GENECLEAN@ kit. This is available from BIO 101 Inc., PO Box 2284, La Jolla, CA, 92038-2284, or from Stratech Scientific, 61-63 Dudley Street, Luton, Bedfordshire, LU2 ONP, England.

3. Methods 3.1. DNA Preparation 1. Harvest the cells from 5 mL of over-night broth culture by centrifugation (see Note 1). 2. Resuspend the pellet m 100 J.JLof lysozyme buffer and transfer to a 1.5-mL microcentrifuge tube, then incubate on ice for 30 min (see Notes 2 and 3) 3. Add 200 pL of alkaline SDS. Vortex, then incubate on ice for 5 mm.

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4. Add 150 pL of 3M sodium acetate, pH 4.8. Vortex, then incubate on ice for 20 min. 5. Centrifuge at 12,000g in a microcentrifuge for 10 mm and transfer 0.4 mL of supernatant to a new tube. 6. Extract twrce with an equal vol of phenol-chloroform.

3.2. DNA Purification

(Using GENECLEAN@

Kit)

1. Add three vols of NaI stock solution from the GENECLEAN@ kit (see Notes 7 and 8) to the phenol-chloroform extracted mini-prep. Use a 2-mL microcentrifuge tube if necessary. 2. Add 5 pL of glassmilk suspension (supplied with the kit). Mix, and incubate for 5 min at room temperature. 3. Centrtfuge for 5 s in a microcentrifuge. 4. Remove the supernatant and wash the pellet three times with NEW wash (supplied with the kit). 5. Carefully remove the last traces of NEW wash from the pellet. 6. Elute the DNA in 100 pL of water.

3.3. Alkali

Denaturation

1. To 86 pL of the purified DNA, add 4 cls, 5M NaOH and 10 pL 2 mM EDTA, then incubate at 37OC for 30 min (see Note 9). 2. Neutralize by adding 10 pL of 3M sodium acetate, pH 4.8. 3. Precipitate with two vols of 100% ethanol at -70°C for 15 min. 4. Centrifuge for 2 mm m a microcentrifuge to pellet precipitated DNA. 5. Wash pellet with 70% ethanol. 6. Dry under vacuum and redissolve pellet in 7 pL water.

3.4. Primer-Template

Annealing

Reaction

1. In a small microcentrifuge tube, set up the following reaction: 1 pL primer (0.5 pmol/pL), 2 pL Sequenase buffer (5X) (see Chapter 14), 7 pL denatured plasmid DNA. 2. Place in a 65°C waterbath for 2 mm. Allow to cool slowly to 30°C over a period of about 30 min. Place on ice (see Note 10).

3.5. Sequencing These are performed

Reactions

as described in Chapter 14.

4. Notes 1. It is important to remove all the broth supernatant from the cell pellet following centrifugation of the bacterial culture. 2. It is important to ensure that the bacterial pellet is fully resuspended in lysozyme buffer. This can be achieved by vigorous vortexmg.

Griffin

and Griffin

3. Some authors point out that lysozyme 1sunnecessary for alkaline lys~s based mini-preps (5). We have found that although lysozyme is not essential, better lysis IS achieved by its use. 4. An RNase step is not necessary m this protocol. 5. A small excess of template DNA may be helpful when sequencing plasmid DNA. Aim to use about 5 pg. 6. This protocol is designed for use with the htgh copy-number pUC series of plasmids (8) and their derivatives. If lower copy-number plasmids are to be used (such as pBR322 and derivatives) it may be necessary to scale-up the procedure to achieve the same yield of plasmid DNA. 7. Mini-prep plasmid DNA is often contaminated by small oligodeoxynbonucleotides and ribonucleotides that can act as primers and produce faint background bands, stops, and other artifacts in the gel. Inhibitors of DNA polymerase may also be present. In our experience purification of the DNA prep by the GENECLEAN procedure, combined with the use of Sequenase Version 2.0 for sequencing, alleviates these problems. 8. Other commercial kits such as US Bioclean (available from United States Biochemical Corporation, PO Box 22400, Cleveland, OH, 44122, or from Cambridge Bioscience, 25 Signet Court, Stoursbridge Common Business Centre, Swann’s Road, Cambridge, CB5 8LA, England) or noncommercial procedures and protocols based on similar principles as GENECLEAN (7) may also work satisfactorily. 9. Double-stranded supercoiled DNA is best denatured by the alkaline denaturation method. Linear DNA can be successfully denaturated by boiling (in the presence of primer). 10. Annealing can also be achieved by warming the annealing reaction mrx to 37°C for 15-30 min. 11. The use of longer primers (25-29 nucleotides) may help reduce artifactual bands when sequencing denatured double-stranded DNA. 12. SequenaseVersion 2.0 works well for double-stranded DNA sequencing.

References 1, Sanger, F , Nlcklen, S., and Coulson, A. R. (1977) DNA sequencing with chamtermmating inhibitors. Proc Nat1 Acad. Sci. USA 74,5463-5467. 2 Messing, J , Gronenborn, B , Muller-Hill, B , and Hofschnelder, P H (1977) Fllamentous coliphage Ml3 as a cloning vehicle: Insertion of a Hind111 fragment of the lac regulatory region in Ml3 replicative form in vitro Proc Nat1 Acad SCL USA 74,3642-3646

3 Messing, J. (1983) New Ml3 vectors for cloning. Meth. Enzymol. 101,20-78 4 Zagursky, R J. and Berman, M. L. (1984) Clonmg vectors that yield high levels of single-stranded DNA for rapld DNA sequencing. Gene 27, 183-19 1. 5 Sambrook, J., Fritsch, E. F , and Maniatls, T (1989) Molecular Cloning-A

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Sequencing

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Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 6 Birnboim, H. C. and Doly, J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 7, 15 13-l 523 7. Vogelstein, B. and Gillespie, D. (1979). Preparation and analytical purrfrcatron of DNA from agarose Proc Natl. Acad Sci USA 76,615-619. 8 Vieira, J. and Messing, J. (1982) The pUC plasmrds, an Ml3 mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19,259-2&I.

Plasmid

Sequencing

George Murphy

1. Introduction In double-stranded DNA sequencing the two DNA strands must first be separatedto enable the primer to bind to the priming site. This may be done by treating the DNA with alkali. Conventionally, use of the alkali denaturation method involves neutralization of the sample by acid treatment followed by ethanol precipitation and recovery of the DNA by centrifugation. The time-consuming processof ethanol precipitation may be avoided by neutralizing the alkali-treated DNA and recovering the sample in its original volume by passagethrough a spin-dialysis column. The use of spin-dialysis confers the additional advantage of cleaning-up the template by removing traces of low molecular weight compounds that may interfere with the sequencing reactions. The method used here to prepare plasmid is a variation on the boiled-lysis method (I) using a rapid approach avoiding phenol extraction. 2. Materials 2.1. Plasmid Preparation 1. LB: 1% (w/v) bacto tryptone, 0.5% (w/v) bacto yeast extract, and 1% (w/v) NaCI.

2. Suspensionbuffer 50 n&I Tris-HCl, pH 8.0,25% (w/v) sucrose. 3. MSTET: 50 m&I Tris-HCl, pH 8.0, 50 mZt4EDTA, 5% (v/v) Tnton-X100, and 5% (w/v) sucrose. From Methods In Molecular Biology, Vol 23. DNA Sequencmg Protocols Edited by* H and A Gnffln CopyrIght 01993 Humana Press Inc , Totowa,

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4. Lysozyme solution: 40 mg/mL in 50 mM Tris-HCl, pH 8.0, 50% glycerol (v/v). 5. TE: 10 mM Tris-HCI, pH 8, 1 mM EDTA. 6. Sodium acetate: 3M sodium acetate, pH 5.0. 7. Isopropanol. 8. Ammonium acetate: 7.5M ammonium acetate. 9. 96% (v/v) ethanol. 2.2. Denaturation 1. RNase A: Dissolve 10 mg pancreatic RNase A in 10 mM Tris-HCl, pH 8.0, 15 r&4 sodium chloride. Boil for 15 min and cool slowly. Dispense into 50-pL aliquots and store at -20°C. Dispose of any used solution once thawed. 2. Sodium hydroxide: 1M sodium hydroxide, 1 mM EDTA. 3. TO.lE: 10 mM Tris-HCl, pH 8.0,O.l r&4 EDTA. 4. Sepharose: equilibrate Sepharose-CL-6B (Pharmacia, Piscataway, NJ) m TO.lE and adjust to a packed gel:supematant ratio of 2: 1. Store at 4 “C. 5. Glass beads: 200 p diameter (Sigma, St Louis), washed in deiomzed water and autoclaved. 2.3. Annealing 1. 5X Buffer: 200 ti Tris-HCI, pH 7.5, 100 mM MgC& and 250 mM NaCl. 2. Primer: 10 pg/rnL in deionized water. 3. Methods 3.1. Plasmid Preparation 1. Using a toothpick, place a bacterial colony into 10 mL LB m a flatbottomed screw-top bottle and grow for 15-18 h at 37”C, shaking at 200-250 rpm and using antibiotic selection if required. 2. Centrifuge the cells at 1500g for 10 min. Decant the supernatant and leave the inverted bottle to dram for 5 min on absorbent paper. 3. Resuspend the cells by vortexing vigorously in 100 pL suspension buffer, then transfer the suspension to a 1.5-mL microcentrifuge tube. 4. Add 600 pL MSTET solution then spot 14 pL of lysozyme solution onto the inside of the tube. 5. Cap the tubes, mix the contents thoroughly by shakmg, and transfer immediately to a boiling waterbath for 1 mm. 6. Place the tubes on ice for 1 mm, then centrifuge at 10,OOOgfor 30 mm at 4OC. 7. Remove the gelatmous pellet with a toothpick. Alternatively, to reduce contammation with chromosomal DNA, use a mrcropipet to transfer the supernatant to a fresh tube.

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8. Add 60 pL sodium acetate and 600 pL isopropanol and leave for 5 min before centrifuging at 10,OOOg for 10min. Aspirate off the supematant,recentrifuge for 5 s, and remove all traces of liquid. Resuspend m 200 pL TE. 9. Add 100 pL ammonium acetate. Leave on ice for at least 30 min then centrifuge for 15 min. 10. Remove the supernatant to a fresh tube and add 750 pL ethanol. Leave at -20°C for 30 min then centrifuge as above. Rinse with 80% ethanol, air dry, and dissolve in 50 pL TE (see Note 1).

3.2. Denaturation 1. Mix 18 pL of a plasmid miniprep with 2 pL of RNase A and incubate for 15 min at 37°C. Add 5 pL sodium hydroxide and incubate a further 15 min at 37OC.If using cesium gradient purified DNA, use 15 pg in 20 pL TE without RNase treatment. 2. Pierce the base of a 0.5-mL centrtfuge tube from the inside with a 21-G needle so that about 2/3 of the needle bevel emerges. Place the tube inside a 1S-mL centrifuge tube completely pierced through the bottom with the same needle. 3. Add 25 pL of a slurry of glass beads in water to the bottom of the 0.5 mL tube, followed by 300 pL of the Sepharose slurry, taking care not to disturb the glass beads (see Notes 2 and 3). 4. Place the assembly onto a suitable receiver tube of at least 5 mL vol and centrifuge in a swing-out rotor at 200g for 4 min. Transfer the 0.5-mL tube to an intact 1.5-mL test-tube and use as quickly as possible to prevent drying of the gel matrix (see Note 4). 5. Add the denatured sample to the top of the gel of the spin-dialysis tube, being careful not to disturb the gel layer. Centrifuge at 200g for 4 min and use the dialysate immediately.

3.3. Annealing 1. If sequencing with 35S-dATP, 8 pL of the prepared template is added to 2 pL buffer and 1 pL of primer. For sequencing with 32P-dATP use only 5 pL of template and make the final volume up to 11 pL with water (see Notes 5-7). 2. Incubate at 37°C for 15 min and centrifuge the tubes briefly to spin down any condensation (see Note 8). 3. Dispense 2.4 pL to each of four tubes and continue as in Chapter 13, using T7 DNA polymerase as the sequencing enzyme (see Note 9).

4. Notes 1. The rapid plasmid preparation used here provides template of good sequencing quality. DNA produced by other methods such as alkaline

Murphy lysis may also be used, but the boiled-lysis methods appear to produce templates that generate fewer artifacts upon sequencing. Cesium gradient purified DNA provides a useful control against which other techmques can beJudged, as the DNA is far less contammated than mmiprep DNA. However, some background is often observed when sequencing gradient purified DNA, perhaps because of some nicking of DNA m ethidium bromide solutions when exposed to UV light. 2. Sephacryl S-200 or G50 (Pharmacia) can be substituted for SepharoseC6B. G50 will shrink away from the tube wall on centrifugation, so it is essential to add the sample to the center of the gel surface. 3. Because of possible problems of low recovery of DNA it is unwise to use spin-dialysis with the gel volume described here for samples smaller than a final volume of 20 pL, that IS using 80% of the volumes given above. 4. Use the prepared spin-dialysis tubes as quickly as possible after the first centrifugation. If they are to be kept unused for more than a few minutes the tubes should be capped to prevent the gel from drying out. 5 Denatured and spin-dialyzed templates can be stored frozen at -20°C for several days, after annealing to primer. The quality of sequence obtained will not be as good as when the sample is used immediately. 6. The amount of DNA used m the denaturation provides enough material to sequence a short insert from both ends, leaving sufficient DNA for a further reaction if required. 7. The amount of primer indicated ISequimolar for 5 pg of a plasmrd of 4 kb. Do not use larger amounts of primer, because at higher concentrations priming to sequences of lower specificity may occur, generating ghost bands on the gel. 8. There is no advantage m anneahng at higher temperatures than 37°C and slow-coolmg to room temperature, since this may causeproblems through reannealmg of the complimentary strands and premature termination. 9. If following sequencing the DNA bands on the gel are smeary or bands are observed in all four lanes, it is possible that some sodium hydroxide is passing through the column. This can be tested by preparing a spm-dralysis tube as described and adding 25 pL of 10 mg/nL Blue Dextran 2000 and 10 mg/mL Orange G dye in TE to the top of the Sepharose and centrifuging as above. Transfer the spin-column to a fresh tube, add 25 pL TE, and recentrifuge. More than 90% of the Blue Dextran should be present m the first dialysate, and no Orange G should pass through in the second. Reference 1 Holmes, D S and Qulgley, M. (1981) A rapid bollmg method for the preparatlon of bacterlal plasmlds. Anal. Bmchem. 114, 193

c%XAPTER

20

Direct Sequencing of Inserts Cloned into Lambda

Vectors

Afghan N. Malik

1. Introduction Bacteriophage lambda is widely used as a vector for both genomic and cDNA cloning. However, although lambda is highly efficient for cloning, its use for direct sequencing is limited because of a lack of reliable sequencing methods. Although various methods for sequencing directly in lambda have been described (1,2), most researchers experience difficulties when sequencing in lambda, obtaining variable results and blank or extremely faint autoradiograms yielding unreliable sequences. The relatively large size of lambda (49 kb) when compared to common sequencing vectors, such as Ml3 (7 kb) or Bluescript (3 kb), and the linear duplex genome of lambda pose specific problems for direct DNA sequencing. Larger quantities of lambda DNA are required to achieve optimal primer: template ratios. Efficient denaturation and subsequent primer annealing to single-stranded template in preference to template self-reassociation are essential for direct sequencing. The most common factors contributing to poor sequencing results in lambda are poor quality of template DNA, inefficient denaturation, and self-reassociation of template DNA in preference to primer: template annealing. Although the linear duplex genome of lambda can be readily denatured to produce single-stranded template, it can also reasFrom Methods m Molecular Biology, Vol. 23’ DNA Sequencmg Protocols Edlted by. H and A Gnffm Copyright 01993 Humana Press Inc , Totowa, NJ

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sociate efficiently, often in preference to primer annealing, contributing to poor sequencing results. In order to directly sequence in lambda, the first requirement is the availability of highly purified template DNA in sufficient quantities, For cDNAcloning vectors, such as lambda gt 10, difficulties are often encountered in the preparation of insert DNA once the clones of interest have been purified due to the large size of the lambda genome compared with the average insert sizes (0.5-5 kb). Many methods for lambda DNA purification, including large-scale CsCl step gradients (3), small-scale liquid lysate methods (4), and methods utilizing preabsorbtion of phage to anion exchange matrices (I) have been described. This chapter describes a simple method utilizing double nuclease digestion and PEG precipitation (5). This method is reliable for a wide range of insert sizes, applicable to processing large numbers of clones at the same time, works well with all different lambda vectors we have tested, and routinely yields at least 2 mg of high purity DNA per 100 mL cultures (5). Once highly purified lambda DNA is available, the second major requirement is a reliable denaturation step followed by efficient annealing of single-stranded template to sequencing primer. Two methods of direct lambda sequencing, differing in the denaturation step, are described in this chapter. The first method utilizes routine alkaline denaturation and can give good results but often requires optimization of primer:template ratio. The second method utilizes a property of the enzyme T7 gene 6 exonuclease to produce single-stranded templates from double-stranded molecules with suitable termini (6). T7 gene 6 exonuclease has nonprocessive 5’-3’ exonuclease activity that degrades only double-stranded DNA (7,8), yielding products that are single-stranded half-molecules of the starting material. In order to sequence lambda DNA, the DNA is cleaved with a restriction enzyme near the priming site but outside the region to be sequencedand treated with T7 gene 6 exonuclease. T7 gene 6 exonuclease digests DNA with 3’ single-stranded termini, blunt ends or 5’ single-stranded termini (including the 12 base 5’ single stranded cohesive end of lambda) to yield single-stranded DNA. The single-stranded DNA can then be sequenced by methods identical to those used for Ml3 templates. The entire process can be carried out in l-l.5 h, and does not require precipitation or purification steps, The use of this enzyme removes

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Lambda

Clones

143

many of the usual problems associatedwith direct sequencing of duplex linear lambda DNA. 2. Materials 2.1. Lambda DNA Preparation 1. SM buffer: 5.8 g NaCl, 2 g MgS04a7H20, 50 mL 1M Tris-HCl, pH 7.5, 5 mL of molten 2% gelatin, make up to 1 L with HzO, sterilize by autoclaving, and store at room temperature; 2. SM buffer: 1M MgC12: autoclaved, store at room temperature; 3. SM buffer: 20% maltose filter-sterilize and store at 4°C; 4. SM buffer: NaCl; 5. SM buffer: DNase 1 10 mg/mL made up m sterrle HZ0 and stored at -20°C); 6. SM buffer: RNase A 10 mg/mL, made up in sterile Hz0 and stored at -20°C; 7. SM buffer: PEG-polyethylene glycol 8000; 8. SM buffer: Proteinase K 10 mg/mL, made up in sterile HZ0 and stored at -2OOC; 9. SM buffer: phenol saturated with TE; 10. SM buffer: phenol-chloroform equal vols of TE-saturated phenol and chloroform; 11. SM buffer: chloroform, 100% ethanol; 12. SM buffer: 70% ethanol made up with H,O; 13. SM buffer: TE, pH 7.4 10 n&I Tris-HCl, pH 7.4, 1 nuI4 EDTA, pH 8.0, autoclaved; 14. SM buffer: 3M sodium acetate, pH 5.4 40.8 g of sodium acetate trthydrate in 80 mL of H20 adjusted to pH 5.4 with glacial acetic acid, made up to 100 mL and autoclaved; 15. SM buffer: LB 10 g bactotryptone, 5 g bacto-yeast extract, 10 g NaCl in 1 L HzO, autoclaved and stored at room temperature; 16. SM buffer: LA 15 g bacto-agar/l L LB, make up in 200-mL aliquots and autoclave, store at room temperature; 17. SM buffer: topagarose 0.65% agarose/l L LB, make up m 100~mL ahquots and autoclave, store at room temperature. 2.2. Alkaline Denaturation Method 1. SM buffer: Freshly prepared 2M NaOH 2 rmI4 EDTA in H,O; 2. SM buffer: 3M sodturn acetate, pH 4.7 40.8 g sodium acetate trihydrate made up in 80 mL Hz0 adjusted to pH 4.7 with glacial acetic acid, made up to 100 mL, autoclaved and stored at room temperature;

144

Malik Table 1 Suggested Restriction Enzyme Digestion Sites for Common Lambda Vectors for Use in Conjunction with T7 Gene 6 Exonuclease Flanking sequencing primer

Cloning vector Lambda GTlO Lambda GT 11 Lambda GT18-23 Lambda Lambda Lambda Lambda Lambda Lambda Lambda

ZAP/ ZAPII Fix/ Fix11 DASH/ DASH II GEM 11,12

3. SM buffer: 4. SM buffer:

gtl0 forward primer gt 10 reverse primer gtll forward gt 11 reverse gt 11 forward gtl 1 reverse T7, M13-20 forward T3, Ml3 reverse T7 T3 T7 T3 T7 SP6

Restriction

enzyme

Hind111 BgiII or none SacI/HindIII/BamHIIXbaI KpnI pvuI/HindIII/BamHI KpnIlnone if msert < 3 kb BglIIlnone If insert < 4 kb MlUI KpnI BglIIlnone if insert c 1 kb BglII KpnI KpnIISmaIISfd BglIIlnone if insert < 1 kb

ethanol kept at -20°C; 70% ethanol (kept at -20°C);

5. SM buffer: appropriate primers either flanking the cloning site of lambda or internal primers from the region to be sequenced; sequencing kit; 6. SM buffer: both Sequenase version 2.0 USB, Cleveland, OH and the T7 sequencing kit Pharmacla-LKB, Piscataway, NJ give good results 2.3. Exonuclease Denaturation Method 1. SM buffer: Appropriate restriction enzyme Table 1; 2. SM buffer: T7 gene 6 exonuclease USB; appropriate pnmers either flanking the cloning site of lambda or internal primers from the region to be sequenced; sequencing kit; both Sequenase version 2.0 USB and the T7 sequencing kit Pharmacia-LKB give good results. 3. Methods

To sequence directly from lambda DNA, first prepare high quality template DNA from clone of interest and then proceed with either the alkaline denaturation method or the exonuclease denaturation method. Although the alkaline denaturation method works reasonably well

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and requires less template DNA, the exonuclease method does not require optimization of primer:template ratio, is more consistent, and yields clear and reliable sequences. 3.1. Preparation of Lambda DNA This section describes a simple method for the preparation of high

quality lambda DNA utilizing a double digestion step and PEG precipitation. You will need phage stock that represents a single purified plaque picked into 1 mL of SM buffer containing 10 r-is,chloroform (approx lo7 PFU). 1. Day 1: Set up an overnight culture of a suitable E. coli host stram (1 colony picked into 10 mL LB containing 10 mM MgCl, and 0.2% maltose, incubated with shaking at 37°C for 12-18 h). This can be kept at 4°C and used for plate lysates for l-2 wk. 2. Day 2: Prepare a plate lysate: Mix 100 pL of phage stock with 200 l.rL of an overmght culture of E. coli cells in a sterile bijou bottle. Incubate at room temperature (lo-20 mm). Add 3 mL molten top agarose (48°C) and pour onto an LA plate. Allow to set (10 min) and incubate inverted (37°C 12-18 h). These should give clear lysis by the next day. Set up a fresh overnight 10 mL culture of E. cob. 3. Day 3: a. Add 2 mL of a fresh overnight culture of E. co/i to 100 mL LB (prewarmed to 37OC)containing 10 mM MgC12 and 0.2% maltose m a 500 mL conical flask (use of smaller container will reduce yield). Grow with vigorous shaking at 37°C until AbOsis between 0.45 to -0.6 (approx 2-3 h). b. While the bacterial cells are growing overlay the plate lysates with 3 mL of SM buffer. Leave 1 h at room temperature. Decant mto sterile tubes. c. Add decanted phage to the E. coli when A,,, reaches 0.45-0.6. Grow with vigorous shaking until lysis (approx 3 h). d. Add 0.5 mL CHCl,. Shake for 10 min at 37°C. e. Add 4 g NaCl, 100 pL DNase 1 (10 mg/mL), 100 r-is,RNase A (10 mg/mL). Shake gently at 37°C 1 h. f. Transfer to 50-mL falcon tubes and remove as much of the chloroform as possible using a Pasteur pipet. Centrifuge at 3000g for 10 min to remove bacterial debris. Decant supernatant into a fresh 500mL conical flask and add 10 g PEGEOOO.Dissolve PEG by swirlmg gently. Incubate either on icewater (1 h) or at 4°C (12-18 h or longer) to precipitate phage.

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g. Transfer to 50-mL falcon tubes and centrifuge at 3000g for 20 mm. Discard supernatant and drain tubes well. Resuspend in 2 mL SM buffer and pool samples to give 4 mL per starting 100 mL culture. Add 40 pL DNasel (10 mg/mL), 40 pL RNaseA (10 mg/mL). Incubate 37°C for 30 min. h. Add 80 pL 10% SDS. Mix well then add 40 pL Proteinase K (10 mg/mL). Incubate 37°C for 45 min. i. Extract with equal vols of phenol, phenol/chloroform, chloroform. J. Add 400 pL 3h4 sodium acetate,pH 5.4, and 8 mL of cold 100% ethanol. Precipitate by placing at -2OOC for I h. Centrifuge at 3000g for 20 min to precipitate DNA. Wash once with 70% EtoH. Resuspend in 0.5 mL TE. Determine concentratton by measuring OD at 260 nm (A digest of 20 pL of this should be sufficient to view small mserts) 3.2. Alkali Denaturation Method 1. To 5 pg of lambda DNA m TE add 0.1 vol2M NaOH, 2M EDTA (freshly prepared). 2. Incubate at 37°C for 10 min. 3. Add 20-100 ng sequencing prtmer (see Note 5), 3M sodium acetate, pH 4.7 and 3 vol cold 100% ethanol. Precipitate by snap freezing m a dry ice/methanol bath, 5 min Centrtfuge at 10,OOOgand discard supernatant. Wash with 500 pL of cold 70% ethanol. Spin 5 min and remove all traces of ethanol using a drawn out Pasteur pipet. 4. Resuspend m 10 pL of 1X sequenase buffer and incubate at 37°C for 10 mm to anneal. Proceed Immediately to labeling and termination reactions as for Ml3 templates (see Chapter 14). 3.3. Exonuclease Denaturation Method 1. Restriction enzyme digestion: (see Table 1 to determme which enzyme to use wtth specific lambda vectors). Mix 15 pg lambda DNA, 50 U appropriate restriction enzyme, and 3 pL 5X sequencing buffer. Make up to a total vol of 15 pL. Incubate at 37°C (or other temperature depending on restriction enzyme) for 1 h. 2. Heat at 80°C for 10 min to inactivate the restriction enzyme (this may need to be altered for specific enzymes). 3. Add 50 U of T7 gene 6 exonuclease. Increased amounts of enzyme do not appear to cause any problems to the subsequent procedures. 4. Incubate at 37°C for 15 min and then heat at 80°C for 15 min. Place on ice. At the end of this procedure the duplex genome of lambda should have been digested at the appropriate over hangs to yteld half-molecules that are single stranded, providing a template for DNA sequencing. If neces-

Sequencing Lambda

Clones

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sary l+L aliquots can be removed at the end of the restriction enzyme digest and at the end of the exonuclease digestion and analyzed on an agarose gel. Before loading checker gel heat samples to 70°C for 5 min. 5. Anneal 12 pL of products from step 4 with l-2 pmol of appropriate primer (see Table 1) at 65°C as for ss Ml3 template (heat to 65°C and then allow to cool slowly over 30 min to below 3OOC). 6. Proceed with labeling and termination reactions as for Ml3 templates.

4. Notes 4.1. Preparation of Lambda

DNA

1. The method may be scaled up or down depending on requirements. However, it is vital to aerate the bacteria well during growth and lysis and appropriately large containers should be used for these steps (e.g., a 500-mL flask for a IOO-mL culture). I have found that the yield is significantly reduced if bacteria are grown in tubes instead of flasks, 2. It is best to check the quality of the template DNA prior to sequencmg. This can be done by running a small amount of uncut and cut DNA on an agarose gel. The uncut DNA should not contain any traces of RNA. If RNA is present, it should be removed by a further RNaseA step (using DNase-free RNase A, that can be prepared by boilmg RNaseA for 10 min), followed by phenol, phenol/chloroform, and chloroform extraction and precipitation. 3. If yield of lambda DNA is low, make sure that the host strain you are using is the correct one. 4. If all traces of PEG are not removed after the PEG precipitation step the resulting DNA may be less pure. To avoid this, dram the tubes well and remove all traces of PEG with a drawn out Pasteur pipet. Alternatively, after resuspending in PEG a single chloroform extraction can be included to remove all PEG.

4.2. Alkaline

Denaturation

Method

5. This method requires higher concentrations of primer than other lambda sequencing methods and often it is necessary to optimize the primer:template ratio to achieve the best results. This can be done by carrying out several reactions with the same conditions but altering the primer concentration between a range of 20-100 ng. 6. Always prepare fresh 2M NaOH 2 mM EDTA using NaOH pellets rather than stock solutions to achieve the best results. A 0.5M EDTA, pH 8.0, stock solutton may be used. Proceed to sequencing immediately after annealing as a delay will reduce efficiency.

Malik 7. Using too much template can lead to blank or nearly blank films as the template reassoctates with itself m preference to the primer. Accurate estimation of lambda DNA concentration is therefore highly desirable. 4.3. Exonuclease Denaturation Method 8. Choosing a restriction enzyme to use in conjunction with T7 gene 6 exonuclease: Use a restriction enzyme that cleaves near the sequencmg primmg site, but not within the sequence to be determined. Enzymes used successfully include EcoRI, PHI, BgZII, EcoRI, KpnI, SacI, Hind111 and others. A full list of appropriate enzymes for a range of lambda vectors is given m Table 1 (supplied by USB). 9. If an unmapped restriction site for the enzyme used is present within the insert the sequencewill stop abruptly. In this caseuse an alternative enzyme. 10. Sometimes this method results in distortion of bands at the top of the gel because of the glycerol present from the restriction enzyme. This should not interfere with the sequence, but if it does you may need to precipitate ss DNA after restriction enzyme digestion and exonuclease treatment before proceeding with sequencing reactions. 11. If no sequence is produced, check that the restrictron enzyme digest step and the T7 gene 6 exonuclease digestion step are working by running the products on an agarose gel. 12. At the time of writing this article, USB was the only company supplymg the T7 gene 6 exonuclease enzyme, and a kit for ds sequencing that utilizes this enzyme.

References 1. Manfioletti, G. and Schneider,V. (1988) A new andfast method for preparing high quality lambdaDNA suitablefor sequencingNut. Acids Res. 16,2873-2884 2 Kim, B. S. and Jue, C (1990) Direct sequencingof lambda gtl 1 recombinant clones.Bio. Techniques 8, 156-159. 3 Sambrook, J., Frrtsch, E. F , and Manitais, T (1989) Molecular Clonrng* A laborutory Manual Cold Spring Harbor Press, Cold Spring Harbor, NY 4 Grosberger,

D. (1987) Minipreps

of DNA from bacteriophage lambda Nut

Acids Res. 15,6737. 5. Malik, A. N , McClean, P. M., Roberts, A., Barnett, P. S., Demame, A G , Banga, J. P., and McGregor, A. M (1990) A simple high yield method for the preparation of lambda gtl0 DNA suitable for subclonmg, amplificatron and direct sequencmg. Nut Acids Res 18,403 l-4032 6. Ruan, C. C. and Fuller, C. W. (1991) Using T7 gene 6 exonuclease to prepare single-stranded templates for sequencing. USB Comments 18, l-8 7 Kerr, C. and Sadowski, P. D. (1972) Gene 6 exonuclease of bacteriophage T7, puriftcatron and properties of the enzyme J Bzo. Chem 247,305-3 10 8 Hemkoff, S. (198 1) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28, 35 l-359.

Cosmid Sequencing Molly Cmxton

1. Introduction The expectation that genome sequence analysis will provide an increasingly useful approach to the characterization of biological material has led to suggestions that the complete genome sequences of organisms ranging from Escherichia coli to Homo sapiens should be determined. Work is already underway to determine the genome sequences of some model organisms, such as E. coli, Succharomyces cerevisiae, and Cuenorhubditis elegans, because their small genomes are well characterized both genetically and physically. The much larger project to sequence the human genome is currently at a more preliminary stage. An important prelude to the sequencing of a genome or subregion, is its physical characterization by way of a clone based map. The physical map comprises a set of ordered, overlapping subclones from which suitably located subclones may be selected and sequenced. A variety of cloning vectors are used in the construction of these maps, with lambdas, cosmids, and YACs being among the most common, Of these, cosmids and lambdas are more suitable for sequencing because they are much easier to purify in sufficient quantity. Lambdas and cosmids provide the majority of the subclones of the physical maps of E. From. Methods m Molecular Bfology, Vol 23 DNA Sequencmg Protocols EdIted by H and A Grlffm Copyright 01993 Humana Press Inc , Totowa,

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coli, S. cerevisiae, and C. elegans, and thus provide an ideal starting

point for complete genome sequencing. A pilot project to sequencea 3-megabase stretch of C. elegans genomic DNA was initiated in 1990 and initial results, one year into the project, have been submitted recently (I). This chapter summarizes the strategy and provides detailed methods currently being used to generate the C. elegans sequence, cosmid by cosmid. Most of the methods described here are derived from standard laboratory procedures (2-4) but modified by members of the C. elegans mapping and sequencing group to provide an efficient and coherent system for sequencing cosmids. It is beyond the scope of this chapter to include all of the details required for a beginner in sequencing. These can be

found elsewhere in this volume. (See Chapters 3-7 as excellent introductions to the basic theory and techniques involved and Chapter 1 for an overview of current sequencing technology.) A key consideration in designing a cosmid sequencing strategy within the context of the C. elegans genome sequencing project is that the sequencing should be as efficient as possible. Thus, traditional shotgun strategies with their inherent high redundancy, though reliable, may not be the most attractive. In theory, sequencing of whole cosmid templates using custom oligonucleotide primers, would give an ideal redundancy of 2. In practice, however, this completely directed strategy is too problematic (I). The main problems are the difficulty in obtaining sufficient amounts of unrearranged cosmid DNA, template secondary structures that are inaccessible to short sequencing primers, and repeat sequences that provide multiple priming sites. By using both shotgun and directed methods, the attractive elements

of both strategies may be combined. Initial shotgun sequencing of randomly generated subclones provides suitably spaced startpoints for primer walking that is employed to complete the sequence. By using a range of sizes of subclone inserts the particular attributes of different cloning vectors may be used to advantage. Ml 3 subclones provide easy template preparation, and because their inserts are small they also provide a means for disruptmg extended repetitious or struc-

tured regions of the cosmid. Phagemid subclones can be very large and thus relatively few subclones should provide contiguous cosmid coverage. Linear amplification sequencing reactions allow sequence to be generated from virtually any type of template, that is, single-

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stranded, double-stranded, small, and large clones (7). The point at which shotgun sequencing should finish and primer walking should begin depends on the relative costs and convenience of each method. This will vary from laboratory to laboratory, depending mainly on the availability of synthetic oligonucleotides (see Note 5). Fluorescent sequencing machines (8-10) offer instant storage and retrieval of digitally recorded sequence trace data. Put together with much improved software for the assembly and editing of cosmid sized sequencing projects (II) (see also Chapters 15-27 in DNA Sequencing: Computer Analysis of SequenceData. Griffin, A. M. and Griffin, H. G. (eds) an efficient sequencing system is possible. Although all the methods described in this chapter may be used equally well for fluorescent or radioactive sequencing, the current absence of fully automatic film reading devices means that a radioactive cosmid sequencing project would involve a great deal more effort. The question of when a cosmid sequencing project is complete is not a simple one. Although the sequence is not strictly finished until every base is determined unequivocally on each strand, this ideal may be impractical for various reasons. Extensive compressions, highly structured regions of the template, and multiple reiterated repeat sequences may all lead to lengthy delays in completing a cosmid sequence. Justification for the effort required to solve these problem areas will depend, realistically, on the information content of the cosmid sequence. It is important, however, to recognize that effort will eventually be saved if the sequencesdeposited in sequence libraries are as accurate and complete as possible. 2. Materials (see Note 2) 2.1. Equipment 1. ABI 373A DNA SEQUENCER-for fluorescent sequencing of shotgun clones. Applied Biosystems Inc., 850 Lincoln Center Drive, Foster City, CA 94404, 2. (ALF) SEQUENCER-for fluorescent custom primer directed sequencing. Pharmacia LKB Biotechnology, S-75182 Uppsala, Sweden. 3. SUN SPARCSTATION-for sequence assembly and editing. Sun Microsystems Inc., 2550 Garcia Avenue, Mountain View, CA 94043. 4. TECHNE MWl OR PHC3 THERMAL CYCLERS AND TECHNE HI TEMP 96 MICROTITER DISHES-for sequencing reactions. Techne (Cambridge) Ltd., Duxford, Cambridge CB2 4PZ, UK.

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5. SONICATOR-for shearing of cosmid DNA. Heat Systems-UltrasonICSInc., 38 East Mall, Plainview, NY 11803. 6. CORNING 25850 96 WELL ROUND BOTTOM PLATES-for subclone storage. Corning Glass Works, Corning, NY 14831. 7. A centrifuge capable of spinning microtiter dishes is also very useful,

2.2. Reagents 1. SmaI-New England Biolabs (Beverly, MA), 24 U&L. 2. CIP-Boehringer Mannheim (Mannheim, Germany), 1 U/pL. 3. Ltgase-Boehringer Mannheim, 1 U&L. 4. Mungbean nuclease-Pharmacia LKB Biotechnology, 130 U/pL. 5. Tuq polymerase-Cetus native Tuq 5 U/pL. 6. Fluoreprime TM-Pharmacra LKB Brotechnology. 7. Nucleotides: The lyophilized sodium salts from Pharmacta LKB Biotechnology are much less expensive than the soluttons, but do not have such a long shelf life. 7-deaza-dGTP, 10 mM solution is from Boehrmger Mannheim. 100 miVdTTP: add 1710 pL of 50 mMTris-HCl, pH 8.0, to 100 mg of solid dl’TP. 100 mM dCTP: add 1760 pL of 50 mM Trts-HCl, pH 8.0, to 100 mg of solid dCTP. 100 mM dGTP: add 1690 pL of 50 n&f Trts-HCl, pH 8.0, to 100 mg of solid dGTP. 100 mk! dATP: add 1800 pL of 50 m&I Tris-HCl, pH 8.0, to 100 mg of solid dATP. 10 n&f ddTTP: add 900 pL of 50 mM Tris-HCl, pH 8.0, to 5 mg of sohd ddlTP. 10 rr&f ddCTP: add 400 pL of 50 n&f Tris-HCl, pH 8.0, to 4 pm01 of solid ddCTP. 10 mM ddGTP: add 400 pL of 50 mM Tris-HCl, pH 8.0, to 4 pm01 of solid ddGTP. 10 mM ddATP: add 400 pL of 50 mM Tris-HCl, pH 8.0, to 4 pm01 of solid ddATP. Refer to Sections 3.5.1 .I. and 3.5.2.1. for the compositions of the NTP mixes for sequencing. 8. TY medium: 1% bactotryptone, 1% yeast extract, 0.5% NaCl. 9. TFB: 100 mh4 KCl, 45 mM MnC12.4H20, 10 mM CaC12.2H,0, 5 mM MES, pH 6.2, 3 mil4 hexaminecobaltic chloride. 10. Glucose/EDTA/Tris: 50 n-144glucose, 25 m44 Tris-HCl, pH 8.0, 10 miI4 EDTA. 11. NaOWSDS: 0.2N NaOH, 1% SDS (make fresh each time wtth concentrated NaOH).

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12. 10X mb buffer: 300 mM NaOAc, pH 5.0,500 mM NaCl, 10 mM ZnCl*, 50% glycerol. 13. 10X SmaI buffer: 66 pL of HzO, 20 pI, of 1M KCl, 6 pL of Tris, pH 8.2,6 pL of 1M MgC12, 1 pL of 10% BSA, 1 pL of 2-mercaptoethanol (make fresh each time). 14. 10X hg buffer: 30 pL of water, 50 pL of 1M Tris-HCI, pH 7.4, 10 pL of 1M MgCl,, 10 uL of 1M DTT (make fresh each time). 15. PEG/NaCl solution: 20% PEG, 2.5M NaCI. 16. EtOH/NaOAc solution: 4 mL of 3M NaOAc, pH 4.8, and 125 mL of 95% EtOH. 17. 10X buffer 1: 500 mM KCl, 100 mM Tris-HCl, pH 8.5, 15 mM MgClz. 18. 10X buffer 2: 400 mM Trrs-HCl, pH 8.9, 100 mM Ammonium sulfate, 25 mA4 MgCl*.

3. Methods 3.1. Cosmid Growth Suitably pure cosmid DNA must be prepared in order to generate random subfragments and for any direct cosmid sequencing. Isopycnit centrifugation over 3 mL cesium chloride density gradients is a quick and easy method to obtain in the order of S-50 ~18cosmid DNA. Cosmid clones tend to be unstable and the yield of DNA varies greatly, so it is wise to pick several colonies for large scale growth. To guard

against deletions and rearrangements, the different preparations should be checked by restriction enzyme digestion before they are combined prior to library construction. 1. Streak the cosmid directly from a -70°C stock to obtain single colonies. 2. Inoculate 4 x 200 mL of TY containmg the appropriate drug, each with a single colony. Avoid using very tmy or large colonies. Shake at 37°C until late log phase (18-24 h). 3. Spin at 10,OOOgfor 5 min in 500-n& Sorvall centrifuge bottles. Tip off the medium and then put the bottle on ice for 2 mm. Aspirate any remaining medium, then add 6 mL of Glucose/EDTA/Tris. 4. Transfer with a 10-mL pipet to a 30-mL Sorvall centrifuge bottle, then complete the suspension by vigorous vortexmg. 5. Add 8 mL of NaOH/SDS, mix then leave on ice for 15 min. 6. Add 6 mL of 3M NaOAc, pH 4.8, mtx, then leave on ice for 30 min. 7. Spin at 17,000g for 10 min, then transfer the supernatant (15-18 mL) to a 50-mL Falcon tube 8. Add 2 vols of 95% EtOH, mix, then spin at 9OOgfor 5 min. Tip out the supernatant and drain,

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9. Resuspend the pellet in 20 mL of 80% EtOH. 10. Spin at 6000g for 5 min. Tip out the supernatant and dram for 5 min then vacuum dry. 11. Add 2.5 mL of 10 mM Trrs-HCl, pH 8.0,10 rnM EDTA, and allow the DNA to dissolve overnight. 12. Add 2.8g of CsCl and 0.25 rnL of 10 mg/mL EtBr, mix, then spin at 6000g

for 10 min.

Transfer

the supernatant

to Beckman

TL100.3

polyallomer tubes. Ensure the tubes are full, then seal and place rn a Beckman TL100.3 rotor with spacers. 13. Spin at 265,000g at 20°C overnight (14 h). 14. Collect the cosmid band with a 20-G needle on a 1-rnL syringe, piercing the wall of the tube about 2 mm below the lower (supercooled cosmid) band. 200 & should be sufficient not try to collect more.

to collect 90% of the cosmid but do

15. Add 300 pL of water to the cosmid band material, and extract with lsobutanol

until the organic layer is colorless.

16. Add water to a fmal volume of 400 pL, then add 800 pL of 95% EtOH

to precipitate the DNA. 17. Dissolve the DNA m 20 l.tL of 10 rnM Tris-HCl, pH 8.0,O.1 mM EDTA, and assay each preparation separately by restriction enzyme digestion.

3.2. Library

Construction

The quality of the subclone libraries affects the required redundancy of the final sequence. Careful size selection of randomly sheared subfragments, together with good preparations of cloning vectors, should minimize the redundancy. Physical shearing of the cosmid DNA will give a better distribution of subfragments than partial restriction digestion, but other methods for enzyme directed random fragment generation also exist (12). Shearing of whole cosmid DNA, rather than just the excised insert, helps to ensure that the subfragments are distributed as randomly as possible, and will only add a maximum of 10% extra sequencing work. Two methods for shearing of DNA are presented (see Section 3.2.1.). Shearing by passing whole cosmid DNA through a 30-G needle can give fragment sizes down to 4 kb. Sonication is used to generate smaller fragments (3000-500 bases using the settings m Section 3.2.1.). Care should be taken to make vector preparations with cleanly cut and phosphatased cloning sites. In order to achieve high efficiencies of ligation and low “false white” backgrounds, the procedures in Section 3.2.2. are recommended. The control ligations in Section 3.2.3.

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are useful for monitoring ligation efficiency (control C) and vector background, owing to frameshifts at the cloning site (control A) and uncut vector (control B). Competent TGl cells may be prepared in any convenient way (2). The simple procedure used in this lab is presented in Section 3.3.4. 3.2.1. Construction of Sheared Libraries 1. Sonication: Add 5 lrg of DNA, 6 p.L of 10X mb buffer and water to 60 pL. Sonicate twice for 5 s, at setting 25, continuous output, 100% duty cycle. Mix between each burst. 2. Nedle shear: Add 5 pg of DNA, 20 pL of 10X mb buffer and water to 200 pL. Shear 4 times through a 30-G needle on a I-mL syringe. The needle is passed through the lid of a 1.5-mL screw-capped vial and pressure is applied by means of a weight (4 kg is ideal) or by hand. 3. Keep the solutions on ice while running 1 pL on a mmigel to check the size and concentration of DNA. 4. Add 0.3 pL of mung bean nuclease and incubate at 30°C for 10 mm 5. Add water to 200 pL if necessary, then add 20 pL of 1M NaCl, 200 pL of phenol/CHC13, vortex, then put on ice for 5 min. 6. Spur to separate the phases then add 2.5 volumes of 95% EtOH to the aqueous phase and freeze. 7. Make a 250-mL LGT agarose/TAE gel with 0.5 cm width slots (0.4% agarose for needle sheared DNA and 0.8% agarose for sonicated DNA) and load 2.5 pg of DNA per slot. Load markers, such as lambda Hind111 and phiX174 HueIII, but be sure to keep the latter at least one slot away from the sheared DNA. 8. Run the gel at 42 mA/47 V for 4 h in TAE buffer containing 17 ng/mL EtBr. 9. Cut out the required fractions (e.g., l-2 kb, 6 -9 kb, and 9-14 kb). 10. Melt the agarose at 65°C and divide into 0.5-mL aliquots in snap-cap tubes, diluting the 0.8% agarose slices twofold with 10 mM Tris-HCL, pH 8.0/0.1 mM EDTA. 11. Add 0.5 mL of phenol, vortex, then put on ice for 5 mm (see Note 1). 12. Spin to separate the phases, take the aqueous phase, then repeat the 0.5mL phenol extraction. 13. Add isobutanol to reduce the aqueous phase to 0.3 mL. 14. Add 30 pL of 1M NaCl and 700 pL of 95% EtOH to precipitate the DNA. 15. Dissolve the DNA in 10 pL of 10 mMTris-HCl, pH 8.0/0.1 mMEDTA. 3.2.2. Vector Preparation 1. Add 20 @ of IOX SmaI buffer, 20 l.rgof vector DNA, water to 200 & and 1 pL of SmaI. Incubate for 1 h at 37”C, then put on ice.

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2. Run 2 pL on a minigel to check the extent of digestion. Ideally there should be a small amount of uncut; SmaI overdigestion results in “chew back” of bases around the SmaI site. 3. Add 20 pL of O.lM EDTA and 60 pL gel loading dye to the dtgest, then load 20 @JO.5 cm lane of a 250 mL 0.4% LGT agarose/TAE gel. Run the gel for 5 h at 35 mA/45 V in TAE buffer containmg 17 ng/mL EtBr. 4. Cut out the linear vector bands, melt the agarose at 65°C and aliquot 0.5-n& portions into snap-cap tubes. 5. Add 0.5 mL of phenol per tube, vortex, then put on ice for 5 min (see Note 1). 6. Spin to separate the phases, remove the aqueous phase, and repeat the 0.5-mL phenol extractton. 7. Add isobutanol to reduce the aqueous phase to 0.3 mL, then preciprtate the DNA with 30 pL of 1M NaCl and 700 pL of 95% EtOH. 8. Dissolve the DNA in 5 pL of 10 mM Tris-HCl, pH 8.0,O.l mM EDTA then add 90 pL of 50 rr&f Tris-HCl, 0.1 rnA4 EDTA and 4 pL of CIP. Incubate at 37°C for 30 min. 9. Add 4 pL of 0.5M EGTA and incubate at 65OC for a further 30 mm. 10. Add 100 pL of water, 200 pL of phenol, vortex, then put on ice for 5 min (see Note 1). 11. Spm to separate the phases, remove the aqueous phase, and precipitate the DNA with 20 pL of 3M NaOAc and 600 pL of 95% EtOH. 12. Dissolve the DNA m 40 pL of 10 mMTris-HCl, pH 8.0/O.1 mM EDTA. 3.2.3. Ligations 1. Make a ligation mix containing, per ligation, 40 ng of vector DNA, 1 pL of 10X lig buffer, 1 pL of 10 mM rATP and water to 8 pL, and dispense 8 pL of this mix into the required number of 0.5~mL snap-cap tubes. 2. Add 100-200 ng of sheared fragments m l-3 pL, or 2 ng of phiX 174 HaeIII as control C. Set up two other controls, A and B WITHOUT sheared fragments. Add 0.5 pL of ligase to all tubes except control B. 3. Incubate at 15°C overnight, then store the ligattons at -20°C until required for transformation. 3.2.4. Competent Cells and Transformation 1. Pick a single colony of TGl cells from a glucose minimal plate mto 10 mL of TY medium and grow overnight at 37’C This overnight culture may be stored at 4°C for up to a week. 2. Inoculate 10 mL of TY medium wrth 0.1 mL of overnight culture and grow at 37°C for 1.75 h. 3. Sprn at 9OOgfor 5 mm m a 15-mL Falcon tube, then tip off the supernatant and drain for 1 mm.

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4. Resuspend the cells in 1 mL of TFB and leave on ice for 5 min. 5. Add 70 pL of dimethylformamide directly to the cells, swirl, and leave on ice for 5 min. 6. Add 75 pL of 1M dithiothreitol (in water), swirl, and leave on ice for 5 min. 7. Immediately aliquot 100 pL of competentcells into tubes for transformation. 8. Add 1 pL of ligation and leave on ice for 30 min. 9. Heat shock at 43°C for 2-3 min then Immediately replace on ice. 10. For phagemid clones, add 0.5~mL TY medium and shake at 37OCfor 30 min then add, directly to the cells, Xgal and IPTG to 1.2 mg/mL and 1.5 mg/mL, respectively. Spread 0.3 mL of this mixture on to plates contaming the appropriate drug. For Ml3 clones, plate out the transformed cells directly, in 3 mL top agar containing 200 pg/mL Xgal and 250 clg/ mL IPTG. Transformation efficiencies are usually in the range 5 x 106-5 x lo7 transformants/pg of DNA. 3.3. Template

Preparation

In general, the quality of template required for fluorescent sequencing is higher than that needed for the more sensitive radioactive methods. Although linear amplification of sequencing reactions reduces the stringent template quality requirements of fluorescent sequencing (7,13) cruder template preparations may always be used for radioactive sequencing. Of course, the highest quality sequenceis obtained from the highest quality templates. Ml3 templates with inserts from the size range 1 to 2 kb are routinely prepared by the standard PEG/phenol procedure (5) (see Section 3.3.1.). Ml3 subclones may also be prepared by a semi-automated microtiter method (see 14; also Chapters 6 and 38). Although the yield of template DNA from this method is low compared to the standard method, it is certainly sufficient for radioactive

sequencing (14)

and for high redundancy fluorescent shotgun sequencing (15). For a low redundancy fluorescent sequencing strategy, however, where the accuracy of each sequenceread is crucial, the PEG/phenol method is more likely to produce the higher quality templates required. Phagemid templates with inserts ranging in size from 6-14 kb, should provide contiguous cosmid coverage and serve as the templates in the primer walking phase of the sequencing project. Either single-stranded

or double-stranded

templates may be used. Single-

stranded phagemid preparation is described in Section 3.3-l., and

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double-stranded preparation is described in Section 3.3.2. Compared to the smaller M 13 subclones, the yields of phagemid DNA are rather variable. It is therefore prudent to assay the yield by agarose gel electrophoresis prior to sequencing. 3.3.1. Ml3 and Single-Stranded Phagemid Preparation 1. Toothpick a white plaque into 1.5 mL of a l/100 dilution of an overnight culture of TGl cells in TY medium and shake for 5-6 h at 37°C (no higher, or Ml3 infection is compromised). For phagemids, ptck cells from streaks (see Section 3.4.) mto 1.5 mL of TY containing the appropriate drug and approxtmately 109PFU helper phage, such as M 13K07, and grow as for MI 3 2. Transfer the culture to a 1.5-mL snap-cap tube and microfuge at top speed for 10 min then decant the supernatant mto a fresh 1.5-mL snapcap tube containing 150 pL of PEG/NaCl solutton (PEG/NaCI can be aliquoted in advance using a multiple pipettor). Do not attempt to remove all of the supernatant as this will disturb the cell pellet and contammate the preparation. Ml 3 subclones: Save the cell pellets for subclone storage (Sectton 3.4.). Mix the PEG/NaCI and supernatant thoroughly by vortexing then leave at room temperature for 10 min. 3. Microfuge at top speed for 10 min, then remove the supernatant using a yellow tip attached to a vacuum apparatus (be sure to avoid the phage pellet). 4. Microfuge at top speed for 2 mm, then remove all of the remaining supernatant with a flat duckbill tip attached to the vacuum apparatus (again avoiding the phage pellet). If there is not enough time to complete the preparations, freeze the phage pellets at -20°C until ready to proceed. 5. Resuspend each phage pellet m 100 pL of 10 mM Tris-HCl, pH &O/10 mM EDTA. Ensure that the pellet is fully resuspended, then add 50 l.rL of phenol, vortex thoroughly, and leave at room temperature for approximately 5 mm. Vortex again, then put on ice for 5 mm (see Note 1). 6. Microfuge at top speed for 5 mm to separate the phases then without delay, transfer 60 l.tL of the aqueous phase, avoidmg the interphase area, to a fresh 1.5~mL snap-cap tube contammg 150 pL of EtOH/NaOAc solution. Vortex thoroughly and freeze. 7. Microfuge at top speed for 10 min, then decant the supernatant and add approx 500 pL of 95% EtOH. Leave at room temperature for 10 min, then aspirate the EtOH rinse with a flat duckblll tip attached to the vacuum apparatus. 8. Leave the tubes open until dry, then dissolve the DNA in 100-200 ltL of sterile distilled water to give a concentration of 50-100 pg/rnL.

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3.3.2. Double-Stranded Phagemid Template Preparation 1. Grow a 2-mL culture of cells to the point of maximum plasmids per cell (this will be approx 8-12 hours at 37°C). 2. Transfer the culture to a 2-mL snap-cap tube, microfuge at top speed for 5 min, then aspirate the supernatant with a blue tip attached to a vacuum apparatus. Save some of the cell pellet for subclone storage (see Section 3.3.4.). 3. Put the cells on ice and add 100 pL of Glucose/EDTA/Tris. Thoroughly resuspend the cells with a pipettor set to 50 pL and make sure there are no lumps of cells before proceeding. 4. Add 200 pL of NaOH/SDS with a blue tip or multiple pipettor, dispensing the solution into each tube without touchmg the tube itself. Do not mix with a pipettor, but cap the tubes, vortex to mix, and leave on ice for 15 min. 5. Add 150 pL of 3M NaOAc, pH 4.8 by dispensing as above, cap the tubes, mix by vortexmg, and leave on ice for 15 mm. 6. Microfuge at top speed for 10 min, then transfer 400 pL of supernatant with a blue tip, avoiding any precipitate, to a fresh 1.5~mL snap-cap tube containing 1 mL of 95% EtOH. Vortex thoroughly to mix, then freeze. If there IS not time to complete the preparations, store them at -20°C until ready to proceed. 7. Microfuge at top speed for 10 min, then aspirate the supernatant with a blue tip attached to the vacuum apparatus,avoiding the pellet. Add approx 1 mL of 95% EtOH and leave at room temperature for approxtmately 10 min, then aspirate to near dryness with the vacuum apparatus, first using a blue tip and then using a duckbill tip. 8. Dissolve the pellet m 100 pL of 200 pg/mL RNase in water, incubate at 37°C for approx 60 min, then add 10 pL of 100 mM EDTA and 60 pL of phenol and vortex thoroughly. Leave at room temperature for approx 5 min, vortex again, then put on ice for 5 min (see Note 1). 9. Microfuge at top speed for 5 min to separate the phases then without delay, transfer 80 pL of the aqueous phase, avoiding the interphase area, to a fresh 1.5-n& snap-cap tube containing 200 pL of EtOH/NaOAc solution. Vortex thoroughly and freeze. If there is not time to complete the preparations, store them at -20°C until ready to proceed. 10. Microfuge at top speed for 10 min, then aspirate the supernatant with a duckbill tip and add approx 500 pL of 95% EtOH. Leave at room temperature for 10 min, then aspirate the EtOH rinse with a duckbill tip. 11. Leave the tubes open until dry then resuspend each pellet m 50-100 pL of sterile distilled water to give a concentration of 150-300 pg/mL. This laboratory routmely assembles 300400 sequence reads from Ml 3 tem-

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plates and 100-200 sequence reads from phagemld templates before walking to complete the sequence. Double-stranded phagemid templates allow phagemid inserts to be sequenced from either end using standard primers. This particular combination of shotgun and walking leads to final sequence redundancies around 4. 3.4. Subclone Storage Glycerol stocks of subclones can be stored in rigid microtiter dishes at -70°C. These may become useful at a later stage, for example, for the preparation of gene specific probes. 1. Distribute 0.1~mL aliquots of 50% glycerol to microtiter wells, 2 For Ml3 clones, reserve the cells from step of Section 3.3.1.) resuspend the cells in 70 pL of TY and mix with the glycerol by pipetting briefly. For phagemid clones, use cells from step 1 of Sectlon 3.3.2. and add 70 pL of culture to the glycerol. If smgle-stranded phagemld templates (see Section 3.3.1.) are used, streak colonies onto a fresh agar plate before setting up miniprep cultures. In this case aliquot 70 pL of TY to the mlcrotiter dish wells, then pick up cells from the streaks with a yellow tip and mix with the TY and glycerol by plpetting briefly. 3. When all the wells are done, replace the microtiter dish hd and put an empty dish on top to reduce condensation, then immediately place both dishes in the -7OOC freezer. 3.5. Sequencing Reactions In all cases, fluorescent sequencing primers may be replaced with radioactive 5’ end labeled primers. This laboratory employs two different fluorescent machines for the two phases (shotgun and primer walking) of a cosmid sequencing project, and different sequencing protocols were developed in conjunction with each machine as it became available to our group. These protocols are interchangeable. The ABI 373A with its higher capacity (24 clones/gel) 4 fluor detection, is more suited to the shotgun phase. The sequencing reactions in Section 3.5.1. are used with this machine. The ALF, with its single fluor detection, is more suited to the walking phase. Custom fluorescent oligonucleotide primers for use with the ALF may be synthesized by incorporating a fluorocein phosphoramidite at their 5’ ends (16). Although T7 polymerase reactions were originally used with the ALF (17), the much more convenient linear amplification/cycle sequencing reactions using Taq polymerase have now been modified for use with the ALF (see Section 3.5.2.).

