Laboratory Techniques in Biochemistry and Molecular Biology Vol 24: Hybridization With Nucleic Acid Probes, Part II: Probe Labeling and Hybridization Techniques

Hybridization with nucleic acid probes Part II: Probe labeling and hybridization techniques

LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULAR BIOLOGY P.C. van der VLIET - Department for Physiological Chemistry, University of Utrecht, Utrecht, The Netherlands

Volume 24

ELSEVIER AMSTERDAM

*

LONDON

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NEW YORK

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TOKYO

HYBRIDIZATION WITH NUCLEIC ACID PROBES Part 11: Probe labeling and hybridization techniques

P. Tijssen Institut Armand-Frappier Uniuersite' de Que'bec 531, Boulevard des Prairies ViNe de Laoal, Que'bec, Canada H N I B 7

1993 ELSEVIER AMSTERDAM . LONDON . NEW YORK . TOKYO

ELSEVIER SCIENCE PUBLISHERS B.V. Sara Burgerhartstraat 25 P.O. Box 211, lo00 AE Amsterdam, The Netherlands Library of Congress Cataloging-in-PublicationData Tijssen, P. Hybridization with nucleic acid probes / P.Tijssen. v. (1-2). (Laboratory techniques in biochemistry and molecular biology ; v. 24) Includes bibliographical references and index. Contents: pt. 1. Theory and nucleic acid probes -- pt. 2. Probe labeling and hybridization techniques. ISBN 0-444-89884-0 (v. 1. : acid-free paper). -- ISBN 0-444-89883-2 (v. 1. : acid-free paper : pbk.). -- ISBN 0-444-89886-7 (v. 2. : acid-free paper). -- ISBN 0-444-89885-9 (v. 2. : acid-free paper : pbk) 1. Nucleic acid probes. 2. Nucleic acid hybridization. 1. Title. 11. Series. QP519.E vol. 24 [QP624.5.D73] 574.19’2’ .28 S-dC2O [574.87’328]

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93-15688

CIP

ISBN 0 7204 4200 1 (series) ISBN 0 444 89886 7 (hardbound Part 11) ISBN 0 444 89885 9 (paperback Part 11) ISBN 0 444 89888 3 (hardbound series Part I&II) ISBN 0 444 89887 5 (paperback series Part I&II) 0 1993, Elsevier Science Publishers B.V. All rights reserved

No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior written permission of the Publisher, Elsevier Science Publishers B.V., Copyright & Permissions Department, P.O. Box 521, 1000 AM Amsterdam, The Netherlands. Special regulations for readers in the USA This publication has been registered with the Copyright Clearance Center Inc. (CCC), Salem, Massachusetts. Information can be obtained from the CCC about conditions under which photocopies of parts of this publication may be made in the USA. All other copyright questions, including photocopying outside the USA, should be referred to the Publisher, unless otherwise specified. No responsibilityis assumed by the Publisher for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of the rapid advances in the medical sciences, the Publisher recommends that independent verification of diagnoses and drug dosages should be made. This book is printed on acid-free paper

Printed in the Netherlands

Symbols used and their definition BASH BCIP BCR BFHS BLOTTO BM bP Ci DeS04 dR/dC

s CPm dNTP

dpm a%

IAS ISH L LCR 1 P

N NSB OAc PEG PCR Qp-RS RFE RGE ss sv

Basic aqueous hybridization solution 5-Bromo-4-chloro-3-indolyl phosphate Branch capture reactions (strand displacement assays) Basic formamide hybridization solution Bovine lacto transfer technique optimizer (a blocking agent) Boehringer Mannheim blocking agent Base pairs (kbp, ‘kilo’ base pairs; kb, ‘kilo’ bases) Curie (radioactivity, 3.7 x 10’’ Bq) Dextran sulfate Sensitivity (change in response per unit concentration change) Decay constant (radioactivity) or discrimination ratio (cross/specific hybridization) Counts per minute; equals dpm efficiency (of counting device) Deoxynucleoside triphosphate (a,as in [a-32P]dATP, indicates phosphate group closest to the sugar moiety, gamma is the phosphate position farthest from the sugar) Disintegrations (nuclear transformations) per minute Double-stranded Isothermal amplification system In situ hybridization Length of nucleic acid (shorter of the two annealing strands) Ligase chain reaction Length of oligodeoxynucleotide Ionic strength ( = E c i z ~ci ; is ion concentration, zi its valence) Complexity of nucleic acid (sum of unique sequences in target strand) Nonspecificallybound Acetate Polyethylene glycol Polymerase chain reaction (an enzymatic amplification method) Q p replication system Rotating field electrophoresis Rotating gel electrophoresis Single-stranded Sievert (radiation dosage)