Cosmid

Sequencing

I61

For radioactive sequencing, any of these methods may be used, but it should be remembered that linear amplification/cycle sequencing reactions require only a fraction of the template used in standard T7/ Sequenase/Klenow reactions. Cosmid templates may be sequenced directly using either of the methods described here. 3.5.1. Linear Amplification Sequencing Reactions Per microtiter dish full of sequencing reactions, that is, per 24 clones: 1. Add 2 pL of template DNA to each of 4 base specific reaction wells, Template solution concentration should be in the range of 5-100 nM. This corresponds to approx 15-300 ~.~g/rnLM 13 templates, 24-480 M/ mL single-strand phagemid templates, 48-960 pg/mL double-strand phagemid templates, and 135-2700 pg/mL cosmid templates (the lower amounts can be used for radioactive sequencing whereas the htgher amounts are needed for fluorescent sequencing). 2. Overlay the samples with light mineral or light paraffin oil using a multiple pipettor, such as a 12-channel pipet, and cover with a Falcon 3913 lid. You do not need to use a fresh lid every time. These first two steps can be repeated depending on how many mlcrotiter dishfuls of sequencing reactions are to be set up. Dishfuls of dispensed templates may be stored frozen. 3. For each microtiter dish, label four 1.5mL snap-cap tubes T, C, G, and A, for the 4 base specific reactton mixes. To each tube add 50 pL of 10X buffer 1, 100 pL of appropriate NTP mix (see Section 3.5.1.1.), 6.25 pL of 1 pJ4 primer, 2.5 pL of Tuq polymerase, and 292 pL of sterile distilled water. 4. Vortex very briefly to mrx. Spin briefly, then dispense 18 pL of base specific reaction mix into the appropriate microtrter dish wells. When dispensing, hold the pipet tip to the surface of the oil and expel the 18 pL. The same tip is used for all 24 wells of each individual base specific reaction mix, but different tips are used for the different base specific reaction mixes. 5. Cover with the Falcon 3913 lid, then briefly spm the microtiter dash to ensure mixing and separation of the two phases. 6. Place the microtiter dish in the Techne cycler and cycle the reactions 20 times through a 1 min denaturation at 95OC, a 1 min annealing at 55°C and a 3 mm extension at 72°C (see Note 3). Note that the annealing temperature wrll depend on the r,,, of the primer used. T,,, can be estimated usmg the equation 7’, = 2(A + T) + 4(G + C). An annealing temperature around 2OClower than the 7’, should be used. Completed reactions may be stored frozen until ready to proceed.

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7. For fluorescent samples, the 4 base specrfic reactions for each clone are pooled and precipitated prior to gel loading: Transfer approx 30 @, from the bottom of each of 3 rows of base specific reactions to the fourth row using a 1Zchannel pipet. Transfer the pooled reactions using a duckbill tip, to labeled OS-mL snap-cap tubes containing 200 & of EtOH/NaOAc solution. Mix by vortexing, freeze, then microfuge at top speedfor 10 min. Aspirate the supernatant with a duckbill tip attached to a vacuum apparatus. Add approx 300 pL of 95% EtOH and leave at room temperature for up to 10 mm. Aspirate with a duckbill trp and vacuum apparatus, then leave the tubes open to dry. Dry sequencmg reactions may be stored frozen until the sequencing gel is ready for loading. 8. Add 4-6 pL of gel loading solution and vortex thoroughly to dissolve the DNA. Heat at 80°C for 5-10 min, then immediately put on ice. 9. For radioactrve samples, ahquots of the sequencmg reacttons are mixed wrth gel loading solution then heated to concentrate and denature prior to gel loading. 3.5.1.1. NTP MIXES T: 12.5 pL of a mrxture of 1 mil4 each dNTP mix, 62.5 pL of 10 mM ddlTP, and 425 JJLof water. C: 12.5 pL of a mixture of 1 miI4 each dNTP mix, 40 pL of 10 rnJ4 ddCTP, and 448 pL of water. G: 12.5 pL of a mixture of 1 rmI4 each dNTP mix, 8 pL of 10 n&f ddGTP, and 480 pL of water. A: 12.5 & of a mixture of 1 mM each dNTP mix, 40 pL of 10 m&I ddATP, and 448 pL of water. 1 nuI4 each dNTP mix: Add 5 pL of 100 n&f dTTP, 5 p.L of 100 mM dCTP, 5 pL of 100 rmI4 dGTP, 5 pL of 100 nnI4 dATP and 480 pL of water. Deaza GTP mixes are made by replacing dGTP with a molar equivalent of 7-deaza-dGTP. 3.5.2. ALF Sequencing Reactions Reactions are carried out in Techne microtiter dishes. The volumes below are for 10 clones. 1. Label 4 tubes A, C, G, and T for the 4 base specrfrc reaction mixes. To each tube add 13 pL of 10X buffer 2,5.5 pL of DMSO, 11 pL of appropriate NTP mix (see Section 3.5.2.1.) and 14.5 pL of diluted Tuq mix (11 pL of Taq plus 49 j.tL of water). As m most cases the ALF IS used for primer walking, the primer 1snot included m this reaction mix. 2. Add 1 pL of 0.25 @I4primer to the bottom of each appropriate mtcrotiter dish well.

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3. Dispense 4 pL of appropriate base specific reaction mix to the sides of the microtiter dish wells so that the same tip may be used for all 10 of each base specific reaction. 4. Add 2 pL of DNA (concentration as for Section 35.1.) to the other side of each well using a repetitive dispenser. 5. Overlay the reactions with light mineral or light paraffin oil, cover with a Falcon 3913 lid, and spin briefly. 6. Place reacttons in the Techne cycler and cycle 20 times through a 0.5 min denaturation at 93”C, a 0.5 min annealing at 55°C and a 1.2 min extension at 70°C (see Note 3). Note that the annealing temperature will depend on the T,,, of the primers (see Section 3.5.1.). 7. Immediately carry out 6 additional cycles of a 0.5 mm denaturation at 93°C and a 2.5 min extension at 70°C. 8. Using the same tip for all of the wells, add 5 pL of gel loading solution to the top of the oil, then briefly spin the dish to combine the loading solution and reaction solution, Using a duckbill tip, remove the lower aqueous layer to the appropriate

well of a microsample

dish such as

Pharmacia (Piscataway, NJ) 2010-700. 9. Denature the samples by placing the microsample dish at 90°C for 2 min and load all of each sample on an ALF gel.

3.5.2.1. NTP MIXES To make the NTP mixes, add an equal volume of dNTP mix to the corresponding ddNTP mix. dNTP mixes: A: 125 /t/WdATP, 500 @4 dTTP, T: 125 @f dTTP, 500 p&! dATP, C: 125 p/V dCTP, 500 @‘t4dTTP, G: 125 l.tM dGTP, 500 @4 dTTP, ddNTP mixes: A: 3.3 mM ddATP. T: 2.85 mM ddTTP. C: 1.53 mM ddCTP. G: 0.28 mM ddGTP.

500 /M dCTP, 500 j.M dCTP, 500 pM dATP, 500 l.tM dATP,

500 @4 dGTP. 500 @4 dGTP. 500 p.M dGTP. 500 p/t4 dCTP.

3.6. Sequence Assembly and Editing The programs TED (19) and XDAP (II) are used to compile the

cosmid sequence. For radioactive projects, only the program SAP (20) is required. Both XDAP and SAP as well as the necessary computer hardware are covered in detail by Rodger Staden in Chapters 15-27 of the companion to this volume, DNA Sequencing: Computer

Craxton Analysis of Sequence Data, Griffin, A. M. and Griffin, H. G. (eds.). A

program OSP (21) for the selection of oligonucleotide primers, will be incorporated into XDAP in the near future. 3.7. Compressions and Other Trouble Spots Steps taken to resolve compressions include replacing dGTP with 7-deaza-dGTP, formamide included in the gel mix (18), and running hot gels. For radioactive sequencing, running hot gels in combination with 7-deaza-dGTP is probably most convenient. As long as the compression is not too extensive, the above procedures should allow both halves of the compressed region to be read by primer extension from both complementary strands of the template. Sequencing problems due to template secondary structure, as opposed to secondary structure in the products of the sequencing reaction, are probably best tackled by using different polymerases, such as T7, Sequenase, Klenow, or Reverse Transcriptase.

3.8. PCR Confirmation PCR amplification is a convenient way to check that the final cosmid

sequenceis colinear with the genomic DNA sequence.The oligonucleotide primers used in the walking phase can be used as PCR primers. By

choosing lo-20 pairs of appropriately oriented primers spaced approx 3 kb apart, an overlapping series of PCR products spanning the length of the cosmid insert can be obtained. The PCR products from cosmid

DNA and genomic DNA should be assayed side by side by agarose gel electrophoresis, and their sizes compared to that predicted from the cosmid sequence. This procedure should uncover any sizeable deletions or rearrangements in the cosmid sequence. PCR reactions are carried out in Techne microtiter dishes. 1. Per well, add 10 $ of 0.5 pg/mL cosmid or 2 clg/mL genomlc DNA, 2.5 pL of 10 pA4primer 1,2.5 pL of 10 @I primer 2, and 10 pL of water. 2. Overlay with light paraffin or light mmeral oil and cover with a Falcon 3913 lid. 3. Spin briefly, place the dish in the Techne cycler, and denature at 95OC for 5 min. then place the dish on ice. 4. Dispense 25 pL of enzyme mix mto wells and respm briefly. Enzyme mix, per well: 5 pL of 10X buffer 1, 10 pL of 1 mM each dNTP mix (see Section 3.5.1 .l.), 0.5 pL of Tuq polymerase and 9.5 pL of water. 5. Cycle the reactions 30 times through a 0.7 min denaturation at 95OC, a

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165

0.7 min annealing at 55*C, and a 3.5 min extension at 72*C. Note that the annealing temperature will depend on the 7’, of the primers (see Section 35.1.).

4. Notes 1. In all the methods involving a phenol extraction step, the extractions are chilled prior to phase separation to minimize contamination of the aqueous phaseby the organic phase.Unbuffered phenol is used throughout. 2. In designing these methods, reagent and equipment costs are kept to a practical minimum. This is very important for any laboratory undertaking very large scale sequencing. There are, of course, other alternatives-although generally more expensive, they may be more suitable for other laboratories. 3. The times and temperatures of the various segments of the sequencing reactions may require alteration, depending on exactly which cycling machine is used. The denaturation and annealing times should be kept to a minimum and extension times, under these conditions of low concentrations of nucleotides, should be kept long. 4. Both the template preparation and sequencing reaction protocols have several points at which samples may be stored. It is therefore relatively easy to organize a daily routme where template preparation, sequencing, data assembly, and editing may be in progress. The number of templates conveniently prepared per day will depend on the capacities of the microfuges available. 5. After the shotgun phase, template preparation ceases and much more time is spent on data editing and completing the sequence using oligonucleotide primers. With the procedure recommended here, this walking phase will probably take at least twice as long as the shotgun phase. The amount of time spent on the shotgun phase compared to the walking phase can be adjusted according to individual preference, by either increasing or decreasing the number of shotgun subclones sequenced, 6. Improvements to make cosmid sequencing more efficient are likely to include smaller scale template preparations, removal of the need for ethanol precipitation of sequencing reactions (ABI), the use of fluorescent dideoxynucleotides in combination with the ABI 373A and further improvements to software which will reduce the amount of time spent interactively with the computer.

Acknowledgments I am extremely grateful to all members of the C. ekgans mapping and sequencing group (I), several of whom were involved in developing these methods. I am particularly grateful to John Sulston for

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his enormous contribution. I thank Trevor Hawkins for providing the sequencing protocol in Section 3.5.2. in advance of publication. I thank all who have given me constructive criticism. I am supported, in part, by grants from the NIH Human Genome Center and the MRC HGMl? References 1. Sulston, J , Du, Z , Thomas, K , Wilson, R , Hillier, L , Staden, R., Halloran, N , Green, P., Thterry-Mieg, J., QIU, L., Dear, S., Coulson, A., Craxton, M., Durbm, R., Berks, M., Metzstem, M., Hawkms, T., Amscough, R., and Waterston, R (1992) The C elegans sequencing project a begmnmg Nature 356, 37-4 1 2. Sambrook, J. Frttsch, E. F., and Maniatis, T. (1989) Molecular Clonrng A laboratory manual. Cold Sprmg Harbor Laboratory Press, Cold Spring Harbor, NY. 3 Ausubel, F M , Brent, R., Kmgston, R E , Moore, D D , Seidman, J. G , Smith, J. A , and Struhl, K. (eds) (1987,1988) Current protocols in molecular biology. Greene Publishing Assoctates and Wiley-Intersctence, New York 4. Berger, S. L. and Kimmel, A R (eds ) (1987) Gutde to molecular clonmg techniques Meth. Enzym. 152. 5. Bankter, A. T and Barrel], B. G. (1983) Shotgun DNA Sequencing. Tech Life SCL B5, 1-34. 6 Barrell, B G (1991) DNA sequencing: present limitations and prospects for the future. FASEB J. 5,40-45 7. Craxton, M. (1991) Linear amphfication sequencing: a powerful method for sequencing DNA. Methods. a companion to methods in enzymology 3,20-26 8 Smith, L M , Sanders, J Z , Kaiser, R. J , Hughes, P., Dodd, C , Connell, C. R , Heiner, C., Kent, S B. H., and Hood, L E (1986) Fluorescence detection in automated DNA sequencing analysis. Nature 321,674-679. 9. Ansorge, W., Sproat, B., Stegemann, J , and Schwager, C (1986) A non-radioactive automated method for DNA sequence determination J. Biochem. Biophys

Meth 13,3 15-323.

10. Prober, J M., Tramor, G L , Dam, R J , Hobbs, F W, Robertson, C W , Zugursky, R. J., Cocuzza, A. J., Jensen, M. A., and Baumeister, K. (1987) A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238,336-341 11. Dear, S. and Staden, R. (1991) A sequence assernbly and editmg program for efficient management of large projects Nut Acids Res 19, 3907-39 11 12 Anderson, S. (1981) Shotgun DNA sequencing using cloned DNaseI-generated fragments. Nut. Acids Res. 9,3015-3027 13 Edttorial(l991) Thermal cyclmg with a new twist adds versatility to sequencing automation ABI Blosystems Reporter 11, 6-7 14 Smtth, V , Brown, C M , Bankier, A T , and Barrell, B G (1990) Semiautomated preparation of DNA templates for large scale sequencing proJects DNA Seq. 1,73-78.

15 Smith, V , Craxton, M., Bankrer, A. T., Brown, C M , Rawlinson, W D., Chee, M. S , and Barrell, B. G. (1991) Meth. Enzym. (in press).

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16. Hawkms, T. L. and Sulston, J E. (1990) Automated fluorescent primer walking. Technique 2,307-3 10. 17. ALF sequencer manual 1990. Parmacia LKB Biotechnology, Upssala, Sweden 18. Hawkins, T. L. and Sulston, J. E. (1991) The resolution of compressions in automated fluorescent sequencing. Nut. Acids Res. 19,2784. 19. Gleeson, T. and Hillier, L. (1991) A trace display and edttmg program for data from fluorescence based sequencing machines Nut. Acids Res. 19,6481-6483. 20. Staden, R. (1990) An improved sequence handling package that runs on the Apple Macintosh Computer Applications in the Biosciences 6,387-393 21. Hillier, L. and Green, P. (1991) OSP: A computer program for choosing PCR and DNA sequencing primers. PCR Methods and Applications 1, 124.

Genomic

Sequencing

Gerd I? Pfeifer and Arthur

D. Riggs

1. Introduction The cloning of eukaryotic DNA in bacteria or by the polymerase chain reaction (PCR) inevitably results in the loss of much information, Protein-DNA contacts and cytosine methylation patterns, both important in gene regulation and other cellular processes,are not reproduced in these cloning procedures. To retain information on cytosine methylation and protein footprints, the DNA has to be sequenced in its native genomic state. Direct sequencing of genomic DNA followed by detection of specific sequences is referred to here to as “Genomic Sequencing.” In 1984, Church and Gilbert introduced a method for sequencing genomic DNA without cloning (I). Total genomic DNA was subjected to base-specific chemical cleavage according to the Maxam-Gilbert DNA sequencing protocol (2). Specific sequences were detected by indirect end-labeling: A restriction cut defines the reference point (endpoint) of the sequence and a hybridization probe is constructed to be close to this end-point. Because of the complexity of mammalian genomes, genomic sequencing as originally described was generally a technically challenging procedure. Attempts have been made to devise methods that are more sensitive than the original method. One way to increase the specific signal relative to background is to From Methods m Molecular Biology, Vol. 23. DNA Sequencmg Protocols Edited by: H and A Gnffm Copynght 01993 Humana Press Inc , Totowa,

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prefractionate the genomic DNA and to enrich for target sequences (3,4). Repeated primer extension with Tuq polymerase from a genespecific primer has also been used for genomic sequencing ($6). The most sensitive method usesthe ligation-mediated poiymerase chain reaction (LM-PCR) to exponentially amplify all fragments of the genomic sequence ladder (7,8). The unique aspect of LM-PCR is the ligation of an oligonucleotide linker onto the 5’ end of each DNA molecule. This provides a common sequence on the 5’ end, and in conjunction with a gene-specific primer, allows conventional, exponential PCR to be used for signal amplification. Thus, by taking advantage of the specificity and sensitivity of PCR, one needs only a microgram of mammalian DNA per lane to obtain good quality DNA sequence ladders, with retention of DNA sequence, methylation, DNA structure, and protein footprint information. The LM-PCR procedure is outlined in Fig. 1. Briefly, the first step is cleavage of DNA, generating 5’ phosphorylated molecules. This is achieved, for example, by the p-elimination step of chemical DNA sequencing. Next, primer extension of a gene-specific oligonucleotide (primer 1) generatesmolecules that have a blunt end on one side. Linkers are ligated to the blunt ends, and then an exponential PCR amplification of the linkerligated fragments is done using the longer oligonucleotide of the linker (linker-primer) and a second gene-specific primer (primer 2). After 15-20 amplification cycles, the DNA fragments are separated on a sequencing gel, electroblotted onto nylon membranes, and hybridized with a gene-specific probe to visualize the sequence ladder. By rehybridization, several gene-specific ladders can be sequentially visualized from one sequencinggel (8). Alternatively, amplified fragments can be detected by primer extension from a 32P-labeled third primer (7). LM-PCR is generally suitable for detection of specific DNA strand breaks. The method has been used for sequencing genomic DNA and for determination of DNA cytosine methylation patterns (8-12). Methylated cytosines are recognized by their failure to react with hydrazine that results in a gap in the C-specific sequencing ladder. LM-PCR also provides adequate sensitivity to map rare DNA adducts, like those formed after UV irradiation (12). To obtain information about protein binding and other aspects of DNA structure, in vivo footprinting experiments can be done on intact cells using dimethylsulfate (7,9) or on permeabilized cells using DNaseI (13).

Genomic Sequencing A

171

P----

chemrcal

P--

cleavage

1 Pi?”

i

primer

extension

ligation

of linker

p-1

1

,==Eq”8

PCR

I

B

321

Fig. 1. (A) Outline of the LM-PCR procedure. (B) Arrangement of LM-PCR primers to sequence both strands of a 200 bp region (hatched area). Primer 1, Sequenase primer; primer 2, PCR primer; primer 3 is used to make a single-stranded hybridization probe (see Note 3)

2. Materials

2.1. Preparation

of Genomic DNA

1. Phenol, equilibrated with O.lM Tris-Cl, pH 8.0. 2. Chloroform. 3. Ethanol. 4. 3M sodium acetate, pH 5.2.

2.2. Chemical

Cleavage

1. DMS buffer: 50 mM sodium cacodylate, 1 m&f EDTA, pH 8.0. 2. DMS (dimethylsulfate, 299%. Aldrich, Milwaukee, WI). DMS is a highly toxic chemical and should be handled in a well-ventilated hood. DMS waste (including plastic material) is detoxified in 5M NaOH. DMS is stored under nitrogen at 4°C. 3. DMS stop: 1.5M sodium acetate, pH 7.0, 1M 2-mercaptoethanol.

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and Riggs

4. Formic acid (Fluka). 5. Hydrazme (anhydrous, Aldrich). Hydrazine is a highly toxic chemical and should be handled m a well-ventilated hood. Hydrazine waste (including plastic material) is detoxified in 3M ferric chloride. Hydrazme is stored under nitrogen at 4°C. The bottle should be replaced at least every 6 mo. 6. Hz-stop: 0.3M sodium acetate, pH 7.5,O.l mM EDTA. 7. Ethanol, precooled to -70°C. 8. 3M sodium acetate, pH 5.2. 9. 75% ethanol. 10. Prperidine (299%, Fluka), stored under nitrogen at -20°C. 2.3. First Primer Extension 1. 5X Sequenase buffer: 250 mM NaCI, 200 mM Trts-HCl, pH 7.7. 2. The primers we have used as prtmer 1 (Sequenase prtmer) are 17- to 19-mer ohgonucleotides with a calculated 7’,,,of 50-56°C. Calculation of Tm is done with a computer program (14). 3. Mg-DTT-dNTP mix: 20 mM MgCl*, 20 mM D’IT, 0.25 mM of each dNTP. 4. SequenaseTM2.0 (U.S. Biochemicals, Cleveland, OH), 13 U/pL. 5. 300 mJ4 Trrs-HCl, pH 7.7. 2.4. Ligation 1. 2M Tris-HCl, pH 7.7. 2. Linkers are prepared in 250 mZt4Tris-HCI, pH 7 7, by annealing a 25mer (5’-GCGGTGACCCGGGAGATCTGAATTC, 20 pmoles/pL, gelpurified) to an 1I-mer (5’-GAATTCAGATC, 20 pmoles/& gel-purified) by heating to 95°C for 3 min and gradually coolmg to 4°C over a time period of 3 h. Linkers can be stored at -2O’C for at least 3 mo. They are always thawed and kept on ice. 3. Ligation mix: 13.33 mM MgC12, 30 rniV DTT, 1.66 mM ATP, 83 pg/ mL BSA, 3 U/reaction T4 DNA ligase (Promega, Madison, WI), and 100 pmoles linker/reaction (=5 pL linker). 4. 3M sodium acetate, pH 5.2. 5. E. coli tRNA. 2.5. PCR 1. 2X Taq polymerase mix: 20 mMTris-HCl, pH 8.9,80 mM NaCI, 0.02% gelatin, 4 r&Z MgCl*, and dNTPs at 0.4 mM each. 2. Primers: 10pmolesof the gene-specificprimer (primer 2) and 10pmoles of the 25-mer linker-primer (5’-GCGGTGACCCGGGAGATCTGAATTC) are used per reaction along with 3 units Taq polymerase, and these can be included in the 2X Taq polymerase mix. The prtmers used m the amplification step (primer 2) are 23- to 26-mers (calculated T, between 63

Genomic Sequencing

173

and 70°C). They are designed to extend 3’ to primer 1. Primer 2 probably should overlap several bases with primer 1, but we have also had good results with a second primer that overlapped only two bases with the first. All primers used in this section should be gel-purified. 3. Mineral oil. 4. 3M sodium acetate, pH 5.2. 5.400 mM EDTA, pH 7.7. 6. E. coli tRNA.

2.6. Gel Electrophoresis 1. Formamide loading dye: 94% formamide, 2 mkf EDTA, pH 7.7,0.05% xylene cyanol, 0.05% bromophenol blue. 2. 1M TBE: 1M Tris, 0.83M boric acid, 10 mkf EDTA, pH 8.3.

2.7, Electrwblotting 1. 1M TBE: 1M Tris, 0.83M boric acid, 10 rnk! EDTA, pH 8.3. 2. Whatman 3MM and Whatman 17 paper. 3. GeneScreenTMnylon membranes (New England Nuclear, Boston, MA).

2.8. Hybridization 1. Hybridization buffer: 0.25M sodium phosphate, pH 7.2, 1 mM EDTA, 7% SDS, 1% BSA. 2. Washing buffer: 20 mM sodium phosphate,pH 7.2,l mA4EDTA, 1% SDS.

3. Methods In Sections 3.1. and 3.2. we will describe the preparation and chemical sequencing of genomic DNA. Sections 3.3.-3.5. contain a detailed protocol of the ligation-mediated PCR procedure that is used to amplify gene-specific fragments. Sections 3.6.-3.8. comprise the sepa-

ration of amplified fragments and their detection. 3.1. Preparation of Genomic DNA 1. Isolate DNA by standard procedures using phenol/chloroform extraction and ethanol precipitation. The DNA preparation should be mostly free of RNA. 2. Digest the DNA with a restriction enzyme that does not cut within the region to be sequenced. This is done to reduce the viscosity of the solution. After digestion, extract the DNA once with phenol/chloroform and once with chloroform and then ethanol precipitate. DNA is dissolved m water, the amount of DNA is determined by OD measurement or agarose gel electrophoresis, and the concentration is adjusted to 2-10 pg/pL,

Pfeifer

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3.2. Chemical Cleavage Genomic DNA is chemically cleaved according to published methods (1,2,15) with some modifications. The conditions below work well for lo-50 l.tg DNA. 3.3.1. G-Reaction 1. MIX, with caution, on ice: 5 pL genomlc DNA (lo-50 DMS buffer, 1 pL DMS. 2. Incubate at 20°C for 3 min. 3. Add 50 pL DMS stop. 4. Add 750 pL precooled ethanol (-70°C). 3.3.2.

G + A Reaction

1. Mix, with caution, on ice: 11 pL genomic DNA (lo-50 formic acid 2. Incubate at 20°C for 10 min. 3. Add 200 /.tL DMS stop. 4. Add 750 pL precooled ethanol (-70°C). 3.3.3.

pg), 200 pL

pg), 25 p.L

T + C Reaction

1. Mix, with caution, on ice: 20 pL genomic DNA (10-50 pg), 30 pL hydrazme. 2. Incubate at 20°C for 20 min. 3. Add 200 l.tL Hz-stop. 4. Add 750 pL precooled ethanol (-7OOC). 3.3.4.

C-Reaction

1. Mix, with caution, on ice: 5 pL genomic DNA (lo-50 5M NaCl, 30 pL hydrazine. 2. Incubate at 20°C for 20 min. 3. Add 200 pL Hz-stop. 4. Add 750 pL precooled ethanol (-70°C).

kg), 15 pL

Process all samples as follows: 5. Keep samples m a dry ice/ethanol bath for 20 mm. 6. Spm 15 mm at 14,000 g m an Eppendorf centrifuge at 0-4”C. 7. Take out supernatant, respm. 8 Resuspend pellet m 225 pL water 9. Add 25 pL 3M sodium acetate, pH 5.2. 10. Add 750 pL precooled ethanol (-70°C). 11. Put on dry Ice, 15 mm. 12. Spm 10 mm at 14,000 rpm in Eppendorf centrifuge at 0-4’C 13. Take out supernatant, respm.

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14. Wash with 1 mL 75% ethanol, spin 5 min in Eppendorf centrifuge. 15. Dry pellet in Speedvac. 16. Dissolve pellet in 100 pL 1M piperidine (freshly diluted). 17. Secure caps with TeflonTM tape. 18 Heat at 90°C for 30 mm in a heat block (lead weight on top). 19. Transfer to a new tube. 20. Add l/l0 vol3M sodium acetate, pH 5.2. 21. Add 2.5 vols ethanol, 22. Put on dry ice, 20 min. 23. Spin 15 min at 14,000 g m Eppendorf centrifuge at 0-4OC. 24. Wash twice with 75% ethanol. 25. Remove traces of remaining piperidine by drying the sample overnight in a SpeedVac concentrator. Dissolve DNA m water to a concentration of about 1 pg/pL. 26. Determine the cleavage efficiency by running 1 pg of the samples on a 1.5% alkaline agarose gel. There should be fragments below the 100 nt size range.

3.3. First Prher

Extension

1. Mix in a sihconized tube: 0.5-5 pg of cleaved DNA, 0.6 pmoles of primer 1, and 3 pL 5X Sequenase buffer in a final volume of 15 pL. 2. Incubate at 95°C for 3 min, then at 45OC for 30 min. 3. Cool on ice, quick spin. 4. Add 7.5 pL cold, freshly prepared Mg-DTT-dNTP mix. 5. Add 5 U SequenaseTM2.0. 6. Incubate at 48OC, 15 mm, then cool on ice. 7. Add 6 pL 300 mil4 Tris-HCl, pH 7.7. 8. Incubate at 67OC, 15 min (heat inactivation). 9. Cool on ice, quick spin,

3.4. Ligation The primer-extended molecules that have a 5’ phosphate as a remnant from the chemical sequencing are ligated to an unphosphorylated synthetic double-stranded linker. 1. Add 45 pL of freshly prepared ligation mix. 2. Incubate overnight at 18°C. 3. Incubate 10 min at 70°C (heat inactivation). 4. Add 8.4 pL 3M sodium acetate, pH 5.2, 10 pg E. coli tRNA, and 220 pL ethanol. 5. Put samples on dry ice for 20 min. 6. Centrifuge 15 mm at 4 OCin an Eppendorf centrifuge.

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7. Wash pellets with 950 pL 75% ethanol. 8. Remove ethanol residues m a SpeedVac. 9. Dissolve pellets in 50 pL HZ0 and transfer to OS-mL silicomzed tubes.

3.5. PCR 1. Add 50 pL freshly prepared 2X Tuq polymerase mix containmg the primers and the enzyme and mix by ptpetting. 2. Cover samples with 50 pL mineral oil and spin briefly. 3. Cycle 16-20 times at 95°C 1 mm, 63-66OC, 2 min, and 76”C, 3 min. 4. To completely extend all DNA fragments and uniformly add an extra nucleotide, an additional Tuq polymerase step is performed. One unit of fresh Taq polymerase per sample is added together with 10 pL reaction buffer. Incubate 10 min at 74OC. 5 Stop the reaction by adding sodmm acetateto 300 mM, EDTA to 10 mM, and add 10 pg tRNA. 6. Extract with 70 l,tL of phenol and 120 pL chloroform (premixed). 7. Add 2.5 vols of ethanol and put on dry ice, 20 min. 8. Centrifuge samples 15 min m an Eppendorf centrifuge at 4°C. 9. Wash pellets m 1 mL 75% ethanol. 10. Dry pellets in a SpeedVac.

3.6. Gel Electrophoresis 1. Dissolve pellets in 1.5 pL of water and add 3 pL formamide dye. 2. Heat samples to 95°C for 2 min prior to loading. Loading is performed with a very thin flat tip. Load only one half of the sample. The gel IS 0.4 mm thick, 50-95 cm long, consisting of 8% polyacrylamide and 7M urea m O.lM TBE. The gel is run until the xylene cyan01 marker reaches the bottom. Fragments below the xylene cyan01dye hybridize only very weakly.

3.7. Eikctroblotting Electroblotting (see Notes 1 and 2) is done with a simple homemade apparatus (Fig. 2) essentially as described by Saluz and Jest (15). Stainless steel plates from a BioRad (Richmond, LA) gel drier are used as electrodes (obtainable as spare parts from Hoefer Scientific, San Francisco, CA). They are connected directly with a platinum wire and a cord to the power supply. A homemade plastic tank is the buffer chamber. The size of the chamber is 50 x 40 x 14 cm (length x width x height). Opening the lid interrupts the current (safety precaution). A BioRad 200/2.0 power supply is sufficient. 1, After the run, cut the gel into one or two pieces, transfer to Whatman 3MM paper and cover with Saranwrap@‘.

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50

cm

Fig. 2. Schematic drawing of the electroblotting

apparatus.

2. On the lower electrode, which is resting on three plastic incubation racks (height about 3 cm), 12 layers of Whatman 17 paper, 47 x 19 cm, presoaked in 90 n-&I TBE, are piled and squeezed with a rolling bottle to avoid air bubbles between the paper layers.

3. Place the gel pieces onto the paper pile and remove all air bubbles between gel and paper by wiping over the Saranwrap@using a soft tissue. 4. When all air bubbles are squeezed out, remove the Saranwrap@ and cover the gel with a nylon membrane cut somewhat larger than the gel and presoaked in 90 mM TBE. 5. The upper paper pile consists of 12 layers of Whatman 17 paper presoaked and cut as mentioned above. The upper electrode is placed and pressed onto the upper pile by putting 2 kg lead weights on top of it. 6. Fill the electroblotting apparatus with 90 rnM TBE until the buffer level is about 5 layers of paper below the gel. The electroblotting procedure is performed at 1.6 A and 30 V. After 1 h, remove the nylon membrane and mark the DNA side.

3.8. Hybridization 1. Dry the membrane briefly at room temperature or at 37°C and bake it at 80°C for 20 min in a vacuum oven. 2. Crosslink the DNA by UV irradiation. UV irradiation is performed by mounting six 254 nm germicidal W tubes (15 W) into an inverted transilluminator from which the upper lid is removed. The distance between membrane and UV bulbs is 20 cm; the UV irradiation time is 30 s. Calibrating the UV irradiation time for different batches of nylon membranes was found to be unnecessary. 3. The hybridization IS done m rotating 250-mL plastic or glass cylinders in a hybridization oven. Soak the nylon membranes in 90 r&I TBE and

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roll them into the cylinders so that the membranes stick completely to the walls of the cylinders without air pockets between wall and mem-

brane. This can be easily done by rolling the membranes first onto a 25mL pipet and then unspooling them into the cylinder. 4. Prehybridize

with 15 mL hybridization

buffer for 10 mm.

5. Dilute the labeled probe (20-80 @i, see Note 3) into 5 mL hybridrzation buffer and hybridize overnight. Prehybridization as well as hybridizatron are performed at 68°C for probes with a G + C content of 60-75%. 6. After hybridization, wash each nylon membrane with 2 L of washmg

buffer at 60°C. Perform several washing steps at room temperature with prewarmed buffer. 7. Dry the membranes at room temperature, cover with Saranwrap@ and expose to Kodak XAR-5 X-ray films. If the procedure has been done without error, a result can be seen after 0.5 to 8 h of exposure with Intensifying screens at -70°C. 8. Nylon membranes can be used for rehybrrdization if several sets of prim-

ers have been included in the primer extension and amplificatron reactions. Probes can be stripped from the nylon membranes by soakmg m 0.2M NaOH for 30 min at 45OC. 4. Notes LM-PCR has been used for analysis of methylated cytosines and for in vivo footprinting (7-13). The method should also be applicable for sequencing into unknown DNA regions adjacent to any known sequence (walking approach), although this has not yet been formally proven. An example for methylation analysis is shown in Fig. 3. Methylated cytosines at CpG dinucleotides are indicated by a gap in the sequence ladder. 1. Some technical

notes on the hybridization

approach: We are currently

using electroblotting and hybridization instead of directly extending a 32P-labeled primer followed by gel electrophoresis (7). The reasons are: (i) Labeling the amplification primer does not provide sufficient specrficity, so an additional labeled primer (third primer) has to be made that

will compete wrth the unlabeled primer from the amplification reaction. (ii) Hybridization introduces an additional level of specificity if a probe that does not overlap with the ampltfication primer is used. (iii)

The hybridization approach as described here results in significantly less exposure of the worker to radioactivity. (iv) Longer single-stranded probes provide a higher specific activity than end-labeled oligonucle-

otides. (v) Nylon membranes can easily be rehybridrzed after inclusion of multiple primer sets in Sequenase reaction and PCR (multiplexmg).

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GC

Fig. 3. Example for genomic sequencingandmethylation analysisby LM-PCR. Lanes labeled G, G + A, T + C, andC are genomic sequencingreactionsperformed on HeLa cell DNA. The sequencesare from the promoter region of the humanXlinked PGK-I gene(9). Lanes 14 are C-specific reactions:lanes 2 and 3, active X chromosomalDNA (unmethylatedat CpG dinucleotides); lanes 1and4, inactive X chromosomal DNA (methylated at CpG dinucleotides). Arrows indicate the position of CpG dinucleotides. 2. Transfer to a nylon membrane can also be accomplished by vacuum blotting using a gel drier, as described (16). Transfer by vacuum blotting was about 50% efficient for longer fragments, and we are not currently using it. However, many may find it adequate, since less efficient transfer can be easily compensated for by longer exposures. 3. We have been using single-stranded DNA probes made by subcloning of defined fragments into expression vectors (17). Other hybridization probes can be used and might be much easier to produce. A very useful method to prepare labeled single-stranded probes is to use repeated primer extension (about 35 cycles) by Tuq polymerase with a single primer (primer 3) on a double-stranded template of the respective cloned DNA (18). The length of these probes can be easily controlled by an appropriate restriction cut. The primer that is used to make a probe

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(primer 3, see Fig. 1) should be on the same strand just 3’ to the amplification prrmer (primer 2). It should not overlap more than a few bases. Even if a cloned DNA 1snot available, smgle-stranded probes can be made from PCR products (19).

Acknowledgments This work was supported by National Institute of Aging grant (AG08196) to A. D. R., and a fellowship from the Deutsche Forschungsgemeinschaft (Pf2 12/l- 1) to G. P P.

References 1, Church, G. M. and Gilbert, W. (1984) Genomic sequencing. Proc NutE. Acad. Sci. USA 81, 1991-1995 2. Maxam, A. M and Gilbert, W (1980) Sequencing end-labeled DNA with basespecific chemical cleavages. Methods Enzymol. 65,499-560. 3. Kochanek, S., Toth, M., Dehmel, A., Renz, D., and Doerfler, W. (1990) Interindividual concordance of methylation profiles m human genes for tumor necrosis factor alpha and beta Proc. Natl. Acad. Scr USA 87,8830-8834 4. Mirkovitch, J. and Darnell, J. E., Jr. (1991) Rapid m VIVO footprinting technique identifies protems bound to the TTR gene m the mouse liver. Genes Dev. 5,83-93.

5. Axelrod, J. D and Majors, J. (1989) An improved method for photofootprintmg yeast genes in vivo using Tag polymerase Nucl. Acids Res. 17, 171-183 6 Saluz, H. and Jost, J. P. (1989) A simple htgh-resolution procedure to study DNA methylatton and in vivo DNA-protein mteractions on a single-copy gene level in higher eukaryotes. Proc Natl. Acad Sci. USA 86,2602-2606. 7. Mueller, P R. and Wold, B. (1989) In vivo footprinting of a muscle specific enhancer by ligation mediated PCR. Science 246,780-786. 8. Pfeifer, G. P , Steigerwald, S. D., Mueller, P. R., Weld, B., and Riggs, A. D (1989) Genomic sequencing and methylation analysts by ligation medtated PCR. Science 246,810-813. 9 Pfeifer, G. P., Tanguay, R. L., Steigerwald, S. D , and Rtggs, A D (1990) In vivo footprint and methylation analysis by PCR-aided genomic sequencing* comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1. Genes Dev. 4, 1277-1287. 10 Pfeifer, G P., Steigerwald, S. D , Hansen, R S., Gartler, S. M., and Riggs, A D. (1990) Polymerase chain reaction aided genomtc sequencing of an X chromosome-linked CpG island. Methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanatton of activity state stability Proc Nat1 Acad. Sci. USA 87,8252-8256.

11 Rideout, W. M., III, Coetzee, G. A., Olumi, A. F , and Jones, P A. (1990) 5Methylcytosine as an endogenous mutagen in the human LDL receptor and ~53 genes. Science 249, 1288-1290 12. Pfeifer, G. P., Droum, R., Riggs, A. D., and Holmqmst, G. P. (1991) In vwu

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mapping of a DNA adduct at nucleotide resolution. detection of pyrimidine (64) pyrimidone photoproducts by hgation-mediated polymerase chain reaction Proc. Nat1 Acad. Sci USA 88, 1374-1378 13 Pfeifer, G. P. and Riggs, A. D. (1991) Chromatin differences between active and inactive X chromosomes revealed by genomic footprintmg of permeabilized cells using DNase I and hgation-mediated PCR. Genes Dev. 5, 1102-l 113. 14. Rychlik, W., and Rhoads, R E (1989) A computer program for choosing optimal ohgonucleotides for filter hybrtdlzation, sequencing and in wtro amplification of DNA. Nucl Acids Res. 17,8543-855 1. 1.5. Saluz, H. P. and Jost, J. P. (1987) A Laboratory guide to genomic sequencing Birkhauser, Boston 16 Gross, D S., Collins, K. W , Hernandez, E. M , and Garrard, W. T. (1988) Vacuum blotting: a simple method for transfering DNA from sequencing gels to nylon membranes. Gene 74,347-356. 17 Weih, F., Stewart, A F., and Schtitz, G. (1988) A novel and rapid method to generate single stranded DNA probes for genomic footprmtmg. Nucl Acids Res. 16, 1628 18 Stiirzl, M. and Roth, W. K. (1990) “Run-off’ synthesis and application of defined single-stranded DNA hybridization probes. Anal. Blochem. 185, 164-169. 19 Tormanen, V T. and Pfeifer, G. P. (1992) Mapping of UV photoproducts within ras proto-oncogenes in UV-irradiated cells: correlation with mutations in human skin cancer. Oncogene 7, 1729-1736.

c%UPTER

23

Sequencing of Double-Stranded PCR Products Susannah

Gal

1. Introduction The polymerase chain reaction (PCR) is fast becoming a standard protocol in many laboratories for cloning and analysis of small quantities of DNA (I). It involves the exponential synthesis of linear DNA molecules from double-stranded DNA templates using specific oligonucleotide primers for the DNA synthesis reaction. Because of its unique sensitivity and general applicability, the PCR procedure has been used in a variety of systems. In some cases, sequencing information from the PCR product is the next step in the analysis and I would like to inform the reader of two protocols for sequencing double-stranded DNA PCR products. The examples where this might be useful include analysis of a single gene mutation in a cell (2,3), analysis of plant DNA samples for viruses and viroids (4,5), and analysis of mitochondrial DNA (6). The first method is a procedure for the preparation of the PCR DNA sample for the sequencing protocol. The following section describes two protocols that can be used to sequence the double-stranded DNA PCR products directly. Other protocols for sequencing are as well described in other chapters in this volume. Protocols for the PCR reaction are described elsewhere (7). From. Methods m Molecular Bology, Vol. 23 DNA Sequencing Protocols Edlted by: H and A Griffm Copyright 01993 Humana Press Inc., Totowa,

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2. Materials 2.1. Method 1 1. Redistilled phenol equilibrated with Tris buffer, pH 8.0 as described (7). 2. Chloroform: isoamyl alcohol, 24: 1, v/v. 3. Centricon microconcentrator column (Amicon Dtvision, Danvers, MA), size depends on size of DNA PCR fragment (see Note 1). 4. Sterile distilled water. 5. 3M Na acetate, pH 4.8. 6. Ethanol. 7. TE buffer: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA. 2.2. Method 2 1. Sequenase@ sequencmgkit (US Biochemical Corporatton, Cleveland, OH). 2. Single-stranded DNA from an Ml 3 clone of part of the sequence of the PCR fragment. For preparation of single-stranded DNA see Chapters 5 and 6. 3. 68OC waterbath. 4. 3M Na acetate, pH 4.8. 5. Ethanol. 6.2M NaOH. 7. Sterile distilled water. 8. PCR primer or other sequencing primer. 9. 35S-dATP for sequencmg. 2.3. Method 3 1. 32P-labeled PCR primer synthesized by kmasmg oligonucleotide with polynucleotide kinase as described (7). 2. Polyacrylamide gel containing urea as described see Chapter 16 and Note 2. 3. Chemicals for Maxam and Gilbert sequencing, see Chapter 31-33. 4. Elution buffer (0.5M ammomum acetatepH6,lOmM magnesium acetate). 5. Ethanol.

3. Methods 3.1. Method 1: Purification of the PCR Products As the PCR procedure requires the use of enzymes and other proteins, buffers, and primers that interfere with the sequencing of the PCR products, these must first be removed. I found a two-part procedure for this to be adequate in most cases. The first step removes the proteins and the second step removes the buffers and primers. The

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NCENTRATED

CENTRIFUGAL FORCE -

Fig 1. Use of the CentrIcon mlcroconcentrator in the concentration mode Figure courtesy of Amicon Division, W R Grace and Co , CT

DNA may be too dilute at the final stage and may need to be concentrated with a precipitation step using ethanol. 1. Remove the PCR mix (leaving the overlay of paraffin oil) to a clean Eppendorf tube, add an equivalent vol of phenol:chloroform/isoamyl alcohol (1: l), and mix for 1 min on a vortex mixer. 2. Spin for 5 min in a microfuge to separate layers and remove the top aqueous phase to a new tube. 3. Repeat the extraction if there is a thick protem Interface. 4. Load the aqueous phase onto a Centricon microconcentrator column. Add 2-mL sterile distilled water to the sample reservoir. 5. Spin the column as shown in Fig. 1 and as described in the operating instructions until less than 300 pL of sample volume is left in the reservoir. 6. Add 2 mL more water and spin again. 7. Remove the filtrate cup and mvert the column for recovery mode as shown in Fig. 2. Spm briefly to bring the sample into the retentate cup. 8. Remove the sample and precipitate the DNA using 0.2 vols 3M Na acetate, pH 4.8, and 2-3 vols ethanol at -7OOC. 9. Wash the pellet with 70% ethanol, dry briefly, and bring the pellet up m a small vol of TE buffer. 10. Determine concentration of DNA by gel electrophoresis and ethldlum bromide staining described m volumes two and four of this series (see also ref. 7).

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CONCENTRATED

t-

CENTRIFUGAL

FORCE

SAMPLE

-

Fig 2. Use of the Centricon mxroconcentrator in the recovery mode. Figure courtesy of Amicon Division, W R Grace and Co , CT.

3.2, Method 2: Sequencing of Double-Stranded DNA PCR Products Using a Single-Stranded DNA from an Ml3 Clone For this protocol, you need a clone of PCR product in a vector that can produce single-stranded DNA, such as an Ml3 phage plasmid. The basis for the protocol and the orientation of the PCR product, the PCR primers (PCR Pl, PCR P2) and the single-stranded M 13 DNA (Ml 3 ssDNA) are shown in Fig. 3. The idea is to use the single-stranded DNA clone in excess to remove one of the strands of the PCR double-stranded DNA product. This “freed” strand can then be sequenced using the standard protocols. The sequencing primer can be one of the PCR primers as shown, and should not have any homology to the Ml3 DNA clone (see Note 3). 1. Place approx 0.5 pmoles of the double-stranded DNA PCR product m a reaction tube. Add water to a vol of 10 pL. 2. Add 2 pL 2M NaOH and let sit at room temperature for 5 min to denature the DNA. Add 3 pL 3M Na acetate to neutralize the base. 3. Add 20 pmole sequencing primer and approxrmately a five-fold molar excess of the single-stranded DNA (see Note 4). Precipitate with 2-3

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+

KxPl s-

8 hr sequendng +

l?

3

Fig. 3. Representation of Method 2, sequencing of double-stranded PCR products using a smgle-stranded DNA from an Ml3 clone vols of ethanol at -70°C. Spin for 10 min in a microfuge, wash the pellet with 70% ethanol, and dry briefly. 4. Dtssolve the pellet m 10 pL of the 1X sequencing buffer from the sequencing kit 5. Denature the DNA by mcubatmg the tube at 68°C for 5 min and allow

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it to cool slowly back to room temperature over the next 30 min to anneal the strands. The easiest way to do this is to take a small beaker of the water from the waterbath and leave the tubes in this until it reaches room temperature. 6. Continue with the standard sequencing protocol described m the manufacturer’s instructions for the Sequenase@kit using 35S-dATP as label (see Chapter 14). 7. Load onto a standard sequencing gel as described in Chapters 16 and 17.

3.3. Method 3: PCR Using a Labeled Primer and Sequencing using Maxam and Gilbert Chemical Sequencing In this procedure, the fragment for sequencing is labeled during the PCR reaction using one labeled PCR primer. The labeled fragment is then purified on a denaturing polyacrylamide gel and sequenced using the Maxam and Gilbert chemical sequencing method. 1. Run the PCR reaction using one labeled and one unlabeled primer 2. Purify the PCR product as described above and dissolve the DNA pellet in 20-30 pL of 90% formamide. 3. Heat the DNA for 5 min at 75°C to denature the strands and keep on ice until loading onto the gel. 4. Load onto a denaturing polyacrylamide gel containing urea and run for an appropriate length of time to have the DNA band m the lower half of gel using dyes as approx size markers (see Note 2). This gel should be run essentially as a sequencing gel, Chapter 17. 5. Determine the localization of the radioactive band by autoradiography and cut it out from the gel, and place the gel piece m an Eppendorf tube. 6. Add 400 pL of the elution buffer to the tube and elute the radiolabeled DNA from the gel by shaking overnight. 7. Spin down the gel pteces and remove the supernate to a new tube. 8. Precipitate the DNA by adding 3 vol of ethanol at -7OOC. 9. Wash the DNA pellet with 70% ethanol and dry briefly. 10. Sequence the radiolabeled PCR fragment using the Maxam and Gilbert method as described m Chapter 32 and load onto a standard sequencing gel (see Chapters 16 and 17) 4. Notes 1. The Centricon column used will depend on the size of the PCR fragment you wish to purify. The Centricon- has a 3000 mol-wt cut off, the Centricona 10,000 mol-wt cut off, Centricona 30,000 mol-

Sequencing of PCR Products wt cut off and the Centricona 100,000 mol-wt cut off. I previously used a Centriconcolumn for a PCR fragment of size 1000 bp. 2. The denaturing polyacrylamide gel is essentially the same as a sequencing gel containing 7M urea and a 29:l ratio of acrylamide to bisacrylamtde. It is however thicker than a sequencing gel, about OSmm and the concentration of acrylamide used to purify the labeled fragment will depend on the size of the DNA. A general rule is shown in the table below. The migration of the two dyes m the sequencing samples [xylene cyan01 FF (XC) and bromophenol blue [BPB)] are used as approximate size markers for double-stranded DNA as below (from ref. 7). Effective size xc Acrylamlde cont. range for separation BPB 3.5% lOOO-2000bp 460bp 1OObp 5.0% 80-500 260 65 8.0% 60-400 160 45 12% 40-200 70 20 15% 25-150 60 15 3. The Ml3 phage clone should have internal homology to the PCR product as shown in Fig. 3. I used an Ml3 clone lacking about 40 bases on both ends compared to the PCR product and that worked well with this protocol. I used one of the PCR primers as the sequencing primer. 4. Sequencing using the Ml 3 single-stranded DNA method requires some empirical determination of the optimal ratio of double-stranded PCR product to Ml3 DNA. At the outset, I determined this by doing several reactions with mcreasmg concentration of the single-stranded DNA from one- to five-fold and the same amount of the PCR product. 5. I was generally able to sequence more than 500 bp using method 3, whereas method 2 is limited to around 300 bp. 6. Using the first sequencing protocol (that using single-stranded DNA from an Ml3 clone), I occasionally observed evidence of multiple bands at specific positions in the sequence suggesting either heterogeneous samplesor polymerase template switching during the sequencing reaction. 7. Generally, sequencing with 35S vs 32P gives sharper, more readable sequence. And as the former tsotope has a longer half life, sequencing reactions can be kept for a longer time. It is also generally safer to work with 35Sas it produces a lower energy emission, 8. The protocols described can only be used if the PCR product is homogeneous in its sequence. If there are multiple alleles of the DNA, then it is better to clone the DNA into a plasmid vector and sequence mdividual clones separately (as described in Chapter 3).

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9. Several other protocols have been described in the literature that show dtrect sequencing of PCR products (3,8-10). I have tried some of them with only limited success. As well, several compantes (Promega, Madison, WI and Glbco BRL, Gatthersburg, MD) are now advertising kits and special protocols for sequencing PCR products that may be useful to try.

References

_

1. Saiki, R. K., Scharf, S., Faloona, F., Mullis, K. B , Horn, G. T., Erlich, H. A , and Arnheim, N A. (1985) Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnoses of sickle cell anemia. Science 230, 1350-1354. 2. Wong, C., Dowlmg, C. E., Saiki, R K., Higuchl,

R G., Erlmh, H A., and Kazazian, H. H., Jr. (1987) Characterization of beta-thalassaemta mutations using direct genomic sequencing of amplified single copy DNA. Nature 330,

384-386. 3 Gyllensten, U. B. and Erlich, H. A. (1988) Generation of single-stranded DNA

by the polymerase chain reaction and Its application to direct sequencing of the HLA-DQA locus. Proc Natl. Acad. Scr USA 85,7652-7656 4 Gal, S. and Hohn, B. (1990) Direct sequencing of double-stranded DNA PCR products via removing the complementary strand wtth single-stranded DNA of an Ml3 clone. Nucl. Acids Res. 18, 1076. 5. Puchta, H. and Saenger, H. L. (1989) Sequence analysis of minute amounts of viroid RNA using the polymerase chain reaction (PCR). Arch. Vwology 106, 335-340. 6 Wrischnik, L. A., Higuchi, R. G., Stoneking, M., Erhch, H. A., Arnheim, N., and

Wilson, A. C. (1987) Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA. Nucl Acids Res. 15, 529-542. 7. Sambrook, J., Fritsch, E. F., and Mamatis, T. (1989) Molecular Clonmg. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 8 Ward, M A., Skandalis, A., Glickman, B. W., and Grosovsky, A. J. (1989) Rapid generation of specific single-stranded template DNA from PCR-amplified material. Nucl. Acids Res. 17, 8394. 9 Green, A , Roopra, A , and Vaudin, M. (1990) Direct single stranded sequencing from agarose of polymerase chain reaction products Nucl. Acids Res. l&6163. 10. Bachman, B., Lueke, W , and Hunsmann, G. (1990) Improvement of PCR amplified DNA sequencing with the aid of detergents Nucl Acids Res 18, 1309

&APTER

24

Sequencing Double-Stranded Linear DNA with Sequenase and [af5S] d.ATP Jean-Laurent

Casanova

1. Introduction The Polymerase Chain Reaction (PCR) products are doublestranded linear DNA molecules. Although hybridization may provide some information on the amplified products, clearcut identification of nucleic acids is best achieved by sequencing. When PCR fragments are heterogeneous, cloning in vectors is compulsory for sequencing. In some cases, however, PCR products are homogeneous and direct sequencing without cloning may be undertaken. We have developed a simple and fast method for directly sequencing linear double-stranded DNA molecules, such as PCR products (I), that is described in detail below. This method is direct, since it allows the sequencing of a PCR product without an intermediate cloning step and without the generation of a single-stranded linear DNA template by an additional step, such as asymmetrical PCR or separation of a biotynilated strand. This method is also simple, fast, and inexpensive, since it makes use of the most common sequencing reagents, namely the Sequenase kit (USB) and [cx-~~S]dATP, without any modification of the primer or of the reaction buffer. The sequencing of double-stranded linear DNA by this method is achieved mainly by optimization of incubation times and/ From. Methods m Molecular Bology, Vol. 23 DNA Sequencing Protocols Edited by H and A Grlffm Copynght 01993 Humana Press Inc , Totowa,

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or temperatures. Advantages and disadvantages, when compared to other sequencing strategies and protocols, are discussed in the notes section. The major problem in sequencing double-stranded linear DNA is the renaturation of the template that affects one or several steps of the sequencing reaction, such as the annealing of the primer to the single-stranded template, the labeling of the newly synthesized fragment with [a-3$1 dATP, or the extension and termination of the synthesis in the presence of dideoxy nucleotides. The following three conditions have been considered in this method to minimize the renaturation or the effect of renaturation on the sequencing process. 1. Annealing of the primer to the template and httle renaturation of the template is best achieved by rapid transfer of the sample from +lOO”C to -70°C. 2. A lo- to lOO-fold primer to template molar excess favors annealing of the primer to the template during rapid cooling. 3. Labeling IS optimal at room temperature between 15-45 s Extension condrtions (time and temperature) have less mfluence on the outcome of the sequencing reaction than those applied to the annealing and labeling steps. 2. Materials 2.1. Specific Materials 1. Sequenase version 2.0 kit (United StatesBrochemrcals, Cleveland, OH), including O.lM DTT, 5X dNTP mix for dGTP sequencing, 4 ddNTP mixtures, IX enzyme dilution buffer, 5X reactron buffer, 1X Mn*+ buffer, and stop solution. 2. [a-35S] dATP: 1 mCi/pmole/80 pL (e.g., Du Pont, Boston, MA). 3. 96-well round bottom plate and lid (e.g., Dynatech, Chantilly, VA). 4. Electroelutor (International Biotechnologies Inc IBI, New Haven, CT), 1-mL syringes, and 18-G 1l/2 needles (1.2 x 40 mm). 5. Linear polyacrylamide (see Note 1) or other carrier (glycogen, tRNA). 6. Metallic racks for Eppendorftubes, metallic water bath resistant to boilmg, dry ice ethanol bath. 7. Salad (lettuce) spurner or 96-well plate centrifuge. 2.2. Optional Materials 1. Centricon 100 microconcentrators (Amicon, Danvers, MA) 2. Qiagen PCR purification kit (Diagen, Dusseldorf, Germany).

Sequencing Double-Stranded 2.3. Common

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Materials

1. Agarose. 2. Ethidium bromide 10 mg/mL. 3. Urea. 4. Acrylamide:bisacrylamide (19:1, W: W) 40%. 5. Ammonium persulfate 25%. 6. N,N,N’,N’-Tetramethylethylenediamine (TEMED). 7. 3h4 sodium acetate, pH 5.2. 8. 1OM ammonium acetate (colored with bromophenol blue). 9. Ethanol and ethanokwater (80:20). 10. SpeedVac or similar vacuum dryer. 11. Oven. 12. Sequencing gel device. 13. 10X TBE buffer: 54 g Tris base, 27.5 g boric acid, 20 mL OSM EDTA pH 8.0/L. 14. 6X DNA loading buffer: Ficoll 15%, bromophenol blue 0.25%, xylene cyan01 0.25%. 3. Methods 3.1. Purification of the PCR Product 1, Precrpitate the PCR products with 0.1 vol of 3M sodium acetate pH 5.2, 3 vols of ethanol (carrier if needed, such as 2 pL of linear acrylamtde 0.25%) and freezing at -70°C for 15 min. 2. Centrifuge the sample at 10,OOOgfor 15 min, discard the supernatant, rinse the pellet with 80% ethanol, spin down agam at same speed for 3 min, discard the supernatant and dry the pellet under vacuum for 3 min. 3. Resuspend the pellet in 15 pL of 1X DNA loading buffer, load onto a 2% agarose gel (for a PCR product of 200-800 bp) stamed with ethidium bromide (2 pL for 50 mL), and run at 5 V/cm for 1 h in 1X TBE buffer (see Note 2). 4. Cut out the band(s) of interest under UV transilluminatlon and store it in a labeled Eppendorf tube(s) at 4°C. Elute DNA from gel slice. We obtain excellent results with an IBI electroelutor, as follows in steps 5-7, but other methods (Biotrap, Gene Clean, and so forth) may be used. 5. Set up the electroelution device by pouring 500 mL of 1X TBE mto the curve and by removing air bubbles from the V shaped channels (see instruction manual for detailed procedure). 6. Place the agarose slices, add 150 pL of ammonium acetate (10M) to each channel (with a l-mL syringe and a 18-Gl ‘iZ needle 1.2 x 40 mm), and run at 100 V for 45 min.

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7. Empty the device carefully and recover each salt cushion (approx 300 uL) with a separate syringe, rinse the channel with 100 pL 1X TBE, and pool thts volume with the initial 300 & from each channel. 8. Precipitate the eluted DNA with 2 pL linear polyacrylamide (0.25%) and 1 mL of ethanol as described earher, and resuspend in 10 pL water. 3.2. Sequencing of the PCR Product 1. Set up a dry-ice ethanol bath, a boiling waterbath, several ice boxes, and thaw out the reagents of the Sequenase version 2.0 kit (not the enzyme). Label the lid of the 96-well plate with the names of the templates at the emplacement correspondmg to the last 4 wells of each line. 2. For 8 PCR products to be sequenced, prepare 8 Eppendorf tubes each contammg 6 pL water, 2 pL 5X reaction buffer, 1 pL primer (10 piV) and 2 pL template (ideally around 0.5 pmoles) (see Notes 3-5). 3. The vols indicated in this section are for 8 sequencmg reacttons. Prepare in an Eppendorf tube a sequencing mix containing 8 pL DTT (O.lM), 16 @ 1X dNTP mix for dGTP sequencmg, and 4 pL [a-35S] dATP. In a second Eppendorf tube, prepare 14 pL of 1X enzyme dilution buffer. Keep both tubes on ice. 4. Put 2.5 pL of each ddNTP mixture in the last 4 wells of each lme of the plate, for example in the following order: ddGTP, ddATP, ddlTP, and ddCTP. 5. Place the 8 Eppendorf tubes in a rack that maintains the lids firmly closed and incubate the rack in the closed boiling water bath for 3-5 min (see Note 6). 6.Add8pLofMn 2+ 1X buffer to the sequencmg mix while the samples are being boiled. 7. Transfer the rack from the boiling waterbath to ice, then immediately transfer the Eppendorf tubes to the dry-ice ethanol bath (see Note 7). 8. Add 2 pL of Sequenase to the 14 uL enzyme dilution buffer, transfer this diluted Sequenase to the sequencing mix, and homogenize by pipeting. Keep the tube on ice. 9. Take the first tube from the dry-ice ethanol bath, wipe off the alcohol from the external walls of the tube with paper towel, turn the tube up side down when opening the lid to prevent anythmg falling into the tube, and add 5.5 pL of the sequencing mix on the top of the frozen pellet. 10 Warm up the tube in your hand and help to thaw and mix the sample by rotating the pipet tip. 11. When the sample is melted, that IS, as soon as you cannot see any ice particles, incubate the tube at room temperature for 15-45 s. 12. Transfer 3.5 uL to the left border of each of the 4 wells of a lme con-

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taining the ddNTP mixtures, cover the plate with the lid, and spin down briefly in the salad spmner (see Note 8). 13. Incubate the plate at room temperature (or at 37°C) until step 15. 14. Repeat the procedure for each of the remaming 7 tubes (see Note 9). 15. Immediately add 4 pL of stop solution to the right border of each of the 32 wells and spin down. 16. Heat the plate in an oven at 80°C for 2 min, load 4 J.ILof the sequencing reaction on a 8M urea, 6% acrylamide sequencing gel, run at 50°C in 1X TBE buffer, dry the gel after fixation, and expose overnight (see Note 10-16).