vi t f0.5 Td

Tdr

Ti Tm Tm,obs

TAFE

Time in seconds Time required to hybridize half of the target nucleic acid; or, in the context of radioactivity, the half-life of a radioisotope Temperature at which 50% of oligonucleotide duplexes are dissociated Duplex retention temperature at which 50% of oligomer hybrids remain intact (during wash) Incubation temperature for annealing reaction (Melting) temperature at which 50% of duplex molecules become dissociated Experimentally observed melting temperature (oligomers) Transverse alternating field electrophoresis Stacking temperature, indicative of stability of hybrids of two succeeding bases

Contents ...................... Chapter 7. Labeling of probes and their detection . . . . . . . . . .

Symbols used and their definition

7.1. Choice of label and labeling method .......................... 7.2. Radioisotopic labeling and detection of nucleic acids . . . . . . . . . . . . . . 7.2.1. Principles of radioactivity and units .................... 7.2.2. Maximum and optimum radioactivity of probes . . . . . . . . . . . . 7.2.3. Chemical stability of radiolabeled probes . . . . . . . . . . . . . . . . 7.2.4. Choice of radiolabel ............................... 7.2.5. Detection of radioactive probes ....................... 7.2.5.1. Principles of autoradiography and use of films . . . . . 7.2.5.2. Autoradiographywith nuclear track emulsion . . . . . 7.2.6. Safety considerations .............................. 7.3. Nonradioactive primary labels .............................. 7.3.1. Cherniluminescence ............................... 7.3.1.1. Acridinium ester probes ..................... 7.3.1.2. Acridinium labeling of probes . . . . . . . . . . . . . . . . 7.3.2. Fluorochrome labels ............................... 7.3.2.1. Fluorochrome labeling of nucleic acid probes . . . . . 7.3.2.2. Fluorochrome labeling of streptavidin or antibody . 7.3.2.3. Time-resolved fluorescence . . . . . . . . . . . . . . . . . . 7.3.3. The use of enzymes as primary labels . . . . . . . . . . . . . . . . . . . 7.3.3.1. Labeling density of enzymes on probes . . . . . . . . . . 7.3.3.2. Impact of solid phase on enzyme kinetics . . . . . . . . 7.3.3.3. Characteristics of major enzymes . . . . . . . . . . . . . . 7.3.3.3.1. Properties of alkaline phosphatase (APase) ........................ 7.3.3.3.2. Properties of horseradish peroxidase (POase) ....................... 7.3.3.3.3. Properties of P-galactosidase . . . . . . . . 7.3.3.4. Colorimetric detection of enzymes . . . . . . . . . . . . . 7.3.3.4.1. Chromogenic detection of alkaline phosphatase ........................ 7.3.3.4.2. Chromogenic detection of horseradish peroxidase

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V

269 269 272 272 274 277 278 280 280 284 286 287 289 290 293 293 294 296 297 298 298 300 302 302 303 304 305 305 310

viii

7.4.

7.5. 7.6.

7.7.

7.8.