4. Notes 4.1. Technical Comments 1. Prepare a 5% acrylamide solution (without his-acrylamide) m 40 mZt4 Tris-HCl, 20 mM sodium acetate, 1 mM EDTA, pH 7.8. Add l/100 vol of 10% ammonium persulfate and l/1000 vol of TEMED, and let polymerize for 30 min. When the solution has become VISCOUS, precipitate the polymer with 2.5 vols of ethanol, centrifuge, and redtssolve the pellet in 20 vols of water by shaking overnight. The 0.25% linear polyacrylamide solution can be stored in the refrigerator for several years. 2. The purification of the PCR products is also possible with Centricon100 filters or Qiagen columns. These procedures separate the primers from all PCR products. Therefore, they may be applied only when a single product is generated by PCR, whereas the agarose gel electrophoresis allows the independent purification and subsequent sequencing of several PCR products from the same reaction. In addition, the presence of a smear or even of infravisible PCR products may alter the sequencing reaction. Altogether, we would recommend as a first choice to purify PCR products on an agarose gel, and to compare (for each particular type of PCR) the other methods to this reference purification, 3. The amount of template should be ideally around 0.5 pmoles (as determined by agarose gel quantification). If lower, load the entire sequencing reaction on gel. 4 The amount of sequencing primer is dependent on the amount of template and should stay between a lo-fold and loo-fold molar excess. 5. The amount of linear polyacrylamide per sample should stay below 1 cog in the course of the sequencing process. This information is not known for the other carriers, such as glycogen or tRNA. 6 Do not heat the samples on a solid heating block or the sample will evaporate. Covering the boiling water bath creates uniform temperature conditions, preventing any evaporation of the samples.

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7. To prevent renaturation of the template, freeze the samples as raptdly as possible, that is, in a dry-ice ethanol bath and not m dry ice or in a freezer. 8. The salad (lettuce) spinner has three major advantages over a classical centrifuge: It is also vortexmg the samples, it is much faster to start and to stop, and it is less expensive. If you do not find a salad spinner, use a classical centrifuge and spm down the plates as briefly as possible. Alternatively, tap the plate vigorously on the benchtop to bring the drops to the bottoms of the wells. 9. The protocol described above can be adapted for handling 1-12 tubes and posstbly more. Indeed, the extension time and temperature appear to be of little importance, at least in the reading of the first 150 bp. 4.2. General Comments 10 The quality of the sequence is primarily dependent on the purity and homogeneity of the PCR product. If your sequence is poorly readable, investigate first the PCR conditions. Check that you wish to sequence the specific product of a single gene from a single cell type and that your primers are specific for that gene only. 11. Some DNA molecules harbor strong secondary structures (caused by, for example, a high GC content), and therefore may not be sequenced under these conditions (2). You may try to label the primer with 32P(to avoid the labeling step) and to otherwise apply the same protocol. However, cloning in vector or generation of single-stranded linear template by asymmetrical PCR or separation of a biotmylated strand may still be required in some cases. 12. Products ranging m size from 200-800 bp have been sequenced with this method. Above or below may be possible but has not been attempted. The length of the readable sequence is at least 150 bp and in most cases around 200 bp. 13. Because PCR also amplifies mishybridizations of the primers, the expected size of a PCR product is only a good indication of specificity, but not as good as classical nonamphfied hybridizations. Thus, hybridization with an internal probe, or ideally, sequencing of all or part of the product, should be performed m all cases.This method allows rapid and simple identification of any PCR product for that purpose. 14. This procedure 1salso the method of choice when you want a sequence information that does not extend over 200 bp. A very good application is, for example, the sequencing of immunoglobulin or T cell receptor junctional regions (3), or the sequencing of polymorphic genes (4,5). 15. Because there is no cloning step,most Tuq polymerase mismcorporations

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are not detected by the sequencing of this polyclonal product. Sequencing several independent PCR products is therefore not necessary tn most cases. 16. The advantages over other direct methods of sequencing linear doublestranded PCR products (such as biotinylated primer or asymmetrical PCR) are mainly the simplicity, rapidity, and low cost of the procedure.

References 1 Casanova, J.-L, Pannetier, C., Jaulin, C., and Kourilsky, P. (1990) Optimal conditions for directly sequencing double-stranded PCR products with sequenase. Nucl. Acids Res l&4028. 2 Musette, P , Casanova, J.-L., Labbaye, C., Dorner, M., Kourilsky, P , and Cayre, Y (1991) Wegener’s autoantigen and leukemia Blood 77, 1398-1399. 3. Casanova, J.-L , Romero, P., Widmann, C., Kourilsky, P., and Maryanski, J. L. (1991) T cell receptor genes m a series of class I Major Histocompatibility Complex restricted cytotoxic T lymphocyte clones spectfic for a Pfasmodwm berghei nonapeptide: tmplications for T cell allelic exclusion and anttgen-specific repertoire. J, Exp. Med. 174, 1371-1383. 4. Jager, R. J., Anvret, M , Hall, K., and Scherer, G (1990) A human XY female with a frame shaft mutation in the candidate testis-determming gene SRY. Nature (London) 348,452-454.

5 Nakayama, K.-I., Tokito, S., Pannetier, C , Nakauchi, H., and Gachelm, G. (1991) MHC gene Q8/gd of the BALB/cJ mouse stram cannot encode a Qa-2,3 class I antigen. Immunogenetics 33,225-234.

&IAFI’ER

25

Solid Phase PCR Sequencing of Biotinylated Products Andrew

Green and Mark

Vaudin

1. Introduction The dideoxy sequencing method (I) has been universally employed and utilized for a wealth of sequencing projects such as small (2) and larger viral genomes (3), and requires purified M 13 or plasmid DNA templates. Numerous improvements have been made to obtain more DNA sequence from a single clone, including improved electrophorelic resolution and rapid computer assisted data entry. Inevitably, as these systems facilitate gel running and data entry, template preparation has become a limiting procedure. Attempts to improve this by the precipitation of M 13 phage with acetic acid and the recovery and subsequent disruption of the phage on glass fiber disks have been described (4). The DNA is eluted with a low salt buffer and requires neither phenol extraction nor ethanol precipitation, although bacterial culture and DNA purification are still required. Therefore, since the development of the polymerase chain reaction (PCR) (5), it has been used as a means of circumventing bacterial growth to prepare DNA templates. The main advantage is that template preparation becomes a simple biochemical process that can be readily automated if required. A major problem, though, is that the dNTPs and the two oligonucleotide primers from the PCR are present in great excess and From* Methods m Molecular B/ology, Vol 23 DNA Sequencrng Protocols E&xl by. H. and A Gnffm Copynght 01993 Humana Press Inc., Totowa,

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must be removed either by electrophoresis or column chromatography (6). Another problem with double-stranded PCR products is that the two DNA strands reanneal rapidly after denaturation. Several methods have been developed to overcome this. Asymmetric PCR produces an excess of one strand (7). However, becauseof unexplained template and primer variability the method is not very reliable. Others have used 32Pend-labeled internal primers prior to sequencing (&IO). Alternative methods of single strand template production include the phosphorylation of one of the PCR primers, which then targets this strand to be digested following PCR by h exonuclease III (11) and the competing out of one strand of the PCR product by the addition of complementary single-stranded DNA from an M 13 clone (12). An improved method depends on the immobilization of DNA on a solid support and the removal of one strand. The presence of biotin at the 5’ end of one of the PCR primers allows a single strand of DNA to be purified by binding to streptavidin coated magnetic beads and washing with alkali, see Fig. 1 (13). However, the disadvantage of this method is that large amounts of primer and Taq polymerase are used and a labeled primer is required for sequencing. The method has been modified (14) and now requires only small scale PCR, can be performed direct from the bacterial colony, phage plaque, or frozen bacterial glycerol stock, and does not require a labeled sequencing primer. The templates can be used for both radioactive and fluorescent dideoxy sequencing. The problem encountered when a single PCR product is not produced has been overcome by the direct single-stranded sequencing from agarose of the desired band (1.5). The problem of optimal annealing temperature for the sequencing reaction in low melting point agarose (16) is thus overcome by the production of a single-stranded sequencingtemplate and offers a convenient way of sequencinggenomic PCR products and products of PCR derived from degenerate primers when multiple products have been produced. of PCR Primers PCR now has multiple applications, including in vitro mutagenesis, expression cloning, sequencing, detection of mutations, high specificity nested PCR, introduction of unique restriction sites, analysis of messenger RNA, and the detection of homologous genes by ambiguous primers based on conserved protein sequences. All these 1.1. Design

Solid Phase PCR Sequencing

1.

201

PCR with one nomul primer one biotinylrted primer

Add magnetic beads coated with streptavidin

Separate DNA with external magnet

Denature DNA in alkali

Wash in I-JO to remove single strand

5.

Add sequencing primer Perform sequencing reaction

Fig. 1. Sequencing PCR products produced with biotin labeled primers.

diverse methods require specific types of primers. With such a variety of applications, the formulation of rules governing the design of PCR primers is extremely arbitrary. However, certain fundamental concepts do apply to the design of primers for specific DNA sequences. In general, primers should be of 17-24 bases in length, with 40-60% GC content and comparable annealing temperatures. Annealing between pairs of primers should be avoided, as this gives rise to false PCR products, and also consumes the excess of primer that is necessary for reannealling. Similarly, there should be no selfcomplementarity for individual primers, where the 3’ end of each primer anneals to itself. The predicted secondary structure of each primer should not allow significant folding upon itself, which would make the primer unavailable for annealing to a template. The primers should not falsely anneal at other sites on the template, and this is especially important for the 3’ end of the primer, where polymeriza-

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tion initiates. When designing primers for cDNA, it is helpful for the expected amplified product to span an intron-exon boundary. Amplification of any genomic DNA would then give rise to a larger than expected fragment. There is no universal rule for the calculation of annealing or melting temperatures for PCR primers. The annealing temperatures are dictated by the length of the primers, their relative GC content, the length of the amplified product, and the secondary structure formed by primers. In general, the higher the annealing temperature, the greater the specificity of the PCR. The simplest rule is that of Suggs (17): Primer melting temperature in “C = 2 x (number of A or T bases) + 4 x (number of G or C bases). Although this formula was originally intendedfor hybridization of short oligonucleotides, rather than PCR, it is still valid. Other formulas incorporate the size of the expected PCR product into the equation (18): Primer melting temperature in “C = 0.041(% G + C) - 5OO/amplified length. Others also include the helix structure (19). Unfortunately, none of these take into account further factors such as the variety of buffer composition, and the annealing temperature is often optimized experimentally for eachparticular setof primers. It is advisable to set the annealing temperature of the PCR at least 5°C below the lower of the calculated melting temperatures of each primer. Several computer programs for the design of PCR primers are now available for use with the IBM PC, Macintosh, and Atari (18,20,21). Computer aided design of PCR primers is a useful tool, but experimental validation of oligonucleotides and analysis of the PCR product is essential to verify the accuracy of the primers. 1.2. Biotinylation of Oligonucleotides The initial methods of biotinylation of oligonucleotides required the synthesis of a primer with an aminolink moiety on the 5’ end. This primer was then purified either by precipitation or HPLC, and then a biotin molecule was chemically attached to the aminolink in an overnight reaction, and the reaction product purified again to remove nonbiotinylated primer. Ninety-five percent pure biotinylated preparations compatible with phosphoramidite chemistry that can be directly added at the 5’ end of a primer as p,art of the oligonucleotide synthesis protocol, and purified as with normal oligonucleotides, are now available from Amersham (Arlington Heights, IL), DuPont (Wilmington, DE), and Cambridge Research Biochemicals (Wilmington, DE). However, these

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methods require the synthesis of two oligonucleotides, one normal, and one biotinylated, with twice the expense. The synthesis of an aminolink coupled primer will give a normal and a biotinylated primer for approximately the same expense.

2. Materials 2.1. Biotinylation of Aminolink Coupled OZigonucZeotides 1. 50 nmol of purified oligonucleotide to be biotinylated, synthesized with an aminolink group at the 5’ end, and the final trityl group on. 2. 100 mM solution N-hydroxysuccinimido biotin (Sigma, St. Louis, MO) in dry dimethylformamide. Store at -2OOC. 3. lit4 sodium bicarbonate buffer pH 9.0. Dissolve 840 mg NaHCOs (Sigma) in 10 mL sterile distilled HzO, and check the pH. Store at -20°C. 4. Ammonia solution (BDH). 5. 3M sodium acetate, pH 5.2. 6. Absolute ethanol. 7. 70% ethanol. 8. Double distilled sterile water.

2.2. PCR with Biotinylated

Primers

1. Double distilled sterile water. 2. 2 nut4 deoxynucleotides made up from 100 mM individual stocks of each base (A,C,G,T, Pharmacia, Uppsala, Sweden) in double distilled sterile water. Store at -20°C. 3. 10X PCR buffer consisting of 15 mM MgCl*, 15 mM KCl, 500 n&f Tris-HCl, and 0.1% Gelatin (Sigma). This is usually provided with Taq polymerase enzyme. If not, make up in double distilled H,O, filter sterilize, and store in aliquots at -20°C. 4. Taq thermostable polymerase. A wide variety is available, both cloned and purified. Second generation thermophilic enzymes are now also coming onstream. 5. Light mineral oil (Sigma). Store at room temperature. 6. Streptavidin coated magnetic beads (Dynal, Oslo, Norway). Supplied in sodium azide. Store at 4°C. 7. A strong magnet (magnets designed to hold tubes are supplied by Dynal or Promega [Madison, WI], but any ordinary one will do). 8. 0.15M NaOH prepared fresh from 1OM stock. 9. Template can be from a variety of sources (e.g., a toothpick of recombinant colony), 1 pL glycerol stock of recombinant bacteria, 1 pL liquid bacterial culture, a toothpick of recombinant bacterial plaque, diluted

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liquid phage lysates, purified genomic DNA, or cDNA (single- or double-stranded). 2.3. Direct

Sequencing of Gel Purified PCR Products 1. PCR reagents as described above. 2. Low melting point agarose (BRL) 3. 50X TAE electrophoresrs buffer. 2h4 Tris-acetate, 50 rnM EDTA, pH 8.0. To make up, mix 242 g Trts base, 57.1 mL glacial acetic acid, and 100 mL 0.5M EDTA, pH 8.0 and bring to 1 L with distilled H,O. 4. Sharp sterile scalpel. 5. Dry heating block set at 80°C.

3. Methods 3.1. Biotinylation of Aminolink Coupled Oligonucleotide 1. Synthesize required primer on olrgonucleotrde synthesizer (250 M column) with Ammolink 2 at 5’ end, Trityl on. 2. Elute the oligonucleotide from the synthesiscolumn with 2 mL of ammoma. 3. Incubate the eluate at 55OCfor at least 8 h, preferably overnight. 4. Dispense 5OO+L alrquots of the solutron m 2-mL tubes, and add 80 l.tL of 3M sodium acetate, pH 5.2, and 1.4 mL absolute ethanol to each. 5. Freeze m dry ice for 30 min, or overnight at 20°C 6. Centrifuge the mixture at 13,000 rpm for 30 mm. 7. Remove the supernatant, and add 300 uL of 70% ethanol. 8. Centrifuge the pellet again for 5 mm at 13,000 rpm. 9. Air or vacuum dry the pellet, and resuspend m a small volume (50-100 j.L) of H,O. 10. Check the concentration of the ohgonucleotide by spectrophotometry. 11. Reaction mix: Oligonucleotide (50 nmol) 150 pL 1M bicarbonate buffer pH 9.0 2.5 pL 100 mM biotin 25 w 250 /IL H,O to Incubate overnight at room temperaturein the dark (wrap in silver paper). The volume of reaction can vary with the concentration of oligonucleotide. The final concentration of biotm and bicarbonate is 10 mM each. 12. Ethanol precipitate the obgonucleotide agam, using steps 4-10 above, and check the concentration of DNA again by spectrophotometry. If necessary, check purity on HPLC or acrylamide gel electrophoresis (see Note 1).

Solid Phase PCR Sequencing 3.2. PCR with

Biotinylated

205 Primers

1. Make up PCR mix: 5.9 l,tL double distilled sterile water 1 pL 2 mM deoxynucleotides (final cone 200 @4) 1 pL 10X PCR buffer 1 pL 10 @Y biotinylated primer (final cone 1 w) 1 pL 10 l.tiU normal primer (final cone 1 piJ4) 0.5 units cloned/purified 7’aq polymerase Volumes quoted are for a lO+L reaction/sample (20-30 ltL reactions are also possible, depending on the amount of final reaction product). 2. Add template (toothpick of plasmid/phage colony or 0.5-l .O pL liquid culture or DNA) 3. Vortex the sample. 4. Overlay with 40 pL of mineral 011,and briefly centrifuge. 5. Amplify m thermal cycling machine using temperatures of: 95°C 2 min denaturing initially then 35 cycles of: 95OC 0.5 min denaturing 55°C 0.5 min annealing 35 cycles 1 72°C 1 min extension 1 followed by a final extension step of 72°C for 5 min (see Note 2). 6. Check l/10 of reaction product on agarose gel against known concentration DNA markers to ensure adequate amount of a single band (total product needed to sequence is approx 1 pg). 3.3. Binding of PCR Product to Streptavidin Beads 1. Resuspend magnetic beads in their container by vortexing. 2. Add 30 pL of beads to whole PCR product ensuring that the bead suspension drops through the oil into the PCR product. 3. Incubate at room temperaturefor 15 min to allow streptavidin-biotin binding. 4. Immobilize the beads using a strong magnet and remove supernatant by aspiration. 5. Add 20 l.tL 0.15M fresh NaOH, mix well using a pipet. 6. Incubate at room temperature for 5 min. 7. Immobilize the beads with the magnet, and aspirate supernatant. 8. Wash the beads in 20 pL of 0.15M NaOH, by resuspending the beads rn the solution, and immediately immobilizing the beads with the magnet, and aspirating the supernatant. 9. Wash the beads 3 times with 50 pL of double distilled sterile H,O, ensuring thorough mixing

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10. Resuspend the beads and DNA m 7 pL of double distilled sterile HZ0 and transfer to 1.5mL tubes prior to sequencing. At this point, the samples can be stored almost indefinitely at -2OOC. 11. Proceed with the sequencing reaction using USB Sequenase IITM kit, but use 1 pL of 10 pM sequencmg primer. 12. Stop the termination reactions using 100 pL of TE, immobilize the beads, and aspirate the supernatant. 13. Resuspend the beads in 4 pL of stop solution. Freeze the sample if necessary at this point. 14. Denature the reactions at 80°C for lo-15 mm from frozen 15. Immobilize the beads with the magnet. 16. Load the supernatant on a 6-8% polyacrylamide sequencing gel (see Note 3).

3.4. Direct Sequencing of Gel Purified PCR Products If multiple bands are seen on an agarose gel after a reaction under optimal PCR conditions, it is possible to sequencethese bands directly, provided there is sufficient DNA (about 1 B) in each band. 1. Perform the PCR reaction under the same condrtions as for Section 3.2., but in a 50 l.tL volume. 2. Load the entire PCR reaction in a 1% low melting temperature agarose gel made up m 1X TAE, and run it m 1X TAE buffer (see Note 4). 3. Examine the gel under UV fluorescence with ethidium bromide, and cut out the band of interest from the gel with the scalpel. 4. Place the gel fragment in a 1.5~mL Eppendorf tube, and add 1 mL of H,O. 5. Place the tube for 5 mm in a heating block preset at 80°C. 6. Check that the gel fragment has melted, and then add 30 pL of magnetic beads. 7. Incubate the sample at 80°C m the heatmg block for a further 5 mm. 8. Remove the tube from the block, and immediately immobilize the beads with the magnet. 9. Wash the sample 3 times with 50 pL HzO. 10. Resuspend the beads m 8 pL H,O. 1I. Proceed with sequencing reaction as for Section 3.3., step 11. 4. Notes 1. The biotmylation reaction will usually go to completion, but occasionally the biotmylated strand may need to be purified by HPLC or acrylamide gel purification. Biotmylation of the primers can also be checked at the stage of extracting the DNA from the PCR product by magnetic

Solid Phase PCR Sequencing

207

beads. The supernatant can be saved, and checked for UV fluorescence in an agarose gel. If all the DNA has been extracted, then the solution should not fluoresce. If the solution fluoresces, then either separation or biotinylation has been ineffective. This technique can also be used to check extraction from agarose gel slices. 2. The annealing temperature of the PCR reaction depends on the length of the primer and its base composition. Primers should ideally have similar annealing temperatures, to optimize the PCR. See the formulas outlined in the introduction for more details. The extension time of the third step of a PCR cycle depends on the expected length of the PCR product. Normally allow about 1 second extension time for every 20 bases of expected PCR product. 3. Occasionally bands in all four lanes can be seen at the site of annealing of the sequencing primer. This tends to occur when the sequencing primer is the same as one of the PCR primers, and can be improved by using an internal primer to sequence, or by using the manganese buffer provided in the USB Sequenase II TM kit. It is important to pull the beads down completely with the magnet, It is more effective to pull the beads to the side of a tube, and aspirate the fluid from the bottom of the tube, which gives cleaner results. 4. The percentage of the agarose gel can be varied from 0.8-l .75% according to the expected size of the PCR products. It is advisable, however, to keep the percentage as low as possible, to improve DNA yields.

References 1. Sanger, F., Nicklen, S., and Coulson, A. R. (1977). DNA sequencing with chain terminating inhibitors. Proc. Natl. Acad. Sci. USA 74, 5463 2. Vaudin, M., Wolstenholme, A. J., Tsiquaye, K. N , Zuckerman, A J., and Hamson, T. J. (1988) The complete nucleotide sequence of the genome of a hepatltls B virus isolated from a naturally Infected chimpanzee. J. Cm. Virol. 69,1383-1389. 3. Baer, R., Bankier, A T., Biggin, M. D., Deninger, P. L., Farrell, P. J., Gibson, T. J., Hatfull, G., Hudson, G. S., Satchwell, S. C., Sequin, C., Tuffnell, P. S., and Barrell, B. G. (1984) DNA sequence and expression of the B95-8 EpsteinBarr virus Genome. Nature 310,207-211. 4. Knstensen, T., Voss, H , and Ansorge, S. (1987) A simple and rapid preparation of Ml3 sequencing templates for manual and automated dideoxy sequencing. Nucl. Acids Res. 15,5507-5516. 5. Saiki, R. K., Scharf, S., Faloona, F , Mulhs, K., Horn, G. T , Erlich, H. A , and Arnheim, N. (1985) Enzymatic amplification of P-globm genomic sequences and restriction site analysis for diagnosis of sickle cell anaemia. Science 230, 1350-1354. 6. Kretz, K. A., Carson, G S., and O’Brien, J. S. (1989) Direct sequencing from low melt agarose with Sequenase. Nucl Acids Res. 17,5864.

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7. Gyllensten, U. B. and Erhch, H A. (1988) Generation of single-stranded DNA by the polymerase cham reaction and its applicatton to direct sequencing of the HLA-DQA locus Proc. Natl. Acad. Set. USA 85,7652-7656. 8 Wrischnik, L. A., Htguchi, R. G., Stoneking, M , Erlich, H. A., Arnheim, N., and Wilson, A. C (1987) Length mutations in human mitochondrial DNA: Direct sequencing of enzymattcally amphfied DNA. Nucleic Acrds Research 15,529-542 9 Wong, C., Dowlmg, C E , Salki, R K., Higuchi, R G., Erhch, H A., and Kazazian, H. (1987) Charactertzation of P-thalassemia mutations using direct sequencing of amplified single copy DNA Nature 330,384-386 10 Craxton M. (1991) Linear amplification sequencing, a powerful method for sequencing DNA. Methods: A Companion to Methods tn Enzymology Academic Press, 3:1, pp. 20-26. 11. Higucht, R G. and Ochman, H (1989) Productton of single-stranded DNA templates by exonuclease digestion following the polymerase cham reaction Nucl. Acids Res 17,5865

12 Gal, S. and Hohn, B. (1990) Direct sequencing of double stranded DNA PCR products via removing the complementary strand with single-stranded DNA of an Ml3 clone. Nucl Acids Res. 18, 1076 13. Hultman, T , Stahl, S , Hornes, E., and Uhlen, M (1989) Direct solid phase sequencing of genomic and plasmtd DNA using magnetic beads as solid support. Nucl. Acids Res 17,4937-4946. 14 Jones, D. S. C., Schofield, J. P , Vaudin, M. (1991) Fluorescent and radtoactrve solid phase drdeoxy sequencing of PCR products in mrcrotitre plates DNA Sequence-J. DNA Seq. and Mapping 1,279-283

15 Green, A., Roopra, A., and Vaudin, M. (1990) Direct single-stranded sequencing from agarose of polymerase chain reaction products Nucl Actds Res 18, 6163-6164 16 Casanova, J-L, Pannetier, C., Jaulm, C., and Kourilsky, P. (1990) Opttmal conditions for directly sequencing double-stranded PCR products with sequenase Nucl. Acids Res. 18,4028 17 Suggs, S. V., Wallace, R. B , Htrosa, T , Kawashina, E. H Itakura, K. (1981) “Using Purified Genes” in Developmental Biology Using Purified Genes, vol 23, ICN-UCLA Symposium on Molecular and Cellular Biology (Brown, D. D., ed.), Academic, New York, pp 683-693 18. Lowe, T. L , Sharefkin, J , Yang, S Q., and Dteffenbach, C W (1990) A computer program for selection of ohgonucleotrde primers for polymerase chain reactions Nuclecc Acids Res 18, 1757-l 761 19. Rychlik, W , Spencer, W J., and Rhoads, R E. (1990) Optimtsation of the annealing temperature for DNA amplification rn vttro. Nucletc Actds Res. 18, 6409-64 12

20 Rychlik, W and Rhoads, R. E. (1989) A computer program for choosing optimal oligonucleotides for filter hybridization, sequencing and in vitro amphfrcation of DNA. Nucleic Actds Res. 17, 8543-855 1. 21, Bridges, C. G. (1990) Olga-ohgonucleottde primer design program for the Atarr ST CABIOS 6,124-125

&APTER

26

Cycle Sequencing Robert W. Blakesley

1. Introduction Cycle sequencing (1,2) is a simple, yet powerful tool for conveniently sequencing double-stranded DNA (dsDNA). As in other dideoxy sequencing methods (3-5) dsDNA is denatured, a primer is annealed, then a complementary oligonucleotide is synthesized by a DNA poIymerase until extension is terminated by incorporation of a dideoxynucleotide. The difference is that in cycle sequencing this series of events occurs not once, but 20-30 times in succession under the control of a thermal cycler. The result is more, clearer, and stronger sequence from dsDNA for less effort. The procedure improves the reliability and efficiency of sequencing dsDNA, and eliminates an often troublesome and time-consuming step of preparing DNA in single-stranded form either in vivo, by utilizing the single-stranded bacteriophage Ml3 (6); or in vitro, by amplifying one strand asymmetrically (7), by separating strands (5), or by denaturing dsDNA (4). In cycle sequencing dsDNA is not denatured until placed in the thermal cycler. Here standard dideoxy sequencing reactions are interspersed with brief periods of heat-denaturation and primer annealing. In this way more DNA is effectively available for sequencing, counteracting template renaturation that may occur over From Methods m Molecular B!ology, Vol 23: DNA Sequencing Protocols Edited by: H and A Gnffm Copynght 01993 Humana Press Inc , Totowa,

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the synthesis period. The series of 20-30 reaction cycles also accumulates more reaction products per template, creating a signal amplification. Less template is needed in cycle sequencing, about 510% of the molar amount required in a standard sequencing reaction. A thermally stable DNA polymerase is required to perform cycle sequencing easily and economically. Taq DNA polymerase, which remains active after repeatedexposure to 95OC,is optimally active at 70°C. The benefits from sequencing at high temperature include reduced false stops from template secondary structure and less nonspecific background through higher stringency of primer annealing. Signals resulting from false priming events are eliminated by employing end-labeled primer instead of incorporating labeled nucleotide in the synthesis reaction. Another advantage of employing an end-labeled primer is realized by reading sequencebeginning severalbasesfrom the 3’-endof the primer, The properties of cycle sequencing allow the use of less pure dsDNA as template, for example, from abbreviated mini-preparations of plasmids (g-10), cosmids (10,11), hgtl0 and hgtll (IO), or partially purified in vitro amplified (PCR*) products (12,13). In fact, readily detectable sequence was obtained directly from lysates of single cosmid- and plasmid-containing colonies and from bacteriophage h and Ml 3 plaques (8,14). Opportunities are available through cycle sequencing for rapid and direct screening of clones, such as for point mutants, and for reducing the complexity of automation of large-scale sequencing projects. 2. Materials 2.1. Endlabeling of Primer 1. Labeling buffer: 300 mM Tris-HCl, pH 7.8, 50 m/t4 MgCl*, 1M KCl. Autoclave prtor to use. Store at -20°C. 2. Kinase dilution buffer: 50 mM Tris-HCl, pH 7.6, 25 n-&Z KCl, 5 rnM dithrothreitol (DTT), 0.1 pM ATP, 0.2 mg/mL bovine serum albumm (BSA), 50% (v/v) glycerol. To prepare, add separate filter-sterile solutions of DTT, ATP, and BSA to an autoclaved mixture of the salts and glycerol. Store at -20°C. 3. Sequencing primer (see Note l), for example, for pUC 18 or pUC 19: 5’-

CCCAGTCACGACGTTGTAAAACG-3’ (23 b), andT4 polynucleotrde kinase are available commercially. *Methods of amphfymg nucleic acids by Polymerase Cham Reaction (PCR) are covered by US Patent No. 4,683,202 issued to the Perkm-Elmer Cetus Corp , Norwalk, CT.

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Cycle Sequencing

4. Labeled adenosine triphosphate: [y-32P]ATP (L3000 Ci/rnmol, 10 mCi/ mL), is available commercially.

2.2. bSequencing Reactions 1. Sequencing buffer: 300 mM Tris-HCl, pH 9.0 (25OC), 50 mJ4 MgCl,, 300 mM KCl, 0.5% (w/v) W-l (Sigma, St. Lotus, MO). Autoclave prior to use. Store at -2OOc’. 2. Formamide dye solution: 95% (v/v) deionized formamide (15), 10 miI4 EDTA, pH 8.0,O. 1% (w/v) bromophenol blue, 0.1% (w/v) xylene cyanol. Store at -20°C. 3. 10X TE buffer: 100 mM Tris-HCI, pH 7.5, 1 mM EDTA. Autoclave prior to use. Dilute sorne TE buffer to 1X with autoclaved distilled water. Store at 4°C. 4. Deoxynucleoside triphosphate (dATP, dCTP, TTP), 7-deaza-deoxyguanosme triphosphate (7-deaza-dGTP), and dideoxynucleoside triphosphate (ddATP, ddCTP, ddGTP, ddTTP) stock solutions, Taq DNA polymerase, cloning vectors (e.g., pUC19 DNA), and mineral oil are available commercially. 5. Deoxynucleoside triphosphate (dNTP) working solutions: Make separate 0.5 mM solutions of dATP, dCTP, 7-deaza-dGTP, and TTP m 1X TE buffer. Store at -20°C. 6. Dideoxynucleoside triphosphate (ddNTP) working solutions. If not available commercially at this concentration, then make from the solid separate 10 mM working solutions of ddATP, ddCTP, ddGTP, and ddTTP in 1X TE buffer. Store at -20°C. 7. Sequencing mixes: Make 200~pL mixtures from the working dNTP and ddNTP solutions, according to Table 1 (volumes are m microliters). Store at -20°C.

2.3. Equipment 1. Thermal cycler: A temperature cycling incubator capable of executing two consecutive programs over the temperature range of 45-95OC, with an incubation chamber for 0.5-mL microcentrifuge tubes. 2. Water baths or heater blocks: Capable of temperatures of 37, 55, and 90°C. Alternatively, ihe thermal cycler can be used for the single temperature, timed incubations. 3. Microcentrifuge for 0.5-mL microcentrifuge tubes. 4. Microcentrifuge tubes: Polypropylene, 0.5 mL, with lockmg caps; caps must not open during the high temperature incubations. Autoclave prior to use. 5. Automatic pipets: Capable of dispensing 0.5-20 l.tL, and 10-100 l.rL. 6. Electrophoresis equipment and supplies for DNA sequencing (15).

212

Blakesley Table 1 Preparation of SequencingMixes A C 0.5 mM dATP 0.5 mM dCTP 0.5 mM 7-deaza-dGTP 0.5 mM TTP 10 mM ddATP 10 mM ddCTP 10 mM ddGTP 10mM ddTTP 1OX TE Buffer Autoclaved distilled water

20 20 20 20 40

20 20 20 20

G

T

20 20 20 20

20 20 20 20

20 4 20 60

20

80

20 96

40 20 60

3. Methods 3.1. Endlabeling of Primer 1. One unit of kinase is sufficient to label 1 pmol of primer. Depending on the sourceof enzyme,excesskinase may causereduced labeling efficiency. It is recommended to dilute the concentrated enzyme solution to I U&L with kinase dilution buffer before use, and use only 1 U/pm01 of primer. The diluted enzyme is stable for several months when stored at -20°C. Approximately 1 pmol of sequencing primer per set of sequencmg reactions is end-labeled using T4 polynucleotide kinase and fresh [y-32P]ATF. Thus reaction can be directly scaled for sequencing multiple templates with the same primer. Dilute the sequencing primer with 1X TE buffer to a fmal concentration of 0.5 pmol/pL (see Table 2). 2. Combine the following in order in an autoclaved OS-mL microcentrifuge tube: 2 pL of 0.5 pmol/pL sequencing primer, 1 pL of labeling buffer, 1 pL of 10 mCi/mL [Y-~*P]ATF, 1pL of 1 U&L T4 polynucleotide kmase; the total volume is 5 pL. Cap the tube and gently mix the contents. Collect the reaction volume by brief centnfugation in a microcentrifuge. 3. Incubate the reaction for 30 mm at 37°C. 4. Incubate the tube for an additional 5 mm at 55°C to terminate the labeling reaction. Centrifuge the tube briefly to collect the contents. Place the tube in wet ice. Although there is an excessof labeled ATP remaming, no cleanup of the end-labeled primer 1srequtred for performing cycle sequencing reactions. Labeled primer is usable for 1 wk when stored at -20°C 3.2. Sequencing Reactions 1. For convenience and to provide umformrty between the four dtdeoxy sequencing reactions a prereaction mixture is first prepared for each template. Dilute the template to a concentratron of approximately 2 fmol/

Cycle Sequencing

213 Table 2 Weight of 1 pmol of PrimeIa

Primer length, b

Weight, ng

18

5.9

20 22 24 26 28 30

6.6 73 7.9 8.6 9.2 9.9

@Weightof 1 pmol ISestimated by multtplymg the primer length m bases by 0.33 ng.

pL with 1X TE buffer. Compared to other methods, less DNA is introduced into the cycle sequencing reactlons (see Notes 2-5), 50 fmol being typical (see Table 3). 2. Prepare a prereaction mix by addmg the followmg in order directly to the tube containing 5 & of end-labeled primer: 4.5 pL of sequencing buffer, 26 pL of - 2 flmol/pL DNA, 0.5 pL of 5 U&L Tuq DNA polymerase; the total volulme IS 36 pL. 3. Cap the tube and gently mix the contents. Collect the contents of the tube by brief centrifugation in a microcentrlfuge. Place the tube in wet Ice. 4. Label the caps of four 0.5~mL microcentrifuge tubes A, C, G, and T; mmera1 oil may remove markings on the tube wall. According to Table 4 prepare IO-& sequencing reactions by placing the components in the appropriately labeled tubes (volumes in microliters). 5. Mix the contents of each tube gently. Place lo-15 JJLof mineral oil in each reaction tube. Securely lock the caps on all four tubes. Centrifuge briefly to collect the volumes and separate the phases m the reaction tubes. Mineral oil must completely cover the aqueous reactions, otherwise blank lanes may result. Place the tubes in wet ice. 6. Start the thermal cycler on a program consisting of a soak for 3 min at 95OC to denature the template, followed by 20 cycles of denaturation for 30 s at 95”C, anne:almg for 30 s at 55”C, and synthesis for 60 s at 70°C, and then followed by 10 cycles of denaturatlon for 30 s at 95°C and synthesis for 60 s at 70°C (see Note 6). When the temperature for the soak reaches 95”c’, place the securely capped sequencing reaction tubes m the incubation chamber. 7. After completion of the program, about 2-2.5 h, remove the reaction tubes carefully from the incubation chamber. Terminate the reactions by addmg 5 pL of formamlde dye solution to each tube. Cap the tubes, mix well, and collect the volumes by brief centrifugatlon. Store at -20°C.

214

Blakesley Table 3 Weight of 50 fmol of Double-Stranded DNA length, kb

DNA0 Weight, pg 0 016

05 1.0 2.5 50 7.5

0.033 0.08 016 025 0.33 0.82 1.48

100 250 450

OWelght of 50 fmol of dsDNA ISestimated by multlplymg the total DNA length in kb by 0 033 pg

Table 4 Sequencma Reactions Tube A

Component

Tube C

Tube G

Prereactron Mix Sequencing MIX-A Sequencing Mix-C

-

Sequencing Mix-G

-

-

2

Sequencing Mix-T

-

-

-

8 2

Analyze the reaction optimal results.

8

2

Tube T

8

8

-

products by gel electrophoresis

2

within

24 h for

3.3. Gel EZec trophoresis 1, Cycle sequencmg reaction products are separated on a standard sequencmg gel descrrbed elsewhere m this volume. To visualize from a few bases to several hundred bases from the 3’-end of the primer gels are prepared as 6 or 8% polyacrylamlde in Tris-borate-EDTA buffer with urea as a denaturant. The bromophenol blue dye m the samples 1s allowed to reach within a few centtmeters of the gel bottom before electrophoresls 1s terminated. 2. Heat denature the completed sequencmg reactions at 90°C for 5 mm. Collect the lrqurd by brief centrifugatlon of the tubes m the microcentrifuge. 3. Load l-4 pL from each reaction (lower, blue aqueous phase) in separate lanes of the sequencing gel. Store the unused portrons of the reacttons at -20°C.

Cycle Sequencing

215

4. Followmg completion of electrophorests, transfer the gel to chromatography paper. Dry the gel under vacuum. 5. Expose X-ray film to the gel overnight at room temperature; an intensifying screen is not used.

4. Notes 1, Primer length, GC content, and the annealing temperature must be chosen carefully, as for PCR reactions. Sequencing with short primers, for example, 15- to 17-mers, necessitates reducing the annealing temperature in a cycle from 55 to 45OC. Reducing the lowest temperature of a cycle, however, increases the chance of false priming and spurious sequence data. It is preferred to maintain the highest primer hybridization stringency so a minimum length of 20 b and a GC content between 50 and 60% is recommended. For sequencing AT-rich DNAs, utilize a primer that anneals to a region of locally high percent GC. 2. Sequencing data quality is dependent on the mtegrtty and purity of the template DNA. However, unlike other procedures cycle sequencing is less sensitive to contaminating biomolecules, generating from impure samples usable sequence for several hundred bases. For example, plasmid DNAs are readily sequenced when isolated by an abbreviated minipreparation, that is, excluding RNase treatment, chromatography, or cesmm chloride banding (9). When purer DNA is used, there are more bands that are clearly read. 3. A great advantage of cycle sequencing is the signal increase caused by the inherent linear amplification. This reduces the demand for scale-up of DNA preparations, especially of larger DNAs, such as cosmids (II). It should be noted that rntroducing too much DNA into a cycle sequencing reaction rapidly depletes the nucleotide pool, creating a large amount of randomly terminated short oligos and indeterminable sequence. In this case, dilute a fresh aliyuot of template five- to tenfold m 1X TE buffer and sequence again. 4. DNAs produced by PCR are readily sequenced by this method, even from one of the amplification primers (12,13). An aliquot of the PCR reaction should be partially purified from residual nucleotides, primers, and salts by selective isopropanol precipitation (16), or glass matrix chromatography (12). 5. The attributes of cycle sequencing can be used advantageously to sequence DNAs isolated directly from single plasmid-containing bacterial colonies and bacteriophage plaques from an agar plate (8,14). The lysis supernatant (9 l.rL) is diluted to 26 pL with 1X TE buffer and cycle sequenced as described above.

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Blakesley

6. Operation of the thermal cycler affects the results of cycle sequencing reactions similar to, but to a lesser extent than those of PCR. Changes in the recommended program may be attempted in order to optimize the results for a particular system,such as shorter run times, fewer cycles, or different annealing temperatures. For example, tt was found that a minipreparation of plasmld DNA and the 23 b primer gave excellent results with a simplified program; 20 cycles of 30 s at 95”C, 60 s at 65OCwith a temperature ramp of 30Wmin (IO). In this example, however, a transition between temperatures faster than 30Wmm reduced the signal mtensity and quality. Not all thermal cyclers perform equivalently under the same program. If the program fails to generate good sequence data, then use a program proven to generate PCR product.

References 1. Murray, V. (1989) Improved double-stranded DNA sequencing using linear polymerasechain reaction Nucl Acids Res. 17, 8889. 2 Craxton, M (1991) Linear amplification sequencing,a powerful method for sequencing DNA. Methods: A Companion to Methods m Enzymology 3,20-26 3 Smith, A J H (1980) DNA sequence analysis by primed synthesis Meth Enzymol. 65,560-580. 4 Chen, E. Y. and Seeburg, P. H. (1985) Supercoil sequencing* A fast and simple method for sequencing plasmid DNA. DNA 4, 165-170. 5. Hultman, T , Stahl, S., Hornes, E , and Uhlen, M. (1989) Direct solid phase sequencing of genomtc and plasmid DNA using magnetic beads as solid support Nucl. Acids Res. 17,4937-4946. 6. Messing, J. (1983) New Ml3 vectors for cloning. Meth. Enzymof 101,20-78 7 Gyllensten, U. B. and Erhch, H. A. (1988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus Proc. Nat1 Acad Sci. USA 85,7652-7656 8 Krishnan, B. R., Blakesley, R W , and Berg, D. E. (1991) Linear amplrfication DNA sequencing directly from single phage plaques and bacterial colonies. Nucl. Acids Res. 19, 1153 9. Adams, S. M and Blakesley, R (1991) Lmear amplification DNA sequent ing. Focus 13,56-58 10. Blakesley, R., unpublished observations 11 Craxton, M (1993) Cosmid sequencing. This volume, Chapter 21 12. Krishnan, B. R., Kersulyte, D , Brtkun, I., Berg, C M , and Berg, D. E. (1991 > Direct and crossover PCR amphftcatron to facilitate TnSsupF-based sequencmg of h phage clones. Nucl &tds Res. 19,6 177-6 182. 13 Adams, S. M. and Blakesley, R W (1992) Sequencing a PCR-amplified DNA with the dsDNA Cycle Sequencing System Focus 14, (in press) 14. Young, A. and Blakesley, R (1991) Sequencing plasmrds from single colonies wtth the dsDNA Cycle Sequencmg System. Focus 13, 137.

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Cycle Sequencing 15 Owen, M. J (1984) DNA sequence determination Methods

in Molecular

Biology

using dldeoxy analogs, m

Vol. 1: Nucleic Acids (Walker, J M., ed ),

Humana, Chfton, NJ, pp. 351-366. 16. Brow, M. A. D. (1990) Sequencing with Taq DNA polymerase, in PCR Protocols: A Guide to Methods and Applications (Inms, M. A., Gelfand, D. H., Sninsky, J. J , and White, T. J , eds ), Academic, San Diego, CA, pp. 189-196.

ckM’TER

Direct

Blotting

27

Electrophoresis

Stephan

Beck

1. Introduction The analysis of membrane-immobilized macromolecules such as DNA/RNA and proteins presentsa common analytical method with widespread applications. The macromolecules are either spotted directly onto a membrane or, if they are a complex mixture, they are separatedby electrophoresis prior to the transfer. Over the last 15 years a variety of procedures and equipment has been developed to achieve such transfer or blotting of gel-separated macromolecules onto an immobilizing matrix. The first transfer method was described by Southern in 1975 and was based on capillary action (I). Since then, procedures have been reported based on electro, vacuum, and pressure blotting, among them direct blotting electrophoresis (DBE) (2). DBE requires special equipment but it allows to combine electrophoresis and electroblotting into a single procedure. The principle of DBE is shown schematically in Fig. 1. The same electric field that separates applied macromolecules in one dimension is utilized for electro-blotting them onto an immobilizing matrix that is moved across the bottom of the gel by a conveyor belt. DBE has been used for Southern blots, Western blots, DNA sequencing, and other applications (2-11). Since this volume is dedicated to DNA sequencing, the following chapter will be limited to the use of DBE for this particular application. From Methods m Molecular Biology, Vol 23 DNA Sequenong Protocols Edited by H and A Griffin Copynght 01993 Humana Press Inc , Totowa,

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A

Fig. 1 Schematic diagram of vertical (A) and horizontal (B) direct blotting electrophoresis (DBE). (S), stepping motor drive, optional under manual or computer control, (R) rollers to guide the conveyor belt, (G) gel; (M) immobilizing matrix or membrane; (C) conveyor belt, (+) anode, (-) cathode

2. Materials 1. The most important item is the DBE apparatus itself. ,Commercial DBE devices are available from e.g., ,GATC, Konstanz, FRG; Hoefer Scientific Instruments, San Francisco, CA; Betagen, Waltham, MA sand construction drawings of homemade devices are available as indicated m the original publications (Z-10). 2. Standard glass plates are used for casting the gel but their bottom edge should be polished at an angle of about 25 degrees (see Fig 1A) m order to achieve optimum transfer. 3. Immobilizmg matrix, e.g., nylon membrane Biodyne A (BNRG3R, 0.2 pm, Pall Biosupport, Portsmouth, UK). 4. Bind-silane, dimethyldichlorosilane, e.g., BDH.

Direct Blotting

Electrophoresis

221

5. Repel-silane (prepared according to ref. 12). Mix in a dark glass bottle: 100 mL ethanol, 0.5 mL 3-methacryloxypropyltrimethoxysilane (GF3 1, Wacker Chemie, Munich, Germany), 0.3 mL acetic acid, 3 mL H20. 6. 25% APS: 2,5 g ammonium persulfate in 10 mL HzO. Store at 4°C and make fresh monthly. 7. TEMED: iV,N,N’,N’-tetramethylethylenediamine, e.g., BDH. 8. Electrophoresls buffer 10X TBE, pH 8.8, 162 g/L Tris base, 27.5 g/L boric acid, 9.2 g/L Na2EDTA. 9. 0.5X TBE gel mix (6%): 460 g/L urea, 150 mL/L 40% acryl/bisacrylamlde (19/l) stock solution, 50 mL/L 10X TBE pH 8.8. Filter and store at 4°C.

3, Method 1. Clean a matching set of glass plates with detergent followed by a Hz0 rinse. Dry plates in an oven or with a paper tissue. While handlmg the glass plates take special care not to damage the pohshed bottom edge or “blotting surface” (see Note 1). 2 Apply 1 mL of “repel-silane” to the inner surface of the notched glass plate under a fume hood and distribute equally on the entire surface with a paper tissue (wear gloves). Allow solvent to evaporate for 5 min. 3. Apply 1 mL of “bind- silane” to the inner surface of the unnotched glass plate as described above. 4 Wipe both treated glass surfaces clean with ethanol and assemble the plates separated by 0.04 cm spacers (see Note 2). Clamp the plates and carefully tape the blotting surface and both sides. 5. Add 50 mL of 0.5X TBE gel mix (6%) into a lOO-mL beaker. Add 100 pL of 25% APS and 100 pL of TEMED and mix well. 6. Take the gel solution up in the syringe and inject it between the glass plates. Control the flow by lrftmg or lowering the plates. 7. Insert comb and allow the gel to polymerize for 30 min in almost horizontal position. Polymerization should start after =5 min. 8. Fill 0.5X TBE buffer into DBE and place a precut nylon membrane into the start position. 9. Carefully remove comb and the tape from gel plates and rinse blotting surface with Hz0 (it should be absolutely smooth). 10. Insert the gel into DBE, attach upper buffer chamber, and fill it with 0.5X TBE buffer. Take special care not to trap any air bubbles at the blotting surface. In order to avoid a “smiling effect” an alummum plate can be clamped onto the gel plate. 11. Rinse the wells thoroughly with buffer and load l-2 pL of denatured sample per well.

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12. Set the Voltage, Vmax = 40 V/cm; Amperage, Amax = 35 mA; Blotting Speed = 10 cm/h. Start electrophoresis. 13. Start blotting process when bromophenol marker (fast dye) reaches blotting surface after =50 minutes. Run DBE until enttre membrane has passed the blottmg surface (see Note 3). 14. Remove membrane for autoradiography or further processing (e.g., Chapter 23, Sections 3 and 4 or Chapter 24, Section 3) and rinse DBE with Hz0 to prevent the rollers from becoming stocky. 15. Scrape the gel off the glass plates and remove the remaining gel by soaking the plates in OSM NaOH for about 1 h.

4. Notes 1. The quality of the blottmg surface is critical for good results. Use only

glass plates with a perfect bottom edge. Test the plates by carefully running a finger along the edge. It should feel sharp but smooth and no cracks should be felt. 2. The band sharpness is mainly determined by the thickness of the gel at the blotting surface. The thinner the gel the sharper the bands. However, at the sametime the casting of the gel and the loading of the samples become more sophisticated. In general, 20-30 cm long and 0.4-mm thick gels are easy to handle and give adequate resolution for most purposes. Reverse wedge gels (wide at top, narrow at bottom) have also been proposed for DBE (8). 3. In DBE the electrophoretic mobility is expressed as the elutton ttme. By separating each molecule/band over a constant distance (DBE) instead of a constant period of time (standard electrophoresis), the elution time becomes proportional to the molecular weight of the molecule/band resulting m a linear band spacing over the entire separation range of the gel (e.g., nucleotides ~50-500 for a 6% gel).

References 1. Southern, E. (1975) Detectton of specific sequences among DNA fragments separated by gel electrophoresis. J Mol. Biol 98,503-517 2 Beck, S. and Pohl, F. M (1984) DNA sequencing with drrect blotting electrophoresrs. EMBO J. 3,2905-2909. 3. Beck, S. (1986) Direct blotting electrophoresis. Electrophoresis ‘86 (Dunn, M. J , ed.), VCH Verlagsgemeinschaft Weinheim, pp. 173-183. 4 Beck, S. (1987) Colorrmetrtc detected DNA sequencing. Anal. Biochem. 164, 514-520 5 Pohl, F M. and Beck, S. (1987) Direct transfer electrophoresis used for DNA sequencmg, in Methods in Enzymology vol 155 (Wu, R , ed.), Academic Press, Orlando, FL, pp. 250-259.

Direct Blotting

Electrophoresis

6 Beck, S. (1988) Protein blotting Biochem

with direct blotting electrophoresls

223 Anal.

170,361-366

7 Beck, S., O’Keeffe, T., Coull, J. M., and Kdster, H. (1989) Chemiluminescent detection of DNA: applications for DNA and hybridization. Nucl. Acids Rex 17,5115-5123.

8. Richtench, P. (1989) Non-radioactive chemical sequencing of biotin labelled DNA. Nucl. Acids Res. 17,2181-2186. 9 Richterich, P., Heller, C., Wurst, H., and Pohl, F. M. (1989) DNA sequencing with direct blotting electrophoresis and calorimetric detection. Biotechniques 7,52-59

10 Heller, C and Pohl, F. M. (1989) Separation of double-stranded linear DNA molecules with direct blotting electrophoresis and field inversion. Proceedings of the efectrophoresisforum ‘89 (Radola, B. J., ed.), VCH Verlagsgemeluschaft Weinhelm, pp. 194-198 11 Thomae, R., Beck, S , ,md Pohl, F M. (1983) Isolation of Z-DNA containing plasmids. Proc. Nat1 Acad. Sci. USA 80,5550-5553. 12. Garoff, H. and Ansorge, W. (1981) Improvements of DNA sequencing gels. Anal. Biochem

l&450-457.

Multipllex

DNA Sequencing Stephan

Beck

1. Introduction Various approaches have been proposed to speed up the process of DNA sequencing in order to facilitate sequencing of large genomes from bacteria (-4 Mbp), yeast (-13 Mbp), human (-3000 Mbp), and other organisms (1-3). The multiplex DNA sequencing strategy (4) presents one of these approaches. It is compatible with the chemical (5) and the enzymatic (6) sequencing chemistry and allows simple scale-up without just multiplying cost and effort. Its principle consists in pooling or multiplexing “n” samples as early as possible in the sequencing process, and in de-pooling or de-multiplexing these samples as late as possible. In between these steps, the samples are processed as pools and, after transfer to a membrane, “n” sequence images can be obtained from the samemembrane by sequential hybridizations with specific ta#g-oligonucleotides. Figure 1 shows this principle and illustrates the difference between the standard and multiplex DNA sequencing strategy. The multiplex approach requires extra work for the sequencing library and two additional steps (transfer and hybridization/wash). However, the amount of work is considerably reduced for the steps of clone growth, template preparation, sequencing reactions, and electrophoresis. Since the first description in 1988 From Methods m Molecular Srology, Vol 23 DNA Sequencmg Protocols Edlted by H. and A Gliffm CopyrIght 01993 Humana Press Inc , Totowa,

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STANDARD

MULTIPLEX

SEQ

Library(m)

Clone

Picking

Growth

Template

Preparabon

SEC Reactions

Electrophoresis

Trancrtrr

HybrldlrationlWarh

Aularadlography

SEC4 Analyrlr

Fig. 1. Schematic diagram of the standard and multiplex DNA sequencing procedure. Each step is represented by an arrow Interrupted arrows indicate steps that do not apply to this particular method

(4) several alternative multiplex concepts and/or vector systems have been developed or proposed (7-11). This chapter will outline a protocol for enzymatic multiplex DNA sequencing using double stranded template DNA. 2. Materials 2.1. Method 1 1. Denaturation buffer: 2M NaOH, 2 mM Na2EDTA. 2. Neutralization buffer: 2M ammonium acetate, pH 4.6. 3. Reactton buffer, 5X: 250 mkf Tris-HCl, pH 8.0, 50 tmI4 MgCl*.

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4. TE buffer: 10 r&4 Trts-HCl, pH 8.0, 1 n&+ Na,EDTA. 5. Nucleotide mixes (dNTP/ddNTP): T-mix: 5 pL 10 ml4 dTTP, 50 pL 10 mM dCTP, 50 pL 10 mM dGTP, 50 pL 10 mil4 dATP, 120 pL 10 mM ddTTP, 725 /.tL TE buffer. C-mix: 50 pL 10 mM dTTP, 5 pL 10 mM dCTP, 50 pL 10 mM dGTP,5OpL10rmI4dATP,32pL10mMddCTP,813pL TE buffer. G-mix: 50 pL 10 mM dTTP, 50 pL 10 mM dCTP, 5 pL 10 rniJ4 dGTP, 501pL 10 rnJ4 dATP, 10 l.iL 10 mM ddGTP, 825 pL TE buffer. A-mix: 50 pL 10 mJ4 dTTP, 50 pL 10 mM dCTP, 50 pL 10 mM dGTP, 5 pI., 10 mA4 dATP, 70 pL 10 mM ddATP, 775 pL TE buffer. 6. Formamide dye: 10 mL 100% deionized formamide, 0.2 mL 0.5M Na2EDTA, 5 mg brornophenol blue, 5 mg xylene cyanol.

2.2. Method 2 1. Electrophoresis/blotting buffer 10X TBE, pH 8.8: 162 g/L Tris base, 27.5 g/L boric acid, 9.2 g/L Na*EDTA. 2.0.5X TBE gel mix (6%): 460 g/L urea, 150 mL/L 40% acryl/ bisacrylamide (19/l) stock solution, 50 mL/L 10X TBE, pH 8.8. Filter and store at 4°C. 3. 5X TBE gel mix (6% ): 115 g urea, 125 mL 10X TBE, pH 8.8,37.5 mL 40% acryl/btsacrylamide (19/l) stock solution, 10 mg bromophenol blue (to visualize the gradient) make up to 250 mL with HzO. Filter and store at 4OC. 4. 25% APS: 2.5 g ammonmm persulfate in 10 mL H,O. Store at 4°C and make fresh monthly. 5. TEMED: N,N,N’,N’-tetramethylethylenediamine (e.g., BDH). 6. Immobilizing matrix (e.g., nylon membrane Biodyne A) (BNRG3R, 0.2 pm, Pall Biosupport, Portsmouth, UK).

2.3. Method

3

1. TdT buffer 5X: 640 pL H20, 150 pL 4M cacodylic acid, pH 7.2, 10 pL 0.5M beta mercaptoethanol, 100 pL BSA (10 pg/pL protease and nuclease free), 100 pL CoCl,. 2. 1X Buffer I, pH 7.2: 100.00 g/L polyethylene glycol 8000, 7.30 g/L NaCl, 2.36 g/L Na2HP04, 1.31 g/L NaH2P0, x 2H20, 50.00 g/L sodium dodecyl sulfate (can be of low grade). 3. 10X Buffer II, pH 7.2: 7.30 g/L NaCl, 2.36 g/L NazHP0,,1.31 g/L NaH,P04 x 2Hz0, 50.00 g/L sodium dodecyl sulfate.

228

Beck 2.4. Method

4

1. 1X Buffer I, pH 7.2: 100.00 g/L polyethylene glycol 8000, 7.30 g/L NaCl, 2.36 g/L Na,HPO,, 1.3 1 g/L NaH2P04 2H,O, 50.00 g/L sodium dodecyl sulfate (can be of low grade). 2. 10X Buffer II, pH 7.2: 7.30 g/L NaCl, 2.36 g/L Na2HP04, 1.31 g/L NaH,PO, 2Hz0, 50.00 g/L sodium dodecyl sulfate. 3. 10X Buffer III, pH 9.5: 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 10 mA4MgC12. 4. Streptavidin (e.g., cat. 5532LB Life Technologies, Inc., Gaithersburg, MD). 5. Alkaline phosphatase in 30 mM TEA pH 7.5: 3M NaCl, 1 mh4 MgCl,, 0.1 mA4 ZnClz, biotin conjugate (e.g., cat. S-927 Molecular Probes, Eugene, OR). 6. Chemiluminescent substrate: 3-(2’-spiroadamantane)-4-methoxy-4-(3”phosphoryloxy)phenyl-1,2-dioxetane disodium salt (e.g., Lumiphos, Lumigen Inc., Detroit, MI, or Tropix Inc., Bedford, MA). l

l

3.1. Method

3. Methods 1: DNA Sequencing

Reactions

1. Add a total of -4 pmoles double-stranded template DNA (consistmg of a pool of up to 20 vectors for radioactive detection, and up to 10 vectors for chemiluminescent detection; see Note 1) to an Eppendorf tube and bring the vol up to 18 pL with H,O. 2. Add 2 pL of denaturationbuffer and incubatefor 5 nun at room temperature. 3. Add 2 pL of neutralization buffer and vortex to mix. 4. Add 75 pL of 100% ethanol, mix, and incubate for 10 min on dry ice. 5. Spin at top speed for 5 min, discard supernatant, and wash the pellet carefully with 200 pL 70% ethanol. 6. Dry the pellet under vacuum and store at -20°C or proceed. 7. Resuspend pellet in 15 l.tL H,O. Add 8 pL 5X reaction buffer, 2 J.IL primer (4 pmoles forward and/or 4 pmoles reverse primer) and incubate for 20 mm at 55°C. 8. Prepare four reaction tubes or a microtlter plate (for T, C, G, and A reactions) and add 2 pL of the appropriate nucleotide (dNTP/ddNTP) per well. Pipet all aliquots to the inside wall of the well/tube. Spm briefly to mix and guarantee uniform incubation times. 9. Add 6 pL of template/primer mix (from step 7) and 2 pL of Taq DNA polymerase (1 U&L) to each well/tube. Spin briefly to mix. 10. Incubate reactions for 2 min at room temperature to extend the primer a bit before incubating the reactions at 75°C for 10 min. 11. Spm briefly to collect any condensation and add 2 pL formamide dye per well/tube. Spin briefly to mix.

Multiplex

DNA Sequencing

2.29

12. Store reactions at -Z!O”C or proceed by heat denaturing the reactions (wells/tubes open) at 80°C for 20 mm. 13. Evaporation will reduce the volume per reaction to 2-3 pL that are then loaded onto the sequencing gel. 3.2. Method .2: Ebctrophoresis and BZotting An alternative method (direct blotting electrophoresis) that combines the electrophoresis and blotting steps is described in Chapter 27. 1. Pouring of a 6% gradient gel (50 x 20 x 0.04 cm). 2. Prepare two beakers (e.g., 100 and 50 mL), a 60-mL syringe, a lo- or 25-mL pipet, and a propipet. Assemble two glass plates (one pretreated with dimethyldichlorosilane; see Note 2) separated by 0.04-cm spacers. 3. Add 45 mL of 0.5X TBE gel mix into a 100-mL beaker and 7 mL of 5X TBE gel mix into a 50-mL beaker. Add 100 l.tL of 25% ammomum persulfate and 100 pL of TEMED to the 100~mL beaker and mix well. Add 15 pL of 25% ammonium persulfate and 15 pL of TEMED to the 50-mL beaker and mix well. The gel will start to polymerize m about 10 min. 4. Take up -35 mL of the 0.5X TBE gel solution in the syringe and put it aside. With a lo- or 25-mL pipet fitted with a propipet take up first 6 mL of the remaining 0.5X TBE gel solution then the 7 mL of the 5X TBE gel solution. Introduce -5 air bubbles with the propipet to form the gradient. 5. Hold the assembled glass plates in front of you at about a 20-30 degree angle and pour the gradient carefully down the edge (or middle). 6. Stop the flow by lowering the glass plates to a horizontal position the moment all gradient solution has left the pipet and before it runs down between the glass plates. 7. Immediately switch to the prefilled syrmge and start injecting the 0.5% TBE gel solution at the same position by lifting the plates up again. 8. Insert comb, clamp along the sides, and allow the gel to polymerize for -30 min m an almost horizontal position. (The quality of the gradient can be checked by the blue color distribution in the gel). 9. Set up electrophoresis apparatus, load sequencing reactions, and run gel in 1X TBE buffer at constant 40 W for about 3.5 h. 10. Set up electroblotter and fill in the required amount of 0.5X TBE buffer pH 8.8 (-12 L for horizontal electroblotter from Polytech Products, 42 x 26 cm, Sommerville, MA). 11. Carefully dismantle the gel plates by inserting a spatula between the glass plates. If the notched glass plate was pretreated with dimethyldichlorosilane (BDH), the gel should stick to the unnotched plate. 12. Trim the gel to 35 x 20 cm with a “pizza cutter” or razor blade and remove the cut-off gel pieces. Place a precut (25 x 40 cm) Whatman

230

Beck

3MM filter paper over the gel. Align filter paper properly before pressing it down onto the gel. Carefully peel off the gel that should now stick to the filter paper. 13. Place filter paper with gel side up onto the Scotch brite (presoaked wtth buffer) of the bottom grid of the electroblotter. Place a prewet nylon membrane (cut to the samedimensions asthe gel; see Note 3) onto the gel. Apply plenty of buffer (0.5X TBE) onto the membrane/gel sandwich and carefully squeeze out all an bubbles. Put on another sheet of filter paper, apply more buffer to wet it, and place the second grid of the electroblotter (presoaked m buffer) on top. Apply rubber bands to hold the sandwich together (see Note 2) and submerge it into the electroblotter. 14. Connect electrodes-gel side to the cathode (-) and membrane side to the anode (+). Electroblot for 20 mm at -100 V (-5 A). 15. Disconnect the electrodes and carefully dismantle the sandwich. Place membrane on a filter paper, bake it at 80°C for 20 min, and UV-crosslink (e.g., with UV Crosslinker, Hoefer Scientific Instruments, San Francisco, CA) the DNA for 0.5 min with -2100 uW/cm* to the membrane (see Note 4). 16. Transfer membrane into a heat sealable plastic bag and store m -250 pL 1X buffer II/cm* membrane.

Radioactive

3.3. Method 3: Hybridization and Detection

1. Probe labeling: Add 1-pL probe (5 pmol/pL), 25 pL [cG~*P]dATP (20 mCi/ml, 4 pmol/pL), 8 @, 5X TdT buffer and 12 U Terminal Transferase to a 1.5-mL Eppendorf tube. Bring vol up to 40 pL with H20 and incubate for 90 min at 37°C. 2. Check the labeling reaction by running an aliquot from to (before adding the enzyme) and tgOmin on a 20% polyacrylamide gel. 3. Prehybridize membrane with -250 pL 1X buffer I/cm* membrane m heat sealed plastic bag for 30 min at ambient or hybridization temperature with moderate shaking. Drain the bag. 4 Add -50 pL/cm* membrane preheated 1X buffer I containmg the labeled probe from step 1(1 nM, final probe concentration; see Note 5). Hybridize membrane at appropriate temperature (e.g , 45°C for 20-mer) for 1 h with moderate shaking (see Note 2). Drain the bag. 5. Wash membrane with -250 pL 1X buffer II/cm* membrane for 10 mm with moderate shaking. The temperature of the wash can vary depending on probe and required stringency (e.g., 21°C for 20-mer). Drain the bag. Repeat wash five times.

Multiplex

231

DNA Sequencing

6. Cover membranewith plasticwrap andexposeover night to anX-ray film. 7. Remove probe by adding -250 pL preheated 1X buffer II/cm2 membrane. Incubate in heat sealedplastic bag for 15 min at 80°C with moderate shakmg. Drain the bag quickly and completely before solution cools down. 8. Wash membrane with -250 pL 1X buffer II/cm2 membrane for 10 min at room temperature with moderate shaking. Drain the bag. Repeat wash once. 9. Store membrane in -100 pL 1X buffer II/cm2 membrane in a heat sealed plastic bag for subsequent probings. Chemiluminescent

3.4. Method 4: Hybridization

and Detection

(Adapted with permission from ref. 12) 1, Prehybridize the membrane wtth -250 pL 1X buffer I/cm2 membrane in heat sealed plastic bag for 30 mm at ambient or hybridization temperature with moderate shaking. Drain the bag. 2. Add -50 u&m2 membrane preheated 1X buffer I containmg 1 nM biotinylated probe (final concentration, see Note 5). Hybridize membrane at appropriate temperature (e.g., 45OC for 20-mer) for 1 h with moderate shaking (see Note 2). Drain the bag. 3. Wash membrane with -250 pL 1X buffer II/cm2 membrane for 10 min with moderate shaking. The temperature of the wash can vary depending on probe and required stringency (e.g., 21 “C for 20-mer). Drain the bag. Repeat wash three times. 4. Add -50 pL/cm2 membrane 10X buffer II containmg 0.5 pg/mL streptavidin. Incubate for 10 min at room temperature with moderate shakmg. Drain the bag. 5. Wash membrane with -250 l,tL 1X buffer II/cm2 membrane for 10 min at room temperature with moderate shaking. Drain the bag. Repeat wash three times. 6. Add 50 l&/cm* membrane 10X buffer II containing 0.5 pg/mL biotinylated alkaline phosphatase. Incubate for 10 min at room temperature s with moderate shaking. Drain the bag. 7. Wash membrane with -250 & 1X buffer III/cm2 membrane for 10 mm at room temperature with moderate shaking. Drain the bag. Repeat wash three times (see Note 2). Drain bag/membrane to completion or blot membrane moist-dry between 2 sheets of 3MM filter paper. 8. Add -50 I& chemiluminescent substrate/cm2 membrane. Incubate for 10 mm at room temperature with moderate shaking. Dram bag/membrane to completion or blot off excess liquid between 2 sheets of 3MM

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Whatman filter paper (do not blot dry). Alternatively, spray the chemiluminescent substrate under hood uniformly over the surface of the membrane using a vaporizmg spray bottle. 9. Seal membrane m a new bag or cover with plastic wrap and expose to X-ray film (see Note 2). The length of the exposure time varies between experiments. Start with a lo-min exposure time. 10. Remove probe by addmg -250 pL preheated 1X buffer II/cm2 membrane. Incubate in heat sealed plastic bag for 15 min at 80°C with moderate shakmg. Drain the bag qutckly and completely before solutron cools down. 11, Wash membrane with -250 pL 1X buffer II/cm2 membrane for 10 min at room temperature with moderate shaking. Dram the bag. Repeat wash once. 12. Store membrane m - 100 pL 1X buffer II/cm2 membrane in a heat sealed plastic bag for subsequent probings.

4. Notes 1. The recommendation to multiplex only up to 10 vectors for the chemiluminescent detection (20 and more are possible for the radioactive detection) is not caused by lower sensitivity but rather by increased background problems. The biotin/(strept)avidin system that is used here as affinity system tends to give unacceptable background levels if exposure times longer than 1 h are needed. Other affimty systems(9) and/or the use of enzyme conjugated probes (12) may overcome this problem 2. Signal problems can manifest themselves in various ways: a. No signal-perform a system check of each individual step (e.g., by using dot blots). b. Uneven signal--check that hybridization solution is equally distributed during the hybridization. Taping down the comers of the bag can help. c. Fuzzy signal-often caused by badly or overloaded gels. Overheating of the gel during electrophoresis and m same cases the use of nucleotide analogs (7-deaza dGTP/dATP) have been associated with fuzzy bands. Given that the electrophoresis and transfer steps went well, sharp bands are achieved by ensuring very good contact between the membrane and the X-ray film during the exposure. Plastic wraps are usually sufficiently thin but heat sealable bags tend to be thicker and can cause light scattering. Also, take special care not to trap any air bubbles between the membrane and the plastic wrap or bag. d. Smeared signal-check that the entire gel/membrane contact area is m good and uniform contact throughout the blotting procedure. In some cases,smeared signal has been associated with the use of silane, which is used for coating one of the glass plates.

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DNA Sequencing

233

e. Spots-some spot problems disappear if the solutions are made fresh and/or are filtered. Others have been put down to local precipitation of the chemiluminescent substrate caused by remaining SDS on the membrane (formatron of a SDSKTAB complex) or a final concentration 210 mA4MgC12 in buffer III. 3. The choice of the membrane is critical for method 4. In general, nylon membranes (e.g., Biodyne A, Pall Biosupport) work fine. Nitrocellulose and polyvmylidene difluoride (PVDF) membranes have been shown to quench the chemiluminescent signal considerably. 4. The conditions for the immobilization of DNA to nylon membranes by UV-irradiation can be quite different depending upon the particular setup. Over/under crosslmking can lead to considerable signal loss. In such a case, determine the optimum conditions for your UV setup by a timecourse experiment with dot blots of known DNA. 5. High background problems are most commonly caused by: a. Too much probe-recheck the used concentratton. b. Too much streptavidm-recheck the used concentration. c. Wrong buffer-recheck SDS concentration in buffers I and II.

References 1 Trainor, G. L. (1990) DNA sequencing, Automatton and the human genome. Anal Chem. 62,4 18-426 2. Barrel& B. G. (1991) DNA sequencing, present hmitatlons and prospects for the future. Faseb J. 5,40-45. 3. Howe, C. J. and Ward, EI S., eds. (1989) Nucleic acids sequencing: apractlcal approach IRL Press, Oxford, UK. 4. Church, G. N. and Kleffer-Higgins, S (1988) Multiplex DNA sequencing. Science 24, 185-188. 5 Maxam, A M. and Gilbert, W. (1977) A new method for sequencing DNA Proc Natl. Acad. Sci. lJSA 74,560-564.

6. Sanger, F., Nicklen, S , and Coulson, A. R. (1977) DNA sequencing with chainterminating inhibitors. Proc. Natl. Acad. Ski. USA 74,5463-5467 7 Yang, M. M. and Youvan, D. C. 1989) A prospectus for multispectral-multtplex DNA sequencing. Biotechnology 7,576-580. 8. Jacobson, K. B., Arlinghaus, H. F., Schmitt, H. W., Sachleben, R. A , Brown, G. M., Thoonard, N , Sloop, F. V., Foote, R. S., Lanmer, F. W., Woychik, R P., Wendy-England, M., Burchett, K. L., and Jacobson, D. A. (1991) An approach to use stable Isotopes for DNA sequencing Genomics 9, 5 l-59 9. Chee, M (1991) Enzymatic multiplex DNA sequencing. Nucl Acids Res 19, 3301-3305 10. Heller, C , Radley, E., Khurshtd, F. A., and Beck, S (1991) M13plex vectors for multiplex DNA sequencing. Gene 103, 131-132.

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11 Creasey, A , D’Angio, L., Dunne, T. S., Kissinger, C., O’Keeffe, T., PerryO’Keefe, H., Noran, L. S., Roskey, M., Schtldkraut, I., Sears, L. E., and Slatko, B. (1991) Application of a novel chemiluminescence-based DNA method to single-vector and multiplex DNA sequencing. B~otechniques 11, 102-109 12. Beck, S. (1993) Non-radioactive detection of DNA using dioxetane chemlluminescence. Recombinant DNA, Part G, m Methods in Enzymology (Wu, R ed.), Academic Press, Orlando, FL, vol 216, 143-153

DNA Sequencing by Chemiluminescent Detection Stepkan

Beck

1. Introduction The development of fluorescent, calorimetric, and more recently chemiluminescent detection systems have opened the way to replace radioactive detection for DNA sequencing (I-3). Although the bulk of DNA sequencing is still carried out using radioactive detection, the availability of better chemistries, equipment, and protocols make nonradioactive detected DNA sequencing increasingly popular. This chapter describes a protocol for standardSanger M 13 DNA sequencing (4,5) using enzymatically triggered 1,2-dioxetane chemiluminescence (6,7) for the detection, The principle of this method is illustrated in Fig. 1. The target DNA is immobilized onto a membrane and labeled with the enzyme alkaline phosphatase via an affinity system, such as the biotin/(strept)avidin or the digoxigenin system. Upon adding the dioxetane substrate to the membrane, the phosphatase will deprotect 1,2-dioxetane molecules by cleaving off the phosphate group, thereby initiating the chemiluminescent reaction that can then be detected as visible light. The main features of this detection system are speed, with detection times between l-30 min, sensitivity, between lo-100 attomoles of target DNA, and compatibility, since the method requires only equipment available in any molecular biology laboratory and From Methods 111Molecular Biology, Vol. 23 DNA Sequencmg Protoco/s E&ted by: H. and A Grtffrn Copyright 01993 Humana Press Inc., Totowa,

235

NJ

236

Beck

b-

SOLID SUPPORT

biotinylated

@J&?g+.

primer

biotinylated alk. phosphatase

E% -

Fig. 1. Schematic representation of chemiluminescent immobilized DNA.

rtreptavidin biotin

detection of membrane

allows the generation of the same data format (e.g., X-ray film hardcopy) as with radioactive detection. Since its first description in 1989 (3) several modifications and improvements have been reported for chemiluminescent detected DNA sequencing (7-11).

2. Materials 2JMethodl 1. TM buffer: 2. TE buffer: 3. Nucleotide T-mix:

C-mix:

G-mix:

100 mh4 Tris-HCl, pH 8.0, 50 mh4 MgCl*. 10 mh4 Tris-HCl, pH 8.0, 1 mM Na2EDTA. mixes (dNTFVddNTP): 5 pL 10 mil4 d’ITP, 50 $ 10 mh4 dCTP, 50 pL 10 mM dGTP, 50 pL 10 mM dATF’, 120 /.IL 10 mh4 ddTTF’, 725 & TE buffer. 50 pL 10 mh4 dlTP, 5 pL 10 mM dCTP, 50 pL 10 mM dGTP,50& lOmMdATP,32@ lOm,I4ddCTP,813j.L TE buffer. 50 pL 10 mh4 d’ITP, 50 & 10 mM dCTP, 5 pL 10 mM dGTP, 50 pL 10 mh4 dATF’, 10 j.L 10 mh4 ddGTP, 825 j& TE buffer.

Sequencing by Chemiluminescent

Detection

237

A-mix:

50 pL 10 r&f dTTP, 50 pL 10 mM dCTP, 50 pL 10 mM dGTP, 5 pL 10 mM dATP, 70 pL 10 mM ddATP, 775 pL TE buffer. 4. Primer: biotinylated at the 5’-terminus (see Note 5). 5. O.lM DTT: O.lM dithiothreitol in H,O. 6. Formamide dye: 10 mL 100% deionized formamide, 0.2 mL 0.5M Na,EDTA, 5 mg bromophenol blue, 5 mg xylene cyanol.

2.2. Method 2 1. Electrophorests/blottmg buffer 10X TBE, pH 8.8: 162 g/L Tris base, 27.5 g/L boric acid, 9.2 g/L Na*EDTA. 2. 0.5X TBE gel mix (6%): 460 g/L urea, 150 mL/L 40% acryl/bisacrylamide (19/l) stock solution, 50 mL/L 10X TBE pH 8.8. Filter and store at 4OC. 3. 5X TBE gel mix (6%): 115 g urea, 125 mL 10X TBE pH 8.8,37.5 mL 40% acryl/bisacrylamide (19/l) stock solution, 10 mg bromophenol blue (to visualize the gradient) make up to 250 mL with H,O. Filter and store at 4OC. 4. 25% APS: 2,5 g ammonmm persulfate m 10 mL H20. Store at 4°C and make fresh monthly. 5. TEMED: N,N,N’,N’-tetramethylethylenediamine (e.g., BDH). 6. Immobiltzing matrix (e.g., nylon membrane Btodyne A) (BNRG3R, 0.2 pm, Pall Biosupport, Portsmouth, UK).

2.3. Method 3 1. 10X Buffer II, pH 7.2: 7.30 g/L NaCl, 2.36 g/L Na2HP04, 1.31 g/L NaH2P04 2Hz0, 50.00 g/L sodium dodecyl sulfate. 2. 10X Buffer III, pH 9.5: 100 mM Tris-HCl, pH 9.5, 100 mM NaCl, 10 mA4 MgCI,. 3. Streptavidin (e.g., cat. 5532LB Life Technologies, Inc., Gaithersburg, MD). 4. Alkaline phosphatase in 30 mM TEA, pH 7.5,3M NaCl, 1 mM MgC&,, 0.1 mM &Cl,, btotm conjugate (e.g., cat. S-927 Molecular Probes, Eugene, OR). 5. Chemiluminescent substrate: 3-(2’~spiroadamantane)-4-methoxy-4-(3”phosphoryloxy)phenyl-1,2-dioxetane disodium salt (e.g., Lumiphos, Lumigen Inc., Detroit, MI, or Tropix Inc., Bedford, MA). l

3. Methods

3.1. Method

1: DNA Sequencing

Reactions

1. Prepare four (T, C, G, and A) primer mixes (calculated for 10 templates = 40 sequencing reactions). Primer T-mix: 15 l.tL T-mix, 2.5 lrL TM buffer, 2.5 pL biotmylated primer (0.5 pmol/pL).

238

Beck

Primer C-mix: 15 pL C-mix, 2.5 pL TM buffer, 2.5 pL biotinylated primer (0.5 pmol/pL). Primer G-mix: 15 pL G-mix, 2.5 pL TM buffer, 2.5 pL biotmylated primer (0.5 pmol/pL). Primer A-mix: 15 pL A-mix, 2.5 pL TM buffer, 2.5 pL biotinylated primer (0.5 pmol/l.tL). 2. Prepare a microtiter plate (e.g., Falcon #3911) and add 2 pL primer T-mix to each T well, 2 pL primer C-mix to each C well, and so forth. (Pipet all aliquots to the inside wall of the microtiter wells. Spin briefly to mix and guarantee uniform incubation times). 3. Add 2 pL single-stranded template DNA (0.1 pmol/pL) to each well, cover plate with plastic wrap, and spm briefly. 4. Incubate reactions at 55°C for 30 mm. 5. Preparation of the enzyme mix (calculated for 10 templates = 40 sequencmg reactions). Mix m an Eppendorf tube on ice: 68 pL H20, 10 pL O.lM DTT, and 4 pL Klenow enzyme (5 U&L). 6. Add 2 pL enzyme mix to each well, cover plate with plastic wrap, and spm briefly. 7. Incubate reactions at 37°C for 30 min. 8. Add 2 pL formamide dye to each well and spin briefly. 9. Store reactions at -20°C or proceed by heat denaturation/evaporation of the reactions (plate uncovered) at 80°C for about 20 min. 10. Load l-2 pL per reaction onto a sequencmg gel 3.2. Method

2: Electrophoresis

and Blotting

An alternative method (direct blotting electrophoresis) that combines the electrophoresis and blotting steps is described in Chapter 27. 1. Pour a 6% gradient gel (50 x 20 x 0.04 cm). 2. Prepare two beakers (e.g., 100 and 50 ml), a 60-mL syringe, a IO- or 25-mL pipet, and a propipet. Assemble two glass plates (one pretreated with dimethyldichlorosilane; see Note 1) separated by O&l-cm spacers. 3. Add 45 mL of 0.5X TBE gel mix mto a lOO-mL beaker and 7 mL of 5X TBE gel mrx into a 50-mL beaker. Add 100 pL of 25% ammomum persulfate and 100 pL of TEMED to the lOO-mL beaker and mix well. Add 15 pL of 25% ammonium persulfate and 15 pL of TEMED to the 50-mL beaker and mix well. The gel will start to polymertze m about 10 mm. 4. Take up -35 mL of the 0.5X TBE gel solution m the syringe and put it aside. With a lo- or 25-mL pipet fitted with a propipet take up first 6 mL of the remaining 0.5X TBE gel solution then the 7 mL of the 5X TBE gel solution. Introduce -5 au bubbles with the propipet to form the gradient.

Sequewing

by Chemiluminescent

Detection

239

5. Hold the assembledglass plates in front of you at about a 20- to 30-degree angle and pour the gradient carefully down the edge (or middle). 6. Stop the flow by lowering the glass plates to a horizontal position the moment all gradient solution has left the pipet and before it is run down between the glass plates. 7. Immediately swrtch to the prefllled syringe and start mjectmg the 0.5% TBE gel solution at the same position by lifting up the plates again. 8. Insert comb, clamp along the sides, and let the gel polymerize for -30 min in an almost horizontal position. (The quality of the gradient can be checked by the blue color distribution in the gel). 9. Set up electrophoresis apparatus and run gel in 1X TBE buffer at constant 40 W for about 3.5 h (see Note 1). 10. Set up the electroblotter and fill in the required amount of 0.5X TBE buffer, pH 8.8 (-12 L for horizontal electroblotter from Polytech Products, 42 x 26 cm, Sommerville, MA). 11. Carefully dismantle the gel plates by inserting a spatula between the glass plates. If the notched glass plate was pretreated with dimethyldlchlorosilane (BDH), the gel should stick to the unnotched plate. 12. Trim the gel to 35 x 20 cm with a “pizza cutter” or razor blade and remove the cutoff gel pieces. Place a precut (25 x 40 cm) Whatman 3MM filter paper over the gel. Align the filter paper properly before pressing it down onto the gel. Carefully peel off the gel that should now stick to the filter paper. 13. Place the filter paper with the gel side up onto the Scotch brite (presoaked with buffer) of the bottom grid of the electroblotter. Place a prewet nylon membrane (cut to the same dimensions as the gel; see Note 2) onto the gel. Apply plenty of buffer (0.5X TBE) onto the membrane/gel sandwich and carefully squeeze out all air bubbles. Put on another sheet of filter paper, apply more buffer to wet it, and place the second grid of the electroblotter (presoaked m buffer) on top. Apply rubber bands to hold the sandwich together and submerge it mto the electroblotter (see Note 1). 14. Connect electrodes-gel side to the cathode (-) and membrane side to the anode (-I-). Electroblot for 20 min at -100 V (-5 A). 15. Disconnect the electrodes and carefully dismantle the sandwich. Place membrane on a filter paper, bake it at 80°C for 20 min, and UV-crosslink (e.g., with UV Crosslmker, Hoefer Scientific Instruments, San Francisco, CA) the DNA for 0.5 min with -2100 pW/cm2 to the membrane (see Note 3). 16. Transfer the membrane into a heat sealable plastic bag and store in -250 pL IX buffer II/cm* membrane.

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3.3. Method

3: Chemiluminescent

Detection

(Adapted with permission from ref. 12) 1. Wash the membrane in a heat sealed plastic bag with -250 pL 1X buffer II/cm* membrane for 10 mm at room temperature with moderate shaking. Dram the bag. 2. Add -50 pL 10X buffer II/cm* membrane containmg 0.5 pg/mL streptavidm. Incubate for 10 min at room temperature with moderate shaking (see Notes 1 and 4). Dram the bag. 3. Wash the membrane with -250 pL IX buffer II/cm* membrane for 10 min at room temperature with moderate shaking. Drain the bag. Repeat wash three times. 4. Add -50 pL 10X buffer II/cm* membrane contaming 0.5 pg/mL biotinylated alkaline phosphatase. Incubate for 10 min at room temperature with moderate shaking (see Notes 1 and 4). Drain the bag. 5. Wash membrane with -250 pL 1X buffer III/cm* membrane for 10 min at room temperature with moderate shaking. Drain the bag. Repeat wash three times (see Note 1). Drain bag/membrane to completion or blot membrane moist-dry between 2 sheets of Whatman 3MM filter paper (Whatman Ltd., UK). 6. Add -50 pL chemiluminescent substrate/cm* membrane. Incubate for 10 min at room temperature with moderate shaking. Drain bag/membrane to completion or blot off excess liquid between 2 sheets of 3MM Whatman filter paper (do not blot dry). Alternatively, spray the chemiluminescent substrate under a hood uniformly over the surface of the membrane using a vaporizing spray bottle. 7. Seal the membrane in a new bag or cover with plastic wrap and expose to X-ray film (see Note 1). The length of the exposure time may vary between experiments. Start with a IO-mm exposure time. 4. Notes 1. Signal problems can manifest themselves in various ways: a. No signal-perform a system check of each mdividual step (e.g., by using dot blots). b. Uneven signal--check that the solutions are equally distributed durmg the streptavidin and alk. phosphatase incubation steps. Taping down the corners of the bag can help. c. Fuzzy signal-often caused by bad or overloaded gels. Overheatmg of the gel durmg electrophoresis and m some cases the nucleotide analogs (7-deaza dGTP/dATP) have been associated with fuzzy bands. Given that the electrophoresis and transfer steps went well,

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sharp bands are achieved by ensuring very good contact between the membrane and the X-ray film during the exposure. Plastic wraps are usually sufficiently thin but heat sealable bags tend to be thicker and can cause light scattering. Also, take special care not to trap any air bubbles between the membrane and the plastic wrap or bag. d. Smeared signal-check that the entire gel/membrane contact area is in good and uniform contact throughout the blotting procedure. In some cases,smeared signal has been associated with the use of silane that is used for coating one of the glass plates. e. Spots-some spot problems disappear if the solutrons are made fresh and/or are filtered. Others have been put down to a local precipitation of the chemiluminescent substrate caused by remaining SDS on the membrane (formation of a SDSKTAB complex) or a final concentration 210 mM MgClz in buffer III. 2. The chemiluminescent detection puts certain restrictions on the choice of the membrane. In general, nylon membranes (e.g., Biodyne A, Pall Biosupport) work fine. Nitrocellulose and polyvinyhdene difluoride (PVDF) membranes have been shown to quench the chemiluminescent signal considerably. 3. The conditions for the immobilization of DNA to nylon membranes by UV-irradiation can be quite different depending upon the particular setup. Over/under crosslmking can lead to considerable signal loss, In such a case, determine the optimum conditions for your UV setup by a time-course experiment with dot blots of known DNA. 4. High background problems are most commonly caused by: a. Too much streptavidin- recheck the used concentration. b. Wrong buffer-recheck SDS concentration in buffer II. 5. Instead of the biotin/(strept)avidin system described here can also use the digoxigenin affinity system (9). References 1. Smrth, L. M , Sanders, J. Z., Kaiser, R. J., Hughes, P , Dodd, C., Connell, C. R., Heiner, C., Kent, S B. H , and Hood, L. E. (1986) Fluorescence detection m automated DNA sequence analysis. Nature 321,674-679. 2 Beck, S (1987) Calorimetric detected DNA sequencing. Anal. Biochem 164, 5 14-520. 3 Beck, S , O’Keeffe, T , Coull, J. M., and Kdster, H (1989) Chemiluminescent detectron of DNA: applications for DNA sequencing and hybridtzation Nucl. Acids Res. 17,5 115-5123 4. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chainterminating inhibitors. Proc Natl. Acad. Sci USA 74, 5463-5467

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5. Bankrer, A. T., Weston, K. M., and Barrell, B. G. (1987) Random cloning and sequencing. Meth Enzymol. 155,5 l-93. 6. Schaap, A. P., Sandison, M. D., and Handley, R S. (1987) Chemical and enzymatte triggering of 1,Zdioxetanes alkaline phosphatase-catalyzed chemiluminescence from an aryl phosphate-substituted dioxetane Tet Lett 28,1159-l 162 7 Beck, S. and Koster, H. (1990) Applications of dioxetane chemiluminescent probes to molecular biology Anal Chem. 62,2258-2270 8 Trzard, R., Cate, R. L , Ramachandran, K L., Wysk, M., Voyta, J C , Murphy, 0. J., and Bronstein, I. (1990) Imagmg of DNA sequences with chemiluminescence. Proc Natl. Acad. Sci. USA 87,4514-4518 9. Chee, M. (1991) Enzymatic multiplex DNA sequencmg. Nucl Acids Res. 19, 3301-3305 10. Creasey, A , D’Angro, L., Dunne, T S., Krssmger, C., O’Keeffe, T., PerryO’Keefe, H., Moran, L. S , Roskey, M., Schildkraut, I., Sears, L. E., and Slatko, B. (1991) Apphcatton of a novel chemilummescence-based DNA method to single-vector and multiplex DNA sequencing Blotechniques 11, 102-109 11 Martin, C., Bresmck, L , Juo, R -R., Voyta, J C., and Bronstein, I. (1991) Improved chemilummescent DNA sequencing. Biotechmques 11, 110-I 13 12 Beck, S. (1993) Non-radioactrve detection of DNA using dioxetane chemrlummescence. Recombinant DNA, Part G, in Methods in Enzymology (Wu, R., ed ), Academic Press, Orlando, FL, vol 216, 143-153.

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30

Reverse Sequencing of Ml3 Cloned DNA AZan II Bankier

1. Introduction When shotgun sequencingprojects are almost complete, continued random sequencing becomes increasingly less fruitful. The sequencemay have regions of data that have only been determined on one strand (and therefore potentially error prone) or there may be gaps in the sequence. In both of these circumstances, it can be of benefit to be able to determine the sequence from the opposite strand of an Ml3 clone, or to be able to get sequence information from the opposite end of a large insert (I,2). The procedure of reverse sequencing was devised to take advantage of the fact that during Ml3 sequencing, large quantities of singlestranded DNA are produced. This single-stranded DNA can be used as a template to generate DNA of the opposite strand by simply performing a primed synthesis in the presence of the four deoxynucleotides. The principle is then identical to the sequencing of linear, double-stranded template (see Chapter 24). The method detailed in this chapter is essentially as first described (3). Perhaps a more convenient method uses the thermal stability of some polymerases, employing polmerase chain reaction protocols (4). Sufficient DNA for sequencing could be produced directly from the From’ Methods m Molecular Biology, Vol 23 DNA Sequencing Protocols Edited by H and A Gnffm Copynght 01993 Humana Press Inc , Totowa,

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original starting material, using very little sample, if specific primers are available. A similar cycling procedure could be used on an M 13 template, with the universal forward and reverse primers. An even better approach is to use asymmetric PCR (5), where the reverse primer is in excess, producing a proportionally larger quantity of the required strand. 2. Materials 1, Synthetic oligonucleotide primers at a concentration of 0 5 pmol/pL in TE.

Both moleculesshouldbe between15and 30 basesrn length.One should hybridize specifically in the 3’ region of the template DNA, relative to the section of interest. For templates derived from the Ml3 series of vectors or vectors having related polylmker cloning regions, this corresponds to the “universal” primer [S d(GTAAAACGACGGCCAGT)]. The other primer (the “reverse” primer) should hybridize to the complement of the template DNA on the 5’ side of the section to be sequenced

[5’ d(CAGGAAACAGCTATGAC) for Ml3 derived vectors]. 2. The template DNA to be sequenced at a concentration of around 0.05 pmol/&. This corresponds to a concentration of around 0.125 pg/l..tL of Ml3 DNA, the approx yield from a standard 1.5 mL preparation being around 5 pg. 3. TM buffer: 100 mM Tris-HCl, pH 8.5, 50 mM MgCl,. 4. Chasemix: 0.5 mMdATP, 0.5 mMdCTP, 0.5 mMdGTP, 0.5 mMdTTP. 5. Klenow fragment DNA polymerase 1 at a concentration of around 5 II/ pL or higher. 6. Distilled or deionized water. 7. 13% PEG: 13% polyethylene glycol (mol wt 6000-8000), 1.6Msodmm chloride. 8. 6.5% PEG: 6.5% polyethylene glycol (mol wt 6000-8000), 0.8M sodium chloride. This can be prepared by diluting the 13% PEG. 9. TE buffer: 10 mM Tris-HCl, pH 8.0,O.l n&f Na,EDTA. 10. Redistilled or ultra pure phenol equilibrated with TE buffer. 11. NaOAc: 3M sodium acetate adjusted to pH 5.0 with acetic acid. 12. In addition, the reagents described in the appropnate chapter (seeChapters 12, 13, 14, or 15) will be required for the subsequent sequence reactions

3. Method 1. In a 1.5-n& microcentnfuge tube, combine 8 pL template DNA (- 0.125 crg/pL); 1 pL forward primer (0.5 pmol/pL); 1 pL TM, and place the tube in an incubator at 55°C for 30-45 min. 2. Add 2 uL chase mix and 0.5 pL Klenow DNA pol 1 (- 5 umts pL) to the tube and mix the reagents and leave at room temperature for 15 mm.

Reverse Sequencing of Ml3 DNA 3. Heat kill the enzyme by placing the tube in a water bath at 70°C for 10 min. 4. Mix m 15 & of 13% PEG and leave on ice for 1 h. Centrifuge the tube in a microcentrifuge for 10 min and carefully remove the supernatant. 5. Wash the DNA pellet in 40 & of 6.5% PEG, centrifuge the tube for 5 min in a microcentrifuge, and remove the supernatant (see Note 1). 6. Redissolve the pellet in 50 & of TE and extract with an equal volume of TE saturated phenol. Remove the upper aqueous phase and transfer it to a clean microcentrifuge tube. 7. Add 5 pL of 3M NaOAc and 150 @Lof 95% ethanol and precipitate the DNA m dry ice for 20 minutes. 8. Centrifuge the tube m a microcentrifuge for 10 minutes, pour off the supernatant, wash the pellet with 1 mL of 95% ethanol, pour off the ethanol, and vacuum dry the pellet for 5 mm. 9. Redissolve the DNA pellet m 9 ~.ILof deiomzed water and place the capped tube m a boiling waterbath for 3 min. 10. Transfer the tube directly to dry ice and leave 1 min (see Note 2). 11. Add to the tube, 1 & of reverse primer, 1 cls,of TM, briefly centrifuge the tube, and place it in a 37°C waterbath for 30 mm. 12. Proceed with the desired DNA sequencing protocol, at the point lmmediately following the annealing step (see Note 3).

4. Notes 1. The PEG precipitation IS intended to remove the deoxynucleotldes from the DNA solution. Any carry-over of these nucleotides would Interfere with the subsequent sequence reactions. 2. Plunging the denatured DNA solution in water, mto dry ice is intended to prevent renaturation of the double-stranded template. By performing the annealing at a reduced temperature, primer annealing 1sfavored. 3. A more complicated procedure for reverse sequencing involves reverse cloning. After primed synthesis, the template derived double-stranded DNA is subjected to a restriction enzyme double-digest to excise the insert. The resulting fragment is then subcloned into a vector carrying an inverted polylinker region (as in the paired vectors mplUmp19)

References 1 Bankler, A. T and Barrell, B. G (1983) Shotgun DNA sequencing,m Techniques m Life Sciences, Nuclerc Acid Biochemutry Vol. B5 (Flavell, R. A., ed.), Elsevier, Ireland, pp l-34. 2. Bankier, A. T , Weston,K. M., andBarrell, B. G (1987) Random cloning and sequencmgby the M 13/dldeoxynucleotidechain terminationmethod,in Methods in Enzymology Vol 155 (Wu, R , ed ), Academic Press,London, pp 51-93

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3. Hong, G. F. (1981) A method for sequencmg single-stranded cloned DNA m both directions. Bioscience Reports, 1, pp 243-252 4. Saiki, R. K , Scharf, S., Faloona, F., Mullis, K., Horn, G. T , Erlich, H. A , and Arnheim, N. (1985) Enzymatic amplificatron of P-globin genomic sequences and restrtction site analysis for diagnosis of sickle cell anemia. Science, 230, 1350-1354 5 Gyllensten, U. B. and Erlich, H A (1988) Generation of single-stranded DNA by the polymerase cham reaction and its application to direct sequencmg of the HLA-DQA locus. Proc Natl. Acad. Sci USA ,85,7652-7656.

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31

Terminal Labeling for Maxam and Gilbert

of DNA Sequencing

Eran Pichersky

1. Introduction There are two basic enzymatic activities that are used to end-label DNA with radioactive phosphate (32P). The enzyme T4-polynucleotide kinase will use the substrate ATP to add a phosphate group (the gamma-phosphate of the ATP molecule) preferentially to the 5’ ends of the molecule. The enzyme DNA polymerase will “fill-in” recessed 3’ ends with the complimentary nucleotides, and can also create recessed 3’ ends from “blunt” ends or even 3’ overhanging ends by its 3’-5’ exonuclease activity, then fill in such ends as well. However, the polymerase enzyme usually used for the fill-in reaction, the Klenow fragment of E. coli DNA Pol I, has very low 3’-5’ exonuclease activity, and therefore its fill-in activity on blunt and 3’ overhanging ends could in principle be ignored for the purpose of end-labeling DNA for sequencing (see Note 1). A linear double-stranded DNA molecule produced by digestion with a restriction enzyme, almost always the starting material for endlabeling DNA (since virtually all restriction enzymes work on doublestranded DNA), has in effect two 5’ ends and two 3’ ends. Thus, labeling with the T4 DNA polynucleotide kinase will label the two 5’ ends (at opposite ends of the double-stranded DNA molecule), and From: Methods m Molecular Brology, Vol 23: DNA Sequencing Protocols Ed&d by- H. and A. Gnffm Copyright 01993 Humana Press Inc., Totowa, NJ

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labeling with the Klenow enzyme will produce two labeled 3’ ends, again at opposite ends of the double-stranded molecule. Note, however, that each strand is labeled on one end only. At this point, there are two alternatives to obtaining labeled DNA suitable for chemical sequencing. One method is to denature the double-stranded molecule and isolate the two strands (each of which is labeled at one end only) separately. Although it is in principle sometimes possible (for example, by a long electrophoretic run on a denaturing gel), the alternative method is much easier and therefore isolating end-labeled singlestranded DNA for sequencing is almost never done. The alternative is to digest the double-stranded DNA molecule, labeled at both ends, with another restriction enzyme that cuts m between the two ends, producing two fragments (hopefully of unequal length) each labeled at only one end of the four ends of the double-stranded molecule. The two fragments can then be separated on a nondenaturing acrylamide gel, reisolated, and used directly for sequencing. Although such a fragment is a double-stranded molecule, only one strand in this molecule is labeled, and that strand is labeled on one end only. All the chemical reactions are carried out as usual. The sample is then denatured and electrophoresed (see Chapter 32); the degradation products of the unlabeled strand do not show up on the autoradiograph and otherwise have no effect on the sequencing process (I). An important issue for consideration is the starting material for the labeling reaction. The DNA of interest may be cloned in a plasmid, or it may be a recombinant phage, or a PCR product. We find that CsCl gradient-purified DNA is the best source, although plasmids obtained by various “mini-prep” procedures are also suitable. The DNA has to be cut with a restriction enzyme, producing a minimum of one linear fragment, or more. If the source of the DNA produces more than one fragment upon restriction digest and one wishes to sequence only one particular fragment, it would appear to be advantageous to isolate this fragment prior to the end-labeling reaction, for example by electroelution from an agarose gel. However, each purification step results in some loss of DNA (and it is always advantageous to reisolate the fragments again after the labeling reaction, to remove the unincorporated label and, in cases where more than one fragment is labeled, to separate the fragments from each other). In

DNA End-Labeling addition, fragments that have been eluted from agarose gels do not label as well as fragments still in the original restriction enzyme reaction tube. Thus, for the highest yield and specific activity, it is best to do all the steps in one reaction tube without any successive purification steps or buffer changes. The fragments are then reisolated by separating them on acrylamide gels, meaning that they should be in the range of 100-1000 nucleotides (see Note 2). How can a DNA fragment labeled on one end only be produced in a single reaction tube without any buffer changes? To label DNA on one end only we take advantage of the fact that the Klenow fragment of E. coli’s DNA polymerase I will label a recessed 3’ end (“fill-in” reaction) but will not label a recessed 5’ end (=overhanging 3’ end) or a blunt end (in principle, the enzyme will label the latter two ends also, but to a much lower extent than the recessed 3’ end, so the fragment is practically labeled at one end only; seeNotes 1 and 3). Thus, if a restriction site giving a recessed 3’ end is present and we wish to label it for sequencing, all that is required is to find a second site nearby that is cut by an enzyme producing ends that will not label. For example, the restriction enzymes PstI, SacI, SphI (3’ overhang), and DraI, HueIII, and RsaI (blunt end) produce sites that under the conditions we use are practically unlabeled in the reaction described below. It should be noted that because the labeled fragments need to be separated on acrylamide gels, there is an upper size limit to the fragment that should be produced (fragments > 1 kb will not be resolved well on the gel, and will also not diffuse out efficiently). When there is no known useful site nearby the site that is being labeled, we simply use a frequent 4-bp cutter, such as HueIII, on the assumption that a site does occur at some not-too-great distance from the site being labeled. If that is not the case, additional 4-bp cutters can be used until the appropriate one is found. Also, when a small fragment is cloned into the polylinker site of a plasmid, one can take advantage of the restriction sites of the polylinker, so that two enzymes are chosen, one at each end of the cloned fragment, with one enzyme producing a 3’ recessed end and the other a 3’ overhanging end. In this way the fragment can be labeled on one end only, and by choosing a different pair of enzymes in a separate reaction, the other end of this fragment can be labeled as well.

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2. Materials 1. DNA: CsCI-purified plasmid, 1 pg/pL. 2. Restriction enzymes. 3. Restriction enzyme buffer (supplied by the manufacturer). 4. Klenow Fragment of DNA Pol I. 5. [a-32P]dATP; Specific activity = 3000 Wmmole. 6. 10 mil4 dCTP. 7. 10 mM dTTP. 8. 10 mM dGTP. 9. Loading dye: 50 mM EDTA, 10 mM HCI, pH 7.5, 0.19 (w/v) bromophenol blue, 0.1% (w/v) xylene cyan01 FF, 50% glycerol. 10 10X TBE buffer: 0.5M Tris-HCl, 0.5M Boric acid, 10 mM EDTA, pH 7.5 (2). 11. Acrylamide. 12. Bis-acrylamide. 13. 100% ethanol. 14. 0.5M NH,Acetate, 1 mM EDTA, pH 7.5. 15. Glass pipets. 16 Glasswool. 17. ddH,O (dd = double-distilled).

3. Method 1. Digest 1 pg of DNA m 30 pL total vol that mcludes l-3 U of each enzyme (e.g., 2 U EcoRI and 2 U HueIII). Incubate at 37°C for 30 mm. 2. Mix the followmg: 1 @ dlTP, 1 pL dCTP, 1 pL dGTP, 1 pL 32P(alpha)dATP, 1 U of Klenow enzyme, then add to the reaction tube and incubate for 20-30 min at 37°C. 3. Stop the reaction by adding 20 pL loading dye (note: this is the same dye used for agarose gels, but is not the dye used m sequencing gels). 4. Load sample on a 15-cm long, 0.3-mm thick nondenaturing 5% acrylamide gel (2O:l acrylamide:btsacrylamide, 1X TBE buffer) with well width of 3-4 cm. Run the gel until the bromephenol blue dye is at the bottom. Stop the gel, take one glass plate off, then cover the gel with SaranwrapTM. In the darkroom, put an X-ray film over the covered gel, mark the film’s position on the gel, and expose for 5-10 mm. Then develop the film. 5. By putting the developed X-ray film against the gel, locate the posrtion of the labeled DNA fragments, cut and remove the appropriate gel region, and place it m a 1.5-mL Eppendorf tube. Do not cut the gel piece mto smaller pieces. Overlay wtth 0.6 mL 0.5M NH4Acetate, 1 mM EDTA,

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pH 7.5 solution, shake well, and make sure the entire gel piece is submerged, then incubate at 37” C for a minimum of 8 h (or overnight). 6. Pipet out the contents of the tube into a second tube, passing the solutton through a pipet fitted with glasswool at the bottom. Add 1 mL 100% ethanol to the tube, shake well, and immediately centrifuge for 10 mm. Decant the supernatant, fill the tube again with ethanol, spin for 2 min, then decant again and aspirate the rest of the solution with a drawn Pasteur pipet. Dry in vacuum for 10 min. Add ddHzO (50-100 pL, depending on the amount of radioactivity) and resuspend the sample. The DNA is ready for sequencing (see Chapter 32).

4. Notes 1. The labeling reaction with the Klenow enzyme is done directly in the restriction digest tube, without any change of buffer. The restriction digests are done with either low-, medium-, or high-salt buffers, according to the requirement of the enzymes, and all these buffers also contam Mg*+. The Klenow enzyme works well in all these buffers, so for labeling all that IS required is the addition of the cold and radioactive nucleotides, and the Klenow enzyme. It is also possible to do the entire process at once-add the restriction enzymes and the nucleotides and the Klenow enzyme together. However, we prefer to do the labeling reaction for only 20-30 mm, and the restriction of 1 pg of DNA should usually be allowed to proceed for about 1 h. Hence, the labeling mix should be added about 30 min after the start of the restriction reaction. We always use 32P-dATP when possible, since this is the radioactive nucleotide we use for all other purposes in the lab. Almost all 3’ recessed sites can be labeled with dATP. It is essential to add the other nucleotides that occur in the site as cold nucleotides. If they occur upstream to the position of the A nucleotide, the latter nucleotide cannot be added until the others are. If they occur downstream to the position of the A nucleotide, it is nevertheless advantageous to include them in the reaction because they are present at much higher concentrations than the 32P-dATP, so once the A nucleotide is added, the positions downstream are added with almost 100% efficiency, and the Klenow enzyme cannot go back and remove the A nucleotide with its 3’-5’ exonuclease activity. As a matter of routine we add all three cold nucleotides (dTTP, dCTP, dGTP) even when one or another of these nucleotides do not occur at the site. The unneeded nucleotide has no effect on the labeling reaction. Another beneficial effect of the cold nucleotides is that at the other end (usually a 3’ overhanging end or a blunt end), even if the 3’-5’

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exonuclease activity of the Klenow enzyme produced a 3’ recessedend, it would instantaneously be filled m with a cold nucleotide, except of course when that position requires an A nucleotide. It would seem that in this latter case one would get a higher background m the sequencing autoradiograph, but we have not found this to be a problem, possibly because of the slowness of the 3’-5’ exonuclease activity of the Klenow enzyme and the relative shortness (20 mm) of the fill-m reaction. 2, We use 5% nondenaturing acrylamide gels to purify labeled fragments The fragments to be isolated are m the 100-1000 bp range. It is not worth the effort to sequence shorter fragments, although it is possible to isolate such fragments on acrylamide gels, and fragments > 1 kb do not resolve well on such gels. Although large fragments can be isolated from agarose gels, m our hands we have never been able to sequence fragments that have been directly isolated from agarose gels. (The DNA could go through agarose gel prior to labeling if it is subsequently reisolated from acrylamide gels prior to sequencing, but fragments sequenced directly after isolation from agarose gels do not yield readable sequence.) To elute the fragments, we simply incubate the gel slice, without any further maceration, in the elution buffer overnight at 37°C. The DNA fragment diffuses out quite well, although larger fragments diffuse more slowly. After overnight incubation, tubes containing fragments of approximately 1 kb will have at least 50% of the radioactivity in solution, and that fraction is higher for smaller fragments. The solution is then passed through glasswool to remove large pieces of acrylamide gel. The remammg acrylamide (monomers or small polymers) does not interfere with any of the sequencing reactions or subsequent manipulations of the DNA, even though it does precipitate together with the DNA. To precipitate the labeled DNA, we add 2 vols of 100% ethanol at RT, mix, and immediately centrifuge. There is no need to use any other concentration of ethanol. The goal is to get the DNA to precipitate with as little salt coprecipitation as possible. Thts is accomplished by aspirating all the liquid after the ethanol precipitation step, and again after the ethanol wash step.The pellet forms mcely on the side of the tube, and it is easy to put the end of the stretchedPasteur pipet all the way to the bottom of the tube and asprrate all the hquid. Repeated cycles of resuspension and precipitations are unnecessary and inadvisable. Prolonged incubation at low temperatures 1salso strongly discouraged. DNA precipitates well at RT, but one gets more salt precipitation at low temperature, thus making things worse, not better (see also Note 3 in Chapter 32).

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3. The DNA-labeling procedure involving T4-polynucleotlde kmase IS lengthy and requires the use of a substantial amount of radioactivity and several changes of buffers. Because of these disadvantages, we never use this enzyme for end-labeling for sequencing.

References 1. Maxam, A. M and Gilbert, W. (1980) Sequencing end-labeled DNA with basespecific chemical cleavages. Method. Enzymol 65,499-560 2. Mama&, T , Fntsch, E F., and Sambrook, J. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

&MI’ER

32

DNA Sequencing by the Chemical Method Emn Pichersky

1. Introduction The chemical method of sequencing DNA (1) has some advantages and some disadvantages compared with the enzymatic method (2). The major disadvantage is that it takes more time to produce the same amount of sequence. This is so for two main reasons. First, the DNA has to be end-labeled and then reisolated prior to the actual chemical sequencing reactions, a process that usually requires an additional day (see Chapter 31). Also, because more DNA is used in the reaction and because the lower specific activity of the sequencedDNA requires the use of an intensifying screen in the autoradiography, bands are not as sharp as in the enzymatic method and therefore it is difficult to obtain reliable sequencepast about nucleotide 250 (unless very long gels are run). Nevertheless, the chemical method is often useful for several reasons, It enables one to begin sequencing anywhere in the clone where a labelable restriction site occurs (see Chapter 3 1 for an easy method for end-labeling) without any further subcloning. The sequence thus obtained can then be used to synthesize oligonucleotide primers for enzymatic sequencing. In addition, in cases of regions that give poor results in the enzymatic reactions (because of secondary structures From Methods fn Molecular Bology, Vol 23: DNA Sequencmg Protocols E&ted by H. and A. Gnffm Copynght 01993 Humana Press Inc., Totowa, NJ

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that inhibit the polymerase enzyme), the chemical method almost always resolves the problem and yields the correct sequence. The chemical sequencing reaction has acquired a reputation of being difficult. We believe this reputation in undeserved. In our hands, the chemical method is consistently successful in producing results as reliable as those obtained by the enzymatic method. It has been our experience that many protocols in molecular biology include unnecessary steps. The likely explanation is probably that when researchers encountered difficulties, they added these steps as a solution to the problem, often on the assumption that the additional steps would not hamper the process, even if they did not help. This is clearly not the case here. In developing the method presented here from preexisting protocols, we have eliminated many steps. In general, we have found that the quality of the sequence has improved with the progressive elimination of these steps. It is still possible that some steps included here are not necessary; certainly no additional steps need to be added. And, of course, the end result has been that the protocol as presented here is very short and the entire process of sequencing (starting with end-labeled DNA) and gel electrophoresis can be accomplished in one (long) day. 2. Materials 1. G Buffer: 50 m1I4sodium cacodylate, pH. 8.0.

2. CT/C Stop: 0.3M sodium acetate,1 mM EDTA, pH 7.0. 3. GA Stop: 0.3M sodium acetate, pH 7.0. 4. G Stop: 1SM sodium acetate, IM 2-mercaptoethanol, pH 7 0. 5. 10% Formic actd. 6. Dtmethyl sulfate (DMS) (see Notes 1 and 4). 7. Hydrazine (95% anhydrous) (see Notes 1 and 4). 8. 100% ethanol. 9.5M NaCl. 10. ddH20 (dd = double disttlled). 11. Carrter DNA: 1 mg/mL m ddHzO (any DNA ~111do; we use plasmtd DNA) 12. 10 mg/mL tRNA in ddH*O (any tRNA). 13. The DNA fragment to be sequenced, end-labeled at one end only,

in ddH,O. 14. 10% piperidine (drlution prepared on the day of the expernnent). 15. Loading buffer: 100% formamrde, 0.1% (w/v) bromophenol blue, 0.1% (w/v) xylene cyan01 FF.

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3. Method 1. For each DNA fragment to be sequenced, mark four 1S-mL Eppendorf “reaction” tubes and add the following solutions: “G” tube: 1 pL carrier DNA, 200 pL G Buffer, 5 pL labeled DNA. “GA” tube: 1 pL carrier DNA, 10 pL ddHzO, 10 pL labeled DNA. “CT” tube: 1 pL carrier DNA, 10 pL ddH*O, 10 pL labeled DNA. “C” tube: 1 pL carrier DNA, 15 pL SM NaCI, 5 pL labeled DNA 2. Mark up four 1.5-mL Eppendorf “stop” tubes and add the following solutions: “G Stop” tube: 2 pL tRNA, 50 pL “G Stop” solution, 1 mL ethanol. “AG Stop” tube: 2 pL tRNA, 200 pL “AG Stop” solution, 1 mL ethanol. “CT Stop” tube: 2 pL tRNA, 200 pL “CT/C Stop” solution, 1mL ethanol. 2 pL tRNA, 200 pL “CT/C Stop” solutron, 1 mL ethanol. “C Stop” tube: 3. To start the reactions (see Note 2), add the following: “G” tube: 1 pL DMS, mix, and let the reactron proceed for 5 mm at RT (see Notes 1 and 4). “AG” tube: 3 pL 10% formic actd and mix (15 min at 37°C) (see Note 4). “CT’ tube: 30 pL hydrazrneand mix (9 mm at RT) (seeNotes 1and 4). “C” tube: 30 pL hydrazme and mix (11 min at RT) (see Notes 1 and4). 4. Stop each reaction by pipetting the contents of the corresponding stop tube mto the reaction tube (use the same Pasteur ptpet; slight crosscontamination of stop solutions has no effect, but do not touch the contents of the reaction solutions with the pipet). Cap the reaction tubes, shake briefly but vigorously, and place in a dry ice-ethanol bath (-80°C) for 3-10 mm (3 min are enough, but the tubes can be left there for up to 10 min rf other reactions are not done yet; do not leave for longer than 10 min) (see Note 3). 5. Centrifuge at 4°C for 7 min, discard supernatant, aspirate the rest of the liquid with a drawn Pasteur prpet, and then add 1 mL of 100% ethanol to the tube, invert twice, and centrifuge for 2 min at RT. Aspirate as before, and dry in a vacuum for 10 min. 6. To each reaction tube, add 100 pL of the 10% piperidine solution (do not shake the tubes as there is no need to resuspend the DNA) and place the uncapped tubes in a 90°C heat-block. After 15-30 s, cap the tubes and let stand for 30 mm. 7. Remove the tubes from the heat-block, let stand at RT for 2-5 min, centrifuge briefly to get the condensation to the bottom. Puncture one hole in the cap with a syringe, then place in dry ice-ethanol bath for 5 mm. 8. Place the tubes in a SpeedVac machine and lyophihze for 2 h. Vacuum should be below 100 milhtorr.

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9. Prior to gel electrophoresis, add 10 pL of loading buffer to each tube, resuspend the sample by shaking and then a brief centrifugation, and place the tubes in the 90°C heat-block for 10 min. Load l-2 pL per sample (see Note 5).

4. Notes 1. Quality of chemicals: In general, the standard laboratory grade chemicals should be used. Some chemicals, however, could be the cause of problems when not sufficiently pure or when too old (presumably degradation products are the culprits). We have only had problems with two chemicals (see below): dimethyl sulfate and hydrazme. Note also that these two chemicals, together with piperidme, are hazardous chemicals. In addition to observing the rules pertaining to the handling of radioactrve chemicals, all reactions involving these three chemicals should be carried out in the hood. 2. Reaction times: We typically do all chemical reactions together, timing them so that they end at the same time. If one is sequencmg 5 different fragments, all 20 tubes can be spun together in a single run (we have a microcentrifuge with 20 slots). When stopping all 20 reactions at about the same time, some reactions are invariably going to run longer than the allotted time. This is usually not a problem, because the reaction times indicated above are general, and they can be extended by up to 30% without much noticeable effect. 3. Precipitation: We always use 100% ethanol. There is no need to use any other concentration of ethanol at any step of the process, and 100% ethanol has the advantage because it evaporates fast. The goal is to get the DNA to precipitate with as little salt coprecipitation as possible. This is accomplished by aspirating all the liquid after the ethanol precipitation step, and again after the ethanol wash step. The pellet forms nicely on the side of the tube, and it is easy to put the end of the stretched Pasteur pipet all the way to the bottom of the tube and aspirate all the liquid. Repeated cycles of resuspension and precipitattons are madvisable. Prolonged incubation m the dry ice-ethanol bath is also strongly discouraged. DNA precipitates well at RT, but one gets more salt precipitation at low temperature, thus making things worse, not better. We almost always precipitate DNA at RT; the only reason Step 4 calls for incubation at -80°C is to inhibit further reaction with the reactive reagents, that at this stage have not yet been removed. 4. Troubleshooting* G reaction: This reaction is usually very clean, but it is the reaction most sensitive to prolonged incubation and to the quality of the reactive reagent, DMS. If reaction proceeds longer than the allot-

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ted time or if old or bad quahty dimethyl sulfate is used, excessive and nonspecific degradation of DNA will occur. Also, when several Gs occur in a row, the 3’-most Gs (lower bands if the 3’ end was labeled with the Klenow enzyme) may appear weaker. AG reaction: This is usually a trouble-free reaction. CT and C reactions: The quahty of the hydrazine should be good (it does not have to be exceptional), otherwise excessive and nonspecific degradation will occur. Sometimes faint bands will be seen m the C and CT lanes when the base IS G (a strong band IS then observed in the G lane). The likely explanation 1sthat the pH m the reaction tubes is too low (there is no buffer m the C and CT reaction tubes, but carryover with the DNA sample might cause this to happen). However, these faint bands are not nearly as strong as the slgnal m the G lane or as bona fide bands of C and T bases. Also, the T bands in the CT lane are often not as strong as the C bands--this is probably caused by mhibltion of the reaction by residual salt (in the C reaction, salt is added specifically to obtain complete inhibition). 5. Gel electrophoresis: We use a 60 x 40 cm (O-3-mm thick) gel of 6% acrylamide (20: 1 acrylamlde:blsacrylamide, 50% urea, 50 mM Tns, 50 miI4 borate, 1 rniV EDTA [3]). We run the gel at constant power (65 W), with an alummum plate to disperse the heat. The samples are loaded twice (a “long run” and a “short run”): The second loading IS done when the xylene cyan01 dye of the first sample is approx two thirds of the way down the gel, then electrophoresls is halted when the bromopheno1 blue of the second sample reaches the bottom of the gel. The complete run takes 5-6 h, and it allows us to read, in the “short run,” the sequence from about nucleotlde 25 to nucleotide 120-150, and in the “long run” the sequence from nucleotide 100 to about 220-250. Addltlonal sequence may be obtained by running longer gels, by loading the sample a third time, or by a variety of other methods if so desired.

References 1. Maxam, A. M andGilbert, W. (1980) Sequencingend-labeledDNA with basespecific chemical cleavages Meth Enzymol. 65,499-560 2 Sanger,F., Nicklen, S.,andCoulson,A R. (1977) DNA sequencingwith chamterminating inhibitors Proc Natl. Acad. Sci USA 74,5463-5467. 3 Maniatis, T., Fritsch, E. F., and Sambrook, J (1982) Molecular Cloning, A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

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DNA Sequencing by Chemical Degradation Using One, Two, and Four Different Fluorophores And&

Rosenthal

1. Introduction There are several chemical methods for sequencing DNA. All of them are based on gel separation of cleaned fragments. However, they use different chemistries to generate these fragments. The classical method, introduced in 1977 by Maxam and Gilbert (I), uses four different chemical reactions to attack in a radioactive end-labeled restriction fragment the heterocyclic bases at guanine (G), adenine and guanine (A + G), thymine and cytosine (T + C), and cytosine (C). In a second reaction with hot piperidine the modified bases are opened and/or removed followed by the cleavage of the DNA backbone at these positions (2). Thus, four sets of nested subfragments are produced and then analyzed in four lanes of a polyacrylamide gel. After autoradiography, the nucleotide sequence can easily be determined from the sequence ladder. Several improvements of this method have been reported over the years. Besides new end-labeling techniques (3,4), they have mainly been focused on the performance of the chemical reactions. Many samples can be processed in parallel using a suitable solid support (5) or microtiter dishes (6). In another approach a single or a combination of two chemical reactions are used to cleave the end-labeled DNA fragment simultaFrom Methods in Molecular B/ology, Vol 23. DNA Sequencmg Protocols Edlted by. H. and A Griffin Copynght 91993 Humana Press Inc , Totowa,

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neously at all four bases and the broken fragments are analyzed in a single lane of the gel (7-11). Under certain conditions different cleavage rates for all four bases can be obtained and the nucleotide sequence is determined by measuring the band intensities only (9). In a third strategy, phosphorothioate internucleotide bonds are enzymatically incorporated into DNA at G, A, T, and C residues using four different primer extension reactions. This sulfur-containing DNA is then cleaved using alkylating agents and the reaction products analyzed in four lanes of the gel (12). Despite these improvements, the overall sequencing rate remains slow and the majority of researchers avoid, whenever possible, chemical methods for DNA sequencing. However, chemical degradation procedures for DNA sequencing will probably remain indispensable for large-scale sequencing projects, including eukaryotic genomes, for several reasons. 1, Genetic information is not only encoded by the sequence of the four common bases G, A, T, and C. Minor bases hke 5methylcytosine, N-methyladenine, and others are used by nature to store additional information and to direct cell differentiation and gene regulation. These unusual bases and their location in the genome can only be determined m uncloned genomic DNA by a combination of chemical sequencing, blotting, and hybridization techniques (13,14) or by combination of chemical sequencing and PCR based amplification schemes (15,16). 2. Protein/DNA mteractrons can easily be studied by chemical methods (15,17,18). 3. Screening for point mutations using the T-specific reaction with manganate, osmium tetroxide, and the C-specific reaction with hydroxylamine (29,20). 4. DNA sequences with strong secondary structure, caused by repetitive elements or high GC or AT content, can be determmed with more accuracy since artifacts owing to polymerase stops are ehmmated. 5. Direct sequencing of PCR products by chemical degradation is easily possible, and for difficult templates is often more accurate than dideoxy sequencing techniques (21,22). Automation of the existing chemical degradation methods is therefore an urgent requirement. The use of fluorophores in chemical sequencing has been delayed for some years because it was assumed that the dye molecules and their linkers would be destroyed under the drastic conditions of chemical cleavage.

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Recently, however, it was reported that fluorescein linked via an aminopropyl anchor group to oligonucleotides or DNA is stable during the reactions commonly used in chemical sequencing (9). Based on this finding, methods for automated sequencing of single-stranded and double-stranded DNA (9) as well as PCR products (21) have been developed that use the Pharmacia ALF sequencer for the on-line detection of the nucleotide sequence in four lanes or one lane during electrophoresis. Recently, the commercially available fluorescein dyes FAM and JOE, as well as the rhodamine dyes TAMRA and ROX, attached via an aminohexyl linker arm to the S-end of DNA have been also found to survive most of the chemical cleavage reactions. This opened the possibility to use them for various chemical sequencing strategies in combination with the ABI 373A sequencer (23). This chapter summarizes all the protocols developed so far for chemical degradation sequencing employing one, two, or four different fluorescent dyes as label.

2. Materials 2.1. Equipment 1. ALF DNA Sequencer (Pharmacla LKB Biotechnology, Uppsala, Sweden): For fluorescent chemical sequencing using one dye and four dlfferent lanes per clone, or using one dye and one lane per clone. 2. ABI 373A DNA Sequencer (Applied Biosystems Inc., Foster City, CA): For fluorescent chemical sequencing using four different dyes and one lane per clone, or using two different dyes and one lane per clone. 3. Techne PHC3 or MW I Thermal Cyclers and Techne Hi Temp 96 Microtiter Dishes (Techne [Cambndge] Ltd., Duxford, UK): For end-labeling by primer extension and PCR. 4. Techne Dry Block (Techne [Cambridge] Ltd.): With three aluminum blocks for 0.5-mL or 1S-mL snap-cap tubes. 5. SpeedVac Centrifuge (Savant Instruments Inc., Hlcksvllle, NY): For removing piperidine from sequencing samples by lyophllization. 6. IEC Centra C4 Centrifuge (International Equipment Company, a dlvlslon of DAMON, Dunstable, Bedfordshire, UK): For microtiter dishes. 2.2. Reagents 1. Ammolink-2: For incorporating amino groups at the 5’-end of an ollgonucleotlde during automated synthesis. ABI Part No. 400808, Applied Blosystems Inc. 2. FAM-NHS, JOE-NHS, TAMRA-NHS, ROX-NHS: Fluorescent dyes as N-hydroxy succimmldyl esters for labeling oligonucleotldes and sub-

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sequent use m the ABI 373A sequencer. The kit contams 5 mg of each dye predissolved in 60 l.tL of dry DMSO. Keep dry at -20°C in the dark. ABI Part Nos. 400985, 400986, 400981, and 400980, Applied Biosystems Inc. 3. 5 (and-6)-carboxy fluorescein-NHS ester, Cat. No. C- 131I, Molecular Probes, Inc., Eugene, OR. 4. Sequenase2.0 at 13 U/pL, United StatesBiochemicals, Cleveland, OH. 5. Taq polymerase(native) Taq at 5 U&L, Perkm Elmer/Cetus,Norwalk, CT. 6. Hybond M & G paper: For soltd-phase chemical degradation of DNA, Cat. No. RPN 1500, Amersham International, Aylesbury, Buckinghamshire, UK. 7. Dimethylsulfate p.A. > 99% (DMS), Cat. No. 41610, Fluka Chemte AG, Buchs, Switzerland. Very toxic by inhalation and contact with skin. Suspected carcinogen. Wear gloves and work only in a well-ventilated fume hood when removmg small amounts of DMS from its original bottle. Dispose aqueous solutions of this agent m 5M NaOH solution. 8. 50 n&J ammonium formate buffer, pH 3.5: Dissolve 315 mg ammomum formate (BDH) in 50 mL of water and adjust pH to 3.5 with formic acid. Make up to 100 mL with water. 9. 1% DMS in ammomum formate buffer, pH 3.5: Mix in a 2-n& snapcap tube under the fume hood 20 l.tL DMS with 2 mL of ammonium formate buffer, vortex, and use immediately. Prepare fresh before use. 10. Formic acid, BDH, or Merck. 11. 66% aqueous formic acid: Mix m a 2-mL snap-cap tube 1.32 mL of formic acid with 0.68 mL of water. Prepare fresh before use. 12. Hydrazine, anhydrous, Cat. No. 21,515-5, Aldrich Chemical Co. Ltd., Gillmgham, Dorset, UK. Very toxic by inhalation, m contact with skin, and if swallowed. Suspected carcinogen. Wear gloves and work only in a well-ventilated fume hood when removing small amounts of hydrazme from its origmal bottle. Dispose aqueous solution of this agent m 2M FeCl, solution. Store hydrazme m its origmal bottle at 4°C. Never aliquot concentrated hydrazine mto plastic tubes. 13.60% aqueous hydrazine: Mix m a 1.5-mL snap-cap tube under the fume hood 40 pL of me-cold water with 60 pL of hydrazme and use immediately. Prepare fresh before use. 14. Hydroxylammonmm chloride (or hydroxylamine hydrochlortde), Cat. No. 10129, BDH Limited Poole, UK, or Merck, Germany. Harmful if swallowed, irritating to eyes and skin. 15. 4M hydroxylamine hydrochloride, pH 6.0: Dissolve 27 g hydroxylamine hydrochloride in 50 mL of water. Add approx 30 mL of trtethylamme

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p.A. > 99.5% (Cat. No. 90340, Fluka AG, Switzerland) and stir solution until amine odor hasdlsappeared.Check pH and adjust to 6.0 with small amounts of the amine. Make up the solution to 100 mL with water.

Properly sealed, the solution can be kept for several months at 4°C. Check pH periodically. 16. Piperidine, Cat. No. 80640, Fluka Chemie AG. Highly flammable, toxic by inhalation and contact with skin. Wear gloves and work only m a well-ventilated fume hood when removing small amounts of piperidine from its original bottle. 17. 10% aqueous plperidine: Mix in a 2-mL snap-cap tube under a fume hood 200 pL of plperidine with 1.8 mL of water and use immediately. Prepare fresh before use.

18. 1M Na2C03/NaHC03buffer, pH 9.0: Dissolve 8.4 g of sodium hydrogen carbonate in 80 mL of water at room temperature. The NaHCO, will not completely dissolve at this temperature and the pH of this solution is around 8.0. Adjust pH to 9.0 by adding sodium carbonate decahydrate (5.4 g of Na,C03

l

10 H,O. Pass the solution

through a 0 2-pm dlspos-

able filter (Nalgene, Nalge). Properly sealed, the solution can be kept for several months at room temperature. Before use check pH and adjust if necessary.

19. 10X PCRbuffer: 500mM KCI, 100mM Tris-HCl, pH 8.3,50 mM MgC&. 20. 0.5M pipendme, 0.5M NaCl: Add to a 2-mL snap-cap tube 1 mL of 10% piperidine, 800 pL of water, and 200 pL of 5M sodium chloride. Mix well and use immediately. 3. Methods

3.1. Synthesis of Homemade Fluorescently Labeled Primers Several fluorescently labeled universal primers for use with the ABI 373A sequencer (-21 forward, reverse, T3, T7, SP6, KS, SK) or the Pharmacia ALF (-2 1 forward) are commercially available. These primers flank the multicloning site of some of the most common cloning/sequencing vectors (e.g., M13mp series, pUC series, pBluescript, pBluescribe) and can be used for end-labeling of recombinant DNA by primer extension or PCR followed by chemical degradation sequencing. For this purpose, however, larger amounts of tfiese primers are needed. In order to reduce costs we have therefore prepared some of the fluorescently labeled universal primers in larger batches. The dye attachment was performed in two chemical steps. First, a

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suitable amino linker molecule is introduced to the 5’-end of the oligonucleotide during automated DNA synthesis. For this purpose, we used a commercially available aminohexyl phosphoramidite derivative known as Aminolink-2. Appropriate dye NHS esters were then coupled to the 5’-amino moiety in an overnight reaction. The dye labeled oligonucleotides were then purified from the excess of dye as well as from the unreacted Aminolink- oligonucleotides by gel electrophoresis that allows processing of up to 12 samples in parallel. Dye oligonucleotides were recovered from polyacrylamide gel by elution with water and purified by filtration using 0.2~p filters. The following protocols summarize the essential steps for synthesis and purification used m our lab. 3.1.1, Synthesis of AmmolinkOlzgonucleotiofes and Ethanol Precipitation

All synthesis was performed using the three column DNA synthesizer 380B from Applied Biosystems. Any other synthesizer with extra phosphoramidite bottle ports can be used. 1. Synthesize oligonucleotides on a 0.2~pm01scale using cyanoethyl phosphoramidites. 2. Dissolve Aminolink- m 3.3 mL of anhydrous acetonitrile and install on an extra phosphoramidite bottle port of your DNA synthesizer. 3. Enter the Ammolink- as a 5’-termmal extra base in the primer sequence. Choose the Trrtyl “ON” ending option. 4. After synthesis and cleavage from the support on the DNA synthestzer incubate the ohgonucleotide/ammonia solutton overnight at 55°C to deprotect. 5. Split the ohgonucleottde/ammoma solutron from a 0.2~pm01 synthesis mto five ahquots of approx 0.5 mL and transfer mto 2-mL snap-cap tubes using a blue tip. 6. Add 50 pL of 3M sodium acetate, pH 5.2, and 1.5 mL of 95% ethanol, vortex briefly, incubate at -80°C for 30 mm, and precipitate oligonucleottde material by spinning the sample at 13,000 g m a mtcrocentrrfuge for 5 min. 7. Remove supernatant and rinse the pellet with 1 mL of 70% ethanol and spin the samples for 2 min at 13,000 g. 8. Remove supernatant and dry oltgonucleotide pellets briefly m a SpeedVac system. 9. Dissolve oligonucleotide material from each tube m 20 pL of water.

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3.1.2. Primer Synthesis Labeled with 5(and-6)-Carboxy Fluorescein (for Use in the Pharmacia A.LF Sequencer) 1. Transfer one 20-l.tL portion of the Aminolink- oligonucleotide (which corresponds to approx 120 pg or 4 Az6eU of a 20-mer oligonucleotide) mto a new 1.5-mL snap-cap tube. 2. Add 50 pL of water and 10 pL of 1M Na$Os/NaHCOs buffer, pH 9.0. 3. Dissolve 2 mg of 5-(and-6)-carboxy fluorescein-NHS ester in 100 pL of dry DMF (or DMSO) in a separate 1.5mL snap-cap tube and add 20 pL of this solution to the Aminolink- oligonucleotide. Incubate overnight at room temperature in the dark. (The dye/DMF solutton should be made fresh before use but five different oligonucleotides can be labeled in parallel.) 4. Add 10 pL of 3M sodium acetate, pH 5.2, and 350 pL of 95% ethanol. Incubate for 30 mm at -80°C. 5. Spin the tube for 10 mm at 13,000 g in a microcentrifuge and remove the supernatant that ISstrongly colored and contams mostof the excessdye. 6. Rinse the pellet twice wtth 500 pL of 80% ethanol, spm the tube for 2 min at 13,000 g, and remove the supernatants. The last supernatant should be nearly colorless. 7. Dry pellet m the SpeedVac centrifuge and redissolve in 20 pL of water.

3.1.3. End-Labeling

of AminolinkOligonucleotide with FM, JOE, TAMRA, and ROX Dyes (for Use in the ABI 373A Sequencer) 1. Transfer four 20-&L portions of the Aminolink- oligonucleotide (see Section 3.1-l., step 9) into four new 1.5-mL snap-cap tubes labeled FAM, JOE, TAMRA, and ROX. Each portion corresponds to approx 120 pg (or 4 A,,, U) of a 20-mer oligonucleotide. 2. Add 6 pL of 1M Na,CO,/NaHCO, buffer, pH 9.0 to each tube. 3. Add 6 pL of each of the four dyes FAM-NHS ester, JOE-NHS ester, TAMRA-NHS ester,and ROX-NHS ester,predissolved in dry DMSO, into the corresponding tubes. Incubate at room temperature overnight in the dark. 4. Add 70 pL water, 10 l.tL of 3M sodium acetate at pH 5.2, and 350 pL of 95% ethanol to each of the four tubes. Incubate for 30 min at -80°C. 5. Spin the tubes for 10 min at 13,000 g in a microcentrifuge and remove supernatants that are strongly colored and contain most of the excessdye. 6. Rinse the pellets twice with 500 pL of 80% ethanol, spin the tubes for 2 min at 13,000 g, and remove supernatants. The last supernatant should be nearly colorless. 7. Dry pellets in the SpeedVac centrifuge and redissolve in 20 pL of water.

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Although a 20-3OM excess of dye over Aminolink- oligonucleotide is used in the coupling reactions, the observed yields are usually between 50-80%. The coupling with JOE-NHS ester is usually much less efficient than those with all the other dyes, and yields of around 25% are observed. However, enough dye labeled primers are obtained for all dyes to be used in chemical sequencing. Before use, the labeled ohgonucleotides must be purified from unlabeled Aminolink- ollgonucleotide material and free dyes still present after ethanol precipitation. Several methods have been reported using either gel electrophores:s, HPLC, or ollgonucleotide purification cartridges (OPCTM , Applied Biosy stems). In our hands electrophoresis

using nondenaturing polyacrylamide gels is the quickest and most efficient method, especially when purifying

many samples m parallel.

1. Prepare a 20% polyacrylamlde gel containing 1X TBE without urea (1 mm thick, 30 cm wide, 40 cm length). Use a comb with at least 20 wells, each approx 1 cm wide. 2. Mix 10 pL of each of the dye ollgonucleotldes (see Sections 3.1.2. and 3.1.3., step 7) with 5 pL of formamide (IBI) and load it mto a single well of the gel. As a control, load 5 & of the appropriate Aminolinkparent compound in formamide adjacent to it. Use the outer lanes of the gels to run bromophenol blue and xylene cyan01as dye markers. Run the gel at 40 W (800-1200 V) for approx 3 h until xylene cyan01 migrated 12-l 5 cm from the well. Separation of the dye oligonucleotldes can be monitored directly through the glass plates under room light. 3. After electrophoresls the dye oligonucleotides can easily be identified under room light as the most brightly colored bands. The FAM primer 1spale yellow, JOE primer 1speach colored, TAMRA is bright pink, and the ROX primer 1spale violet. The fluorescein primer is equivalent to the FAM primer. Excise the appropriate bands and place the gel slices into individual 2-mL snap-cap tubes. If m doubt, place the whole gel section between bromophenol blue and xylene cyan01 on a silica gel TLC plate containing a fluorescence indicator and examine all bands using UV shadowing at 310 nm and 254 nm in the darkroom. The dye ollgonucleotides can be visualized at 310 and 254 nm, but the Aminolinkparent compound can only be seen at 254 nm. The dye ohgonucleotlde should migrate slower than the corresponding Ammolmk-2 compound.

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FAM-, JOE-, TAMRA-, and ROX-oligonucleotides show slightly different mobilities. 4. For elution, add 300 pL of water to each tube and incubate for 3 h at 60°C using the Techne dry block. Transfer the supernatant into a clean tube and repeat elution with another 300 pL portion of water. The dye oligonucleotide is then further purified by passing the combined supernatants through a 0.2~pm disposable filter (Acrodisc). 5. A 50-w aliquot is made up to 1 mL with water and a spectrum between 200and 650nm is recorded. From each dye oligonucleotide a 1@Vand a 10 w solution is made and kept, together with the stock, at -20°C in the dark.

3.2. Fhorescent End-Labeling of DNA by Primer Extension (see Note 1) End-labeling of recombinant DNA for chemical sequencing can easily be achieved by extending a dye labeled primer/template complex in the presence of a polymerase and all four deoxynucleoside

triphosphates (9). The resulting DNA fragments differ m length but have a defined 5’-end. All fragments are labeled at one end only. In a single extension reaction microgram or pm01 quantities of single-stranded DNA must be used. Double-stranded templates must first be denatured using alkaline treatment followed by ethanol precipitation. If Taq polymerase is used, however, end-labeling can also

be performed by a cycled extension reaction (linear amplification), or by the polymerase chain reaction (PCR). This has the advantage of needmg much less DNA (fmol range) and any single- or double-stranded template can be used without alkaline denaturing. Even single plaques or colonies or a few copies of total genomic DNA can directly be used as templates for an end-labeling reaction by PCR, All primer extension reactions can be performed in single tubes or in an individual well of a microtiter dish. 3.2.1. End-Labeling with One, Two, or Four Different Using a Single Primer Extension Reaction

Dyes

In this protocol a disposable microtiter dish (Falcon 3911) is used for the primer extension reactions. This allows labeling of up to 96, 48, or 24 different single-stranded templates if one dye (carboxy fluorescein), two different dyes (FAM and JOE), or four different dyes (FAM, JOE, TAMRA, and ROX) are used, respectively.

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Double-stranded plasmid DNA is first denatured with 0.2M NaOH for 5 min at room temperature, neutralized with 3M sodium acetate at pH 5.2, and ethanol precipitated. The pellet is rinsed with 70% ethanol, briefly dried in a SpeedVac system, and resuspended in water. 1. Labeling with carboxy fluorescem: Add 2 pL of denatured plasmid template (at a concentration of 2 pg/pL) or 2 pL of single-stranded Ml3 template (at a concentration of 1 pg/pL) to each well of a microtiter plate kept on ice. 2. Labeling with FAM and JOE: For each template to be labeled, assign two wells of the mtcrotiter dish as FAM and JOE. To each of these wells add 2 pL of the appropriatetemplate DNA. 3 Labeling wtth FAM, JOE, TAMRA, and ROX: For each template to be labeled, assignfour wells of the microtiter dish as FAM, JOE, TAMRA, and ROX. To each of these wells add 2 pL of the appropriate template DNA. 4. Depending on the number of different dyes mcorporated during primer extension, one annealing mix is needed for the use of carboxy fluorescein, two different annealing mixes are needed for the use of FAM and JOE, and a set of four mixes are needed for the use of the four dyes

FAM, JOE, TAMRA, and ROX. For eachtemplate to be labeled, to a 1S-mL snap-cap tube add: 4 pL water, 2 pL 5X sequenase buffer, and 2 $ of the appropriate dye primer (1 w). Scale this up by the number of templates to be labeled. Vortex very briefly to mix. 5. Dispense 8 pL of the appropriate annealing mix on the side of each appropriate well of the mtcrotiter dish using a repetitive pipet (i.e., add carboxy fluorescein mtx to all wells, FAM mix to FAM wells, JOE mix to JOE wells, etc.). The same tip is used to dispense an individual annealing mix but different tips are used for the different mixes. Cover the wells with a layer of Saranwrap@and centrifuge the dish briefly to mtx reagents. 6. For annealing, incubate the mtcrotiter dash m an oven at 55°C for 30 min. Centrifuge the dish to concentrate any condensate, remove the Saranwrap@, and place the dish on ice. 7. In a 0.5-mL snap-cap tube on Ice prepare an extension mix containing* 1 pL of O.lM DTT, 2 pL of 2M dNTP, and 2 pL of dtluted sequenase 2.0 (1 U&L). Scale this up as required. 8. Without delay add 5 pL of thus extension mix on the side of each well of the microttter dish using a repetitive ptpet. Cover the wells with a layer of Saranwrap@ and centrifuge the dish briefly to mtx reagents. 9. Incubate the dish at 37°C for 3 min. 10. Heat denature the covered extension reacttons for 5-10 mm in an oven at 80°C centrifuge briefly to concentrate any condensate, and place tray on ice. Remove Saranwrap@

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3.2.2. End-Labeling with One, Two, or Four Different Dyes Using a Linear Amplification Reaction In this protocol a thermostable Taq polymerase is used to extend the dye labeled primer and the reaction is repeated several times (cycled) using a suitable thermocycler. This leads to a linear amplification of the target DNA that is labeled at one end only. The use of a disposable thermostable polycarbonate microtiter dish (Hi Temp 96, Techne) allows labeling of up to 96 different templates with one dye, 48 different templates with two dyes, or 24 different templates with four dyes. 1. Labeling with carboxy fluorescein: Add 2 pL of single-stranded or double-stranded DNA template (Ml 3 DNA at a concentration of approx 0.1-0.2 pg/pL plasmid DNA at a concentration of approx 0.2-0.4 pg/ pL, cosmid DNA at a concentration of 2-3 pg/pL) to each well of a microtiter plate kept on ice. 2. Labeling with FAM and JOE: For each template to be labeled asslgn two wells of the mlcrotlter dish as FAM and JOE. To each of these wells add 2 & of the appropriate template DNA. 3. Labeling with FAM, JOE, TAMRA, and ROX: For each template to be labeled assign four wells of the microtiter dish as FAM, JOE, TAMRA, andROX. To each of thesewells add 2 pL of the appropriate template DNA. 4. Depending on the number of different dyes incorporated during primer extension, one amplification mix is needed for the use of carboxy fluorescein, two different mixes are needed for the use of FAM and JOE, and a set of four mixes are needed for the use of the four dyes FAM, JOE, TAMRA, and ROX. For each template to be labeled, to a 2-mL snap-cap tube kept on ice add: 13 pL water, 2 w 10X PCR buffer, 2 pL 2.5 mM dNTP mix, 1 pL of the appropriate 10 w dye primer, and 1 U of Taq polymerase. Scale this up by the number of templates to be labeled. Vortex very briefly to mix. 5. Without delay dispense 18 pL of the appropriate amplification mix on the side of each appropriate well of the microtiter dish using a repetitive pipet (i.e., add carboxy fluorescein mix to all wells, FAM mix to FAM wells, JOE mix to JOE wells, and so forth). The same tip is used to dispense an individual amplification mix, but different tips are used for the different mixes. Overlay the samples with three drops (50 pL) of light mineral 011dispensed with a blue tip and centrifuge the dish briefly to mix reagents. 6. Place the microtlter plate in the Techne thermocycler preheated to 95°C and incubate the samples for 90 s. Thereafter, immediately start the following 25 cycles: (95°C for 30 s, 50°C for 1 mm, and 72°C for 1 min).

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7. After completton, denature the samples by heating the microtiter dish on the cycler for 3 min at 95°C. Place dish on ice for 5 mm. If necessary,keep the plate at -20°C until ready to proceed wtth the chemtcal reactions 3.2.3. End-Labeling with One, Two, or Four Different Dyes Using PCR PCR amplifications use two primers that hybridize to opposite strands of a specific DNA fragment, and a thermostable DNA polymerase that catalyze the strand copying reaction. The primers are oriented so that elongation proceeds inward across the region between the two primers. Repetitive cycles of denaturing the newly synthesized strands, annealing the primers, and DNA synthesis lead to an exponential amplification of the target DNA. Typically, a pair of universal primers is used that flank the DNA cloned into the multicloning site of a cloning vector. Since one of the primers IS labeled with a fluorescent dye, the resulting PCR product is labeled at one end only and can be directly used for the chemical sequencing reactions. The labeling protocol is nearly identical to that described in the previous section except for the following points: 1. Two pL of recombmant DNA at a concentration of 2-4 ng/pL is added to each appropriate well of a microttter dish. However, inserts of M13, single-stranded or double-stranded phagemtd, or lambda templates should not exceed 3-4 kb. In addition, white plaques or colonies can be directly toothpicked into the PCR reactton. Genomic DNA (100 ng/pL) may also be used as template (see Note 2). 2. For each template to be labeled, to a 2-mL snap-cap tube kept on ice add: 12 pL water. 2 pL 10X PCR buffer. 2 pL 2.5 mM dNTP mix. 1 pL of the appropriate universal dye primer at 10 l.W. 1 pL of a second unlabeled universal primer at 10 @4. 1 U of Tuq polymerase. Scale this up by the number of templates to be labeled. Vortex very briefly to mix 3. Place the microtiter plate m the Techne thermocycler preheated to 95°C and incubate the samples for 90 s. Thereafter, start the following 35 cycles: (95°C for 30 s, 50°C for 1 mm, and 72°C for 3 mm). 4. After completion, denature the samples by heating the microtiter dish on the cycler for 3 min at 95°C. Place dish on ice for 5 mm. If necessary,keep the plate at -20°C until ready to proceed with the chemical reactions.

Fluorescent

Chemical

3.3. Chemical

Sequencing

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273 Strategies

(see Note 1)

3.3.1. One Dye IFour Reactions IFour Lanes-Method The chemical degradation method described by Maxam and Gilbert (1,2, see Chapters 31-33) employs a minimum of four separate basespecific modification reactions followed by a strand cleavage reactlon using hot piperidine. The piperidine is then removed by repeated lyophilization. The original method involves numerous ethanol precipitations to remove excess reagents after modification. It is therefore tedious and not suitable for sequencing large numbers of samples. There are, however, two modifications to the original method that can be used to process many samples in parallel. Church and Kiefer-Higgins have used microtiter dishes to perform the degradation reactlons for up to ninety-six samples (6). In this setup, ethanol precipitation is no longer a problem because there is no need to handle individual tubes. In the other method, developed by Rosenthal et al. (5), the chemical reactions are carried out while the labeled DNA is immobilized on a suitable two-dimensional carrier matrix like Hybond M & G paper. In this way, many samples can be immobilized and processed in parallel because ethanol precipitation is not needed. Excess reagents are simply washed away, During piperidine reaction the anchor group used to link the DNA to the solid support is chemically cleaved and the DNA fragments are recovered in the supematant. Piperidine is then removed by lyophilization. This solid-phase chemical degradation method has originally been used to sequence radiolabeled DNA (5). It can also be applied to degrade DNA labeled with one, two, or four different fluorescent dyes (9,21,23). In the following protocol the essential steps of this method are described. 1. Preparing the carrier support and lmmobilizatlon: One single sheet of Hybond M & G, 50 x 50 mm in size, is cut into sixteen strips of 3 x 50 mm. Four strips, one for each of the four chemical modlficatlon reactions, are needed to sequence five samples. Sixteen strips thus provide sufficient space to sequence twenty different DNAs (see Note 3). On each strip, five squares, 3 x 3 mm in size, are marked by pencil allowing a safety distance of about 5-7 mm between squares. A number is assigned to each template to be sequenced and to each square of a strip. The same number is used on the other three strips to identify the template. Each strip is also labeled with one modification reaction: G, A + G, T + C, or C.

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Each DNA template to be sequencedand labeled with carboxy fluorescein (see Section 3.2.) ts immobilized by applying 1.5-w aliquots of it onto the four mdividual carrier segments (one on each of the four strips) possessing the appropriate number (see Note 4). After all templates are rmmobilrzed, loadmg is repeated with another 1.5~pLaltquot. Another two 1.5~pL ahquots are loaded onto the T + C segmentsonly, m order to balance losses of DNA m the reaction with aqueous hydrazme. 2. Washing: All rmmobrlized paper strrps are washed together m a glass beaker with 100 mL of water, followed by another wash with ethanol. Finally, the strips are placed onto filter paper and dried at room temperature for 5 min. Paper strips labeled with the same reaction, for example G, are then folded along their centerline and placed into a 2-mL snap-cap tube labeled G. Up to five different G strips (20 fragments) can be modified at the same trme m one tube Srmilarly, the A + G and C paper strips are placed in the appropriate tubes. The T + C paper strips, however, are placed between two glass slides. 3. Base-specific modification reactions: The followmg four modification reactions are carried out: G with 1% dimethylsulfate in 50 mM ammomum formate, pH 3.5 for 1 mm, A + G with 66% formic actd for 3 min, T + C with 66% aqueous hydrazine for 3 mm, and C with 4M hydroxylamine, pH 6.0 for 5 min. For the G, A + G, and C reactions 2 mL of the appropriate solutron is added to the individual tube. The paper strips must be completely covered by the solutron. For the T + C reactions approximately 20 pL of the aqueous hydrazine IS added to each paper strip so that they are Just moistened. 4. Washing: G, A + G, and C reactrons are termmated by removing the approprrate paper strips with fme forceps from the reaction tubes and washing them in a glass beaker three times with 100 mL of water, followed by one wash with 100 mL of ethanol. Each set of strtps is mdividually washed to avoid contammatron. The C + T strips are first washed three times with 100 mL of ethanol, followed by one wash wtth water, and finally one wash with ethanol. All strtps are then placed onto filter paper and dried at room temperature for 5 mm. 5. Sorting the carrier segments: The strips are cut into their 3 x 3 mm squares with a pair of scissors, and each square 1splaced accordmg to its pencil written code (template number and type of reaction) into an 1S-mL snap-cap tube labeled with the same code. 6. Pipertdine reaction and lyophilizatton: To each tube 100 uL of 10% prperidme is added to completely cover the carrier segment. All tubes are heated in a Techne dry block at 90°C for 30 mm. After this mcubation, the samples are briefly centrrfuged to collect the condensates, the

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tubes are opened, and the carrier segments removed using the tweezers. All samples are then lyophilized in a SpeedVaccentrifuge. Lyophilization is repeated with 50 ~.ILof water. Finally, the samples are resuspended in 8 pL of 50% aqueous formamide contammg 1% dextran blue. 7. On-line detection: For each clone, 2-pL aliquots are loaded onto a set of four lanes of a polyacrylamide gel. Samples are analyzed using the Pharmacia ALF sequencer. Raw data are plotted m four channels and 250-350 nucleotides per clone are manually determined. 3.3.2. Four Dyes /Four Reactions I One Lane-Method

In this method, a set of four fluorescent dyes (FAM, JOE, TAMRA, and ROX) is used to label DNA templates (see Section 3.2.) Each dye is assigned to one of the four common chemical cleavage reactions. The following two combinations are recommended: (i) G = FAM, A +G=JOE,T+C=TAMRA,andC=ROX,or(ii)G=ROX,A+G = TAMRA, T + C = JOE, and C = FAM. Solid-phase chemical degradation is performed as described m Section 3.3.1. but with the following two modifications. During immobilization, FAM-DNA is applied to the appropriate squareof the G-strip, JOE-DNA is applied to the corresponding square of the A + G-strip, TAMRA-DNA to its squareof the T + C-strip, and so on. For the piperidine reaction, the four paper segments of one template are placed together in one 1.5~mL snap-cap tube and simultaneously treated with 10% piperidine at 90°C. The papers are then removed from the tube and the piperidine is lyophilized. Because of the four color code, the degradation products of all four reactions can be analyzed in a single lane of the ABI 373A sequencer.

Analyzed data are plotted in four colors within one channel. With the present software, accurate base calling is not possible for these data. Typically, 200-350 nucleotides are manually determined from the plot using the four color code. 3.3.3. Two Dyes/Four

Reactions I One Lane-Method

This method is based on the idea that, because of the redundant information contained in the chemical cleavage pattern, two fluorescent dyes and signal intensities are sufficient to obtain sequenceinformation from one clone within a single lane. Use the fluorescein dyes FAM and JOE to label DNA templates (see Section 3.2.): Combine FAM with the G and the A + G reaction, and JOE with the T + C and the C reaction. Chemical cleavage is performed as described in Sec-

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tions 3.3.1. and 3.3.2. The degradation products of all four reactions are combined and analyzed in a single lane of the ABI 373A sequencer. Analyzed data are printed using only two colors (FAM and JOE), and the nucleotide sequence is manually determined from the plot using a combination of the two color code and signal intensities. Guanine and adenine bases have the same color (FAM), but Gs show approximately twice the intensity of neighboring As. Cytosme and thymine bases are displayed in the second color (JOE) but Cs are twice as intense as neighboring Ts. Typically, we can manually read up to 250 bp from a clone in a single lane. This method has some advantages over the technique applying four dyes. DNA labeling requires only two separate primer extension reactions and is therefore less tedious. In addition, the fluorescein dyes FAM and JOE show similar mobility characteristics and can be detected on-line with high sensitivity. There is no need to create mobility files for new sequencing primers so homemade dye labeled primers can be used to walk and sequence along a longer insert. We have been

using this technique for direct sequencing of genomic PCR products. 3.3.4. One Dye ITwo Reactions I Two Lanes-Method (see Note 5)

Instead of four separate reactions one can also use a set of two degradation reactions for each clone, such as the A + G reaction with 66% formic acid and the A > C reaction with 1.2M sodium hydroxide

(see Note 6). After piperidine cleavage the products are loaded onto two lanes of the gel. Samples are analyzed using the Pharmacia ALF sequencer and raw data are plotted in two channels. Using this method,

guanine, adenine, and cytosine bases can easily be determined by individual peaks. Thymine bases, however, have to be identified by gaps or very small signals. The amount of sequence information is therefore limited to 100-200 nucleotides for each clone.

The following protocol describes the experimental details of this method. The A > C and the A + G reaction are performed in solution using 1.5mL snap-cap tubes (see Note 7). 1. Dispense 5 pL of each carboxy fluorescein labeled template mto two 1.5mL. snap-cap tubes labeled A > C and A + G. Add 20 pL of 1.2M NaOH to the A > C tube and heat for 5 min at 60°C. Add 20 pL of 66% formic acid to the A + G tube and incubate for 3 min at room temperature.

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2. Place the A > C tubes on ice and add 10 pL of 3M sodium acetate, pH 5.2. Add 65 & water and 350 $ of ethanol. MIX well and incubate for 10 min on ice. Spin the tubes at 13,000 rpm for 5 min and remove supernatant. Rinse the pellet with 70% ethanol and dry the pellet briefly in a SpeedVac centrifuge. 3. Place the A + G tube in the SpeedVac centrifuge and remove the formic acid by lyophilization. 4. Add 100 pL of 10% piperrdine to the A > C and the A + G tube and incubate them for 30 mm at 90°C. Remove prperidine by lyophrlrzatton in a SpeedVac centrifuge. Lyophilization is repeated with 50 & of water. Finally, the samples are resuspended in 8 pL of 50% aqueous formamide containing 1% dextran blue and loaded onto two lanes of the ALF sequencer.

3.3.5. One Dye I One Reaction I One Lane-Method Fluorescein-labeled DNA is chemically degraded using only one reac-

tion comprising the methylation of G-residuesfollowed by a partial cleavage with piperidine in the presence of sodium chloride at all four bases (9). The cleavage products are loaded onto one lane of the gel and analyzed using the Pharmacia ALF sequencer.The nucleotide sequenceis manually determined from the plot by measuring different signal intensities following the rule G > A > C > T. Setting the G-specific fluorescence intensity at lOO%, the intensity values for A, C, and T residues are between 40 and 60%, 20 and 30%, and 5 and 10%. Intensities of neighboring peaks for the same base differ by less than 10%. Typically, lOO-200 nucleotides can be obtained from the labeled end. There are two advantages of this method. First, the chemical cleavage is very simple and many different templates can be processed in parallel. Second, on-line analysis is done in a single lane of the gel. Since the ALF sequencer has 40 lanes, 40 different clones can be analyzed simultaneously. This method should be used when only relatively short stretches of sequence are needed from many clones. 1. Transfer 5 @ of a DNA template labeled with carboxy fluorescern (see Section 3.2.) into a 0.5-mL snap-cap tube. 2. Add 10 pL of 1% dimethylsulfate in 50 mM ammonmm formate, pH 3.5, and incubate 2 min at room temperature. 3. Without delay, add 90 @Lof an aqueous 0.5M prpertdine solution contaming 0.5M sodium chlortde and incubate samples for 4-5 h at 90°C in a Techne dry block.

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4. Centrifuge sample briefly to collect condensate and add 20 pL of 3M sodium acetate, pH 5.2, and 350 pL of ethanol. Vortex the tube brtefly to mtx and Incubate the sample for 30 min at -80°C. The cleavage products are precipitated by centrifugatton for 10 min at 13,000 t-pm. After removal of the supernatant, the pellet is rinsed once with 70% ethanol, dried briefly in a SpeedVac centrifuge, and resuspended m 8-10 pL of 50% aqueous formamide containing 1% dextran blue. 5. Load 2-3 pL of each clone onto a single lane of the ALF sequencer.

4. Notes 1. In chemical sequencing, fluorescently labeled DNA may be replaced with radioactive labeled DNA. All protocols for end-labeling by primer extension

can also be performed

usmg a 32P-labeled

primer.

Radloac-

tive labeled DNA can be subjected to chemical degradation by the methods described m Sections 3.3.1.) 3.3.4., and 3.3.5., and the cleaved DNA fragments are then loaded onto four lanes, two lanes, or one lane of an ordinary polyacrylamide gel, respectively Sequence ladders are generated by autoradiography. 2. PCR products from genomtc DNA were usually separated from mmor bands by LMP agarose electrophorests. Products containing bands were cut out, melted, and isolated by phenol/chloroform extraction and ethanol precipitation. 3. Immobilization can also be performed on larger squares of Hybond M & G paper. Four sheets, 50 x 50 mm in size, provide sufficient space to immobilize eighty different samples for the four reactions. 4. Before immobilizatton of samples from labeling reactions generated by PCR or linear amplificatton, remove as much of the light mineral oil as possible. This can be done by either transferrmg all samples from beneath the oil into a new tube or microtiter dish, or by placing the mtcrotiter dish mto a freezer at -20°C for some time until the aqueous phase IS frozen and then removing the 011from the wells usmg a multichannel pipet. Although small amounts of light mineral oil do not affect the immobilization, try to avoid carrying over too much oil onto the carrier segment. 5. This method can also be used m combmation with two different dyes, such as FAM and JOE. The AC reaction IScarried out with FAM-labeled template DNA and the A + G reaction with JOE-labeled template DNA. After ptperldme reaction the cleavage products are combined and analyzed m one lane of an ABI 373A sequencer (two dyes/two reactions/ one lane-method).

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6. Other combinations, such as the G reaction with DMS and the A > C reactlon with NaOH, can also be used. 7. Microtiter dishes can also be used for these reactions. Use a separate dish for each reaction. Before incubating at 65”C, cover the A > C mlcrotiter dish with Saranwrap@.

References 1 Maxam, A. M. and Gilbert, W. (1977) A new method for sequencing DNA Proc. Natl. Acad. Sci. USA 74,5&I-564

2. Maxam, A M and Gilbert, W (1980) Sequencing end-labeled DNA with basespecific chemical cleavages. Meth. Enzymol. 65,499-560 3. Volckaert, G. (1987) A systematic approach to chemrcal DNA sequencmg by subcloning in pGV45 1 and derived vectors. Meth. Enzymol. lSS,23 l-250. 4. Eckert, R L. (1987) New vectors for rapid sequencing of DNA fragments by chemical degradation Gene S1,247-254 5 Rosenthal, A., Jung, R., and Hunger, H.-D. (1987) Sohd-phase methods for sequencing ohgonucleotides and DNA Meth. Enzymol. lSS,301-33 1. 6. Church, G M and Kiefer-Higgins, S (1988) Multiplex DNA sequencing. Science 240, 185-188. 7. Ambrose, B J. B. and Pless, R C. (1985) Analysis of DNA sequences using a single chemical cleavage procedure. Biochemistry 24,6194-6200.

8 Ambrose, B. J. B. and Pless, R. C. (1987) DNA sequencing chemical methods. Meth. Enzymol. 152,522-538. 9. Rosenthal, A , Sproat, B., Voss, H., Stegemann, J., Schwager, C., Erfle, H , Zimmermann, J., Contelle, Ch , and Ansorge, W (1990) Automated sequencing of fluorescently labeled DNA by chemical degradatron DNA Sequence 1, 63-77

10. Negri, R., Costanzo, G., and Di Mauro, E. (1991) A single-reaction method for DNA sequence determination. Anal. Biochem. 197,389-395. 11. Testoff, M. A. and Pless, R. C (1991) Sequence analysis of end-labeled DNA fragments by solvolysis m aqueous solutions of different amines. Anal. Biothem. 197,3 16-320

12. Gish, G. and Eckstein, F. (1988) DNA and RNA sequence determination based on phosphorothioate chemistry. Science 240, 1520-1522. 13 Church, G. M. and Gilbert, W. (1984) Genomic sequencing Proc. Nutl. Acad Sci USA 81,1991-1995

14. Saluz, H. P., Jiricny, J., and Jost, J. P. (1986) Genomrc sequencing reveals a positive correlation between the kinetics of strand-specific DNA demethylatron of the overlappmg estradiol/glucortrcoid-receptor binding sites and the rate of avian vitellogenin mRNA synthesis. Proc. Nutl. Acud. Sci USA 83,7 167-7 17 1, 15 Saluz, H. P. and Jost, J. P. (1989) A simple high-resolution procedure to study DNA methylation and in vivo DNA-protein interactrons on a single-copy gene level in higher eukaryotes. Proc. Natl. Acad. Sci. USA 86,2602-2606.

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16 Pfeifer, G P , Stergerwald, S., Mueller, P R., Weld, B , and Riggs, A D (1989) Genomic sequencing and methylatron analysis by hgatron medrated PCR. Science 246,780-786. 17. Saluz, H P., Wrebauer, K , and Wallace, A. (1991) Studying DNA modifications and DNA-protein interactions. Trends Gen 7,207-211 18 Gogos, J. A , Tzertzmis, G , and Kafatos, F C (1991) Binding sue selection analysrs of protein-DNA interactrons vra solid phase sequencing of ohgonucleottde mixtures. Nucl. Acids Res 19, 1449-1453 19 Cotton, R G , Rodrrgues, N R., and Cambell, R D (1988) Reactivity of cytosme and thymme m single-base-pair mismatches with hydroxylamme and osmrum tetroxide and its applrcatron to study mutatron Proc Nut1 Acad See USA 85,4397-440 1 20 Gogos, J., Karayiorgou, M , Aburatani, H., and Kafatos, F. C (1990) Detectron of single base mismatches of thymine and cytosme residues by potassmm permanganate and hydroxylamine in the presence of tetraalkylammonium salts Nucl Acids Res 18,6807-6814 21 Voss, H , Schwager, C., Wirkner, U., Sproat, B , Zimmermann, J , Rosenthal, A., Erfle, H., Stegemann, J., and Ansorge, W (1989) Direct genomrc fluorescent on-line sequencmg and analysis using in vrtro amplification of DNA. Nucf. Acids Res 17,2.5 17-2527 22 Tahara, T , Kraus, J P , and Rosenberg, L E (1990) Direct DNA sequencing of PCR amphfted genomrc DNA by the Maxam-Gilbert method. Biotechnrques 8,366-368. 23. Rosenthal, A. and Bankier, A (1990) Fluorescent DNA sequencing by chemrcal degradation. on-line detection of one or two DNA clones in one lane using four different fluorophores. Cold Spring Harbor Meeting “Genome Mapping and Sequencing” Abstracts, p 15 1

CHAPPER

34

Linear Amplification Sequencing with Dye Terminators And& Rosenthal and D. Stephen Charnock-Jones 1. Introduction Present day sequencing technology is mainly based on the dideoxy method introduced by Sanger m 1977 (I; seeChapter 1). DNA sequencing is a complex process requiring many different complicated steps like subcloning, template preparation, sequencing reactions, gel electrophoresis, reading the nucleotide sequence into the computer, and data handling. Various modifications may involve synthetic primers, enzymatic manipulations, and polymerase chain reactions. The last five years have seen efforts by academic institutions (e.g., CalTech, Pasadena, CA; EMBL, Heidelberg, Germany) and commercial companies (Applied Biosystems, Foster City, CA; Du Pont, Wilmington, DE; Pharmacia, LKB Biotechnology, Uppsala, Sweden; Hitachi, Japan) to replace the conventional radioactive label with fluorescent dyes. These fluorophores, attached to the sequencing primer or to nucleotides, are incorporated into the DNA chain during the copying reaction catalyzed by DNA polymerases (e.g., Klenow fragment of DNA polymerase I, modified T7 DNA polymerase [SequenaseTM] or Tizq DNA polymerase). During separation of the base-specific polynucleotides on a polyacrylamide gel, a laser beam excites the dyes. The emitted fluorescence is collected by detectors (photomultiplier, From Methods m Molecular Biology, Vol. 23’ DNA Sequencing Protocols Edlted by Ii. and A Grlffm Copyright 81993 Humana Press Inc., Totowa,

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CCD elements, or cameras) and sent to a computer, where appropriate software converts the data into nucleotide sequence. A number of such instruments are now commercially available and are increasingly used in large-scale projects (2). The vast majority of sequence thus generated utilizes sequencing primers that are fluorescently labeled at their S-ends. The Pharmacia ALF uses a single fluorophore as a label and therefore the four reactions must be run in separate lanes (3), whereas the Applied Biosystems 373A uses four different fluorescent labels (and therefore four primers) allowing the reaction products to be analyzed in a single lane (4). Although the ABI 373A has a higher capacity, the ALF permits convenient “primer walkmg” (m particular, gap closing in shotgun projects), because a new primer can be readily synthesized using a fluorescently labeled phosphoramidite (5,6). An alternative solution to this problem is to use dye labeled terminators. These have several potential advantages over labeled primers. The most important is that any unmodified primer can be used. Thus, the usual “walking primer strategy” can be applied to sequence a clone completely or to close gaps after random projects. In addition, genomic PCR products can also be rapidly sequenced with the primers used to generate them. A second significant advantage of dye terminators is that only molecules that have terminated specifically will be labeled. Therefore, enzyme “pauses” caused by secondary structure are not detected. The sequencing reactions are also much simpler to perform (and potentially to automate) because the four base-specific terminations can all be carried out in the same tube and then analyzed in the same lane of a gel. Dye labeled terminators have been used in the Du Pont Genesis 2000TM (7,8) and the ABI 373A. In the last two years new protocols for Tuq cycle sequencing have been developed and attracted much attention because of a number of important advantages over traditional dideoxy techniques. In particular, any single-stranded or double-stranded template can be used. The reactions can be easily set up in microtiter dishes, involving little manual manipulation, and requiring only small amounts of template (fmole scale). In cycle sequencing, a single primer is used to linearly amplify a region of template DNA in the presence of Tuq polymerase and a mixture of deoxynucleotide triphosphates and chain terminating dideoxynucleotide triphosphates. Most protocols recommend the

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use of either radioisotopes or fluorescent labels attached to the 5’-end of the primer. In fact, dye primer cycle sequencing using ABI’s four color code and the 373A sequenceris very reliable and usually gives 400-500 bp of automatically called sequencedataof high quality with a wide range of templates (9). It has also been proven very useful for large-scale sequencing projects (10). However, this particular configuration with a throughput of 24-36 samples can only be applied to shotgun projects. Tuq cycle sequencing can also be performed using the ABI dye terminators and the 373 sequencing machine. Here we present a package of protocols for template preparation, dye terminator cycle sequencing, and removal of the excess of dye terminators. Most of the protocols have been either developed in our laboratories or slightly modified from methods recommended by ABI (II). Modifications have been made in order to reduce the reagent costs or allow a higher throughput of samples. All protocols described have been successfully used with the specified templates. They are recommended for shotgun as well as walking primer projects. 2. Materials 2.1. Equipment 1, ABI 373A DNA Sequencer(Applied BiosystemsInc., Foster City, CA): For fluorescent sequencing using dye terminators and any primer (shotgun and primer directed sequencing). 2. Techne PHCZ, PHC3, or MWl Thermal Cyclers and Techne Hi Temp 96 Microtiter Dishes (Techne [Cambridge] Ltd., Duxford, UK): For cycle sequencing reactions. 3. IEC Centra C3 Centrifuge (International Equipment Company, a division of DAMON, Dunstable, Bedfordshire, UK): For spin columns. 4. IEC Centra C4 Centrifuge (International Equipment Company): For microtiter dishes. 5. Perspex block of 24-36 minicolumns with teflon smters (Homemade, see Methods, Section 3.2.2.): For quick removal of excess of dye termlnator by gel filtration. 6. SpeedVac Concentrator(Savant Instruments Inc., Hicksville, NY): For lyophllizing sequencing samples after removal of excessof dye terminators.

2.2. Reagents 1. Tuq Dye Deoxy TMTerminator Cycle Sequencing Kit (see Note 1): For cycle sequencing of 100 templates. Contains AmpliTaqTM DNA poly-

merase,four dye terminators, deoxynucleotlde mix, sequencingbuffer,

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uruversal (-21) sequencing primer, control template. (ABI part number 401150, Applied Biosystems Inc.) 2. Dye DeoxyTM Terminator Kit: For cycle sequencing of 100 templates, Contains four dye terminators (ABI part number 401095, Applied Biosystems Inc.). 3. Tuq polymerase: Cetus native Tuq, 5 U/pL. 4. 5X Cycle sequencing buffer: 400 mM Tris-HCl, 10 mA4 M&l,, 100 rnM (NH&S04, pH 9.0. 5. Nucleotide mix for cycle sequencing: 750 pit4 dITP, 150 l.uV dATP, 150 l.t~VdTTP, 150 @4 dCTP. All deoxynucleotide triphosphates were purchased as solutions from Pharmacia LKB Biotechnology, Uppsala, Sweden. 6. Loading buffer: Prepare a mixture of deiomzed formamtde/water/50 mM EDTA at pH 8.0 (5:l:l; v/v/v). 7. Commercial 1mL spin columns: Select-DTMG-50 from 5 Prime to 3 Prime. 8. Sephadex G50 suspension: A 5% w/v slurry of SephadexTMG50 superfine (Pharmacia LKB Biotechnology) is prepared in 50 mM NaCl, 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA, and autoclaved; once cool, Triton X-100 is added to 0.05% final concentration. This solution is then stored at 4°C until use. 9. U-well microtiter plate, Falcon 3918. 10. TB: 12 g/L tryptone, 24 g/L yeast extract, 4 mL glycerol. Autoclave and add 100 mL of a phosphate solution containmg 2.33 g KHZP04 and 16.43 g K2HP04 3HzO. Il. 2xTY: 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl. Adjust pH with NaOH to 7.4 and autoclave. 12. GTE solution: 50 mM glucose, 20 mil4 Tris-HCI, pH 7.4, 10 mM EDTA. 13. GTE/lysozyme/RNase solution: 50 mM glucose, 20 mM Tris-HCI, pH 7.4, 10 mM EDTA containing 3 mg/mL lysozyme (Sigma, St. Louis, MO) and 1 mg/mL boiled RNase A (Sigma). l

3. Methods 3.1. Template Preparation-General Protocols As is true with all molecular biological techniques, DNA sequencing reactions work best with good quality DNA. Thus, the preparation method that produces the cleanest DNA will ultimately yield the best sequence. Ml3 DNA as well as being single stranded can be readily isolated in a pure state by standard PEG precipitation (12,13). Plasmid DNA can be prepared by a variety of modifications to the original alkaline lysis method of Birnboim and Doly (14). In the standard miniprep

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protocol, alkaline lysis is followed by RNase treatment, phenol extraction, and ethanol precipitation (13). Another miniprep protocol utilizing an additional PEG precipitation has been reported to reliably yield good results (II). Plasmid and cosmid DNA can also be extracted and purified using column chromatography. Sequence data from M 13 templates are of higher quality than those obtained from plasmids. Usually, 400-450 bp with an accuracy of about 99% can be obtained. Plasmid DNA obtained using Qiagen columns (15) or Magic TMMinipreps (Promega Corp, Madison, WI), is of higher quality than that from minipreps. The accuracy for the first 400 bp often exceeds 98%. However, this involves considerable time and expense and is therefore only worthwhile when performing multiple walks on a single clone. The plasmid minipreps described above are more suited to process 12-24 clones in parallel, but also involve many steps and take several hours. Here we present additional protocols for crude preparations of plasmid DNA. These involve fewer steps, do not use phenol or chloroform, and can be performed in a microtiter plate. They are therefore suitable for sequencing a large number of clones. 3.1.1. Plasmid Miniprep Method The major difference between this and the standard method is that no RNase digestion and phenol/chloroform extraction steps are used. There is only one ethanol precipitation step that uses one volume of ethanol (16). This, apart from allowing the precipitation to be performed in a single tube, inefficiently precipitates short contaminating DNA fragments that by false priming contribute to the background in sequencing reactions, Prior to sequencing, RNA is degraded by treatment with NaOH followed by neutralization with HCI. 1, Inoculate a single “tooth-picked” bacterial colony into 3 mL of a rich growth medium like TB or 2xTY, containing antibiotic as appropriate, and incubate with vigorous shaking at 37°C overnight. 2. Decant 2 mL of the overnight culture into a 2-mL snap-cap tube, and spin in a benchtop centrifuge at 13,000 g for 1 min. 3. Completely remove the supematant from the bacterial pellet by aspiration. 4. Carefully resuspend the pellet in 200 uL GTE solution. 5. Add 400 j.tL of a freshly prepared solution of 0.2M NaOH/l% SDS, and invert several times before placing

on ice for 5 min.

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6. Add 300 pL, of 3M potassium acetate, pH 4.8, and Invert several times before replacing on ice for 5 min. Do not vortex. 7. Centrifuge at 13,000 g for 5 min, and transfer supernatant with a blue tip to a clean 2-mL snap-cap tube, avoiding the white precipitate. 8. Add 1 vol(900 $) of absolute ethanol to the supernatant, vortex briefly, and immediately centrifuge at 13,000 g for 5 min. 9. Carefully discard the supernatant and wash the nucleic acid pellet with 2 mL of 70% ethanol before a final centnfugatlon at 13,000 rpm for 2 min. 10. Discard the supernatant and dry the pellet under reduced pressure. 11. Resuspend the pellet in 40 pL of deionized sterile water. 12. Two microliters of the DNA are then used for sequencing. The RNA present 1s degraded by the addition of 3 pL of 0.4M NaOH and incubated at 65°C for 5 mm. This IS then neutralized by the addition of 3 pL of 0.4M HCl (see Note 2). This DNA (total volume of 8 pL) can then be used to give acceptable results (97-98% accuracy for the first 400 bp). If necessary, a conventional RNase treatment followed by phenol/chloroform extraction and ethanol precipitation can be carried out. This certainly improves the reliability of the sequencing and is performed as follows: a. Boiled RNase A (Sigma) is added to a concentration of 50 pg/mL and incubated at 37OC for 20 mm. b. The DNA is phenol extracted and then chloroform extracted. c. Five microliters of 3M potassium acetate, pH 4.8, and 110 pL ethanol are added. d. The DNA recovered by centrifugation (13,000 g for 10 mm) and the pellet washed with 100 pL of 70% ethanol and dried in VQCUO. e. The DNA is redissolved m 40 pL of sterile deionized sterile water and 2-3 pL used for sequencing. 3.1.2. Plasmid Microprep in Microtiter Plate (See Note 3) This entire protocol is performed in a microtiter plate, and the growth and DNA preparation in a rigid U-well plate (Falcon 3918). Because of the fact that the alkaline lysis step is performed in the presence of lysozyme and RNase, the final DNA is RNA free. The sequencing can be carried out in a Techne thermostable polycarbonate plate if desired. 1 Inoculate a single “tooth-picked” bacterial colony mto 250 & of TB or 2xTY, containing antibiotic as appropriate, and incubate at 37°C for 24 h. 2. Spin in a benchtop microtiter plate centrifuge at 4,000 g for 1 min. 3. Remove the supernatant from the bacterial pellet by aspiration. 4. Resuspend the pellet in 30 pL, GTE/lysozyme/RNase solution. Incubate at 37°C for 10 min.

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5. Add 60 pL of a freshly prepared solution of 0.2MNaOH/l% SDS, and rmx by sharply tapping the plate several times before placing on ice for 5 mm. 6. Add 45 pL of 3M potassium acetate, pH 4.8, and mix with a pipet (by stirring or gentle pipetmg up and down) before replacing on ice for 5 min. 7. Centrifuge at 4,000 g for 5 min, and remove 90 pL of the supernatant to a clean microtiter plate (the Techne thermostable plate may be used from this pomt). Take care to avoid any of the white cellular debris that may not have pelleted completely. 8. Add 1 vol(90 pL) of absolute ethanol to the supernatant, mix, and immediately centrifuge at 4,000 g for 5 min. 9. Remove the supernatant by aspiration, and wash the plasmid pellet with 100 pL of 70% ethanol (there is no need to centrifuge at this point, Just add the 70% and remove it by aspiration). 10. Dry the pellet under reduced pressure. 11. Resuspend the DNA pellet in 6 pL of sterile delomzed water. The DNA is then ready for sequencing. All the DNA from a single well is used for sequencing. 3.1.3. Preparation of PCR Templates from Genomic DNA The following protocol has been used extensively to isolate and purify genomic PCR products in the 0.3-3 kb range prior to cycle sequencing with dye terminators. The genomic templates were obtained by a newly developed PCR amplification technique for chromosome walking (17,181. The first 350-400 bp of sequence from both ends of the PCR fragment were obtained with the same pair of primers that had been used to amplify the fragment. Further sequence information was obtained by using custom made sequencing primers. The accuracy of the first 300-400 bp was greater than 98%. 1. Genomic PCR products obtained by symmetric PCR m a total of 20-50 pL are purified from other nonspecific DNA fragments as well as from prrmers and nucleottdes by separation on 1% LMP agarose mmrgels contammg 1X TAE buffer and ethrdium bromide. 2. Excise appropriate PCR DNA band from gel with a scalpel and place the slice mto a 2-mL snap-cap tube. Minimize the time of UV exposure as much as is practical. 3. The DNA IS recovered by dissolving the agarose in sodium iodine and binding the DNA to glassmilk. For this purpose we are usmg the GenecleanTM kit (BIO 101 Inc., La Jolla, CA), but have slightly modtfied the elution procedure. Weigh the agarose together with the tube and add 3 vols of NaI stock solution provided m the kit. Incubate the tube for 5 min at 55OC.

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4. Add 5 pL of the glassmilk provided with the kit, vortex the tube briefly, and incubate it for 5 mm on ice. 5. Pellet the glassmilWDNA by spinnmg the tube for 10 s at 13,000 g. Remove the supernatant completely with a blue tip. 6. Wash pellet three times with 700 pL of NewWash solution provrded in the kit and kept at -2OOC. During each washmg the pellet should be resuspended completely. After the supernatant from the last wash has been removed, spin the tube again and remove remaining liquid with a yellow tip. 7. For elution of the DNA from the glass support, resuspend the pellet m 20 pL of sterile deronized water and incubate the tube at 55°C for 5 mm. Spin the tube for 30 s and remove 18 pL of the supernatant mto a new tube avordmg the pellet. Repeat the elutton with another 20 pL of water. In order to remove small amounts of the glassmilk that has been carried over, spm the tube agam for 30 s at 13,000g and transfer the supernatant mto a new tube. The volume is reduced by a brief lyophiltzation in a SpeedVac system. Run an allquot on agarose and determine the amount of DNA by comparison with a control. Dilute the DNA to 0.5 pg/pL. 3.2. Linear Amplification Sequencing with Dye Terminators-General Protocols During sequencing with dye terminators fluorescent dyes are incorporated at the 3’-end of many nested polynucleotides. These fluorophores can interact with neighboring bases and lead to different secondary structures that might not always be resolved during electrophoresis in the presence of 8M urea. In order to reduce this effect, known as band compressions, deoxyinosine triphosphate (dITP) must be used in the nucleotide mix instead of the deoxyguanosine derivative. It has also been found that longer extension times at 60°C are more effective than shorter times at 72°C (II). 3.2.1. Cycle Sequencing Using a Single Primer The following protocols describes terminator cycle sequencing using a thermostable polycarbonate microtiter plate designed for use with the MWl or PHC3 thermocyclers (Techne). 1. Add 2 pL of single-stranded Ml3 template (at a concentration of 0.25 p&L), 2 pL of plasmid, cosmld, or PCR product (at a concentration of 0.5 pg/pL), or 6 pL of crude plasmid mimprep (see Sections 3.1.1, and 3.1.2.) to each well of a microtiter plate kept on Ice.

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Table 1 SequencingMl 3 Templates 1 x mix 12 x mix

Reagent Sterile deionized water 5X Cycle sequencingbuffer Deoxynucleotide mix Dye terminator mix Sequencingprimer (0.8 pmol/pL) Cetus Tag polymerase(5 U/$) Total

SW

4w 1cIL 4w lc1L 0.2 j.IL 18.2 $

218.4 pL

24 x mix

96 x mix

192 96 24 96 24 4.8

768 384 96 384 96 19.2

pL ClL w W w w

436.8 pL

pL pL w pL CLL j.lL

1747 2 pL

2. Depending on the DNA template different reaction premixes are prepared in a 2-mL snap-cap tube on ice, Volumes are given for 1, 12, 24, or 96 different templates (see Tables l-3; Note 4). After adding Taq polymerase, vortex very briefly to mix, and spin briefly. 3. For all templates except crude plasmtd mnnpreps add 18 p.L of the appropriate reaction premix to each well of the plate. Use the same tip for all wells. For the crude plasmid minipreps add 14 pL of the appropriate premix to each well. In all cases the final reaction volume is 20 pL. 4. Overlay the samples with 40 pL of light mineral oil using a multtchannel pipet. 5. Place the microtiter plate in the Techne thermocycler preheated to 95°C and incubate the samplesfor 90 s.Thereafter, start rmmediately the following twenty-five cycles: (95*C for 30 s, 50°C for 15 s, and 60°C for 4 min). After completion, keep the plate at -20°C until ready to proceed. 6. The excess of terminators is removed either by using home-made spin columns (see Section 3.2.1.), or by gel filtration using an array of 2436 minicolumns fixed m a block of perspex (see Section 3.2.2.). 7. Finally, the samples are resuspended into 5 pL of loading buffer, heated for 3 min at 90°C, transferred immediately onto ice, and loaded onto a ABI 373A sequencer. 3.2.2. Cycle Sequencing Using Multiple Primers In order to generate extremely long reads with dye terminators we have developed a modified cycle sequencing protocol for the ABI dye terminators that consists of two steps. First, a sequencing primer

is extended in the absence of dideoxynucleotides by linear amplification with Taq polymerase. This generates a heterogeneous mixture of polynucleotides that all have common S-ends but vary in length. Sec-

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Table 2 Plasmids, Cosmids, and Symmetric PCR Templates Reagent

1 x mrx

12 x mix

Sterile deronized water 5X Cycle sequencing buffer Deoxynucleotrde mix Dye terminator mix Sequencing primer (0.8 pmol/j.tL) Cetus Taq polymerase (5 U&L)

5w 4cIL 1P4cIL 4 uL 0.2 pL

48 ClL 12 FL 48 W 48 @ 24W

Total

18.2 pL

218.4 pL

60 r-IL

24 x mix

96 x mix

120 96 24 96 96 4.8

480 384 96 384 384 192

pL ClL w W W J.IL

436 8 J.IL

)iL /.L & j.L pL pL

1747.2 ~.IL

ond, dye terminators arethen addedand cycle sequencing performed. All the different primer molecules produced during precycling are specifically terminated with dye terminators. If a reasonabledistribution of primers across the template length is generatedin the first amplification, the resulting sequencepattern will exceed 400-500 bp and strong signal intensities can be observed up to 800 bp. The following protocol describes the method for single-strandedMl3 templates in single tubes. 1. Add 2 pL of single-stranded M 13 template (at a concentratton of 0.5 pg/pL) into a OS-mL snap-cap tube kept on ice. 2. Prepare the following reaction premix in a OS-mL snap-cap tube on ice. Volumes are given for 1, 12, or 24 different templates (see Table 4). After adding Tuq polymerase, vortex very briefly to mix, and spin briefly. 3. Add 14 pL of this mix to each tube on ice, mix the contents by prpeting, and overlay it wtth 40 pL of light mineral oil. 4. Place the tubes in the Techne PCH-2 thermocycler preheated to 95°C and incubate

the samples for 90 s. Thereafter,

immediately

start the

following fifteen cycles of linear amplification (95°C for 30 s, and 50°C for 1 s) followed by rapid ramp to 4°C for 5 mm. 5. Meanwhile, mix all four dye terminators m equrmolar amounts together with 0.5 U of fresh Tuq polymerase per clone to be sequenced. While the tubes are incubating at 4°C in the thermal cycler, 4 pL of the dye terminator mix are added quickly beneath the or1of each tube and briefly

mixed by pipeting (seeNote 5). 6. Cycle sequencing IS then continued with the following twenty-five cycles: (95°C for 30 s, 50°C for 15 s, and 60°C for 4 min). After completion, keep the tubes at -20°C until ready to proceed.

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Sequencing

Table 3 Crude Plasmid Minipreps (see Sections 3.1.1 and 3.1.2.) Reagent

1 x mix

Sterile deionized water 5X Cycle sequencing buffer Deoxynucleotide mix Dye terminator mix Sequencing primer (0.8 pmol@L) Cetus Tuqpolymerase (5 U&L)

1* 4c1L 1cIL 4& 4 pL 0.2 pL

Total

14.2 pL

12 x mix

24 x mix

24 12 w 48 CrL 96 12 w 24 48 w 96 96 48 FL 4.8 241.1~ 170.4 & 340.8

96 x mix

96 W

w & cl~ W W /IL

384 96 384 384 192

)IL

1363.2 pL

j.L ClL /AL pL, *

7. The excess of terminators is removed either by ustng homemade spin columns (see Section 3.2.1.), or by gel filtration using an array of 24-

36 minicolumns fixed in a block of perspex (see Section 3.2.2.). 8. Finally, the samples are resuspended into 5 pL of loading buffer, heated for 3 min at 9O”C, transferred immediately onto ice, and loaded onto a ABI 373A sequencer. 3.3. Removal

of Excess Dye TerminatorsGeneral Protocols

The large excess of unincorporated dye terminators must be removed prior to loading of the gel. The method originally recommended for this required individual spin columns to be run for each sample. Recently, several alternative methods have been developed (II, 19). These rely on either precipitation with isopropanol or cetyl-trimethyl ammonium (CTAB), or extraction with phenol/chloroform. All of these methods have some advantagesover spin columns but the results are not as good. We therefore have given below a detailed method for

using spin columns (see Section 3.3.1.). However, we have also developed a small-scale gel-filtration system that is capable of handling between 24 and 96 samples in parallel. This system requires an array of columns formed in a block of perspex (Fig. 1). The volumes required for packing, washing, and elution of the array of columns are given below and should be rigidly adhered to (see Section 3.3.2.). The parallel gel filtration process gives results of comparable quality to spin columns, but it is much less time consuming.

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Table 4 Sequencmg M 13 Templates Reagent

1 x mix

12 x mix

Sterile deionized water 5X Cycle sequencing buffer Deoxynuclcotide mix Sequencing primer (0 8 pmol/pL) Cetus Tuq polymerase (5 U&L)

2w 0.1 j.lL

72 48 24 24 1 24

Total

141 pL

1692&

6W

4cIL 2+

cl~ W cl~ ll~ w

24 x mix 144 96 48 48 2.4

pL W FL W @

3384&

3.3.1, Use of Homemade Single Spin Columns A number of different commercially available 1-mL spin columns (Select-DTM G50 from 5 Prime + 3 Prime, Inc. (West Chester, PA), Nu-CleanTM D50 from IBI, Quick SpinTM from Boehringer Mannheim, Mannheim, Germany) have been recommended (II). In our opinion, they are very expensive if used on a daily basis. We have also found a great deal of variability among spin columns of the same batch from a single supplier. These disadvantages can be avoided, if homemade spin columns are prepared. The following protocol has been used for two years without a single failure. 1. Remove the Sephadex G50 suspension (see Section 2.2., step 8) from the refrigerator and allow to warm up to room temperature. Mix thoroughly and add sequentially 1 mL into twelve or twenty-four empty 1-mL, core columns (e.g., the Select-D TMG50 type) mounted onto the collection tubes. Allow excessbuffer to drain from the column. The flow is initiated by gravity. Repeat the filling process until all columns are properly filled with 1 mL of gel bed. Discard the buffer from the collection tubes. 2. Add twice 500 & of deionized sterile water to the top of each column and allow to dram completely by gravity. This step removes excess salt and detergent present in the buffer. Discard the buffer from the collection tubes after each wash. 3. Add 20 pL of sterile deionized water beneath the oil of all sequencing samples to be processed. 4 Place twelve spin columns

together with the collection

tubes Into mdl-

vidual 15-mL Falcon tubes and spin them at 1200 g for 90 s using the Centra 3C centrifuge (see Note 6) 5. Carefully remove 35 pL of the reaction mixture from beneath the 011 avoiding mineral oil and load it on the top of the Sephadex material. Try to place it in the center of the gel.

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Fig. 1. Layout and design of the multicolumn block. The protocol described herein for the preparation of the columns must be rigidly adhered to. A S%(w/v) slurry of Sephadex TM G50 superfine is prepared in STE (50 mM NaCl, 10 mM Tris, pH 7.4, 1 mJt4 EDTA), and autoclaved; once cool, Triton X-100 is added to 0.05% final concentration. This is then stored at 4’C until use. Immediately before use, the Teflon sinter in each column is prewetted with 50 FL of 1% Triton X-100 in H20. The Sephadex slurry is thoroughly mixed, and 600 pL added to each column. The bed is allowed to settle by gravity flow. As soon as the bed has run dry, the column is washed with 250 j.tL HzO, and again run dry. The reaction mix (20 l.tL) from the Tuq cycle sequencing is removed from under the oil, loaded directly onto the Sephadex, and allowed to run in. The column is then washed with 100 l.tL of H20, and the eluate discarded. An additional 100 pL of Hz0 is added, and this time the eluate is collected. The flourescent sequencing products are in this fraction, while the excess terminators remain in the column. The products are then lyophilized to dryness, redissolved in 7 p.L gel loading buffer (5 l.tL formamide, 2 pL 25 mM EDTA), denatured (95°C 3 min), chilled, and loaded on the gel as described by the manufacturer. A multichannel pipet can be used throughout for all the washing and elution steps.

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6. Transfer columns to clean collection tubes and reassemble the setup into 15mL Falcon tubes. Spin at 1200 g for 90 s usmg the Centra 3C centrifuge. 7. Place the collection tube into a SpeedVac system and dry down the sample within 45 min. 8. If necessary, proceed with the next twelve spm columns from step 4. 3.3.2. Use of a Fixed Array of Twenty-Four

Minicolumns

1. Immediately before use, the TeflonTM smter in each column is prewet with 50 pL of 1% Triton X- 100 in sterile deionized water. 2. The Sephadex G50 slurry (see Section 2.2., step 8) is thoroughly mixed and 600 pL added to each column. The bed is allowed to settle and run by gravity flow. 3. As soon as the bed has run dry the column is washed with 250 pL sterile deionized water and agam run dry. 4. The reaction mix (20 pL) from 7’aq cycle sequencing is removed from under the oil, loaded directly onto the Sephadex, and allowed to run in. The presence of small amounts of oil does not effect the performance of the column appreciably. 5. The column is then washed with 100 pL of sterile deionized water and the eluate discarded. 6. The perspex block of mm1 columns is placed above a microtiter dish (Falcon 3911, Becton Dickinson Labware, Oxnard, CAP). An additional 100 pL of sterile deionized water is added to each column and this time the eluate is collected. The fluorescent sequencing products are m this fraction while the excess terminators remam in the column. 7. This fraction is then lyophihzed to dryness using a SpeedVac system. A multichannel pipet can be used throughout for all the washing and elution steps.

4. Notes 1. If necessary, the core component of the kit-the four dye terminatorscan be purchased separately from ABI. We have used native Cetus Taq polymerase for two years and found that with a wide range of different templates, less units are required for cycle sequencing than with AmpliTaq. No further difference between these enzymes has been observed in cycle sequencing. Tuq polymerase from other suppliers or home made products should be carefully tested before using for routine sequencing. The cycle sequencing buffer and the nucleotide mix can be easily prepared as described m Section 2.2.

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2. In order to obtain a neutral pH after neutralization it is important to use the NaOH and HCI solutions at exactly the same concentrations. Therefore, we recommend the use of commercially obtained solutions of 1M acid and base. 3. This method can also be scaled to standard 2-pL snap-cap tubes simply by multiplying all the volumes by a factor of 10. 4. All premixes can also be prepared using 2-mL instead of 1 pL of the deoxynucleotide mix per clone. In this casethe volume of deionized stenle water per clone has to be reduced by 1 pL. This modification results in a more balanced distribution of the raw data and can improve base calling. 5. The conditions presented here are appropriate for use with a Techne thermocycler PHC-2 with maximum heating and cooling rates. The molar ratio between short sequencing primer and template is crucial for the protocol. Weak raw data in the 30-200 bp range indicates that too little short primer is left after precycling to allow for sufficient termination in this range. This problem can be solved by adding more primer to the reaction (before or after precycling). The method can easily be adapted to process 96 clones in a microtlter dish. 6. Although twenty-four or more spm columns can be set up initially, do not process more than twelve spin columns at a time during steps 4-6. Work quickly but precisely to prevent the columns from drying excessively during these steps.

References 1. Sanger,F., Nicklen, S., and Coulson, A. R. (1977) DNA sequencing with chamterminating inhibitors. Proc Natl. Acad. Sci. USA 74,5463-5467. 2. Hunkapiller, T., Kaiser, R. J., Kopp, B F , and Hood, L. (1991) Large-scale and automated DNA sequence determination. Science 254,59-67. 3. Ansorge, W., Sproat, B., Stegemann, J., and Schwager, C. (1986) A non-radioactive automated method for DNA sequence determination. J. Biochem. Biophys. Methods 13,3 15-323.

4. Smith, L. M., Sander, J. Z., Kaiser, R. J., Hughes, P., Dodd, C., Connell, C. R., Heiner, C., Kent, S. B H., and Hood, L. E. (1986) Fluorescence detection in automated DNA sequencmg analysis Nature 321,674-679 5. Schubert, F., Ahlert, K., Cech, D , and Rosenthal, A. (1990) One-step labelling of ohgonucleotldes with fluoresceme during automated synthesis. Nucl Acids Res. 18,3427.

6. Hawkins, T. L. and Sulston, J. E. (1990) Automated fluorescent primer walking. Technique 2,307-3 10. 7. Prober, J. M., Trainor, G. L., Dam, R. J., Hobbs, F. W , Robertson, C. W., Zugursky, R. J., Cocuzza, A. J., Jensen, M. A., and Baumeister, K. (1987) A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238,336-341

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8. Jones, D. S. C., Schofield, J. P., and Vaudin, M. (1991) Fluorescent and Radioactive solid phase dideoxy sequencing of PCR products in mrcrotiter plates. DNA Sequence 1,219-283.

9. Craxton, M. (1991) Linear amplification sequencmg a powerful method for sequencing DNA. Methods: a companion to methods in enzymology 3,20-26. 10. Sulston, J., Du, Z., Thomas, K., Wilson, R., Hillier, L., Staden, R., Halloran, N., Green, P., Thierry-Mieg, J., Qiu, L., Dear, S., Coulson, A., Craxton, M., Durbin, R., Berks, M., Metzstein, M., Hawkins, T., Ainscough, R., and Waterston, R. (1992) The C. efegans sequencing proJect: a beginning. Nature 356,37-41. 11. Taq DyeDeoxy Terminator Cycle Sequencing Kit Description and Manual 1992.

Applied Biosystems, Inc., Foster City, CA. 12. Bankier, A. T., Weston, K. M., and Barrel& B G. (1987) Random cloning and sequencing by the M13idideoxynucleotide chain termination method. Meth. Enzymol. 155,5 l-93.

13. For a detailed protocol see also Craxton, M., (1993) Cosmid Sequencing. This book Chapter 2 1. 14. Birnboim, C. and Daly, J. (1979) Nucf. Acids Res. 7, 1513-1523. 15. The Qiagenologist Application Protocols Diagen Gmbh, Dusseldorf, Germany, October 1990. 16. Jones, D. S. C. and Schoefield, J. P (1990) A rapid method for isolating high quality plasmrd DNA suitable for DNA sequencing Nucl. Acid Res. 18, 7463-7464. 17. Rosenthal, A. and Jones, D. S C. (1990) Genomic walking and sequencing by oligo-cassette mediated polymerase chain reaction. Nucl. Acids Rex 18, 3095-3096.

18. Rosenthal, A., MacKinnon, R. N., and Jones, D. S C. (1991) PCR walking from microdissection clone M54 Identifies three exons of the human gene for the neural cell adhesion molecule Ll (CAM-Ll). Nucl. Aczds Res. 19,5395-5401. 19. DNA Sequencing Model1 373A, User Bulletin 20, Applred Biosystems, Inc., December 1991. Applied Biosystems, Inc. Foster City, CA

Sequencing Reactions for the Applied Biosystems 373AAutomated DNA Sequencer Nicolette Ha&ran, Z@n Du, and Richard K Wilson 1. Introduction Efficient completion of large DNA sequencing projects has been greatly facilitated by the development of fluorescence-based dideoxynucleotide sequencing chemistries and instruments for real-time detection of fluorescence-labeled DNA fragments during gel electrophoresis (L-6; see Chapter 1). Besides eliminating the use of radioisotopes, these systemsautomate the task of reading sequencesand provide computerreadable data that may be directly analyzed or entered into a sequence assembly engine. In this chapter, we describe DNA sequencing using the first commercially-available fluorescent instrument, the Applied Biosystems, Inc. Model 373A Automated DNA Sequencer. This sequencer, originally introduced in 1987 as the Model 370A, utilizes a multi-spectral approach in which four distinct fluorescent tags are detected and differentiated in a single lane on the sequencing gel (6). The four tags are incorporated during the DNA sequencing reactions and may be present on either the 5’ end of the sequencing primer (“dye-primers”) or on the dideoxynucleotide triphosphate (“dye-terminators”). Since the 373A requires only one lane per sample, up to thirty-six samples may be analyzed per run. By comparison, the From. Methods m Molecular Wology, Vol. 23. DNA Sequenong Protocols Edited by. H and A. Griffin Copynght 01993 Humana Press Inc , Totowa,

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DuPont Genesis system currently supports only a four-dye, one-lane dye-terminator chemistry and allows analysis of up to twelve samples per run. The Pharmacia ALF sequencer supports only a one-dye, fourlane approachwith dye-labeledprimer, and can analyze up to ten samples per run. The ability of the 373A to use both dye-primer and dyeterminator chemistries greatly facilitates the completion of projects that require both shotgun and primer-directed sequencing phases. We have used the 373A for DNA sequence analysis of M13, plasmid, and phagemid subclones, as well as templates produced by PCR. Protocols are described for preparation of both single- and doublestranded template DNA. Additionally, a protocol in which we have used PCR amplification as a means of producing template DNA directly from cosmid and lambda clones is included. A sequencing protocol using modified T7 DNA polymerase and fluorescent dyeprimers is described for use with single-stranded Ml 3 and phagemid DNA. A second sequencing protocol described here utilizes Taq DNA polymerase and thermal cycling, and may be used with dye-primers and both single- and double-stranded templates. A third sequencing protocol utilizes T7 DNA polymerase and fluorescent dye-terminators and may be used with single-stranded M 13 and phagemid DNA. This third method requires that special hardware be installed in the 373A. An alternative dye-terminator chemistry using Taq DNA polymerase is available from Applied Biosystems, although it has not been reliable in our hands and hence is not included in this chapter. 2. Materials 1. A thermal cycler 1s required for the linear amphfrcatron sequencing method and for the PCR template preparation methods. In our hands, the GeneAmp 9600 andDNA Thermal Cycler mstrumentsfrom PerkmElmer (Norwalk, CT) have worked very well. The 9600 instrument carries the advantages of a 96-well mtcrotiter plate format, significantly faster ramping times, and a heated plate cover that eliminates the need to overlay reactions with mineral oil. The Techne MW-1 thermal cycler also works well for linear amplification sequencing reactions m 96-well microtiter plates (M. Craxton, personal communication). 2 The automated fluorescent DNA sequencing system described here is the Applied Biosystems, Inc. (Foster City, CA) Model 373A. The current configuration includes a Macintosh 11~1computer with 8 MB RAM and a 100 MB fixed disk drive.

ABI 373A DNA Sequencing

Reactions

aquaticus (Tuq) DNA polymerase may be purchased from one of several enzyme suppliers. It is our experience that the recombinant AmpliTaq enzyme from Perkin-Elmer gives the best results. 4. Modified T7 DNA polymerase (Sequenase) should be purchased from United States Biochemical (Cleveland, OH); version 1.Oenzyme is best for dye-primer sequencing, version 2.0 enzyme containing pyrophosphatase is best for dye-terminator sequencing. 5. Deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs): a. Stocks: 100 n&f dNTP solutions and 5 mM ddNTP solutions may be purchased from Pharmacia (Piscataway, NJ). Prepare 20 mM stocks of each dNTP in TE buffer (see below). Store at -20°C. Nucleotide analogs, such as 7-deaza-dATP and 7-deaza-dGTP, are useful for resolvmg some sequence compressions and may be purchased as 10 mM solutions from Boehrmger-Mannheim Biochemicals (Indianapohs, IN). b. For PCR: Prepare a mix containing 1.25 mM of each dNTP m TE buffer. Store at -20°C. c. For dye-primer sequencing with modified T7 DNA polymerase: 1. Prepare 8 mA4dNTP mixes: 100 pL each of 100 mM dATP, dCTP, dGTP, and dTTP in TE buffer to a final vol of 1.25 mL (store at -2OOC)* ii. Prepare 50 @4 ddNTP solutions: ddA: 2 pL of 5 mM ddATP + 198 pL of TE buffer. ddC: 2 pL of 5 mM ddCTP + 198 pL of TE buffer. ddG: 4 pL of 5 n&f ddGTP + 396 pL of TE buffer. ddT: 4 pL of 5 mM ddTTP + 396 pL of TE buffer. iii. Sequencing mixes: Combine equal vols of the 8 mM dNTP mix and one of the four 50 @l4ddNTP solutions. For 100 reactions, prepare 100 pL of A and C mixes and 200 pL of G and T mixes. d. For dye-primers sequencing using linear amplification: i. Prepare stock dNTP mixes (sufficient for 200 reactions): dATP mix: 1.25 pL 20 mM dATP, 5.0 pL each 20 mM dCTP, dGTP, dTTP, 184.75 pL TE. dCTP mix: 1.25 pL 20 mM dCTP, 5.0 pL each 20 mM dATP, dGTP, dTTP, 184.75 l.tL TE. dGTP mtx: 2.50 pL 20 rniV dGTP, 10.0 pL each 20 mM dATP, dCTP, dTTP, 367.50 pL TE. dTTP mix: 2.50 pL 20 mM dTTP, 10.0 pL each 20 miWdATP, dCTP, dGTP, 367.50 pL TE. Note: 7-deaza-dATP and 7-deaza-dGTP may be substituted for dATP and dGTP m all mixes. For dATP mix, use 2.5 pL of 7dcdATP and 10 pL of 10 mM 7dc-dGTP; for dCTP mix, use 10 pL 3. l’hermus

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each of 10 mM 7dc-dATP and 10 mM 7dc-dGTP; for dGTP mix, use 5 JJLof 5 rniV 7dc-dGTP and 10 pL of 10 mM 7dc-dATP; for dTTP mix, use 20 pL each of 10 mM 7dc-dATP and 10 mM 7dc-dGTP. ii. Prepare ddNTP solutions (sufficient for 167 reacttons): ddATP (3.0 r&l): 100 pL of 5 mM ddATP + 67 pL of TE. ddCTP (1.5 n&f): 50 pL of 5 mM ddCTP + 117 $L of TE. ddGTP (0.25 rnM): 16.7 pL of 5 mMddGTP + 317.3 /JL of TE. ddTTP (2.5 mM): 167 l.tL of 5 mM ddTTP + 167 pL of TE. tn. Prepare dNTP/ddNTP working rruxes(sufficient for 100 reactions): 50 pL dATP mix + 50 pL 3.0 mM ddATP. 50 pL dCTP mix + 50 pL 1.5 n&! ddCTP. 100 pL dGTP mix + 100 pL 0.25 mM ddGTP. 100 pL dTTP mix + 100 pL 2.5 mM ddlTP. e. For dye-terminator sequencing wrth modified T7 DNA polymerase: i. 2 rniV [aS]dNTPs, 10 mM Trts-HCI (pH 7.2), 0.1 rniV EDTA. it. ZL 1.I. 1.2 dye-terminator mix: 2.2 pil4 ddT-6fam, 9.0 @4 ddCSzoe, 6.0 pA4 ddA-lou, 16.0 l&! ddG-nan (purchase from ABI; store at -2OOC). 6. Oligonucleotide primers: for Ml3 and phagemtd sequencing, universal, reverse, T7, and SP6 primers are available from Applied Biosystems. The sequences of these primers are: (-21 M13) 5’ TGT-AAA-ACGACG-GCC-AGT 3’, (M13RPl) 5’ CAG-GAA-ACA-GCT-ATG-ACC 3’. Note: Some preparattons of pUC 18 and pUC 118 have deleted one nucleottde at the 3’ end of the pnming site for the M13RPl pnmer; for sequencmg subclones based m these vectors, a reverse primer that lacks the 3’ C should be used, (T7) 5’ TAA-TAC-GAC-TCA-CTA-TAG-GG 3’, (SP6) 5’ ATT-TAG-GTG-ACA-CTA-TAG 3’. 7. 2X YT media: 16 g bacto-tryptone, 10 g yeast extract, 5 g NaCl, distilled water to 1 L, autoclave to sterilize. 8. 20% PEG (6000-8000 MW), 2.5M NaCl. 9. 10 mM Trts-HCl, pH 7.5. 10. Phenol: Saturated with 10 mM Trts-HCl, pH 8.0, 0.1 mM EDTA. 11. TE buffer: 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA. 12. GET buffer: 0.9 g dextrose, 2.5 mL of 1M Tris-HCl, pH 8.0, 2 mL of 0.5M EDTA, distilled water to 100 mL. 13. 10 mg/mL RNase A: Dissolve RNase in 10 mM Tris-HCl, pH 8.0,15 mM NaCl; boil for 15 min to inactivate DNase. Store in aliquots at -20°C. 14. Lysis solution: 10 mL of 2MNaOH, 10 mL of 10% SDS, distilled water to 100 mL.

ABI 373A

DNA Sequencing

Reactions

15. 3M KOAc solution: 60 mL 5M KOAc, 11.5 mL glacial acetic acid, 28.5 mL H,O. 16.2N NaOH. 17. 2M ammonium acetate, pH 7.4. 18. 10X PCR buffer: 500 mM KCI, 100 m&f Tris-HCI, pH 8.3, 15 mM MgC12, 0.1% (w/v) gelatin. 19. 3M sodium acetate, pH 4.8. 20.40% (w/v) PEG-8000, 10 ti MgC&. 21, 40% A&B: 380 g acrylamide, 20 g bisacrylamide, distilled water to 1 L. Deionize by stirring for 1 h with Amberlite MB-l (50 g/L), filter, store at 4°C in an opaque bottle. 22. 20X TEB buffer: 324 g of Tris base, 55 g of boric acid, 18.6 g of EDTA, distilled water to 1 L. 23. 15% Ammonium peroxysulfate. 24. 5X Hind/DTT (+Mn2+; make fresh): a. 50 rnA4 Tris-HCl, pH 7.5, 300 mA4NaCl, 5 mM DTT. b. Immediately before sequencing, add 3 @, of 1M MnCl, to 197 pL of 5X HindlDTT buffer. 25. 5M ammonmm acetate (pH 7.4). 26. 5X LASR buffer: 400 mA4 Tris-HCl, pH 8.9, 100 n-uJ4(NHJ2 SO,, 25 mM MgC12. 27. 10X Mn2+ buffer: 50 n&f MnC12, 150 mM sodium isocitrate. 28. 10X MOPS buffer: 400 mMMOPS, pH 7.5,500 mMNaC1, 100mMMgC12. 29. 9.5M ammonium acetate.

3. Methods 3.1. Preparation of Template

DNA

3.1.1. Large Scale Preparation Ml3 or Phagemid DNA 1. a. For phagemids: Using a sterile toothpick, pick a white colony and inoculate 10 mL of 2X YT medium (with ampicillin to 50 pg/mL) media in a 50-mL orange cap centrifuge tube (Corning). Grow the culture for 2-6 h (until the be,, measures0.5-1.0; 1Aesr,= 1 x lo8 cells/ mL). Add M13K07 helper phage (1 x 10” pfu/mL) at an moi of 10. Incubate the culture overnight at 37OC (addition of Kanamycin at a final concentration of 70 pg/mL is optional). b. For Ml3 clones: Using a sterile toothpick, pick a white plaque and maculate 10 mL of 2X YT medium in a 50-mL orange cap tube (Corning). Grow the culture for 8-16 h. of Single-Stranded

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2. Centrifuge the cultures in a Beckman GPKR centrifuge (or equivalent) at 3700 g for 15 mm to pellet the cells. 3. Transfer the supematant to 50-mL tubes containing 2.0 mL of 20% PEG (6000-8000), 2.5MNaCl. Cap the tubes tightly and mix by gently invertmg approx ten times. Let stand at room temperature for 30 mm. 4. Centrifuge at 4200 g for 30 min to pellet the phage. Carefully remove all of the supernatant by aspiration. A second spin followed by additional aspiration may be useful to remove all traces of PEG. 5. Resuspend the phage pellets m 500 pL of 10 n-M Tris-HCl, pH 7.5, by vortexing. Rinse all material from the sides of the tube and transfer the phage solution to a 1.5-mL microcentrifuge tube. 6. Add l/2 vol (250 pL) of phenol (saturated wtth 10 mM Tris-HCl, pH 7.5). Vortex vigorously for 15 s. When preparing several samples, the use of a multttube vortexer IS useful. 7. Centrifuge at 13,000g for 5 min at room temperature to separate the phases. Carefully transfer the top (aqueous) phase to a clean microcentrifuge tube. 8. To the aqueous phase, add an equal volume (500 pL) of chloroform or water-saturated ether. Vortex vigorously. 9. Centrifuge for 1 mm to separate the phases. Carefully remove and discard the organic phase. Let the tubes stand open at room temperature for a few minutes to allow any remaining solvent to evaporate. 10. Add l/10 vol(50 pL) of 3M sodium acetate, pH 5.2, and 2 vol (1 mL) of absolute ethanol. Invert several times to mix, and precipitate the DNA overnight at -20°C. 11. Pellet the DNA by centrifugation at 13,OOOgat room temperature for 15 min. Discard the supernatant and wash with 1 mL of 70% ethanol. 12. Dry the DNA pellets briefly under vacuum. Dissolve in 100 pL of TE buffer. 13. Quantitate the yield of single-stranded DNA by measurmg the absorbance at 260 nm. 3.1.2. Preparation of Double-Stranded Plasmid DNA 1. Spin for 15 s to pellet the cells from 1.5 mL of overnight culture; resuspend the cells in 100 pL of GET buffer. 2. Add 200 pL of freshly prepared lysis solution, mix gently by inverting a few times (do not vortex), leave on ice for 5 min. 3. Add 150 pL of 3M KOAc solution, mix, leave on ice 5 min. Spin for 5 min to pellet the chromosomal DNA and cell debris, remove 400 pL of supernatant to a clean tube. 4. Add 1 mL of absolute ethanol and mix by vortexmg. Place samples on dry ice for 5 min to precipitate the DNA.

ABI 373A DNA Sequencing Reactions 5. Wash once with 1 mL of 70% ethanol. Dry briefly under vacuum. 6. Resuspend the DNA pellet in 100 pL of 200 pg/mL RNase A and incubate at 37OC for 1 h. 7 Extract once with 50 pL of phenol. Transfer the aqueous phase to a new tube and precipitate the DNA by addition of l/10 vol of 3M sodium acetate, pH 5.2, and 2 vol of ethanol. Place dry ice for 5 min. 8. Pellet the plasmid DNA by centrifugation for 15 min at room temperature. Wash once with 1 mL of 70% ethanol, and briefly dry the DNA under vacuum. Dissolve the DNA pellet in 100 pL TE buffer.

3.1.3. Template Production Using PCR 1. PCR is performed using bacteriophage lysate, DNA (0.01-1.0 pg), or bacterial colonies or bacteriophage plaques that have been picked and transferred to a small amount of water or TE buffer. The reactions should contain the following components: distilled water 10X PCR buffer 1.25 mM dNTPs primer 1 (20 ClM) primer 2 (20 piV) template (typically l-5 pL) Taq DNA polymerase (5 U&L) Total volume

XCIL 5cIL 8PJ2.5 /AL 2.5 /IL YW 0.2 J,lL

50 ClL

2. If necessary, overlay each reaction with 80 pL of hght mineral oil. The thermal cycler is preheated to 95”C, and the samples are placed in the preheated block and incubated at 95OCfor 2 min. With the Perkin-Elmer instruments, thermal cycling is performed using the following parameters: DNA thermal cycler 9600 thermal cycler 94°C for 30 s 55°C for 30 s 72°C for 60 s

92°C for 10 s 55°C for 60 s 72OC for 60 s

for 35 cycles

for 35 cycles

At the conclusion of thermal cycling, the samplesare maintained at 4°C. 3. The samples are removed from the reaction tubes and transferred to 1.5~mL microcentrifuge tubes containing 8 pL of 3M sodium acetate, pH 4.8, and 20 pL of 40% (w/v) PEG-8000, 10 mM MgC12. If the samples were covered with mineral oil, it is important to avoid transferring any of the oil. The samples are mixed by vortexing and allowed to stand at room temperature for 10 min.

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4. The DNA is pelleted by centrifugation at 13,000g for 15 min at room temperature. All of the supernatant is carefully and completely removed by aspiration. The DNA pellets are washed twice with 250 pL of 100% ethanol (room temperature) and dried briefly under vacuum. 5. Each sample 1sdissolved m 20 pL of 10 mM Tris-HCl, pH 8.0, 0.1 mM EDTA. One to two pL of each sample may be analyzed on an agarose gel. For DNA sequence analysis using the linear ampliftcation sequencmg method described below, 1 pL of the PCR-prepared DNA is used m the A and C reactions and 2 pL in the G and T reactions.

3.2. Preparation

of Sequencing

Gels

1. Careful cleaning of the glass plates (supplied by Applied Biosystems, Inc.) is crucial with the fluorescent sequencing smce dust and scratches will cause light scatter. The plates should be carefully washed with a dilute solution of Alconox using a soft paper wiper (a large KimwipeTM works well), rinsed with warm water, and then thoroughly rmsed with distilled water. The plates are then rinsed with absolute ethanol, and the inner surfaces are wiped wtth isopropanol and allowed to au dry. Spacers and combs should also be washed and dried thoroughly. It is important to maintain each plate with a designated “mner” stde to minimize gel bubbles caused by small scratches-to do this, the “outer” side of the plate may be marked on one corner by a glass etcher, with tape, or permanent mk. 2. Lay the unnotched plate, outer side down, on a flat stable platform (a Styrofoam tube rack works well). Lay gel spacers along each long edge of the plate. Gently place the notched plate over the unnotched plate and spacers. Make sure the plate edges are all flush and that the spacers are properly positioned between the plates. Using two large binder clamps on each long side, clamp the assembly together. 3. Prepare the gel solution by combining the followmg m a 250-mL beaker: 22 g urea, 8 mL of 40% A&B, 2.5 mL of 20X TEB, and distilled water to 50 mL. Warm the solution at 55°C for a few minutes to dissolve the urea, and stir until the solution becomes clear. Filter the gel solution through a 0.45~pm membrane. (If sequencing gels are to be run daily, a working gel solution may be prepared and stored at 4°C. Combine 110.3 g urea, 40 mL of 40% A&B, 13 mL of 20X TEB, and 125 mL of distilled water. Drssolve the urea and filter as above. If stored m an opaque bottle, this solutton can be used for 2-3 wk. Use 50 mL/gel.) 4. To the gel solution, add 245 pL of 15% ammonium peroxysulfate and 30 pL TEMED. Swirl the beaker to mix and fill a 60-mL syringe with the solution. With the gel plate assembly flat on the platform, inject the

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gel solution between the plates along the notched edge of the top plate. Start at one side of the assembly and slowly move the syringe across the top, allowing capillary action to pull the solution into the form. If bubbles begin to form, tap on the top of the plates with a finger to force gel solution across the trouble spot. When the front edge of the gel solution flows to the open end of the gel form, cease injecting solution and insert the straight-edged casting comb into the notch at the top of the gel form. Place three binder clamps across the top of the notched plate. Allow the gel at least 2 h to polymerize. 5. Remove the clamps and the casting comb. There will be a thm strip of polyacrylamide along the inner bevel of the notched plate; remove this by runmng the edge of the casting comb along the bevel. Rinse the outside surface of the plates with water to remove any gel material, rinse again with absolute ethanol and allow to air dry. Wipe the outside of the plates with TexwipeTM glass cleaner or isopropanol. Place the gel assembly in the electrophoresis chamber of the 373A DNA sequencer. 6. Restart the Macintosh computer. Make sure that the ABI Data Collectton and Data Analysis programs are launched automatically upon startup (automatic startup of Multifinder, Data Collection, and Data Analysis should be preconfigured under “Set startup”). On the 373A sequencer, perform the plate check function to ensure that the glass plates have been properly cleaned and are free of dirt or gel material. The scan of the plates should be observed m the “Scan” window of the Data Collection program. Four flat lines, one for each “color” of the four fluors, should be observed. If blue peaks are observed, these indicate dust or smudges on the glass plates; carefully clean the dirty area of the plates with isopropanol or TexwipeTM glass cleaner and repeat the plate check as needed. If four-color peaks are observed, these indicate light scattermg caused by contaminants present in the gel itself. In this latter case, the peaks may disappear as the gel is prerun. However, if contaminant peaks persist after cleaning the plates and prerunning the gel, it is recommended that the gel be replaced or that samples not be loaded mto the wells that correspond to the problem area. If wells are skipped, be sure to check sample tracking when the run is completed to ensure that the contaminant peak has not been tracked and analyzed. 7. Set the upper buffer chamber and the lucite alignment brace in place. Carefully insert the sharkstooth comb between the glass plates, centering the comb with the numbers printed on the alignment brace. The teeth of the comb should just barely pierce the top of the gel. Tighten the two screw clamps on the upper chamber to secure the alignment brace. Fill the upper and lower buffer chambers with 1X TEB. Using a

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Pasteur pipet, flush urea out of the sample wells. Connect the buffer chamber electrodes to the 373A and close the electrophoresrs chamber door. Prerun the gel at 30 W constant power and 40°C for 30 min. At some point during the prerun, a new Sample Sheet (under the “File” menu) should be opened, and the appropriate data entered. 3.3. DNA Sequencing Reactions 3.3.1. T?/mT? DNA Polymerase Sequencing Method with Fluorescent Dye-Primers

In this procedure, reactions are performed using the modified T7 DNA polymerase chemistry (7,8) as adapted for sequencing with fluorescent dye-primers (9). The reactions are conveniently performed in 96-well U-bottom plates (Falcon, Becton Dickinson Co., Rutherford, NJ, No. 391 l), either by hand or using an automated pipeting station (10). Since the fluorescent dyes used in the G and T reactions produce a weaker signal, the reaction volumes are doubled. 1. Annealing reactions should be set up as follows: A

G T ---C 5X Hmd/DTT (+Mr?+; make fresh) G 1w 2w 2cIL template DNA (from aboveAPCR) 3W 3cIL 6@ 6cIL dye-prtmer 1cIL 1cIL 2cIL 2cIL The reactions are heated to 55°C for 3-5 mm, then cooled slowly to room temperature for lo-15 mm. If the reactions are performed m a 96-well plate, a dry block heater may be modified to effectively heat all 96 wells (II, 12). 2. To each annealing reaction is added: 8 mA4dNTPs + 50 w ddXTP mix modified T7 DNA polymerase (1.5 U/pL)

----A 2 pL

C

G

T

2cIL

4%

4cIL

1.5pL

1.5pL

3pL

3l.L

The reactions are incubated at 37°C for 5-10 min. 3. At the conclusion of extension and termination, the four reactions must be stopped before they may be combined and concentrated for electrophoresrs. A simple method for stopping the reactions is to place 6 pL of

5M ammonium acetate,pH 7.4, and 120 pL of 95% ethanol in 1.5~mL microcentrifuge tubes (one tube for each template). For each template set, 8 pL of the A reaction is transferred to the tube and two quick

ABI 373A DNA Sequencing Reactions

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cycles of up-and-down pipeting are used to rmse the tip and mix the sample with the ethanol solution. Without changmg the pipet tip, 8 pL of the C reaction and 16 @ of the G and T reactions are transferred to the tube, with up-and-down pipeting followmg each addition. The ethanol solution effectively stops further enzymatic activity. This procedure ISrepeated for each template set. The combmed reactions are placed at -2OOC for 30 min to precipitate the DNA products. 4. The DNA is pelleted by centrifugation at 13,000g for 15 min at room temperature, washed once with 300 pL of 70% ethanol (room temperature), and dried briefly under vacuum. The dried sample may be stored at -20°C for several days. 3.3.2. Linear Amplification Sequencing Method (Taq DNA Polymerase) with Fluorescent Dye-Primers 1. Sequencing reactrons should be set up in either 0.5 mL mtcrocentrifuge tubes or in 0.2-mL Micro-Amp TMtubes and should contain the following components: C T --A -- G 5X LASR buffer dNTP/ddXTP mtx dye primer (0.4 pmol/pL) template DNA (approx 250 ng/pL) 7’aq DNA polymerase (0.7 U/pL)

lPJlc1L lI.IL 1 pL w

1w 1w 1$ 1 pL 1w

Total volume

2N2w 2cIL 2lJ4 2cIL

loli

2w 2cIL 2& 2cIL 2$

1opL

2. If necessary, overlay each reaction with 50 JJL of light mmeral oil. The thermal cycler is preheated to 95°C and the samples are placed in the preheated block. With the Perkin-Elmer instruments, thermal cyclmg is performed usmg the followtng parameters: DNA thermal cycler 95°C for 30 s 55°C for 30 s 70°C for 60 s for 15 cycles, then

9600 thermal cycler 95°C for 4 s 55°C for 10 s 70°C for 60 s for 15 cycles, then

95°C for 30 s 95°C for 4 s 70°C for 60 s 70°C for 60 s for an additional 15 cycles for an additional 15 cycles At the conclusion of thermal cycling, the samples are maintained at 4°C.

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Note: If the fluorescent dye-labeled SP6 sequencing primer is used, the annealing temperature should be reduced from 55 to 50°C. Similarly, for other sequencing primers, the meltmg temperature (T,) should be calculated, and the thermal cycling conditions adlusted accordmgly. 3. As described for the modified T7 DNA polymerase chemistry, the four sequencing reactions must be stopped before they may be combined and concentrated for electrophoresis. Again, a simple method for stopping the reactions is to transfer each sample to a 1S-mL microcentrifuge tube containing the ammonium acetate/ethanol solution. After ethanol precipitation, the dried samples may be stored at -20°C for several days. 3.3.3. mT7 DNA Polym-erase Sequencing Method with Fluorescent Dye-Terminators This procedure requires that the 373A DNA sequencer be equipped with a special five-color (53 l/545/560/580/610) filter wheel. The sequencing reactions require single-stranded DNA, either Ml3 or phagemid. 1. For each sample, set up an annealing reaction in 1.5 mL microcentrifuge tubes as follows: 10X Mn2+ buffer 2ctL 10X MOPS buffer 2cIL ssDNA (2.5 clg) XW primer (3.2 pmol/pL) 0.5 pL ddHzO YctL Total 11 w 2. Incubate at 55°C for 5 min, then cool to room temperature and let stand for 7 mm. Briefly centrifuge to collect the condensation. 3. Add the following reagents to the annealing reaction: 2 mM [aS]dNTPs ZL 1.1 ,1.2 dye-termmator mix Sequenase/pyrophosphatase (13 U&L)

4c1L 4w 1 pL

4. Mix gently and incubate at 37°C for 10 mm. 5. To each reaction, add 20 $ of 9.5M ammonium acetate and 90 pL of ethanol. Mix gently and place on ice for 10 min. 6 Pellet the reaction products by centrifugation at 13,000g for 15 mm at room temperature. Wash twice with 300 p.L of 70% ethanol, and dry briefly under vacuum. Typically, a DNA pellet will not be observed.

ABI 373A DNA Sequencing Reactions 3.4. Electrophoresis

309

and Data Collection

1. Dissolve the dried sequencing reaction products in 5 pL of loadmg solution. Heat at 98°C for 3-5 min. 2. Carefully flush urea out of all sample wells using a Pasteur pipet. Load all odd-numbered samples mto the odd-numbered wells (e.g., 1, 3, 5). Close the electrophorests chamber door and electrophorese samples into the gel at 30 W constant power and 40°C for 2-5 min. Open the electrophoresrs chamber door, and load the even-numbered samples mto the even-numbered wells (e.g., 2,4,6). Close the electrophoresis chamber door and begin electrophoresis at 30 W constant power and 40°C for lo-14 h. Because of salt effects as samples enter the gel, this odd/even loading scheme will leave a small space between lanes, thereby facilitating proper lane tracking by the analysis software. 3. Using the mouse, chck on the “Collect” button to begin data collectton. The computer will collect data for lo-14 h. During thts time, it is recommended that the host Macintosh not be used for any other function. 4. At the completion of the run, open the electrophoresis chamber door and siphon some of the buffer from the upper buffer tank. Disconnect the electrode cables and unlatch and lower the laser stop. Carefully lift the upper buffer tank and gel assembly out of the instrument. Buffer remaining m the upper tank can be discarded. The glass plates should be carefully separated, cleaned, and stored in a safe location. Avoid using a razor blade to remove the gel; instead, press a paper towel against the gel, then peel the towel and gel off of the plate and discard. Rinse the upper and lower buffer tanks and invert on paper towels to dry.

3.5. Data Analysis and Assessment At the conclusion of data collection, the Macintosh computer will automatically launch the Data Analysis program. At this time, a gel file and a Results folder containing individual sample and sequence files will be created, Occasionally, problems will arise during the ini-

tial analysis phase that result in the absence of analyzed data. If this occurs, data can be salvaged using the recovery procedures described in the Troubleshooting section below. Data can be viewed by opening any of the sample files, selecting “Analyzed data” from the “Window” menu and using the custom view magnifier located on the floating “Analysis” controller. If the host Macintosh has been connected to a color printer or plotter, the analyzed data may be printed. Alternatively, data may be archived by transferring

sample files over a net-

work connection to another Macintosh, or a Sun computer using a

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communications program such asNCSATelnet. If data must be retrieved later for further work on a Macintosh, sample files should be transferred with the MacBinary protocol selected. Typically, data analysis begins immediately following the large fourcolor mass that represents unincorporated dye-labeled sequencing primer. However, the occasional sample will be analyzed from an earlier point if an extraneous fluorescent signal is detected. These samples may be reanalyzed manually; a new starting coordinate may be determined by viewing the raw data for that sample. “Call bases” is selected from the “Analysis” menu, and the new starting coordinate is entered in the appropriate box. The “Analysis queue,” located under the “Window” menu may be opened to monitor the analysis process. Note that several samples may be reanalyzed simultaneously. Clues as to data quality may be found in several places. The most obvious is the appearance and range of the analyzed data. Fairly uniform signal intensity, decreasing slightly over the length of the read, should be observed. Although base calls will be made for the entire read, experience has shown that the accuracy of machine base assignment falls off significantly after 400 bases. In a sample of fairly high quality, “no calls” (“N”) should be infrequent in the first 400 bases. Background noise, visible as small peaks along the baseline, should not be so high as to interfere with base calling. Excessive noise often is caused by impure sequencing template as discussed in greater detail below. Additional information as to data quality can be obtained from the “File info” window. Here, the signal and base spacing data is displayed. Typically, signal strength for a sample of high quality should be greater than 40-50 for the lowest of the four dyes. Base spacing should be 9-l 1; if base spacing falls outside of this range, accurate base calling can be affected. Base spacing can be optimized by adjusting gel and running buffer composition and electrophoresis conditions. The gel file, which contains a computer-generated color “autoradiograph” of the entire run, is also useful for assessment of data quality. Here, straight and separatelanes should be observed, with easily visible bands extending late into the run. Gel-related problems are often most apparentupon observation of the data contained in the gel file. For example, lanes that overlap, for reasons discussed below, may cause tracking problems and poor analysis of otherwise satisfactory data. Tracking may be assessedby observing the white tracker lines that should bisect

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all visible samples. If a tracker line does not seem to properly follow sample bands, the sample should be tracked manually. This can be done by choosing “Track a lane” from the “Analysis” menu and clicking on several points along the sample lane. At the conclusion of retracking, the sample will be analyzed automatically. 4. Notes 4.1. Equipment Problems 1. No scan lines appear in the Scan window. Make sure “Start scan” button on the 373A has been pressed. Main menu window should read “Stop scan.” If scan has been started and no scan lines appear, abort the run and restart. If scan lures still do not appear, reset the 373A by pressing the delete key on main keypad and selecting “total reset” on the LCD menu (Note: This reset will erase two settings-the current date and time, and the PMT voltage; reenter these values after the reset has been completed). If resetting the 373A does not solve the problem, check the cable connecting the 373A to the host computer. If none of the above solves the problem, ABI field service should be contacted. 2. Scan lines are less than 25% full scale. When the scan window is viewed at the beginning of a run, the flat scan lines should 25-33% of full scale. If the scan lines are lower than this, PMT voltage should be increased. The PMT voltage setting 1s accessed by selecting “calibration” and then “configure” on the LCD menu, and moving through the next few selections. The proper PMT voltage setting will differ by machine and may change as the laser ages. Typtcally a setting of 540560 works well. 3. Electrophoresis power failure. When electrophoresis power fails, the 373A will alert the user with a loud beep. Stop the run or prerun and restart electrophoresrs. If no current value IS observed, open the electrophoresis chamber door and check electrode cable connections. Check the platinum wires and their connections. If the electrophoresis power still fails, press the delete key on the 373A main keypad and select “elect only” on the LCD. If none of the above solves the problem, ABI field service should be contacted. 4. Stage motor failure. The electric motor that drives the scanning stage has a finite lifespan. Failure of this unit is often preceded by screeching sounds. This problem is not eastly remedied by the user; ABI field service should be contacted. 5. Laser failure. The argon laser also has a finite lifespan, although there appears to be a wide range to this lifespan. To ensure against sudden laser failure, a weekly diagnostic test should be performed. Under the

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“Self test” menu on the 373A LCD, press “more” until the “laser” test option is displayed. Press “laser” and enter “40” as the target power (e.g., 40 mW); record the actual laser output and the current drawn by the laser. As the laser ages, it ~111draw more current to produce the necessary power. When laser failure is imminent, the amount of current the laser draws will increase noticeably. At this point, ABI field service should be contacted. 6. Computer problems. Problems with data analysis can occur when the fixed disk drive of the host Macintosh computer becomes over 75% full. To avoid these problems, sample files should be backed-up, either on a floppy disk or by transfer to a remote fixed disk, and promptly removed. Additionally, the host Macintosh should be vtewed as a dedicated computer and should not be used for word processing, graphics, or any other purpose. Placing varrous INITs and cDevs (control panel devices) on the computer can cause severe problems. To avoid problems caused by disk fragmentation, a disk repair program, such as Apple’s Disk First Aid, should be run at least once a month. If problems arise with data analysis, check the “log file” located under the “Windows” menu in either the Data Collection or Data Analysis programs. This file contams an event list of data collection and analysis and can provide clues as to intermittent computer problems. If data analysis has failed and an error message 1s present, complete recovery of data is possible using one of several recovery procedures. A complete list of error messages and their causes is available from Applied Biosystems, along with detailed instructions for data recovery; it is highly recommended that all 373A users obtain this list and post it near the sequencer.

4.2. Gel-Related

Problems

7. Incomplete polymerization. Characterized by distorted patterns in the gel file. The sequencing gel should be allowed to polymerize for no less than 2 h. If sufficient polymerization time had been allowed, preparation of fresh 15% APS and TEMED may remedy the problem. 8. Sample crosstalk. This can be seen by observing the gel file, and can be caused by failure to properly insert the sharkstooth comb. The comb should be inserted so that the points of the teeth pierce about l-2 mm mto the gel. Addttionally, leakage can occur between wells if the comb was inserted, partially removed, and reinserted. If the comb damages part of the gel, avoid loading m that regton or replace the gel. Sample leakage also may occur with mcompletely polymerized gels (see above).

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9. Sample lanes overlap. This can occur when samples are not loaded in alternating lanes as described above. If samples overlap, lane tracking can be impeded. Additional problems can occur if too much time elapses between the second loading. Here, the fluorescence signal that accompanies the primer/salt front preceding the reaction products can interfere with proper detection and tracking. No more than 5 min should elapse before the even-numbered samples are loaded. 10. Gel runs too fast or too slow. Unincorporated dye-primers or dye-terminators should become visible in the Gel view after 60-80 min. If these peaks are seen much earlier or later, data analysis can be severely affected. Check the electrophoresis conditions on the 373A; we routinely run gels at 30 W constant power. The gel and buffer compositions also should be checked. Polyacrylamide gel compositions other than 6% are incompatible with the 373A.

4.3. Problems Relating to Template Preparation and Sequencing Reactions 11, Sequence data termmates early. Several factors can be responsible for truncated sequence data. If an RNase step was used in the template preparation, the presence of contaminating DNase will result in truncated sequence data. This problem often is accompanied by an increase in background peaks. Reactions performed with insufficient template DNA result in truncated data; a significantly decreased signal strength also will be observed in the File info window. Too much template in the sequencing reaction can also produce truncated data. If certain resins are used to remove RNA from the template preparation, contaminants may co-purify with DNA that intensify this problem. Here, the proper amount of DNA necessary for obtaining high quality sequence data should be titrated. Another common causeof truncated data is incorrectly formulated dNTP/ddNTP mixes. 12. Low signal strength. This problem may be caused by insufficient template DNA or insufficient primer in the sequencing reaction. Check that the concentration of primer is correct. For some sequencing primers, the standard 55°C annealing temperature may be too high, and should be reduced. This is especially true for the linear amplification sequencing method. For example, the SP6 primer gives very good results with an annealing temperature of 50°C, but almost no signal is observed when the primer is used with a 55°C annealing temperature. Low signal also can be caused when sequencing reagents are incorrectly formulated or stored.

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13. High background. DNA that contains a large amount of RNA will produce sequence data with high background. This is especially true for the linear amplification sequencmg method. Another common cause of high background is the presence of more than one template in the DNA preparation. This often happens because more than one colony or plaque has been inadvertently picked, and can be avoided by carefully picking only single colonies or plaques. Other background problems can originate when contaminated reagents are used for either template preparation or sequencmg reactions.

Acknowledgments The authors wish to thank R. Waterston and J. Sulston for advice and support, all of the members of the C. elegans genome sequencing project, and C. Chen for technical support.

References 1. Smith, L. M., Sanders, J. Z., Kaiser, R J , Hughes, P , Dodd, C., Connell, C R., Heiner, C., Kent, S. B. H., and Hood, L. E. (1986) Fluorescence detection in automated DNA sequencmg. Nature 321,674-679. 2. Ansorge, W., Sproat, B. S., Stegemann, J., Schwager, C., and Zenke, M. (1987) Automated DNA sequencmg: Ultrasensitive detection of fluorescent bands during electrophoresis. Nucl. Acids Res. 15,4593A602. 3. Prober, J M., Trainor, G L., Dam, R. J., Hobbs, F. W., Robertson, C. W , Zagursky, R. J., Cocuzza, A J , Jensen, M. A., and Baumeister, K. (1987) A system for rapid DNA sequencmg with fluorescent chain-terminating dideoxynucleotides. Science 238,336341. 4. Brumbaugh, J. A., Middendorf, L R , Grone, D. L., and Ruth, J L. (1988) Continuous, on-line DNA sequencmg using ohgodeoxynucleotide primer wrth multiple fluorophores. Proc. Nutf Acad. Sci. USA 85,5610-5614 5 Kambara, H., Nrshikawa, T., Katayama, Y., and Yamaguchi, T. (1988) Optimization of parameters m a DNA sequenator using fluorescence detectron. Biotechnology 6, 8 16-821. 6. Connell, C. R., Fung, S., Hemer, C , Bridgham, J , Chakerian, V , Heron, E , Jones, B., Menchen, S , Mordan, W., Raff, M , Recknor, M., Smith, L , Springer, J , Woo, S., and Hunkapiller, M. (1987) Automated DNA sequence analysis BioTechniques

$342-348

7. Tabor, S. and Richardson, C. C. (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc. Natl. Acad. SCL USA 84,4767-477 1. 8. Tabor, S. and Richardson, C C (1989) Effect of manganese tons on the mcorporation of drdeoxynucleotides by bacterrophage T7 DNA polymerase. Proc. Nat1 Acad Sci USA 86,4076-4080

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9. Wilson, R. K., Chen, C., and Hood, L. (1990) Opttmization of asymmetrrc polymerase chain reaction for fluorescent DNA sequencing. BloTechniques 8, 184-189. 10 Wilson, R K , Chen, C , Avdalovic, N., Burns, J , and Hood, L. (1990) Development of an automated procedure for fluorescent DNA sequencing. Genomlcs

6,626-634. 11. Koop, B F., Wilson, R, K., Chen, C., Halloran, N., Sciammis, R., and Hood, L. (1990) Sequencing reactions in microtiter plates. BioTechniques 9,32-37. 12 Seto, D. (1991) A temperature regulator for microtiter plates. Nucl Acids Res

19,2506.

&API’ER

36

Sequencing Reactions for ALF (EMBL) Automated DNA Sequencer Wilhelm Ansorge, Jiirgen Zimmermann, Holger Erfze, Neil Hewitt, Thomas Rupp, Christian Schwager, Brian Sproat, Josef Stegemunn, and Hartmut Voss 1. Introduction Automated fluorescent DNA sequencing (1-3) has turned into a subject of great interest during the last few years, and has successfully been applied to large-scale sequencingprojects, like sequencing of the human HPRT gene locus (4). Most actual genome sequencing efforts, such as sequencing the C. elegans genome, are performed on automated devices. Figure 1 shows the principle of the ALF DNA Sequencer that is based on the device developed at European Molecular Biology Laboratory (EMBL, Heidelberg, Germany) (1,5). It combines the advantages of the standard one label, four tracks Sanger sequencing methodology, with advanced features like continuous on-line detection and direct storage of data in a computer readable form. Besides DNA sequencing, design and flexibility of the system allow a wide range of applications like restriction fingerprinting (6), clinical diagnostics, and analysis of DNA-protein interactions. Methods m Molecular S/ology, Vol 23: DNA Sequencing Protocols From Edited by. H. and A Grlffm CopyrIght 01993 Humana Press Inc., Totowa,

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Ag 1. Prmcrple of the ALF (EMBL) DNA sequencer A water thermostated polyacrylamide gel is used for fragment separation At a fixed distance (e.g ,20 cm from top of the gel), a laser beam IS projected across the enttre width of the gel. This beam acts as a finish line. As soon as fluorescently labeled fragments enter the beam, they become excited and emit fluorescence light that is continuously measured by the optical detector for each smgle lane. Off-line evaluation allows automated sequence determmatton

Fluorescent labeling of molecules for DNA sequencing is achieved by extension of fluorescently labeled primers or incorporation of fluorescently labeled deoxynucleotides. The samples are loaded wrthout further purification in four different tracks on the gel. Figure 2 demonstrates a typical sequence output. All protocols and hints listed here are optimized for the ALF (EMBL) DNA Sequencer and have been wrdely used in the EMBL DNA sequencing service and various sequencing projects. The chapters are derived from the EMBO practical course manual DNA sequencing: Advanced approaches, automated methods and analysis.

DFi1s.C Run 901222A \AMOATA\AMIDITEQ Nematoden

Corn DhaSemid

6-Skb

DNA, ALF Lauf

insert

Clone 350 min1. a04f6

92 Time w 00

47

05 52

Ih mIDate

22P/i2% /

OPS

Fig. 2 Sequence output of a phagemid DNA with 9 kbp insert with fluorescently labeled primer and T7 DNA polymerase.

2.1. Manual

2. Materials Preparation of Single-Stranded

DNA

with Glass Fiber Filters 1. Fresh overnight culture of, for example, E. coli TG 1. 2. 2xYT medium: 16 g/L tryptone, 10 g/L yeast extract, 5 g/L NaCl. 3. Whatman GF/C glass fiber filter (7 mm diameter). 4. Glacial acetic acid (HAc). 5.4M NaC104 in TE. 6. 70% ethanol. 7. 0.1X TE. 8. Sterile toothpicks. 9. Spin X cartridges (Costar, Cambridge, MA, cat. no. 8162) carrier (0.45 urn cellulose acetate filter removed). 10. Filtration unit (Frg. 3).

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Fig. 3. Filtration unit, designed at EMBL, for preparation of single-stranded DNA loaded with Spin-X-Cartridges and GFK glass fiber filters. The unit allows parallel handling and processing of twenty-four samples. The unit consists of the suction unit (designed to fit twenty-four cartridges in microtiter format arranged as a Biomek 1000 adapter for twenty-four tubes) and a lid that allows the removal of all twenty-four cartridges together.

2.2. Automated Preparation of Single-Stranded DNA with Glass Fiber Filters 1. For biochemicals see Section 2.1.1. 2. Biomek 1000 system with EMBL filtration unit and vacuum pump.

2.2.1. Robot Configuration 1. Tools: PlOOO,P200, MP200, 1 fold Bulk, Biomek 1000 pipeting tools. 2. Tips: 1000~pL or 250~pL Biomek 1000 pipet tips. 3. Tray 1: 15mL culture tubes and 2-n& Eppendorf tubes in Biomek 1000 24 tube racks. 4. Tray 2: Pos. (3) 4M NaC104, Pos. (2) 0.1X TE, Pos. (1) HAc in Biomek 1000 solution reservoirs, 75 mL each. 5. Tray 3: EMBL filtration unit. 6. Reservoir: 70% ethanol. The scheme of the workstation configuration is shown in Fig. 4.

ALF

321

Fig. 4. Schematic view of GF/C process.

2.3. Preparation

of Double-Stranded

DNA

100 mL phosphate stock, 900 mL extract stock. a. Phosphate stock: 0.17M KH2P04, 0.17M K2HP04. b. Extract stock: 1.2 g/L bacto tryptone, 1.2 g/L yeast extract.

1. TB medmm:

2. LB medium: 10 g/L bacto tryptone, 5 g/L yeast extract, 5 g/L NaCl 1 mL 1N NaOH. 3. Buffer Pl : 50 mM Tns-HCl, 10 m&I EDTA, 100 Clg/mL RNase A (heat-

treated), pH 8.0. 4. Buffer P2: 200 mM NaOH,

1% SDS.

5. Buffer P3: 3.OM KAc, pH 5.5. 6. QBT: 750 mM NaC1, 50 mM MOPS, 15% ethanol, 0.15% Triton X100, pH 7.0. 7. QC: 1000 mM NaCl, 50 mM MOPS, 15% ethanol, pH 7.0. 8. QF: 1250 mM NaCI, 50 m44 MOPS, 15% ethanol, pH 8.5. 9. Isopropanol. 10. 70% ethanol.

2.4. Sequencing with T7 DNA Polymerase All solutions have to be prepared on ice, unless stated otherwise, and should be stored at -20°C to prevent evaporation and decomposition. Solutions that are set up from powders are filtrated through a 0.22+m filter. All reactions are performed in microtiter plates from Greiner or Falcon, if not stated otherwise.

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1, Rx buffer (1.5): 1000 miV Tris-HCl, pH 8 100 mM MgCl,. 2. Ex buffer (1.5): 324 n&f DTT, 304 mM citrate, pH 7.0,40 mM MnCI,. 3. DMSO. 4. Termination mixes (16): In all mixes 7-deaza-dGTP is included routmely to avoid compressions during electrophoresis. Solutions have to be set up for each of the four ddNTPs, the asterisk* means that only one of the four is present in the respective A, C, G, or T mix. Best results are obtained with nucleotides from Boehrmger, Pharmacta, or USB. 1 rml4 dATP, 1 mM dCTP, 1 mih! c7dGTP, 1 mM dTTP, 5 @! ddNTP*, 50 mM NaCl, 40 mA4 Tris-HCl, pH 7.4. 5. Labeling mix: 1 l.&f dlTP, 1 luV dCTP, 1 pil4 c7dGTP; 10 luV fluorescein- 15-dATP. 6. NaOH: Take commercial 1M stock solutions and aliquot them into I-mL batches. Solution should be stored m small aliquots because of transient neutralization of the NaOH by atmospheric COZ. 7. HCl: Take commercial 1M stock solution and aliquot into I-mL batches. 8. Stop mix: Deionize formamide by mixing 50 mL commercial Formamide (Merk) with 5 g Biorad AG 501-X8,20-50 mesh on a stirrer for 30 mm at room temperature. Filtrate through a Whatman No. 1 paperfilter. Adjust Dextran blue MW 2 x lo6 concentration to 8 mg/mL and EDTA, pH 8.3, to 20 mM. 9. Enzyme: T7 DNA polymerase may be used from Pharmaciaor USB accordmg to their instructions. Usually the enzymes are delivered together with the appropriate enzyme dilution buffer 10. Primers: For sequencing with the ALF (EMBL) DNA sequencer, either a fluorescently labeled primer or, when using fluorescein-15-dATP, an unlabeled primer IS needed. For primer selection and synthesis refer to Section 5. 11. Sequencing kits: For the ALF (EMBL) DNA sequencer a special designed kit from Pharmacia (order no. 27-1690-04) is available, mcluding fluorescently labeled universal and reverse primer, as well as DMSO to reduce secondary structures during sequencing reactions. 2.5. Automated Single-Stranded DNA T7 Reaction Protocol 1, Biomek 1000 with computer. 2. EMBL thermal controller or Beckman HCB 1000. 3. Beckman Sideloader (if desired). 4. Polystyrol microtiter plates. 5. Biochemtcals as in Section 3.

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ALF 2.51. Robot Configuration 1, Tools: MP20 &fold pipeting tool. 2. Tips: 250 pL pipet ups. 3. Tray 1: Stock plate, kept permanently at 4°C. 4. Tray 2: Second EMBL thermal block. 5. Tray 3: First EMBL thermal block or HCB 1000.

2.6. Cycle Sequencing 1. Sequencing buffer 5X: 250 m&I Tris-HCl, pH 9.0, 100 n&I (NH,),S04, IO mM MgCl,. 2. Taq DNA polymerase (Perkin Elmer, Cetus). 3. Fluorescein-1ZdCTP (Boehringer, Mannheim). 4. Termination mixes: c7dGTP dTTP ddNTP Mix dATP dCTP A 20 20 20 20 350 j.lM C G

T

20 20 20

20 20 20

20 20 20

20 20 20

200 pM 30 Mf 600 pM

5. Label mix: 10 @4 fluorescein-l2-dATP. 6. Stop mix: Deionized formamide, containing dextran blue 8 mg/mL 10 mJ4 EDTA, 5 mA4 NaOH. For preparation, see Sectron 3.1.

2.7. Single-Stranded DNA TaqlTtZ Protocol Using End-Labeled Primer 1. Reaction buffer: 260 mM Tris-HCl, pH 9.5, 65 mM MgCl,. 2. Primer: Fluorescently labeled universal sequencing primer UP-40 (2 pmol/pL). 3. Thermostable DNA polymerases: a. A Taq DNA polymerase, Version 2.0, 32 U/j.& b. Tth DNA polymerase, Pharmacia 8 U&L. 4. 5X Labeling mrx: 5 @I dATP, 5 j.tA4dCTP, 5 w dGTP, 5 pJ4 fluorescein-12-dUTP, dilute 1:4 just before use. 5. Terminatron mixes: a. A mix: 15 @4 each dATP, dCTP, dGTP, dTTP 300 @%4 ddATP. b. C mix: 15 @4 each dATP, dCTP, dGTP, dTTP 225 piI ddCTP. c. G mix: 15 @f each dATP, dCTP, dGTP, dTTP 22.5 WddGTP. d. T mix: 15 @!I each dATP, dCTP, dGTP, dTTP 450 pJ4 ddTTP. 6. Chase mix: 500 w each dATP, dCTP, dGTP, dTTP. 7. Stop mix: Deionized formamide cont. 8 mg/mL dextran blue.

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Most reagents (except the fluorescently labeled primer, the labeling mix, and the stop mix) are included in the TAQence Version 2.0 sequencing kit from USB, order no. 71070. The protocol described below requires 15 min. 2.8. Single-Stranded DNA Bst Protocol Using End-Labeled Primer 1. 5X reaction buffer: 100 mM Trts-HCI, pH 8.5, 100 mM Mg,CI. 2. Primer: Fluorescently labeled untversal sequencing primer UP-40 (2 pmol/mL). 3. Bst DNA polymerase 1 U/pL. 4. Termination mixes’ Mix A C G T

dATP 620 nM 800nM 800 nM 800 nM

dCTP 62

W

8 pM

SOW 80 W

dGTP

62 w

dTTP

62 w

80 w

80 w

4W 8OiJ-M

80 w SW

ddNTP 25 JJMddATP 50 f.tM ddCTP 75 uJ4 ddGTP 150 @4 ddTTP

All mixes contain 1.5 mM Tris, 0.15 mM EDTA, pH 8.0. 5. Chase mix: 500 w each dATP, dCTP, dGTP, dTTP.

6. Stop mix: Deronrzedformamrde contams 8 mg/mL dextran blue. All reagents (except the fluorescently labeled primer and the stop mix) are included in the BstTMpremixed standard sequencing kit from BioRad, Catalog No: 170-3407. 2.9. Template Purification and Sequencing Using Biotinylated PCR Products on Magnetic Beads 1. 10X PCR buffer: 100 mMTris-HCl, pH 8.3 (20°C), 15 mMMgC12, 500 mM KCl, 1% Tween 20, 0.1 mg/mL gelatin. 2. PCR nucleotide mix 200 pM dNTP. 3. Lysis buffer: 10 mMTris-HCl, pH 8.3 (2O”C), 5OmMKCl,O.l%Tween20. 4. Magnettc beads: Dynabeads M-280 Streptavtdin (Dynal AS Norway, Prod. No. 112.05), aliquoted into 200~pL batches. 5. Binding solution: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2M NaCl. 6. TE buffer: 10 mM Tris-HCl, pH 7.5, 1 mM EDTA.

7. Tuq DNA polymerase: Amph-Taq (Cetus). 8. Sequencing solutions as described in Section 3.1, 9. PCR primers (biotmylated and nonbtotinylated). 10. Magnet MPC-E (Dynal AS, Norway).

A.LF

325 2.10. Purification of Genomic PCR Products for Dideoxy Sequencing

1. Chemicals for PCR. 2. SeaKem Agarose gel 1.5% in TBE. 3. CentriconMicroconcentrator, Amtcon for spm dialysis.

2.11. Chemical Degradation of Fluorescently End-Labeled Crude PCR Products on Solid Support 1. 5 x 50-100-n& beakers. 2. 1 pair of fine tweezers. 3. Absorbent filter paper. 4. Scissors. 5. Fluorescently end-labeled PCR products. 6. Hydroxylamine hydrochloride 99%. 7. Potassium permanganate >99%. 8. Dimethyl sulfate (DMS) 99% (Use fresh DMS and store it in aliquots of 50 mL at 4°C). 9. Piperidine 98%. 10. Ammonmm formate 99%. 11. Formic acid 98-100%. 12. Triethylamme 99%. 13. Hybond M&G solid support (Amersham RPN.1500). 14. T > Pu/T + Pu-modiftcatton: Potassium permanganate stock solutton. Dissolve 200 mg of potassium permanganate in 100 mL of distilled water. Store the solution sealed and light protected at 4OC. 15. G-modtfication: Ammonium formate buffer, pH 3.5. Dissolve 31.5 mg of ammonium formate in 5 mL of distilled water and adjust pH to 3.5 with formic acid. Finally make the solution up to 10 mL with distilled water. The solution is stable for several months at 4°C. 16. A + G modification: Formic acid solution. An 88% (v/v) aqueous solution of formtc acid is used for sequencing oligonucleottdes, a 66% (v/v) aqueous solution for long DNA fragments. These soluttons should be prepared fresh prior to use. 17. C-modificatton: Hydroxylamme hydrochloride, pH 6.0. Dissolve 2.75 g of hydroxylamine hydrochloride in 5 mL of distilled water. Add 3 mL of triethylamine and star the mixture thoroughly. Adjust to pH 6.0 with triethylamine. Finally, make the solution up to 10 mL with distilled water. The solution is stable for several months in a sealed bottle at 4OC. 18. Piperidine solution for cleavage and elutton: The 10% (v/v) aqueous piperidine should be prepared fresh before use.

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2.12. Labeling and Purification of Prinzers 1. Oligonucleotide, wtth an ammolink at 5’ position in deprotection solution (i.e., ammonia). 2. 5,-6-carboxy-fluorescein-succmimidyl-ester (Boehrmger, Mannheim, order no 1055089 or Molecular Probes Inc., Eugene, OR, order no. C-l 311). 3. 3M Sodium acetate, pH 5.2. 4. Ethanol. 5. 70% Ethanol. 6. 250 mM Sodium carbonate/sodium btcarbonate, pH 8.5-9.0. 2.13. Preparation of Gels 1. Gel set up (consisting of thermal-notched plate, thermal-cuppling plate, comb, and spacers). 2. Secusept (Henkel) or equivalent detergent. 3. Distilled or deionized water. 4. Lint free tissues (Kimwipes, Kimberly & Clark) and Q-tips. 5. Macromould, Pharmacia, or an equivalent support. 6. 0.22 pm 15-mL filter unit. 7. Syringe (50 mL). 2.13.1. Chemicals 1. Premixed acrylamide/btsacrylamide (29: l), Biorad. 2. Trishydroxymethylaminomethane. 3. Boric acid. 4. EDTA. 5. APS. 6. TEMED. 7. Urea, BRL (enzyme grade), USB Gold. 2.13.2. Stocks and Solutions 1. 1OX TBE stock:Tns 108 g, boric acid 55 g, EDTA 7.45 g, add water to 1 L. 2. 30% Acrylamtde stock (29:l): The solution has to be kept at 4OC. 3. 10% APS: Store in aliquots at -2O”C, use a new altquot daily 4. Running buffer: 1.2X TBE.

3. Methods 3.1. DNA Preparation In this chapter we describe two protocols for preparation of singleand double-stranded DNA in sufficient amounts and quality for

sequencing. In general, carefully following the protocol is more important for successful sequencing than the actual protocol. Nevertheless,

ALfF the broad variation in sources of DNA requires a set of optimized protocols for fast and straightforward preparation of DNA. M 13 biology and cloning are described elsewhere (7,8). Cloning in phagemids and helper phage rescue is an alternative to the Ml3 system for single-stranded DNA sequencing (9). The glass fiber filter method (IO,II) described here can be used for either cloning system. 3.1.1. Manual

Preparation of Single-Stranded DNA with Glass Fiber Filters The following solid phase process has been developed for fast and pure DNA preparation of multiple single-stranded DNA samples in parallel. Twenty-four samples can easily be manually processed within 30 min. 1, Dilute a fresh overnight culture of TGl loo-fold with 2X YT medium and dispense 2-mL aliquots into 15mL Falcon tubes, one tube for each Ml 3 plaque to be sequenced. 2. Pick one Ml3 plaque per tube using sterile toothpicks, make sure that a plug of agar is transferred. 3. Grow cells 5-6 h at 37OC (preferably on a rotary wheel). Transfer cells to 1S-mL microcentrifuge tubes, spin 5 min in microcentrifuge. 4. Transfer the supernatant to a new microcentrifuge tube without disturbing the pellet. Add 15 pL of HAc to precipitate the phages. At this stage the precipitated phages can be stored at least for 1 d at 4OC. 5. Add the supernatant onto the Whatman GF/C filters placed in a spin-x filter carrier in the EMBL filtration unit, using gentle suction (Fig. 5). The precipitated phages will stick onto the glass fiber filter. Several filters for different samples can be handled simultaneously without cross contamination. The filter carrier can be reused an unlimited number of times. 6. Apply 1 mL 4M NaC104 in TE to each filter. The phage coats break, and the single-stranded DNA sticks to the filter in the presence of high salt concentrations. 7. Wash the filter with 1 mL 70% ethanol to remove all impurities. Let the filter air dry for 5 min or spin dry for 30 s. Extended drying can result in strand breakage, but usually does not interfere with the sequencing reactions. 8. Place the filter into a 1.5~mL microcentrifuge tube. Add 20 pL 0.1X TE to the filter and spin for 30 s in a microfuge. Check 1 pL on an agarose gel. Store the DNA at -2OOC until used in sequencing reactions. Ten pL solution is sufficient for one sequencing reaction.

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apply 1.

par HAc

prrclpl GFiC

flltw:

Z 1 ml 4 M 3. 1 ml 70 %

4. rptn

5. aaa 6. rpm

et al.

phagas

NaCIO, Ethanor

flltar dry ror 30s 0.1 x TE

201.d ONA

mto

mwoluga

IUD*

Fig. 5. Layout of the robotic workstatlon with tools, 1000 pL tips, trays, sample rack, and filtration unit.

3.1.2. Automated Preparation of Single-Stranded DNA with Glass Fiber Filters

For high throughput applications an automated process for the preparation of single-stranded DNA is available (12). The process is based on the commercial pipeting robot Biomek 1000 from Beckman Instruments (Fullerton, CA) and the EMBL filtration unit, described in Fig. 4. The system can produce twenty-four samples in about 30 min. 1. Phages are grown in lo-mL

tion (seeSection 2.1.3.).

Falcon culture tubes under vigorous

agita-

ALF 2. Bacteria are pelleted by centrifugation at 5OOOgfor 10 min. Tubes are placed on the Biomek, tray 1 position. 3. Two milliliters of each phage supernatant are transferred from the culture rack 1 to the respective 2-mL tube in the precipitation rack (PlOOO tool). Each tube is already preloaded by the robot with 40 pL of glacial acetic acid. The solution is mixed by aspiration and dispensing. Tips are changed after each pipeting step to prevent any cross contamination. The process is paused for 5 min to allow precipitation of the phages. 4. The culture tube rack is replaced with the filtration unit and vacuum is applied. The precipitated phages in the supernatant are transferred from the 2-mL Eppendorf tube (Tray 1) to the respective position in the filtration unit (Tray 3) and the culture medium is gently sucked through the GF/C filters. 5. Phages, sticking to the filter, are disrupted by twice adding 1 mL of 4M NaC104 (PlOOO tool) to each cartridge. 6. DNA, which remains bound to the filter, is washed with 2 mL of 70% ethanol (Pl 000 tool) 7. Processis pausedfor 5 min to allow drying of the filters in the filtration unit. 8. The robot transfers 40 pL of 0.1X TE from the reservoir to each filter cartridge. 9. DNA is eluted from the GFK filters by spinning the sample into 1.5 mL microfuge tubes at 3000g for 1 min. In general, 15 pL of the DNA solution is sufficient for one sequencing reaction. 3.1.3. Preparation

of Double-Stranded

DNA

Double-stranded DNA preparation using ion exchange columns, such as Qiagen and Nucleobond-AX are extensively used in our laboratory. Columns are available for capacities of 20, 100,500, and 2000 (Nucleobond-AX) E. Other common plasmid purification protocols may also work for automated DNA sequencing. The procedure described here combines high reproducibility and reliability. We recommend the use of high copy number plasmids (i.e., pUC and pBluescriptI1 series) for DNA sequencing. Other vectors may require larger culture volumes to achieve a sufficient DNA yield. Cloning for DNA sequencing should be done in ret- strains like XL- l-Blue, to prevent rearrangements. The protocols are written for a minipreparation (~25 ltg). The values in brackets are related to maxipreparations [>0.5 mg]. The protocol requires 80 min. 1. Grow plasmtd m proper host (i.e., XL-l-BLUE) overnight in 4 mL (200 mL) TB-medium with the appropriate antibiotics m a 50-mL Falcon

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tube [lOOO-mL Erlenmeyer flask]. Shake at about 300 rpm. Amplification with chloramphenicol is not necessary. TB-Medium allows higher cell densities and provides faster growing of bacteria. Yield of plasmid DNA is strain and plasmid dependent. Best yields are achieved with pUC and pBluescript vectors. 2. Spin down bacteria at 4000g 5 mm, pour off the supernatant, and leave tube inverted on a piece of towel for drying. It IS important to get rid of all culture supernatant, 3. Resuspend bacterial pellet in 500 pL [lo mL] buffer PI with a pipet and transfer suspension mto a 2-mL Eppendorf tube (40-mL Sorvall tube). Keep Pl on ice and store at 4°C after adding RNase. Homogeneous resuspension is essential for complete lysis in step 4. 4. Add 500 pL [lo mL] buffer P2 and mix by inverting the 2-mL Eppendorf tube 3-4 times and incubate at 25°C for 5 min. Lysis should be visible at once and the solution should get homogenous without any particles. In larger volumes lysis can take longer. Extend lysis up to 15 mm and/or to increase temperature to 37°C. 5. Add 500 l.tL [lo mL] buffer P3, mix by inverting several times, and spin at 4OCfor 15 min at 13,OOOg[20 mm at 20,OOOgl.Transfer supernatant to a fresh tube and spm again for 10 mm. Sometimes a layer of slimy material will remain on top of the solution. It will not interfere with quality of the preparation but can block the column. If there is any turbid layer visible, transfer the supernatant to a fresh tube and spm again for 5 mm at 4°C or remove the layer with a pipet tip. 6. Pour the supernatant immediately into a fresh tube. At this stage the samples can be stored at 4’C at least overnight. 7. Equilibrate Qiagen Tip-20 [Pack 5001 with 1 mL [ 10 mL] QBT. In most cases the column runs without forcing the hquid. 8. Apply sample. 9. Wash with 2X 2 mL [2X 15 mL] buffer QC. 10. Elute DNA with 1 mL (15 mL] buffer QF mto a 2-mL Eppendorf tube [30-mL Sorvall centrifuge tube] 11. Add 800 l.tL [ 10.5 mL] of isopropanol (25’C), vortex and spin at 4°C for 30 min at 13,000g. 12. Pour off supernatant. 13. Wash twice with 2 mL 70% ethanol. 14. Dry sample in SpeedVac for 1 mm. 15. Redissolve sample in 22 pL [200 pL] distilled water. Check 2 uL sample on an agarose gel. Use 7 pL for one sequencing reaction.

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3.2 Fluorescent DNA Sequencing Reactions In general fluorescent DNA sequencing reactions are influenced by the same factors as in radioactive sequencing (e.g., annealing conditions, primer to template ratio, and nucleotide concentrations). In automated on-line detection systems the signal strength is more effected by template amount, since low numbers of labeled molecules cannot be compensated by increasing the exposition times. Therefore we use in our laboratory for each kbp dsDNA construct length 1.5 pg DNA. For sequencing ssDNA, less than 1 pg is needed. In this chapter, protocols for T7, Bst, and Taq DNA polymerase are described. In our hands these enzymes behave superior to other polymerases, like Klenow, reverse transcriptase, or Vent DNA polymerase. We do not see any significant differences in sequencing results when using delta versions of thermostable enzymes. Protocols with fluorescent primers and internal labeling with fluorescein- 15dATP are provided. The handling of single-stranded DNA and doublestranded DNA, as well as PCR templates is discussed. Since its introduction in 1987 (13), T7 DNA polymerase has become the sequencing enzyme of choice, Although there are genetically or chemically modified T7 DNA polymerases available, we do not see any significant difference using either version. Introduction of Mn2+ ions (14) results in uniform peak strength, improving the accuracy of automated sequence readers. Nevertheless, Mn2+ ions have an inhibiting effect on the enzyme, and therefore a proper isocitrate/citrate buffering and deoxy- to dideoxy-nucleotide ratio is required for a suitable overall labeling distribution. 3.2.1. Single-Stranded DNA T7 Protocol Using End-Labeled Primer

The single-stranded DNA T7 protocol using end-labeled primer (17) requires a total of 20 min. 1. For annealing add to a mlcrofuge tube: a. ssDNA (0.4-l pg): 10 pL. b. End-labeled primer UP-40 (OS-IO pmol/mL): 2 pL. c. Rx buffer: 2 pL. d. Heat to 70°C for 3 mm and cool down to 22°C

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2. For strand synthesis and termmation add: a. EX buffer: 1.OuL. b. T7 DNA polymerase (5 U): 1 pL. c. Divide template in 4 aliquots mto a microtiter plate: 5 l.rL, d. (A, C, G, T) termination mtxes to the predtspensed aliquots: 3.0 p.L. e. Incubate at 37°C for 10 mm. 3. stop: a. Add stop solution to each reaction: 4 pL. b. Heat denature for 5 min at 75°C load 5 pL onto the gel. 3.2.2. Single-Stranded DNA T7 Protocol Using Fluorescein-15-dATP The single-stranded DNA T7 protocol using fluorescein- 15-dATP

requires a total of 20 min. 1. Annealing: a. Add ssDNA (400 ng) m a microfuge tube: 10 pL. b. Unlabeled primer UP-40 (0.5-10 pmol/pL): 2 l.tL c. Rx buffer: 2 & d. Heat to 70°C for 3 mm and cool down to 22°C. 2. Labeling and extension: a. Add labeling mix: 1 pL. b. Add T7 DNA polymerase (5 U): 2 pL, c. Incubate at 37°C for 10 mm. d. Add Ex buffer: 1 pL. e. 4 pL DMSO. 3. Termination: a. Add termination mtxes to a microtiter plate (A, C, G, T): 3.0 pL. b. Divide and add labeling/extension reaction 4X: 5 J.IL. c. Incubate at 37°C for 5 mm.

4. stop: a. Add Stop solution to each reaction. 4 pL. b. Heat denature for 5 min at 70°C load 5 pL onto the gel. 3.2.3. Automated Single-Stranded DNA T7 Reaction Protocol Existing robotic workstations allow the automation of the above-des-

cribedprotocols,with minor hardwareandsoftware modifications (18). Automated devices achieve not only higher sample throughput, but also better

reliability and reproducibility. In our laboratory we use a Biomek 1000, Beckman, with a thermal controller (Ftg. 6), constructed at EMBL (Biomek HCB 1000 thermal controller can be used), The additional Sideloader

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Fig. 6. Complete automated DNA sequencing reaction system based on a Biomek 1000 with thermal controller and sideloader.

system, Beckman allows fully automated processing of several hundred sample per day, by automatic disposable- and sample-rack exchange. The Biomek 1000 can handle volumes between 2-1000 pL, using different pipeting tools and plastic tips. We use V-bottom microtiter plates to provide centering of the reaction droplets in the wells. Reproducibility of pipeting steps is mainly dependent on planar plates. Microtiter plates can be obtained from Greiner and Dynatech. The system has to be calibrated for aspiration- and dispensing-height to prevent the tip from hitting the reaction vessel ground. The Biomek system operates in an open environment, therefore evaporation losses in the reaction microtiter plates and in the stock plate have to be compensated for. In the stock plate, usually kept at 4OC, the daily losses are calculated to a volume equivalent to one reaction cycle. General mixing of chemicals is done by pipeting with mid/ high piston speed and additional injection of air. Mixing of sequencing buffer and T7 DNA polymerase is provided by explicit aspiration and dispensing in the same well at minimum depth of the tip. In liquids with higher viscosity the actual volumes dispensed are smaller

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than the set values. Volume differences are compensated empirically. Similarly, volumes for liquids wetting the plastic tip surface are correspondingly corrected for deviations from the set values. 1. Stockplate (one cycle = 8 samples in one column). a. Co1 1: Fluorescently labeled prrmer (4 pmol/pL), 8 &/cycle. b. Co1 6: Sequencing buffer (Rx:Ex+ktH20 = 1: 1:l), 12 &/cycle. c. Co1 7: T7 DNA polymerase (1 U/w), 6 @/cycle. d. Co1 8: ddA termination mix, 6 pL/cycle. e. Co1 9: ddC termmation mix, 6 &/cycle. f. Co1 10: ddG termination mix, 6 w/cycle. g. Co1 11: ddT termination mix, 6 pL/cycle. h. Co1 12: Loading dye, 30 pL 2. Reaction plate at beginning of process (8 samples). Column 1: 8 singlestranded DNA templates, 5 pL. The addrtional 8-16 templates can be loaded on Co1 5 and/or Co1 9. 3. Reaction plate at end of process (8 samples). a. Co1 1: A terminated samples. b. Co1 2: C terminated samples. c. Co1 3: G terminated samples. d. Co1 4: T terminated samples. e. Respective Co1 5-8 and Co1 9-l 2 for the additional 8-l 6 templates. 4. Time and capacity: 8-48 samples per cycle in 20 min. The process is given in Table 1. Sources and targets are specified as

follows: The numeralwithin brackets meansthe tray position; Co1number meansthecolumnposition on the Biomek. Pipeting is done always at maximumdepth. Sampleanddisposabletransferby a Sideloaderis not included. 3.2.4. Double-Stranded DNA T7 Protocol Using End-Labeled Primer

All double-stranded DNA sequencing protocols are based on fast alkaline denaturation and hydrochloric acid neutralization (19). The double-stranded DNA T7 protocol using enlabeled primer requires a total of 20 min. Cosmid and lambda DNA sequencing (20,21) reactions are based on the plasmid protocol described below. To keep the

molar amount of template DNA constant for sequencing (0.245 pmol), more template mass is required. Optimal primer and enzyme concentrations have been determined empirically. 1. Denaturing and annealing in a mtcrofuge tube: a. DNA (1.5 pg/l kbp construct length), 10 +,.

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335 Table 1 Automated DNA Sequencing Process Utilizing

Action

Volume, pL

Temperature setting 4’C Pipet primer to sample 4.0 Tip change 6.0 Pipet buffer to sample Tip change Temperature setting 75°C Pause 3 min Temperature setting 2YC Pause 2 min Pipet T7 polymerase to sample 3.0 10.0 Mrxing by aspiration and dispensing Aliquot 3.5 Temperature setting 37°C Tip change Pipet A termination to A track 4.0 Tip change 4.0 Pipet C termination to C track Tip change 4.0 Pipet G termination to G track Tip change Pipet T termination to T track 4.0 Tip change Pause 15 min 6.0 Pipet stop mixes/w. tip change Temperature setting to 75°C Pause 5 min Temperature setting to 0°C End Samples are ready to load or store at -20°C

Biomek

Source [l]Col

1

[ l]Col6

Destinatron r.31 [3]Col 1 [3]Col 1 131 [31

[l]Col7 [3]Col 1

[3]Col 1 [3]Col 1 [3]Cols l-4

[l]Col

[3]Col 1

8

[ l]Col9 [l]Col

[3]Col2 10

[3]Col 3

[ l]Col 11

[3]Col4

[l]Col

[3]Cols l-4

12

b. End-labeled primer (2-10 CIM), 2 pL. c. NaOH (lM), 1 & d. Heat to 75°C for 3 min. e. Add/mix HCI (lM), 1 pL. f. Rx buffer, 2 pL. g. Ex buffer, 1 pL. h. DMSO, 4 pL. 2. For strand synthesis and termination add: a. T7 DNA polymerase [7 U], 1.0 pL. b. Termmation mixes to a microtiter plate (for A, C, G, T) 3.0 p.L,

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c. Divide annealed template 4X, 4 pL. d. Incubate at 37°C for 10 min. 3. stop: a. Add stop solution to each reaction, 4 pL. b. Heat denature for 5 min at 70°C load 5 pL onto the gel. 4. Modifications in brief: a. Cosmid or lambda DNA, 15-30 pg. b. Fluorescently labeled primer, 10 pmol. c. T7 DNA polymerase, 6 U. Processing of samples is performed as described above. 3.2.5. Double-Stranded DNA T7 Protocol Using

Fluorescein-15dATP

The double-stranded DNAT7 protocol using fluorescein- 15-dATP (22) requires a total of 20 min. 1. For denaturmg and annealing add/mix: a. DNA (5 pg, e.g., from Qiagen mimprep), 7 pL. b. Unlabeled primer (2-10 pM), 2 pL. c. NaOH (lM), 1 pL. d. Heat to 70°C for 3 mm. e. HCl (IM), 1 pL. f. Rx buffer, 2 pL. 2. For labelmg and extension add/mix: a. Labeling mix, 2 pL. b. T7 DNA polymerase (7 U), 1 pL. c. DMSO, 4 uL. d. Incubate at 37°C for 10 min. 3. For termination add: a. Ex buffer, 1.O pL. b. Terminatton mixes to a microtiter plate (A, C, G, T), 3.0 pL. c. Divide and add labeling reaction 4X, 4 l.tL, d. Incubate at 37°C for 5 mm. 4. stop: a. Add stop solution to each reaction, 4 pL. b. Heat denature for 5 mm at 7O”C, load 5 pL onto the gel. 3.2.6. Automated T7 Double-Stranded DNA Sequencing Reaction Protocol Using End-Labeled Primer This section lists only the modifications that are needed for automated double-stranded DNA sequencing (23) in comparison to singlestranded DNA. Basics and system set-up are the same as in Section 3.4.

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1. Co1 1: Fluorescently labeled primer (4 pmol/pL), 8 @+/cycle. 2. Co1 2: NaOH (diluted 1 in 6), 8 &/cycle. 3. Co1 3: HCl (diluted 1 in 6), 8 @,/cycle. 4. Co1 6: Sequencing buffer (Rx:Ex:~~H~O = 1:l :l>, 12 l&/cycle. 5. Co1 7: T7 DNA polymerase (5 U&L), 6 pL/cycle. 6. Co1 8: ddA termination mix, 6 l&,/cycle. 7. Co1 9: ddC termination mix, 6 pL/cycle. 8. Co1 10: ddG termination mix, 6 pL/cycle. 9. Co1 11: ddT termination mix, 6 &/cycle. 10. Co1 12: Loading dye, 30 uL/cycle. The protocol with the modifications for automated double-stranded DNA sequencing can handle 8-48 samples per cycle in 20 min. Here is only the denaturation/neutralization step listed (Table 2). After neutralization follow the process exactly as in Section 3.2.3.

3.3. Troubleshooting

fir T7 Sequencing

In general, each series of sequencing reactions should include one control reaction using high quality DNA. For single-stranded DNA we recommend the use of 1 pg of commercially available M 13mp 18 DNA from Pharmacia or USB. For double-stranded DNA we recommend the use of 5 ltg of a Qiagen maxi prep of pBluescript (without insert) in E. coli XLl-Blue, grown in TB or 2x YT medium. Fluorescent DNA sequencing results with labeled primer strongly depend on the quality of the fluorescently labeled primers. To check the quality when changing batches or when introducing a new primer, prepare seven sequencing reactions, each with 5 l.tg pBluescript KS11 as template. Perform sequencing reactions with the same protocol using 4 pmols of Universal/Universal-4O/Reverse/T7/T3/SWKS primer. In the following troubleshooting guide it is assumed that the control reaction works. 3.3.1. No Peaks

or Very Little

Signals

1. Component missing: Check control reaction and protocol. 2. Wrong primer used: Make sure that correct primer is used. 3. Inefficient priming or labeling: Check whether the respective primer gives strong signals in control reactions. If not, change primer batch. 4. Template concentration too low: Make sure using the correct template concentrations; 1 l.lg for single-stranded DNA or 1.5 ug/kb construct length for double-stranded DNA.

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Table 2 Denaturatlon/Neutralization Step for Automated T7 Double-Stranded DNA Sequencing Action Volume, & Source Destination Temperature setting 4°C [31 Temperature setting 7S’C [31 Pipet primer to sample 40 [3]Col 1 1 ICol 1 Tip change Pipet NaOH to sample 60 [3]Col 1 [ lCol2 Tip change Pause4 min Temperature setting 37°C Pause2 min 6.0 Pipet HCl to sample [3]Col 1 [ lCol3 Tip change 60 Pipet buffer to sample [3]Col 1 1 lCol6 Mixing by aspiration and dispensing Continuewith Table 1 5. Dirty template DNA: Check if control reaction IS alnght. If yes, prepare new template DNA, carefully following the protocols. If necessary, try alternative protocols, that may work better in your hands. 6. Enzyme lost activity: Check control reaction. If necessary,replace enzyme. 3.3.2. Full Stops: Peaks in All Four Lanes at the Sam Position Within a Clone 1. Poor DNA quality inhibits enzyme: Check control DNA, if necessary repeat DNA preparation. In case of single-stranded DNA use GFK method, or when using PEG precipitation, make sure that all PEG is carefully removed from the prep. In general “full stops” mostly occur in plasmid preps. We strongly recommend that you use the QiagenTM miniprep protocol and wash twice with 70% ethanol before resuspending the sample. If necessary, wash once more with 70% ethanol. 2. In case of double-stranded DNA, fast denaturation and neutrahzation protocol failed: Make sure to use fresh 1M NaOH and 1M HCl solutions. Always use commercial preadjusted solutions. Do not use homemade stocks unless they both have exactly the same molarity. Mix very carefully during fast denaturatlon and neutrallzatlon. 3. Enzyme has lost activity: Check control reaction. Resequence with a new batch of T7 polymerase or add more enzyme (10 U). 4. CT-rich region: T7 DNA polymerase tends to have difficulties m CTrich regions. This problem can often be solved by simply resequencmg the sample in presence of more enzyme (10 U).

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5. Strong secondary structure is causing enzyme to dissociate from the template: In caseof double-stranded DNA, preheat template/primer annealing mixture for 5 min at 70°C and use up to 10 U T7 DNA polymerase for one sequencing reaction. When sequencing single-stranded DNA, change sequencing enzyme: perform sequencing reaction with thermostable enzymes like Bst DNA polymerase or Tuq DNA polymerase at 65-72*C. 6. Dirty or old reagents: Prepare fresh reagents. 3.3.3. Compressions Bands at nearly the same position in two or three lanes in GC-rich sequences, followed by an increased spacing in the next bases could be an indication of sequence dependent compression under the conditions of gel electrophoresis. This problem is already reduced to a minimum by routinely using 7-deaza-dGTP in the nucleotide mixes during sequencing reactions. Ifit still occurs try the following: Substitute the 7-deaza-dGTP by dITP or 7-deaza-dITP in the nucleotide mixes. Run the sample again, but use normal 7-deaza-dGTP in parallel to compare, since dJTP may cause problems in areas where 7-deaza-dGTP works. Run the gel at 60°C. In any case,if a compression occurs we strongly recommend to sequence the other strand also. 3.3.4. Double Sequence: Extra Peaks Throughout the Sequencing Run at Many Positions in All Lanes 1. DNA preparation contains two different DNAs that are producing double sequences: Prepare new DNA starting from a single plaque or colony. In caseof Ml 3 DNA resultmg from spontaneous deletion of insert arising during phage growth. Try control DNA and limit phage growth to 6.5 h. 2. Mispriming in the unknown insert DNA: Use another sequencing primer for the same orientation, such as Universal-40 primer instead of universal primer. 3.3.5. Labeling Too Short: Signals Decrease Exponentially 1. Enzyme has lost activity: Check control reaction. Resequence with a new batch of T7 DNA polymerase. 2. Enzyme inhibited by Mn2+ ions: Check annealing buffer. Make sure composition of the annealing buffer is correct. Make sure all reagents are mixed properly. 3. Termination mixes with wrong dNTP:ddNTP ratio: Try new termmation mixes. 4. Wrong DNA concentration: Make sure to use sufficient DNA,

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3.3.6. Missing Bands Weak signals at distinct positions in a good sequencing run could be an indication that the termination reaction time is too long. If the termination reaction is allowed to continue too long, the synthesized DNA may be degraded at specific sequences by extraneous nucleases or pyrophosphorolysis. Try reducing the termination reaction time to 2 min. The effect is particularly noticed when too high concentrations of doublestranded DNA were used. Try to reduce the amount of template DNA. 3.3.7. Bad Resolution Even at Lower Bases (450) 1. Contaminated DNA preparation: Check control DNA; if necessary make new template prep. 2. Poor quality of polyacrylamlde gel: Prepare fresh acrylamide/bisacrylamide stock solutions and buffers using only high quality reagents. Change TEMED and APS. Stock solutions should be stored for not longer than 1 week at 4°C in the dark. Acrylamide should polymerize within 12 min after pouring. 3. Gel run at wrong temperature: Check temperature and adjust to 45-5OOC. 4. Sample not sufficient denatured: Make sure samples are always heated to 90°C for at least 3 mm immediately prior to loadmg onto the gel. 5. Impurities m primer synthesis: Check primer. 3.3.8. Transient

Bad Resolution and /or High Background During the Run in All Lanes 1. Salt front or other impurities co-migrating: a. Change all biochemicals. b. Use T7 DNA polymerase in the highest available concentration (>lO U&L) and dilute enzyme in ice cold TE instead of enzyme dilution buffer to keep glycerol content as low as possible. 2. Wrong loading dye: Make sure there is no bromophenol blue and xylene cyan01 but 8 mg/mL dextran blue as loading aid in the stop solution

3.4. Cycle Sequencing Protocols for Taq DNA Polymrase Cycle sequencing (24,25) is based on the idea to increase the amount of labeled sequencing fragments by repeating the steps of a sequencing reaction (thermal denaturation, annealing, and extension/termination) in a cyclic process. Thermostable enzymes, that is Taq or Tth DNA polymerase will survive the thermal treatment. Typically thirty cycles are performed. Annealing conditions and synthesis time are

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strongly dependenton primer sequenceand template structure. Labeling of strands can be performed either by extension of an end-labeled primer or incorporation of fluorescein-12-dCTP together with an unlabeled primer. 3.4.1. Taq Cycle Sequencing Using End-Labeled Primer The Tuqcycle sequencing using end-labeled primer requires 75 min. 1. Mix sample in 1.5-mL microfuge tube: a. DNA (50 ng M13,500 ng plasmid, 5 pg cosmid), 5 pL. b. End-labeled primer (2-10 CIM), 4 pL. c. 5X cycling buffer, 6 pL. d. DMSO, 3 pL. e. ddH@, 4 a. f. Taq DNA polymerase mix, 1 pL. g. Total, 20 pL. 2. Mix by vortexing and spin briefly. 3. Prepare 4 x MICRO AMPTM reaction tubes with 4 pL nucleotide mixes (A, C, G, T) each. 4. Aliquot sample, four times 5 pL, into the respective A, C, G, T reaction vessels. 5. Cycling conditions: Cycle conditions have to be optimized for the used thermocycling device. Here are the conditions for some primers based on the Perkin Elmer 3600 cycler (three waterbaths with a robot arm for moving samples) with very fast temperature step gradients (>lO”C/s). 6. UP-40 Primer: 30 Cycles (95°C for 35 s, 55°C for 35 s, and 72°C for 70 s).

3.4.2. Taq Cycle Sequencing Using Fhorescein-12-dUTP The Taq cycle sequencing using fluorescein- 12-dUTP (26) requires 75 min. 1. Reaction mix in 1.5-n& microfuge tube: a. DNA (50 ng M13,500 ng plasmid, 5 pg cosmid), 5 pL. b. Unlabeled primer (2-10 CIM), 4 pL. c. 5X cycling buffer, 6 pL. d. Labeling mix, 4 pL. e. Tuq DNA polymerase mix, 1 pL. f. Total, 20 l.tL. 2. Mix by vortexing and spin briefly. 3. Prepare 4 x Micro Amp TMtubeswith 2 pL termination mixes (A, C, G, T) each.

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4. Divide reaction mix, four times 4 pL, mto the of the respective A, C, G, T reaction vessels. 5. Conditions for cycle sequencing wtth fluorescein-12-dCTP are tdentical to those mentioned above. 3.4.3. Single-Stranded DNA TaqlTth Protocol Using End-Labeled Primer Thermostable DNA polymerases may be useful in sequencing templates with strong secondary structures that cannot be resolved using T7 DNA polymerase. In our experience Taq, Tth, and Bst DNA polymerases gave comparable results. Bst DNA polymerase shows best peak uniformity. We describe here sequencing protocols wtth Taq/ Tth and Bst DNA polymerase. 1. Annealing: a. Reaction buffer, 2 pL. b. Single-stranded DNA (400 ng), 10 pL. c. Fluorescently labeled UP-40 (2 pmol/pL), 1 pL. d. Heat to 70°C for 3 min, cool down to 22°C. e. Add A Taq Version 2.0. DNA polymerase (diluted 1:S) or Tth DNA polymerase (diluted 1:2), 2 pL. 2. Extension and termination: Add termmation mix A, C, G, T to four tubes, 4 pL, prewarm at 72°C. divide annealed template 4X, 3.5 pL, to each termination mix and incubate at 72OC for 5 min. 3. Chase: Add chase mix to each reaction A, C, G, T, 2 pL, and incubate at 72OC for 2 min. 4. Stop: Add sequencing stop mix, 4 pL, and heat denature at 70°C for 5 min, load 5 pL on the gel. 3.4.4. Single-Stranded DNA Taq Protocol Using Fluorescein-12-dUTP Time The single-stranded DNA Taq protocol using fluorescein- 12-dUTP requires 20 min. Materials as listed in Section 2.7. 1. Annealing: a. Reaction buffer, 2 pL. b. Single-stranded DNA (400 ng), 10 pL. c. Unlabeled UP-40 (2 pmol/pL), 1 pL, d. Heat to 70°C for 3 mm, cool down to 22°C. 2. Labeling and extension. Add to the annealed primer/template: Labeling mix (diluted 1:5), 2 JL, A Taq Version 2.0. DNA polymerase (diluted 1:8), 2 pL, incubate at 45°C for 5 min.

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3. Termination: a, Add termination mtx A, C, G, T to four tubes, 4 pL. b. Prewarm at 72°C. c. Divide the annealed template 4X, 4 pL, to each termination mix incubate at 72OC for 5 min. 4. Chase: Add chase mix to each reaction A, C, G, T, 2 pL, and incubate at 72°C for 2 min. 5. Stop: Add sequencing stop mix, 4 pL, heat denature at 70°C for 5 min, load 5 pL on the gel.

3.5. Single-Stranded DNA Bst Protocol Using End-Labeled Primer The protocol requires 20 min. 1. Annealing reaction: a. 5X reaction buffer, 2 l.tL. b. Smgle-stranded DNA (400 ng), 7 pL. c. Fluorescently labeled UP-40 (2 pmol/pL,), 1 pL. d. Heat to 70°C for 3 min, cool down to 22’C. e. Add Bst DNA polymerase (I U/l&), 1 pL. 2. Sequencing reaction: a. Add termination mixes A, C, G, T to four tubes, 2 pL. b. Prewarm at 65OC. c. Divide the annealed template 4X, 2.5 pL, to each termmation mix. d. Incubate at 65°C for 2 mm. 3. Chase reaction: Add chase mix to each reaction A, C, G, T, 2 pL, incubate at 65°C for 2 min. 4. Stop reaction: Add sequencing stop mix, 4 pL, heat denature at 70°C for 5 min, load 5 pL onto the gel.

3.6. Sequencing

of PCR Products

3.6.1. Protocol for Template Purification and Sequencing Using Biotinylated PCR Products on Magnetic Beads with Fluorescein-12-dATP or End-Labeled Primers The protocol (27-30) for purifying and sequencing PCR products with magnetic beads is outlined in Fig. 7. The use of magnetic beads allows exchange of buffers and enzymes between PCR and sequencing reaction without centrifugation and/or filtration. PCR is carried out using an appropriate set of one biotinylated and one unmodified primer. The PCR for recombinant material is carried out on a single

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t

>

PCR

BIOM-PIU~W 4

V

41 V

Strand Immobilization

Strand Separation -41

v

mmobdmd

strand

<

Fig. 7. Scheme for sequencmg PCR products with magnetic beads

colony/plaque. The biotinylated PCR product is immobilized by cova-

lent binding to the streptavidin coated magnetic beads. The nonbiotinylated strand is separated by alkaline denaturation. To obtain the two single-stranded sequencing templates, beads are aggregated by a magnet. The biotinylated strand remains attached to the beads, whereas the nonbiotinylated

strand is in the supernatant. Finally, a standard

Sanger sequencing reaction with T7 DNA polymerase is performed on each strand separately. Using the Biomek 1000 robotic work station for the sequencing reactions, the complete method following PCR can be automated (31) taking approx 45 min.

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1. Pick a colony with a pipet tip, suspend in 10 pL lysis buffer and heat to 99OC for 5 min. Spin down the cell debris. Use 1 pL of the supernatant for PCR. 2. Set up 50 pL PCR reaction: a. 10X PCR buffer: 5 pL. b. dNTP mix: 5 pL. c. 5 pmol of each PCR primer: 2 pL. d. ddHZO: 37 pL. e. Supernatant from step 1: 1 pL. f. 1 U Taq DNA polymerase: 0.2 pL. g. Cover the PCR mixture with 20 pL mineral oil. 3. PCR is performed for thirty cycles, with denaturation at 96°C for 30 s, annealing at 65°C for 1 min, and extension at 72OC for 2 min. Annealing temperatures should be optimized in respect to the T, of the used primers. 4. Add 20 pL of Dynabeads M-280 Streptavidm (10 mg/mL) mto a 1.5mL microfuge tube. 5. Wash once with 1 vol of binding solution: Place the tube in the magnet, wait 30 s to collect the beads at one side of the tube. Remove the buffer with a pipet. Add binding solution and mix by repetitive pipeting, with the tube in the magnet. Remove the supernatant again. Resuspend the beads m 40 pL binding solution. 6. Transfer 40 pL of the total 50 pL PCR reaction to a fresh tube and add 40 pL of washed Dynabeads, from step 5. 7. Incubate at room temperature for 15 min. The beads should be resuspended 4 times during the incubation. 8. Collect the beads and remove the supernatant. 9. Wash the beads with 50 pL binding solutton. 10. Resuspend with 50 pL TE buffer. The beads can be stored in TE at 4OC for several weeks. 11. Collect the beads, remove the TE buffer, and resuspend the beads in 10 pL of O.lM NaOH solution, 12. Incubate at room temperature for 5 min. 13. Collect the beads on one side of the tube and transfer the NaOH supernatant (nonbiotinylated strand) to a fresh tube and keep it on ice for later use. 14. Wash the beads with 50 pL of O.lM NaOH. 15. Wash the beads with 50 pL binding solution, 16. Wash beads with 50 pL TE buffer and resuspend in 10 pL distilled water and keep on ice unttl use. 17. Prior to use neutralize the NaOH supernatant (nonbiotinylated strand, from step 11) with 10 pL of O.lM HCl, mix immediately with a pipet.

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In general do not store DNA in nonbuffered solutions. After purification, the two strands of DNA can be processed as single-stranded DNA templates, either with fluorescently labeled primer, with fluorescein1ZdUTP (32) or fluorescein-15dATP (32a) and unlabeled primer.

3.6.2. Protocol for Purification of Genomic PCR Products for Diokoxy Sequencing

This protocol was contributed to the EMBO course by Bernd Dworniczak, Institute of Human Genetics in Miinster, and is based on the original protocol from Gyllenstein and Ehrlich (33). A combination of symmetric PCR, intermediate gel purification, asymmetric PCR, and spin dialysis results in pure single-stranded DNA (Fig. 8). The main advantage of the procedure is that nonspecific side products can be excluded after symmetric PCR. 1. Start with a symmetric PCR reaction in 50 pL according to common procedures. 2. Run 50 pL PCR products on a 1.5% TBE-agarose gel. 3. Cut the selected band out of the gel and place the agarose block mto a microfuge tube. 4. Add 100 l.tL of TE buffer and incubate overnight at 4°C. It is not necessary that the agarose block is completely submerged in buffer. 5. Take 10 pL of the solution for reamplification in an asymmetric PCR using the same primer pair as m step 1. The ratio between the individual primers should be 50: 1. Amplification conditions are identical to those in step 1. 6. Apply PCR solution (without oil) to a CentriconMicroconentrator. 7. Repeat 3-4 times. Perform spin-dialysis, using the Microconcentrator in a fixed angle rotor, according to manufacturer’s recommendation. 8. Residual liquid (40 pL) is removed from the Microconcentrator and dried down in a SpeedVac. 9. Template is resuspended in 14 pL distilled water, and 7 pL are taken for the sequencing reaction.

3.6.3. Protocol for Chemical Degradation of Fluorescently End-Labeled PCR Products on Solid Support The use of solid supports in Maxam-Gilbert sequencing reactions (34) facilitates modification and cleavage reactions avoiding precipitation steps. Figure 9 shows the principle of Maxam-Gilbert solid phase sequencing with fluorescent labels (3.5-37). Fluorescently labeled

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Symetric PCR

Gel Purification

4-J Asymetric PCR

DNA Sequencing Fig. 8. Scheme for purification

of genomic PCR products

templates are prepared by PCR with one labeled and one unlabeled primer. After immobilization of the products to the solid support, base specific modification, cleavage, and elution are performed. 1. Perform modification reactions as shown in Table 3. 2. Remove the paper from the reaction tube. Wash 3 times in distilled water and 3 times in 96% ethanol. Let the paper air dry. Cut the single segments and transfer them into 4 mtcrofuge tubes labeled “A,” “C,” “G,” and “T.” 3. Prepare fresh 10% (v/v) aqueous piperidine solution and add 100 pL into each tube. Close the tubes carefully, heat for 30 min at 90°C to perform chain cleavage, and release the DNA from the paper. 4. Remove paper segments from tubes with a tweezer and dry the solution m a SpeedVac for 30 min. 5. Wash twice to remove all traces of piperidine: Add 100 & distilled

water and dry in SpeedVac.

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single end-labeled DNA-fragments

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349 Table 3 Modification Reactions Reagent Volume 2X KMn04, 0.2 mM 30.0 pL 1.0 mL H2O NH20H, 4M, pH 6.0 l.OmL l.OmL HCOOH, 66% 1.0 mL HCOONH4, 50 mM, pH 3.5 DMS 7.5 pL

Time 2x 10min

Temp 22%

10 min 5 min 2-5 min

22T 22°C 22OC

6. Resuspend the DNA in 10 pL stop mix, heat denaturefor 2 min, and load 4 pL/sample onto the sequencinggel.

3.7. Selection and Preparation of Primers fir Fluorescent DNA Sequencing “Walking primer” approachesseem to be the most effkient strategy for sequencinglarger DNA fragments. Subcloning work and redundancy of the generatedsequenceis reduced to a minimum. In general, success of sequencing mainly depends on proper design of primer for optimal annealing.Standardprimers areincluded in sequencingkits, custom primer can be ordered from several companies (e.g., Clonetech). 3.7.1. Selection of Primers To achieve best sequencing results the following criteria for selection of primers should be considered: 1. Avoid secondary binding along the known template sequence (vector

and insert), under the annealingconditions required. 2. Avoid stable primer dimer formation. 3. Avoid self-complementarity (internal hairpins). 4. Assure stable duplex formation with template, by selection of minimal AG values (38,39) under the appropriate annealing conditions (salt concentration). 5. The annealing site for the new walking primer should not be closer than 50-70 bp to the 3’ end of the region already sequenced, to allow a sufficient overlap in sequence assembly. 6. Primers for sequencing PCR products should be 30 bp downstream from the ends of the product. Primers closer to the ends often result in poor signals.

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The selection of oligonucleotides for DNA sequencing is easily performed with computer programs like OLIGOTM (MedProbe A.S., Postboks 2640, St. Hanshangen, N-013 1 Oslo, Norway), that help to design primers according to the rules mentioned above. The following primer sequences are optimized for DNA sequencing, in our laboratory with commonly used vectors, such as M13, pUC, pBluescriptI1 (PBS) derivatives: 1%mer UP-40: GTT-TTC-CCA-GTC-ACG-ACG UP-20: GGT-AAA-ACG-ACG-GCC-AGT 18-mer GAA-ACA-GCT-ATG-ACC-ATG 18-mer REV: T3: ATT-AAC-CCT-CAC-TAA-AG 17-mer AA-TAC-GAC-TCA-CTA-TAG 18-mer T7: 19-mer SP6: GAT-TTA-GGT-GAC-ACT-ATA-G KS: CGA-GGT-CGA-CGG-TAT-CG 17-mer SK: GCC-GCT-CTA-GAA-CTA-GTG 18-mer PBS-T3: TCA-CTA-AAG-GGA-ACA-AAA-G 19-mer pBST7: GCG-TAA-TAC-GAC-TCA-CTA 18-mer 3.7.2. Labeling

and Purification

of Primers

Detailed description of oligonucleotide synthesis can be found elsewhere (40). We will explain the post synthesis labeling procedure of aminolinked primers and their purification in detail. Fluorescently end-labeled primers can be synthesized on any commercial DNA synthesizer, The label is introduced by coupling a fluorescently labeled amidite analog, (FluorePrimeTM, Pharmacia), as the last residue to the 5’ end of the primer. Alternatively an aminolinker (e.g.,Aminolink-2, Applied Biosystems, Foster City, CA) can be coupled to the S-end and a postsynthesis labeling reaction is performed. The postlabeling procedure is discussed below. 1. Remove the ammonia solution from the fully deprotected oltgo by SpeedVac . 2. Resuspend in 300 $ distilled water, add 30 p.L of 3M sodmm acetate

solution, 900 pL of -2OOCabsolute ethanol and mix the contents by inversion.

3. Precipitate the oligo for 1 h at -2OOCand spin for 5 min to collect the oligo. This precipitation procedure is essential to remove ammonium ions that will otherwise react with the dye. 4. Discard the supernatant and wash the pellet with 300 pL of cold 70%

ethanol.

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5. Dry the pellet in a SpeedVac and dissolve in 200 pL of fresh 250 mM sodium carbonate/sodmm bicarbonate buffer, Check pH before usage because of atmospheric CO2 neutralization of the solution. 6. Add 1 mg of 5,-6-carboxy-fluorescein-succinimldyl-ester and mix by vortexing. The dye is not very water soluble. 7. Leave the reaction at 22OC overnight in the dark. 8. Purification of the oligonucleotide can be performed on a 20% PAGE. On PAGE the fluorescein labeled oligo is observed as the most intensive, slowest migrating green band. This band is excised from the gel and the oligo obtained by electroelution or by soaking out. Purification can also be performed on reversed phase HPLC. The dye labeled primer appears as two bands (monitor at 260 nm and 495 nm), because the commercial dye is supplied as a pair of tsomers. This has no effect on sequencing results. The simplest way of purification is desalting by a size exclusion column, such as NAP-10TM (Sephadex G-25) from Pharmacia, according to manufacturer’s recommendation, but usmg an eluation volume of 1 3 mL. 9. In general for a purified fluorescein labeled primer of average base composition the measured AZ6anrn:Aag5 nm ratio should be about 3:5.

3.8. Preparation

of Gels

3.8.1. Glass Plates Thermostatic-, notched glass plates, spacers, and comb, should be thoroughly cleaned using detergent powder, that is, Secusept. Never use material that can harm the glass surface. After cleaning, all parts must be rinsed with distilled or deionized water and dried with lint free tissues, that is, Kimwipes. 3.6.2. Light Coupler Rinse light coupler with deionized water and dry with Kimwipes. Take special care of the optical polished edges, where the laser beam enters the gel. Wipe these sides with a cotton stick. Check surface for damage. If any scratches are detected take a new coupler. 3.8.3. Assembly of Gel Sandwtch 1. Place thermostatic plate on a support, with gel side up. 2. Insert light coupler in respective spacer. 3. Deposit spacers on the thermostatic plate, spacer with light coupler on the side with the water inlets. 4. Position clean notched plate on top of thermoplate. Be aware that spacers are aligned properly to the glass plates.

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5. Fix sandwich with two clamps on each side. 6. For a 340 x 280 x 0.3 mm gel, 50 mL of the gel solution are needed. a. 7% gel solution 50 mL for 0.3 mm gel: 30% acryl. stock, 11.7 mL, 10X TBE stock, 6 mL, Urea, 21 g, add distilled water to 50 mL. b. Filter through a 0.22~pm filter unit and degas for 2 mm. 7. For polymerization add 150 pL APS and 45 pL TEMED. 8. Fill gel solution into 50-mL syringe and cast gel from top side of gel (where comb will be inserted). Gel solution enters sandwich just by capillarity. To avoid any air bubbles, provide constant flow of liquid and an excessof material in notch. Move syringe across the whole width of the notch. Take care of straight liquid front within gel sandwich. If gel front tends to become uneven, tap the glass plate with a finger. Leave a small excess of gel solution between the “ears” of the glass plate to ensure that no air will be trapped when the comb is inserted. 9. Insert well cleaned comb and attach buffer tank. 10. Polymerization should be between 7-10 min. 11. Electrophoresis settings with ALF: Power = 45 W, current = 42 mA, voltage = 1600 V, running time = 580 min, laser power = 4 mW, sampling rate = 1.75 s.

4. Notes 1. Programs are available from authors upon request. 2. Cosmid and lambda maxi scale preparation: Preparations are done according to supplier’s recommendation. 3. Automatization: For a partial automatization of dsDNA preparations on a Biomek 1000 system, Beckman, or any of the above-mentioned cartridge systems (Nucleobond-AX and Qiagen) are usable. The cartridges fit in a modified EMBL Filtration unit with the lid modified m height for Tip-20. All pipeting steps are programmed as in the ssDNA purification process. All centrifugation steps need manual interference. 4. Programs will be made available upon request. 5. Alternatively the Auto Cyle Kit, Pharmacia can be used. 6. Best results m sequencing of PCR products are obtained using a third internal sequencing primer. 7 The Microconcentrator can be reused. 8. Best results m sequencing PCR products are obtained wtth a third mternal sequencing primer. 9. Sequencmg reactions are performed as recommended for ssDNA sequencing in Section 3.2.

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10. In addition

to conventional polyacrylamide gels we have successfully used HydrolinkTM (AT Biochem). This matrix doubles the separation speed, appears more stable during prolonged electrophoresls, and allows reloading of the gel (41).

References 1. Ansorge, W., Sproat, B., Stegemann, J., and Schwager, C. (1986) A non-radloactive automated methods for DNA sequence determination. J. Biochem. Biuphys. Meth. 13,3 15-323.

2 Smith, L. M., Sanders, J. Z., Kaiser, R. J., Hughes, P., Dodd, C , Connell, C. R., Heiner, C., Kent, S B. H., and Hood, L. E. (1986) Fluorescence detection m automated DNA sequence determination. Nature 321,674-679 3. Prober, J. M., Trainor, G L., Dam, R. J., Hobbs, F. W., Robertson, C. W., Zagursky, R. J , Cocuzza, A. J., Jensen, M. A., and Baumeister, K. (1987) A system for rapid DNA sequencing with fluorescent chain terminatmg dideoxynucleotides. Science 238,336-341. 4. Edwards, A., Voss, H., Rice, P., Civitello, A., Stegemann, J., Schwager, C , Zimmermann, J , Erfle, H., Caskey, T., and Ansorge, W. (1990) Automated DNA Sequencmg of the Human HPRT Locus. Genomlcs 6,593-608. 5 Ansorge, W., Sproat, B., Stegemann, J., Schwager, C., and Zenke, M. (1987) Automated DNA sequencing: ultrasensitive detection of fluorescent bands during electrophoresis. Nucl. Acids Res. 15,4593A602 6. Voss, H., Stegemann, J., Schwager, C , Zimmermann, J , Erfle, H., Hewitt, N A., Rupp, T., and Ansorge, W (1992) High-Speed Automated DNA Fragment Analysis for Genome Mapping by Restriction Fmgerprintmg. Meth. Mol. Cell. Biol. 3,77-82.

7. Messing, J. (1988) New Ml3 vectors for clonmg. Meth. Enzymol 101,20-78. 8 Messing, J , Gronenborn, B., Muller-Hill, B., and Hofschneider, P. H. (1977) Single-strand filamentous DNA phage as a carrier for in vitro recombined DNA. Proc. Natl. Acad. Sci. USA 74,3642-3646.

9 Hawkins, T. and Sulston, J. E. (1990) Automated Fluorescent Primer Walking. Technique 2,307-3 10. 10. Kristensen, T., Voss, H., and Ansorge, W. (1987) A simple and rapid preparation of Ml3 sequencmg templates for manual and automated dideoxy sequencing. Nucl. Acids Res. 15,5507-5516. 11. Krlstensen, T., Voss, H., and Ansorge, W. (1987) A simple and rapid preparation of Ml3 sequencing templates for manual and automated dldeoxy sequencing. Nucl. Acids Res. 15, 5507-5516. 12 Zimmermann, J., Voss, H , Knstensen, T., Schwager, C , Stegemann, J , Erfle, H., and Ansorge, W. (1989) Automated Preparation and Purification of Ml3 Templates for DNA Sequencing. Meth. Mol Cell. Biol 1, 29-34. 13. Tabor, S and Richardson, C C (1987) DNA sequence analysis with a modified bacteriophage T7 DNA polymerase Proc. Natl. Acud. Sci. USA 84,4767-4771.

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14 Tabor, S. and Richardson, C. C. (1989) Effect of manganese ions on the incorporation of dideoxynucleotides by bacteriphage T7 DNA polymerase and Escherichia coli DNA polymerase. Proc. Natl. Acad. Sci. USA 86,4076-4080. 15. Bergh, S , Hultman, T , and Wahlberg, J. (1991) Solid phase sequencing, in EMBO practical course: Solid phase techniques for DNA sequencing, clonrng, andproteinpurification, Royal Institute of Technology, Stockholm, Sweden. 16 Voss, H., Schwager, C , Kristensen, T., Duthie, S , Olsson, A , Erfle, H , Stegemann, J., Zimmermann, J., and Ansorge, W. (1989) One-step reactron protocol for automated DNA sequencing with T7 DNA polymerase results in uniform labeling. Meth. Mol. Cell. Biol 1, 155-159 17. Kristensen, T., Voss, H., Ansorge, W., and Prydz, H (1990) DNA dideoxy sequencing with T7 DNA polymerase improved sequencing data by the additions of manganese chloride. Trends rn Genetics 6,2-3. 18 Zimmermann, J., Voss, H., Schwager, C , Stegemann, J , and Ansorge, W (1988) Automated Sanger dideoxy sequencing reaction protocol. FEBS Lett. 233,432-436. 19. Zimmermann, J., Voss, H , Schwager, C , Stegemann, J , Erfle, H., Stucky, K , Kristensen, T., and Ansorge, W (1990) A simplified protocol for fast plasmid DNA sequencmg Nucl Acids Res. 18,1067 20. Voss, H , Zimmermann, J., Schwager, C , Erfle, H., Stucky, K , and Ansorge, W. (1990). Automated fluorescent sequencing of cosmtd DNA. Nucl. Acids Res. 18, 1066. 21. Voss, H , Zimmermann, J., Schwager, C., Erfle, H., Stucky, K., and Ansorge, W. (1990). Automated fluorescent sequencing of lambda DNA. Nucl. Acrds Res. 18,53 14 22. Voss, H., Schwager, C., Wrrkner, U , Zimmermann, J., Erfle, H , Hewitt, N. A., Rupp, T., Stegemann, J., and Ansorge, W (1991) A new procedure for automated DNA sequencing with multiple internal labelling by fluorescent dUTP. Meth. Mol. Cell. Biol. 3,30-34 22a.Voss, H., Wiemann, S., Wirkner, U., Schwager, C , Zimmermann, J., Stegemann, J , Erfle, H., Hewitt, N. A , Rupp, T., and Ansorge, W. (1992) Automated DNA Sequencing System Resolving 1000 Bases with Fluorescem15-dATP as Internal Label Meth Mol Cell. Biol. 3, (in press). 23 Zimmermann, J., Dietrich, T., Voss, H , Erfle, H., Schwager, C , Stegemann, J., Hewitt, N., and Ansorge, W. (1992) Fully Automated Sanger sequencing protocol for double stranded DNA. Meth Mol. Cell Biol. 3,39-42. 24. Murray, V (1989) Improved double stranded DNA sequencing using the linear polymerase chain reaction. Nucl. Acids Res. 17,88-89 25 Carothers, A M , Urlaub, G , Mucha, J , Grunberger, D., and Chasin, L A (1989) Point mutation analysis m mamahan gene. Rapid preparation of total RNA, PCR amplification of cDNA and Taq sequencing by a novel method Biotechnique

7,494

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26. Ztmmermann, J., Voss, H., Erfle, H., Rupp, T., Dletrtch, T , Hewitt, N A , Schwager, C., Stegemann, J., and Ansorge, W. (1992) Cycle Sequencing Protocol with Fluorescein-12-dCTP for M13, Plasmid and Cosmid DNA. Meth Mol Cell. Biol. 3 (in press). 27. Hultman, T., Bergh, S., Moks, T., and UhlCn, M. (1991) Bidirectional SohdPhase Sequencing of In Vitro Amplified Plasmid DNA. BioTechniques 10, 84-93.

28. Hultman, T., Stlhl, S., Hornes, E., and UhlCn, M. (1989) Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as solid support. Nucl. Acids Res. 17,4937-4946. 29. Lundeberg, J , Wahlberg, J., Holmberg, M., Pettersson, U., and Uhltn, M. (1990) Rapid colortmetric detection of in vttro amplified DNA sequences. DNA and Cell Biol. 9,287-292.

30. Wahlberg, J., Lundeberg, J., Hultman, T., and UhlCn, M. (1990) General colorimetric method for DNA diagnostics allowing direct solid/phase genomic sequencing of the positive samples. Proc. Nat1 Acad. Sci. USA 87,6569-6574. 31 Hultman, T., Bergh, S., Moks, T., and Uhlen, M. (19!91) Bidirectional solidphase sequencing of m vitro-amplified plasmid DNA. Biotechnique 10,84-93 32. Zimmermann, J., Voss, H., Erfle, H., Rupp, T., Dretttch, T , Hewitt, N A., Schwager, C., Stegemann, J., and Ansorge, W. (1992) Direct Sequencing of PCR Products using Magnetic Beads and Fluorescein-12-dUTP. Meth MOE Cell. Biol. 3, 114-l 15. 32a,Zimmermann, J., Voss, H., Erfle, H., Wiemann, S., Rupp, T., Hewett, N. A , Schwager, C , Stegemann, J., and Ansorge, W (1992) Direct Sequencing of PCR Products using Magnetic Beads and Fluorescein-15-dATP (in press). 33. Gyllenstein, U. and Ehrlich, H. (1988) Generation of smgle stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl. Acad. Sci. USA 85,7652-7656. 34. Rosenthal, A., Schwertner, S., Hahn, V., and Hunger, H. D. (1985) Solid phase methods for sequencing nucleic acids. I. Simultaneous sequencing of different oligonucleotides using a new, mechanically stable anion exchange paper Nucl. Acids. Res. 13, 1173-l 184. 35 Ansorge, W., Rosenthal, A., Sproat, B., Schwager, C., Stegemann, J., and Voss, H. (1988) Nonradioactive automated sequencing of oligonucleotides by chemical degradation. Nucl. Acid Res 16,2203-2206. 36 Voss, H , Schwager, C., Wirkner, U. Sproat, B., Zimmermann, J., Rosenthal, A., Erfle, H., Stegemann, J., and Ansorge, W. (1989) Direct genomlc fluorescent on-line sequencing and analysis using in vitro amphfication of DNA. Nucl Acid Res. 17,25 17-2527.

37. Rosenthal, A., Sproat, B., Voss, H. Stegemann, J., Schwager, C., Erfle, H , Zimmermann J , Coutelle, C , and Ansorge, W. (1990) Automated sequencing of fluorescently labelled DNA by chemical degradatton. DNA Sequence 1, 63-7 1

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38. Breslauer, K. J., Frank, R., Blocker, H., and Marky, L A. (1986) Predtctmg DNA duplex stability from the base sequence. Proc Natl. Acad. Sci USA 83, 3746-3750

39 Freter, S M , Kiezek, R., Jaeger, J. A , Sugineto, N , Caruthers, M. H., Neilson, T., and Tuerner, D H. (1986) Improved free-energy parameters for predlctions of RNA duplex stability. Proc. Nat1 Acad. Sci USA 83,9373-9377. 40 Gait, M. J (ed ) (1984) Uligonucleotide Synthesis. A practical approach IRL Press, Oxford, UK. 41. Stegemann, J., Schwager, C , Erfle, H , Hewitt, N , Voss, H , Ztmmermann, J , and Ansorge, W. (199 1) Hugh speed DNA sequencing on a commercial automated sequencer using 0.3-mm thin gels Meth. Mol. Cell Biol 2,292-293

CkIAPTER

37

Sequencing Using the Du Pont GenesisTM 2000 DNA Analysis System* Len Hall

1. Introduction The Du Pont GenesisTM 2000 DNA Analysis System consists of an instrument, reagents capable of labeling DNA with novel fluorescent dideoxynucleotides (I), and a Macintosh@’ II computer to operate the instrument and perform all subsequent data analysis. The instrument houses a vertical electrophoresis apparatus (similar to a standard manual sequencing setup) with associated buffer reservoirs and high voltage power supply, together with a fluorescence detection system. An argon laser sweeps across the 12-lane sequencing gel and excites fluorescently labeled DNA fragments as they electrophorese past a fixed window toward the bottom of the gel, and the emitted fluorescence is detected simultaneously by two photomultiplier tubes with differing filters (Fig. 1). The GenesisTM2000 System is based on a modified form of Sanger sequencing (Z), where the four traditional dideoxynucleotides are replaced by four ddNTP analogs, each possessing a unique succinyl fluorescein moiety (I). Each fluorescently-labeled terminating nucle*Editor’s Note* Du Pont IS no longer marketing the GeneslsTM2000 system. This chapter is included because many laboratories possess this instrument and still wish to use it. From. Methods m Molecular Biology, Vol 23: DNA Sequencmg Protocols Edlted by: H. and A. Gnffm Copyright 01993 Humana Press Inc , Totowa,

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otide can therefore be identified from its unique emission spectrum as determined from the ratio of the two photomultiplier signals (Figs. 1 and 2A). A major benefit of this terminator labeling system over primer labeling systems and conventional manual sequencing, is that only DNA fragments that have incorporated a ddNTP are detected. This totally eliminates artifacts resulting from premature termination caused by regions of template secondary structure, and other things of that nature. Another benefit of terminator labeling is the ability to perform a single sequencing reaction containing all four ddNTPs, rather than four parallel reactions, each with a different ddNTP. Since the four ddNTPs possess different fluorescein groups, with different spectral properties, the two filter detection system is able to distmguish the differently terminated DNA fragments in a single lane. Finally, by using terminator (rather than primer) labeling, standard primers used for conventional manual sequencing can be used with the GenesisTM 2000 System, thereby making rt particularly suitable for modern gene walkmg sequencing strategies using custom primers. The GenesisTM 2000 chemistry includes a number of additional advanced features. Band compressions and other artifacts caused by template secondary structure (e.g., in G + C-rich regions) are markedly reduced by the use of deaza-dGTP and deaza-dATP in place of the corresponding unmodified dNTPs in the sequencing reaction (Fig. 2B).

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The polymerization reaction is carried out by SequenaseTMin the presence of Mn2+ ions, resulting in a more uniform incorporation of the four different ddNTPs, with a subsequentincreasein dataaccuracy (Fig. 2C,D). Other features of the GenesisTM 2000 System include automatic lane finding, automatic lane tracking, and automatic data processing (base calling) at the end of a run. Reagent kits are available (see below) for both single-stranded (Ml 3) and double-stranded (plasmid) sequencing. In the latter case, rapid DNA preparations (e.g., alkaline lysis methods) are perfectly adequate for most purposes, thereby avoiding the need for large-scale CsCl banding methods that are both costly and time consuming. Finally, by using biotinylated primers, both strands of a PCR product may be sequenced directly, without the need for subcloning. PCR reactions are carried out in the presence of one biotinylated and one nonbiotinylated primer, enabling the resulting biotinylated product to be immobilized and rapidly purified using streptavidin-coated magnetic beads. Following alkaline denaturation, the biotinylated (immobilized) and nonbiotinylated (released) DNA strands are independently sequencedin separatereactions. A number of reagentsare now available for generating 5’ biotinylated primers. For example, a biotin phosphoramidite (Du Pont-NEN Research Products, Product No. NEF-707) may be conveniently incorporated during standard oligonucleotide synthesis. 2. Materials 2.1. Sequencing Reactions for Cloned Single-Stranded Templates 1. Du Pont GenesisTM2000 single-strandedsequencingkit (NEK-515 or NEK-525). This kit containsthe following reagents,all of which should be stored at -20°C. a. T7 Reagent tablets (containing dNTPs and fluorescent ddNTPs). Thesearehydratedwith 100pL of Hz0 just prior to use.Each hydrated tablet is sufficient for 15 sequencingreactions.If not all used immediately, the solution may be storedat -2OOCfor up to 2 wk. Repeated freeze-thaw cycles (>3) of the hydrated tablets and exposureto prolonged or intense hght should be avoided. b. SequenaseTM. Supplied frozen and may imtially be stored at -70 or -20°C. However, oncethawed it should subsequentlyonly be stored at -20°C. It should only be removed from -20°C storagejust prior to use and returned immediately.

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ment is still accurate. Note that the sequence-GGGCGCC- (whrch shows bad band compression on manual sequencing gels) is well resolved. Panels C and D illustrate the improvement obtained (more even peak height) when the sequencing reactions are carried out in the presence of Mn2+ (panel D) instead of Mg2+ (panel C).

Hall c. 5X SequenaseTMbuffer: 200 mil4 Tris-HCl, pH 7.5, contammg 100 mil4 MgClz and 250 mM NaCl. d. G-505 Loading solution: A G-505 fluorescently-labeled 20-mer oligonucleotide in 200 mM EDTA solution containing 2 mg/mL crystal violet. A working dilution is prepared by adding 950 pL of deionized formamide to the 50 pL of stock solution. e. Sterile, deionized H20. f. Single-stranded control DNA (Ml3mp18) at 250 ng/pL. g. Control sequencing primer (for M13) at 5 ng/pL. 2. 5M NH,OAc. Store at 4OC. 3. 75% and 100% ethanol. Store at -20°C. 4. TE buffer: 10 r&f Tris-HCI, pH 8.0, 1 mM EDTA. 5. Template DNA m HZ0 or TE buffer at a concentration of at least 250 ng/pL. Store at -2OOC. 6. An appropriate oligonucleotide primer at a concentration of 5 ng/pL m H,O or TE buffer. Store at -2OOC. 2.2. Sequencing Reactions for Cloned Double-Stranded Templates 1. Du Pont GenesisTM2000 double-stranded sequencing kit (NEK-535 or NEK-540). This kit contains the following reagents, all of which should be stored at -20°C. a. T7 Reagent tablets (containmg dNTPs and fluorescent ddNTPs). These are hydrated with 100 cls, of Hz0 just prior to use. Each hydrated tablet is sufficient for 15 sequencing reactions. If not all used immediately, the solution may be stored at -20°C for up to 2 wk. Repeated freeze-thaw cycles (>3) of the hydrated tablets and exposure to prolonged or intense light should be avoided. b. SequenaseTM. Supplied frozen and may mltially be stored at -70 or -2OOC. However, once thawed it should subsequently only be stored at -2OOC. It should only be removed from -2OOC storage just prior to use and returned immediately. c. 5X SequenaseTMbuffer: 200 mM Tris-HCl, pH 7.5, containing 100 mM MgClz and 250 mM NaCl. d. G-505 Loading solution: A G-505 fluorescently-labeled 20-mer oligonucleotide in 200 mM EDTA solution contammg 2 mg/mL crystal violet. A working dilution is prepared by adding 950 pL of deionized formamide to the 50 l.tL of stock solution. e. Sterile, deionized H20. f. Double-stranded control DNA (pDP-1) at 300 ng&L. g. Control sequencing primer (T7 for pDP-1) at 5 ng/pL. 2. 2M NH,OAc, pH 5.0, with acetic acid. Store at 4OC.

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363

3. 2M NaOH. Store at 4°C in a tightly closed bottle. 4. 75 and 100% ethanol. Store at -2OOC. 5. 5M NH,OAc. Store at 4°C. 6. TE buffer: 10 rniI4 Tris-HCl, pH 8.0, 1 mM EDTA. 7. Template DNA in Hz0 or TE buffer at a concentration of at least 250 ng/pL. Store at -2OOC. 8. An appropriate oligonucleotide primer at a concentration of 5 ng/pL m Hz0 or TE buffer. Store at -20°C. 2.3. Direct

Sequencing PCR Products 1. Du Pont GenesisTM 2000 single- or double-stranded sequencing kit (NEK-515, NEK-525, NEK-535, or NEK-540). See previous sections for further detatls. 2. Dynabeads@M-280 Streptavidin (Dynal@) at 10 mg/mL. Store at 4°C. 3. Triton@ wash solution: 0.17% w/v T&on@ X-100, 100 mMNaC1, 10 mM Tris-HCl, pH 7.5, 1 rniI4 EDTA. Sterile solution. 4. 0.39% (w/v) Triton@ X-100. Sterile solution. 5. 0.5M NaOH, 2 r&I EDTA. Store at 4°C in a tightly closed bottle. 6. 3M NaOAc. Store at 4°C. 7. 5M NH,OAc. Store at 4°C. 8. 70 and 100% isopropanol. Store at -20°C. 9. 75 and 100% ethanol. Store at -2OOC.

of Biotinylated

2.4. Preparation of Sequencing Gels 1. 40% acrylamide/btsacrylamide (19:l) stock solutton. Prepared by dtssolving 38 g of acrylamide and 2 g of blsacrylamrde m deionized water (see Note 1) and adjustmg the volume to 100 mL. Use the best quality acrylamide and bisacrylamrde available (e.g., BDH Electran, Grade 1). Store at 4°C in the dark for up to 2 mo (see Note 2). The mix must not be heated to aid dissolutton as this has an adverse effect on subsequent gel quality (see Note 3). Warning: Acrylamide is a known neurotoxin that may be absorbed through the skin. Avoid inhalation of the solid when preparmg solutions and handle solutions wxth extreme care. 2. Urea. (Low m heavy metals, e.g., BRL cat. no. 5505UA). 3. 1OX TBE buffer. Prepared by dissolvmg 121.1 g of Tris-base, 5 1.4 g of boric acid, and 3.7 g of EDTA in deionized water (see Note I) and adjusting to 1 L. Filter (to reduce precipitation during storage) and store at 4°C for up to 2 mo m a tightly stoppered bottle (see Note 4). Discard if the solution starts to precipitate out. 4. 10% (w/v) ammomum persulfate solution. Prepare fresh immediately prior to use.

5. TEMED (As pure as possible, see Note 2, e.g., BRL cat. no. 5524UB). 6. AbsolveTM (Du Pont-NEN) or suitable noniomc detergent. Caution: Some detergents may leave a fluorescent restdue on the glass plates (see Note 5). 7. 100% (“absolute”) ethanol.

3. Methods 3.1. Sequencing Reactions for Cloned Single-Stranded Templates 1. For each DNA template combme the following in a 1.5-n& mlcrocentrtfuge tube: Template DNA (1.3 pmol, 3 pg, see Notes 12 and 14), l-l 2 pL, primer (2.7 pmol, 15 ng; see Note 15), 3 pL, 5X SequenaseTMbuffer, 6 pL, Hz0 to a final total volume of 21 pL 2. Denature the samples at 95-100°C for 2 mm. 3. Anneal at 37°C for 10 min. 4. Meanwhile hydrate a T7 reagent tablet from the Du Pont Genesis 2000TM kit with 100 pL of sterile water from kit. Keep on ice away from strong hghts. 5. To each annealed primer/template add: Hydrated T7 reagent tablet mrx 6.5 pL, SequenaseTM(add Just prior to use) 1 pL. 6. Allow primer extension and termination to occur at 37°C for 5 min. 7. Add 30 pL of 5h4 NH40Ac solution, 150 pL of cold (-20°C) 100% ethanol. Vortex and centrifuge immediately m a mtcrocentrifuge at 12,000g for 15 min (see Note 11). 8. Carefully remove all of the supernatant by asprratrng. Add 500 pL of cold (-20°C) 75% ethanol to the pellet;vortex andcentrifuge at 12,000gfor 10min. 9. Again aspirate away all of the supernatant and dry the pellet under vacuum for 5 min. 10. Resuspend the pellet m 3 pL of formamide/loadmg solutron. Denature the sample at 95-100°C for 2 min then chill on ice, immediately prtor to loading onto gel. 3.2. Sequencing Reactions Double-Stranded Templates 1 For each DNA template combine the following in a 1 5-mL mtcrocentrrfuge tube: Template DNA (2.2 pmol, 4 pg; see Notes 12 and 14), 1-16 pL, 2M NaOH 2 pL, Hz0 to a final total volume of 18 pL. 2 Denature the samples at 37OCfor 5 min. 3. Add 3 pL 2M NH,OAc, pH 5.0,7 pL Hz0 and 75 pL of cold (-20°C) 100% ethanol and mix gently. Centrifuge immediately in a mtcrocentrifuge at 12,000g for 15 min (see Note 11).

for Ctoned

GenesisTM 2000 DNA Sequencing 4. Carefully remove all of the supernatant by aspirating. Add 500 pL of cold (-20°C) 75% ethanol to the pellet; vortex and centrifuge at 12,OOOg for 10 min. 5. Again aspirate away all of the supernatant and dry the pellet under vacuum for 5 min. 6. Resuspend the pellet of denatured DNA by adding: Primer (13.5 pmol, 75 ng; see Note 15) 15 @, 5X SequenaseTMbuffer 6 u.L 7. Anneal at 37OC for 30 min. 8. Meanwhtle, hydrate a T7 reagent tablet from the Du Pont Genesis 2000TM kit with 100 pL of stertle water from kit. Keep on ice away from strong lights. 9. To each annealed primer-template add: Hydrated ‘I’7 reagent tablet mix 6.5 J.L, SequenaseTM(add just prior to use) 1 pL. 10. Allow primer extension to commence at 37OC for 2 min. 11. Add a further 1 J.L of SequenaseTMto each reaction and continue extension and cham termmation at 37°C for 5 min. 12. Add 30 pL, of 5M NH40Ac solution, 150 pL of cold (-20°C) 100% ethanol. Vortex andcentrifuge immediately at 12,OOOgfor 15 mm (see Note 11). 13. Carefully remove all of the supernatant by aspirating. Add 500 pL of cold (-20°C) 75% ethanol to the pellet; vortex and centrifuge at 12,000g for 10 min. 14. Again aspirate away all of the supernatant and dry the pellet under vacuum for 5 mm. 15. Resuspend the pellet in 3 pL of formamide/loading solution. Denature the sample at 95-100°C for 2 min then chill on ice, Immediately prior to loading onto gel.

3.3. Direct of Biotinylated

Sequencing PCR Products

1. Wash sufficient Dynabeads@ M-280 Streptavidin as follows (each sequencing reactton requires 15 pL of washed beads). a. Resuspend the beads in 2 vols of Trrton@ wash solutton m a microcentrifuge tube. b. Collect the beads against one side of the tube with the aid of a magnet. c. Carefully remove the wash buffer with a ptpettor. d. Repeat with a further two washes. e. Finally resuspend the beads in Triton@ wash solution to then ortgrnal volume (i.e., at 10 mg/mL). N.B. Do not allow the beads to dry out between washes. Avoid generating bubbles. Quantitative collection of beads IS important m obtammg an adequate sequencing signal.

366

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2. For each PCR product combme the following in a 1S-mL microcentrifuge tube: Biotmylated PCR product, 20 pL, and washed Dynabeads@ (see Note 15), 15 pL. 3. Incubate at 37°C for 30 min, occasionally resuspendmg the beads. 4. Collect the beads on the side of the tube with a magnet, remove, and discard the supernatant. Wash the beads as in step 1 with 100 pL of Triton@ wash solution. 5. Resuspend the beads by adding: Deionized H,O, 16 pL, and OSM NaOH, 2 r&4 EDTA, 4 pL. 6. Denature at room temperature for 5 mm. 7. Collect the beads on the side of the tube (tube A) with a magnet and transfer the NaOH supernatant to a new tube (tube B). 8. Wash the beads (tube A) with 100 pL of Triton@ wash solutton and discard the supernatant. 9. Resuspend the beads in: Deionized HzO, 12 pL, 5X SequenaseTMbuffer, 6 pL, primer (1.8 pmol, 10 ng; see Note 15), 2 pL. 10. Denature the DNA bound to the beads at 50°C for 5 min, then anneal at 37°C for 7 mm (see Note 15). 11. To the annealed primer-template add: T7 hydrated reagent tablet mix, 6.5 pL, SequenaseTM(add just prior to use), 1 pL. 12. Incubate at 37°C for 5 mm. 13. Collect the beads on the side of the tube and wash three times with 100 pL of Triton@ wash solution, discarding the supernatants. 14. Finally resuspend the beads in 3 pL of formamide/loading solution. Denature the sample at 95-100°C for 2 min then chill on ice, immediately prior to loading onto gel. 15. To the NaOH supernatant containing the nonbiotinylated DNA strand (tube B) add 2 pL of 3M NaOAc, 22 pL of cold (-20°C) isopropanol, and centrifuge at 12,000g for 15 min. 16. Carefully remove all of the supernatant by aspiratmg. Add 500 pL of cold (-20°C) 70% isopropanol to the pellet; vortex and centrifuge at 12,OOOgfor 10 mm. 17. Again aspirate away all of the supernatant and dry the pellet under vacuum for 5 min. Keep on ice until required. 18. Resuspend the pellet of nonbiotmylated DNA by adding: 0.39% Triton@ X-100, 12 pL, primer (1.8 pmol, 10 ng), 2 pL, 5X SequenaseTM buffer, 6 pL. 19. Denature the nonbiotinylated DNA at 95°C for 2 min, then anneal at 37°C for 10 min. 20. To the annealed primer/template add: Hydrated T7 reagent tablet mix, 6.5 pL, SequenaseTM(add just prior to use), 1 pL.

GenesisTM 2000 DNA Sequencing 21. Incubate at 37°C for 5 min. 22. Add 30 pL of 5M NH40Ac solution, 150 pL of cold (-20°C) 100% ethanol. Vortex and centrifuge immediately at 12,OOOgfor 15 min (see Note 11). 23. Carefully remove all of the supernatant by aspirating. Add 500 pL of cold (-20°C) 75% ethanol to the pellet; vortex and centrifuge at 12,000g for 10 min. 24. Again aspirate away all of the supernatant and dry the pellet under vacuum for 5 min. 25. Finally resuspend the pellet in 3 pL of formamtde/loadmg solution. Denature the sample at 95-100°C for 2 min then chill on ice, immediately prior to loading onto gel.

3.4. Preparation

of Sequencing

Gels

1. Clean the plates well with warm water and AbsolveTM or a suitable nonionic detergent (e.g., TeepolTM; see Note 5). Rxnse well with deionized water, wipe dry, wipe briefly with 100% ethanol (see Note 6), and dry again. Ensure that the mirrored area is free of lint or smudges. 2. Place the gel spacers on the mirrored plate so that 1he top of the spacers protrudes about 5 mm above the top of the plate, place the notched plate on top, and tightly tape up the two glass plates along the sides and bottom. 3. Prepare a 6% gel mix by combining the following: 40% acrylamide/ bisacrylamide stock, 4.5 mL, urea, 15 g, 10X TRE, 3 mL, deionized water, 11.5 mL. 4. Stir until completelydissolved.Do not heat above 37’C asthis impairs subsequent gel quality (see Note 3). Prepare fresh, immediately before use. 5. Filter through a 0.45 pm disposable membrane filter (either by using a vacuum filter unit or a filter attached to a 50-mL disposable syringe). 6. Degas the filtered gel solution under vacuum for about 5-10 min (see Note 7). 7. Add 10 pL of TEMED and mix by gently swirling. (N.B. Always add the TEMED and mix before adding the ammonium persulfate). 8. Add 300 pL of freshly prepared 10% ammonium persulfate solution and mix well but avoid unnecessary aeration. 9. Immediately pour the gel solution into the taped-up plates, insert the gel comb, and allow to polymerize by laying the plates almost horizontally with the top raised about 5 cm on a suitable support. If necessary, clamp the sides over the spacers near the top of the gel with foldback clips to ensure that the comb is held tightly (see Note 8). 10. Cover the top with plastic film to exclude oxygen and prevent drying out.

368

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11. Leave for at least 2 h (preferably more) to fully polymerize (seeNote 9). Gels can conveniently be pouredthe day before use,carefully sealed with plastic film to prevent drying out, and stored overrnght at room temperaturein the dark. 12. Removecomb and tape, ensuremirrored region is clean, and clamp gel into GenesisTM2000 instrument. 13. Add freshly prepared1X TBE buffer (seeNote 1) to the electrodechambers and carefully wash out the slots. 14. Prerun the gel, load the samples(seeNote 13), and run as descrtbedin the instrument manual. 3.5. Analysis of Data Analysis of the Genesis 2000TM raw sequencing data and assignment of bases may be carried out automatically at the end of the sequencing run. A simple histogram gives a good indication of the quality, and hence reliability, of the data. The sequence is then available as a simple ASCII file for import into most of the available DNA software packages (Wisconsin, DNAStar@, DNASIS@, MacMolly@, and so forth) for subsequent sequencealignment, translation, and such. In addition, the data is also available as a readily interpreted waveform that can be easily edited. Two types of editing are required; ambiguous base assignments and incorrect base assignments. The software quirks that lead to many of the ambiguous assignments (Y, R, M, N) are soon learned and most ambiguities can be accurately edited very quickly. Incorrect assignments (which are less common) pose more of a problem, particularly in the caseof new DNA sequences.However, determination of both DNA strands and comparison of the resulting aligned sequences will highlight any inconsistencies that can then be checked and are usually easily corrected after examination of the corresponding waveforms. This should not cause any unnecessary work, however, since sequencing both DNA strands should always be a requirement for new sequences (whether determined by manual or automatic sequencing), to avoid sequencing artifacts. In practice the use of fluorescent terminators together with deaza-dGTP and deazadATP analogs (see Introduction), usually results in fewer band compressions and other sequencing artifacts than standard manual sequencing methods.

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4. Notes The simplicity and advanced features of the tablet-based sequencing reactions, and sound design of the GenesisTM 2000 instrument, should enable high quality data to be routinely obtained by most users, provided that the protocols are properly adhered to. It should be stressed, however, that some of the requirements for good automated sequencing may be different from those of good manual (radioactive) sequencing. First, the need to “read” the DNA sequence as it passes a fixed window near the bottom of the sequencing gel results in much longer electrophoresis times for automated sequencing, This

in turn demands higher quality gels to maintain the necessary resolution. Second, the real-time collection of data by automated sequencers requires an adequate signal if accurate data are to be obtained. In the case of manual sequencing, weak signals can be compensated for by longer autoradiographic exposure times. In my experience, most problems arise in the two important areas of: gel resolution and data signal strength. 4.1. Problems

with Gel Resolution

1. Use very high quality water (e.g., milli-QTM) for all solutions, including the 1X electrophoresis buffer. Single distilled water is often inadequate. 2. Use the best quality acrylamide, bis-acrylamide, urea, and TEMED available. Do not keep stock solutions for more than 2 mo. 3. Never heat the gel solution above 37OC when dissolving the urea; this can have a marked effect on subsequent gel quality. 4. Store the 10X TBE at 4°C in a tightly-stoppered bottle and discard if a precipitation appears. 5. Clean electrophoresis plates with AbsolveTM or a suitable nonionic detergent (e.g., TeepolTM). Some detergents leave a residue that can effect subsequent gel resolution. 6. Only use 100% (“absolute”) ethanol to clean gel plates. Some less pure grades leave a fluorescent film that increases the gel background. 7. Always degas the filtered gel solution to remove oxygen that inhibits gel polymerization. 8. When clamping gel plates, ensure that the clips are directly over the spacers.Improper alignment can causeuneven gel thickness and decrease in gel resolving capacity,

370

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9. Always allow the gel to fully polymerize for at least 2 h before use, even though the gel will appear to have polymenzed within about 10 mm. 10. Some users may prefer to sllicomze the glass plates to facilitate gel pouring. If so, the agent used should be chosen carefully since some silanizing reagents (e.g., SigmacoteTM) may produce an increased background. I have used Repelcote TM(BDH Ltd.) for this purpose for a number of years without problems. 11. Residual salt in sequencing reactions has a very adverse effect on gel resolution. To avoid this problem, carry out ethanol precipitations and washes exactly as described in the above protocols. In particular, do not exceed the stated volume of ethanol; centrifuge immediately after ethanol addition and mixing (i.e., do not place at -20 or -70°C prior to centnfugation); remove as much supernatant as possible after centrifugation (by aspiration, do not simply pour). 12. Gel overloading owing to the presence of too much DNA may cause poor resolution (e.g., poor quality rapid plasmid preparations contamlnated with bacterial genomlc DNA). 13. The end lanes on the sequencing gel may produce slightly poorer quality data owing to edge effects. This can be minimized by loading the wider end slots (which are not used for reactions) or flankmg empty lanes (if not all slots are used) with 3 pL of 95% formamide solution containing 10 mM EDTA and 0.1 mg/mL crystal violet. N.B. Do not use G-505 loading solution for this purpose as the presence of lanetracking dye in empty lanes will cause lane finding and tracking problems. When running less than twelve reactions do not leave spaces between data lanes; load all samples in adjacent slots. 4.2. Problems with Weak Signals 14. Insufficient template is probably the major cause of weak signals. If 3 pg of single-stranded (M13) or 4 pg of double-stranded (plasmid) template is used, adequate data signal strength should be obtained. It is therefore imperative that the template DNA concentration is known with some degree of accuracy. This cannot be achieved by absorbance measurement since most template preparations (even after CsCl banding) will contain residual RNA. Estimations based on ethidium bromide stammg in agarose gels are adequate if accurate standards are also run. N.B. Increasing the amount of DNA (template plus contaminating host cell DNA) above about 5 pg may cause a loss of sequencing gel resolution because of overloading. The optimum operating range (about 3-5 pg) 1stherefore quite narrow.

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15. In general, variation in primer sequence, length, concentration, and annealing temperature, and so on, seem to have relatively little effect on signal strength for most Ml 3 and plasmid sequencing, thereby contributing to the simplicity and robustness of these reactions. However, this may not be the case for direct sequencing of biotinylated PCR products. Here, careful optimization of the annealing temperature, amount of Dynabeads@used, and such for a particular PCR product may significantly improve the signal strength and hence quality of the data.

References 1. Prober, J. and Trainor, G. L (1987) A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynucleotides. Science 238,336-341 2. Sanger, F., Nlcklen, S , and Coulson, A. R (1977) DNA sequencing with chamterminatmg inhibitors. Proc. Natl. Acad. Sci USA 74,5463-5467.

&AP!TER

38

The Use of Robotic Workstations in DNA Sequencing Alan

T. Bankier

1. Introduction With the advent of genome sequencing, a much greater impetus has taken effect in expanding the capabilities of DNA sequencing. This is necessary since present limitations make such projects very long term and very expensive. Although this drive shall inevitably bring about a revolution in DNA sequencing rates, it will almost certainly involve expensive equipment. These benefits are likely to only affect a few highly funded genome sequencing centers. More modest, large-scale sequencing facilities can benefit from the automation or semiautomation of current procedures using cheaper, commercially available equipment. The Applied Biosystems 373A, the Pharmacia ALF, and the DuPont Genesis 2ooOTM(Chapters 33-37) automate one aspect of the procedure by removing the need for autoradiography and film reading. The Amersham Autoreader and similar devices remove the need for film reading only, Other aspects that can benefit from automation are template DNA preparation and purification (1,2), and performing the sequence reactions (3,4). Almost any commercially available laboratory robot can be programmed to carry out the more tedious aspects of sequencing, but may involve several months or even years of software developFrom Methods in Molecular 5!ology, Vol23’ DNA Sequencing Protocols E&ted by H. and A. Gnffm Copyright 01993 Humana Press Inc., Totowa,

373

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Bankier

ment. Some devices provide a simple interface that enables indirect programming through easy to use menus. Notable among these is the Beckman Biomek 1000, which has microliter delivery capability in single or eight channel format and can automatically exchange pipeting and aspiration tools. It also has add-on capability for temperature controlled positions and a side-arm able to remove and replace labware. Although it is possible to fully automate some procedures, by careful programming and adjustment to protocols, it may not always make the most sense. Very often, a combination of manual intervention and automated pipeting is the most effective way to carry things out. For example, unless a truly hands-off approach is desired or needed, it makes little sense to use the robot to aspirate the 192 wells of two microtiter trays, taking several minutes, when the trays can be removed and simply turned upside down. This is especially true if the trays have already been removed for some other reason such as centrifugation. In this chapter, the semiautomated use of the Beckman Biomek is considered for purifying template DNA and performing sequence reactions. 2. Materials 1. All of the materials used for phage growth, as described in Chapter 6,

will be required. 2. A Beckman Biomek 1000 laboratory workstation. In addition, appropriate pipeting tools and labware will be needed. Most of the procedures outlined in this chapter can be performed with only the MP200 eight channel pipet tool. A copy of the Biotest open software IS also essential. 3. Sterile polystyrene 96-well microtiter trays (e.g., Cornmg cell wells). There appears to be little advantage to using erther tissue culture treated or nontreated plates. 4. A benchtop centrifuge capable of spinning microtiter trays at speeds up to 4000 g, such as the I.E.C. Centra 4X. If this speed cannot be attained, the centrifugation times will need to be extended. 5. PEG/NaCl: 20 % polyethylene glycol (mol. wt. 6000-8000), 2.5M sodium chloride. 6. TE: 10 n-&f Tris-HCl, pH 8.0, 0.1 mM Na2EDTA. 7. TE/SDS: 1 % sodium dodecyl sulfate in TE. 8. EthanoVNaOAc: 95 % ethanol and 3M sodium acetate pH 5.0 (equihbrated using acetic acid), combmed in the ratio 25: 1. 9. Microtiter tray plate sealers (e.g.. Costar cat. no. 3095).

Using Robots in Sequencing

375

10. Ohgonucleotide primer at a concentration of 0.5 pmol/pL. A single “universal” primer (5’ d[GTAAAACGACGGCCAGT]) can be used for shotgun sequencing in M13. 11. TM buffer: 100 mM Tris-HCl, pH 8.5, 50 mA4 MgC&. 12. The template DNA to be sequenced, at a concentration of around 0.05 pmol/pL, stored in microtiter trays. 13. O.lM dithiothreitol (Cleland’s reagent). Store at -20°C. 14. 35S,32P,or 33Palpha labeled dATP at -400 Ci/mmol, 10 mCi/mL. 15. Klenow fragment DNA Polymerase I at a concentrationof around 5 U&L. 16. Four nucleotide specific dideoxy/deoxy nucleotide mixes (ddNTP/ dNTPs) as follows: ddATP mix: 250 @4 dCTP, 250 @4 dGTP, 250 pil4 dTTP, 10 piV ddATP. ddCTP mix: 12.5 w dCTP, 250 @4 dGTP, 250 rJM dTTP, 80 l,uV ddCTP. ddGTP mix: 250 l.uVdCTP, 12.5 pil4 dGTP, 250 @4 dTTP, 160 it/V ddGTP. ddTTP mix: 250 @VdCTP, 250 @4 dGTP, 12.5 @f dTTP, 500 j&I ddTTP. 17. Using the dideoxy/deoxy nucleotide mixes prepared above, four working nucleotide/buffer/primer solutions are made A solution: ddATP mix/TM/primer/water in the ratio 8: 1:1:6. C solution: ddCTP mtx/TM/primer/water in the ratio 8: 1:1:6. G solution: ddGTP mix/TM/primer/water in the ratio 8: 1:1:6. T solution: ddTTP mix/TM/primer/water in the ratio 8: 1:1:6. 18. Chase mix: 0.5 mMdATP, 0.5 mMdCTP, 0.5 mMdGTP, 0.5 mMdTTP. 19. Formamide dye mix: 100 ml deionized formamide, 0.1 g xylene cyan01 FF, 0.1 g bromophenol blue, 2 ml 0.5M Na2EDTA. 3. Methods The Biomek tablet can hold up to four exchangeable tools, one tip rack, and three modules or microtiter trays. The layout used in these methods uses position 2 for reagents and positions 1 and 3 for the microtiter trays, as shown in Fig. 1. The protocols detailed here require the use of Biotest software for the Biomek. Although the software that is supplied with the workstation can be used, it does not allow the precise control needed to go to very specific heights, and to serially dispense. The Biotest interface software permits direct control of the driving motors through a limited set of commands. Even though Biotest lacks the structure of a

376

Bankier

Fig. 1. A schematic, top view of the Biomek tablet. Positions A-D hold the pipeting tools, although only the MP200 tool is needed. The tip position holds a rack of 96,25O+L tips. Position 2 is used for the reagent reservoirs (either modules or a microtiter tray) and positions 1 and 3 are used for the recipient microtiter trays.

high level language, control can be performed using integer variables and a simple looping structure. Since direct programming involves control of movement to very precise positions, it is not possible (nor advisable) to provide detailed programs for the routines. Each individual instrument needs to be fully set up, according to the labware to be used (see Note 1). For this reason the routines are described in a “skeleton” form. Measurement of precise locations using the Biotest program should be made at a reference well or reservoir at each position to be used, along with the base of each (where the tips just touch the bottom). Movement to other points within each position on the tablet can be made from this reference, for example, by adding well to well jumps. Other important measurements are the height at which the tool/tips can safely negotiate all occupied positions on the tablet, and specific dispense and pipet heights (see Note 2). The outline programs listed in each table demonstrate the use of these values as variables (in capital letters). Looped procedures that make the program structure compact are enclosed within parentheses. Any instructions concatenated on the same line are separated by semicolons. The variables TRAY 1, TRAY2, and TRAY3 refer to the tablet positions 1,2, and 3 as shown in Fig. 1, and is essentially the Y

Using Robots in Sequencing

377

axis variable (TRAY is listed where the positions may change within a loop). COLUMN is the well X axis variable. FILL, ASPIRATE, DISPENSE and SAFE are all used as Z axis variables. FILL should be at the point where the tool and tips are just above the well or reservoir bottom. ASPIRATE is at 50 & depth within a well. DISPENSE can be altered according to the volume already present in the well (see Notes 3 and 4). SAFE should be sufficient to avoid collisions. 3.1. Using Robotics

for Template

Preparation

The robotic preparation of phage DNA described here uses the workstation purely as a pipeting device. Microtiter trays are removed from the tablet for mixing, incubation, and centrifugation. One tray of ninety-six phage cultures is processed, following the procedure described in Chapter 6 and detailed in Fig. 2. Larger numbers of trays can be handled by adding appropriate loops and increasing reagent volumes. The most convenient number of trays to process is two. This provides balanced trays for centrifugation and the total reagent volumes can be held in the labware. Purification of the template DNA is started immediately following phage growth as described in Chapter 6. The procedure is split into two, following PEG precipitation. This is the only point at which the method can be interrupted. Of course, it is best to continue through to completion if possible. Table 1 details supernatant transfer and PEG precipitation. Before and between supernatant transfers the tray should be centrifuged at 4000 g for 10 min. After adding PEG, vortex the tray and leave it at room temperature for 15 min (see Note 5). Centrifuge the tray as before, and throw off the supernatant (see Note 6). Leave the tray to drain on tissues for 5 min. Table 2 outlines the remaining procedure of SDS denaturation, ethanol precipitation and redissolving of the DNA. After adding TE/SDS, remove the tray, seal it with a plate sealer and incubate it at 80°C for 20 min, then return the tray to the tablet. Once the ethanol/acetatehasbeen mixed in, leave the tray at room temperature for 15 min, and pellet the DNA in a centrifuge at 4000 g for 10 min.. Throw off the ethanol, return the tray to the tablet, and resume the program. The ethanol wash can be discarded without centrifugation, and the tray drained on tissue for 5 min before vacuum drying for 5 min. Return the tray to the tablet to dissolve the DNA in TE (seeNote 7).

Bankier

378

Fill Plates

with 250 I toothplck and Grow I Pellet Cells I Transfer 200 pl I Pellet Cells Transfer

150

pl

I Add 30 pl PEG I Preclpltate Centrlfuge

and

Phage I Discard Supernatant

I Add 50

SDS I T.E. I Incubate at 80%

I Add

125 pl Ethanol

I Acetate

I Pellet

DNA

I Add

175 pl Ethanol

Discard

I

Supernatant

I 1 Redissolve

In 20 ~1 T.E. 1

Fig. 2. A summary of the use of the Blomek for semlautomated DNA preparations. Actions performed by the robot are boxed

3.2. Using

Robotics

for DNA Sequencing

If an HCB temperature controlled add-on unit is available, complete sequence reactions can be performed on the Biomek. The major drawback of this approach is the number of templates that can be sequenced simultaneously. The standard tablet is limited to one tip rack position and three other labware positions (Fig. 1). Assuming that one position is required for reagents and one for templates, only

Using Robots in Sequencing

379

Table 1 Outlme Program for PEG Precipitation

of 96 Samples

pick up mp200 tool ; pick up ups ; VOLUME = 200 uL loop 2 times ( loop 12 times ( go to TRAY 1 at COLUMN and SAFE height move down to ASPIRATE height ; sip VOLUME , move up to SAFE height go to TRAY3 at COLUMN and SAFE height move down to DISPENSE height , pipet VOLUME , move up to SAFE height increment COLUMN by 1

1 reset COLUMN

, change tips ; VOLUME

= 150 pL ; pause for centrifugation

>

loop 2 times ( go to TRAY2 at REAGENT and SAFE height move down to FILL height ; sip 180 ~JL ; move up to SAFE height loop 6 times ( go to TRAY at COLUMN and SAFE height move down to DISPENSE height, pipet 30 pL , move up to SAFE height increment COLUMN by 1 change tips , increment REAGENT

, reset COLUMN

; pause for PEG precipitation

one position remains for the sequence reactions. Using conventional microtiter trays, this means a maximum of ninety-six reaction wells or twenty-four standard sequencereactions. Of course, these sequence reactions can be carried out in around one hour and so several sets of twenty-four could be prepared in a single day. An alternative approach, is to consider sequence reactions as three separate processes: Filling reagent plates with nucleotides, primer, and buffer; dispensing templates and annealing; and performing the sequence reactions. Each of these steps can be carried out independently and the reaction trays stored at -20°C for several weeks. Filled reagent trays have been stored in this manner for over two years. These reagent filled trays can even be lyophilized and reconstituted using the template DNA solution immediately before annealing. This concept has been used in a sequence kit by Amersham.

380

Bankier Table 2 Outlme Program for DNA Purification

of 96 Samples

loop 3 times ( go to TRAY2 at REAGENT and SAFE height move down to FILL height , SIP 200 pL ; move up to SAFE height loop 4 times ( go to TRAY at COLUMN and SAFE height move down to DISPENSE height ; pipet 50 pL , move up to SAFE height increment COLUMN by 1 >

) ; reset COLUMN , change tips , VOLUME = 125 pL ; pause for SDS incubation loop 2 times ( loop 12 ttmes ( go to TRAY2 at REAGENT and SAFE height move down to FILL height , sip VOLUME , move up to SAFE height go to TRAY at COLUMN and SAFE height move down to DISPENSE height ; plpet VOLUME , move up to SAFE height increment COLUMN by 1 ); change tips , VOLUME = 175 pL ; increment REAGENT , reset COLUMN , pause 1 loop 3 ttmes ( go to TRAY2 at REAGENT and SAFE height move down to FILL height ; sip 80 pL ; move up to SAFE height loop 4 times ( go to TRAY at COLUMN and SAFE height move down to DISPENSE height ; pipet 20 pL , increment COLUMN by 1 ) , replace tips ; replace tool

The protocol used in this method, is essentially that of sequencing using Klenow polymerase as described in Chapter 12. With modification, any of the sequencing protocols can be used. Table 3 shows the program used for plate filling. The four combined nucleotide/buffer/primer solutions areplaced in adjacentreagent wells of position 2 on the tablet. Aliquots of 4 *of these solutions are dispensed into the wells of two microtiter trays, to provide a 3 x 8 reaction format on each tray (seeNotes 1-5). Many such trays can be produced by looping the program, reusing the same tips to avoid wastage. At this point the reagent trays can be lyophilized, stored frozen, or used straight away.

Using Robots in Sequencing Table 3 Outline Program for Plate Filling of Two Microtiter

381 Trays

pick up mp200 tool , pick up tips loop 4 times ( go to TRAY2 at REAGENT and SAFE height move down to FILL height ; sip 24 pL ; move up to SAFE height loop 2 times ( loop 3 times ( go to TRAY at COLUMN and SAFE height move down to DISPENSE height ; plpet 4 pL ; move up to SAFE height Increment the COLUMN by 4 ) COLUMN = REAGENT , increment TRAY > change the tips ; increment REAGENT by 1 ; COLUMN = REAGENT ; reset TRAY > remove the tips ; remove the tool

The annealing program outlined in Table 4, takes template DNA (24) from a microtiter tray, in position 2 of the tablet, and dispenses 4 x 2 & of each into the appropriate wells of two microtiter trays. Remove the trays from the tablet, seal them with a plate sealer, and incubate at 80°C for 30-45 minutes (see Note 7). Finally the enzyme label mix is added, left for 15 min, and chase solution is added, and the tray is once again left for 15 min as shown in Table 5. The enzyme label mix should be prepared only immediately before use by mixing the following ingredients: 35SdATP/water/DTT/Klenow in the ratio 1: 12:2: 1,The enzyme extension reactions can be performed at higher temperatures using the HCB add-on equipment, or simply by transferring the trays to an incubator. If required, a third loop can be introduced to add formamide dye mix. 4. Notes 1. The Blomek workstation needsto be set up initially with a defined tip height (through the vertical offset and safety margin, set in the utilities menu) to optimize normal routine use.The small volume pipetings used in sequencingmethods requires an even more accuratetip height. The precise position of reagentand reaction well bottoms should be deter-

382

Bankier Table 4 Outline Program for Annealing 48 Templates

pick up mp200 tool , pick up ups loop 2 times ( loop 3 times ( go to TRAY2 at TEMPLATE and SAFE height move down to FILL height , sip 16 pL ; move up to SAFE height loop 4 times ( go to TRAY at COLUMN and SAFE height move down to DISPENSE height ; pipet 4 l.tL ; move up to SAFE height increment COLUMN 1 change the tips ; increment TEMPLATE > increment TRAY ; reset COLUMN

1

remove the tips , remove the tool

mined and used in self-programmed routines. This posttron will have to

be reassessed after any maintenance. 2. Always dummy-run new routines without a tool on the pod and with no labware on the tablet. Programming mistakes are not only common but unavoidable. A simple mistake could be very costly. 3. Ptpeting small volumes mto dry microttter trays can be difficult using the routines already programmed m the Biomek software. Much better results are obtained using the Biotest software, where ptpet tip posrtions can be precisely controlled. 4. A great benefit to using Brotest software ts the ability to program repetitive dispensing. A large volume can be drawn mto the plpet and drspensed serially mto several wells. This approach even has the advantage of pipeting more reproducibly. 5. Mixing of the reagents is possrble srmply by pipeting part of the total volume m and out. This can become costly m terms of tips since their reuse would cause crosscontaminatton. This IS fairly unimportant with the large volumes used in the DNA preparation procedure, but could not be tolerated, under most ctrcumstances, in the sequence reactions. 6. An eight channel bulk dispense tool is available that can also be used for PEG supernatant aspiration. It is quicker to simply “throw” it off. 7. When lyophilized reagent trays are used, the template DNA should be redissolved in a larger volume of TE and dispensed mto the reaction trays at a larger volume. This avoids unnecessarydilution of the reaction.

Using Robots in Sequencing

383

Table 5 Outline Program for Sequence Reactions on 48 Templates pick up mp200 tool , pick up tips loop 2 times ( go to TRAY2 at REAGENT and SAFE height move down to FILL height ; sip 48 pL ; move up to SAFE herght loop 2 times ( loop 12 times ( go to TRAY at COLUMN and SAFE height move down to DISPENSE height ; pipet 2 pL ; move up to SAFE height increment COLUMN ) increment TRAY ; reset COLUMN ) reset TRAY ; reset COLUMN , increment REAGENT ; change tips pause 15 minutes > remove tips ; remove tool

References 1. Smith, V , Brown, C M , Bankier, A. T., and Barrell, B G. (1990) Semiautomated preparation of DNA templates for large-scale sequencing projects DNA Sequence 1,73-78.

2 Zimmermann, J., Voss, H , Kristensen, T., Schwager, C , Stegeman, J., Erfle, H., and Ansorge, W. (1989) Automated preparation and purification of Ml3 templates for DNA sequencing. Merh Mol. Cell Biol 1,29-34. 3. Bankier, A. T. and Barrell, B. G. (1989) Sequencing single-stranded DNA using the chain terminatton method, in Nucleic Acids Sequencing: A Practical Approach (Howe, C. J. and Ward, E. S., eds.) IRL Press, Oxford, pp. 37-78. 4 Mardrs, E R. and Roe, B A. (1989) Automated methods for single-stranded DNA tsolation and dtdeoxynucleotide DNA sequencing reactions on a robotrc workstation. Biolechniques 7,736-746.