7.3.3.5. Chemiluminescent enzyme substrates ........... 7.3.3.5.1. Dioxetane substrates for alkaline phosphatase and p-galactosidase . . . . . . . . . 7.3.3.5.2. Luminol substrates for peroxidase . . . . . 7.3.3.6. Labeling of nucleic acid with enzymes . . . . . . . . . . . 7.3.3.7. Conjugation of antibodies or streptavidin with enzymes .................................. 7.3.4. Electron-dense markers ............................ Recognition systems ..................................... 7.4.1. Streptavidin and biotin-labeling of enzymes or linker proteins . 7.4.2. Antibodies ...................................... 7.4.2.1. Use of anti-nucleic acid antibodies . . . . . . . . . . . . . 7.4.2.2. Hapten antibodies ......................... 7.4.3. Sulfhydryl ligands ................................. Overview of enzymatic incorporation of labels into probes . . . . . . . . . . Uniform incorporation of labels in nucleic acids . . . . . . . . . . . . . . . . . 7.6.1. Nick translation .................................. 7.6.1.1. Radiolabeling by nick translation .............. 7.6.1.2. Nick translation to incorporate secondary labels . . . . 7.6.2. Random-primed synthesis of labeled DNA . . . . . . . . . . . . . . . 7.6.2.1. Radiolabeling by random primer extension . . . . . . . 7.6.2.2. Incorporation of secondary labels by random primer extension ............................... 7.6.2.3. Random-primed DNA synthesis by PCR . . . . . . . . . 7.6.3. Defined primer extension on single-strandedtemplates . . . . . . 7.6.3.1. Upstream priming on single-stranded templates . . . . 7.6.3.2. Downstream priming on single-strandedtemplates . . 7.6.4. Labeling through sequential exonuclease-polymerase activities (‘replacement synthesis’) ............................ 7.6.5. Preparation of nonradioactive probes by PCR . . . . . . . . . . . . . 7.6.6. In vitro synthesis and labeling of RNA by transcription . . . . . . 7.6.7. Reverse transcription of RNA ........................ End-labeling of probes ................................... 7.7.1. Labeling of 5’-ends of DNA or RNA . . . . . . . . . . . . . . . . . . . 7.7.2. Labeling of 3’ ends of DNA or RNA . . . . . . . . . . . . . . . . . . . 7.7.2.1. Radiolabeling of 3’-ends of DNA . . . . . . . . . . . . . . 7.7.2.2. Nonradioactive labeling by tailing of 3’-ends of DNA 7.7.2.3. Labeling of 3’-ends of RNA . . . . . . . . . . . . . . . . . . Chemical modification of probes to introduce labels . . . . . . . . . . . . . . 7.8.1. Introduction of reactive groups in nucleic acid by transamination ................................... 7.8.2. Photohaptens (biotin, digoxigenin) ..................... 7.8.3. Psoralen crosslinkers .............................. 7.8.4. Crosslinking of haptenated or enzyme-labeled basic polymers .

314 314 318 320 321 321 321 323 325 325 325 327 329 330 330 334 335 337 338 340 341 341 342 342 342 347 349 353 353 353 354 358 359 359 361 362 363 365 366

ix 7.8.5. Radioactive labels ................................ 7.8.6. Mercurated nucleic acids for sulfhydryl-hapten ligands . . . . . . 7.8.7. N-Acetoxy-N-2 acetylaminofluorene(AAF)labeling . . . . . . . . 7.9. Universal probes ........................................ 7.10. Biological probes .......................................

Chapter 8. Mixed phase and solution hybridizationformats 8.1. Materials and solutions ................................... 8.1.1. Solid phase systems ............................... 8.1.1.1. Membranes .............................. 8.1.1.2. Paramagnetic beads ........................ 8.1.1.3. Particulate solid phase ...................... 8.1.1.4. Microtiter plates .......................... 8.1.1.5. Affinity columns .......................... 8.1.1.6. Alternative solid phases ..................... 8.1.2. Equipment ...................................... 8.1.3. Solutions and buffers used in hybridization . . . . . . . . . . . . . . . 8.1.3.1. Blocking reagents ......................... 8.1.3.2. (Pre)hybridizationsolutions . . . . . . . . . . . . . . . . . . 8.2. Slot/dot blot hybridization on membranes ..................... 8.2.1. Immobilization of target nucleic acid . . . . . . . . . . . . . . . . . . . 8.2.2. Hybridization conditions ............................ 8.2.2.1. Prehybridization .......................... 8.2.2.2. Hybridization ............................ 8.2.2.3. Acceleration of hybridization by polymers . . . . . . . . 8.2.2.4. Posthybridization washes .................... 8.2.3. Probe detection and analysis ......................... 8.2.4. ‘Fast’ blots of nucleic acid from cells . . . . . . . . . . . . . . . . . . . 8.2.5. Reprobing after stripping original probe from blots . . . . . . . . . 8.2.6. Hybridization to solid phase other than membranes . . . . . . . . . 8.3. (Semiksolution hybridization ............................... 8.3.1. Capture hybridization procedures ..................... 8.3.2. Reverse target capture hybridization . . . . . . . . . . . . . . . . . . . 8.3.3. Sandwich hybridization assays ........................ 8.3.3.1. Sandwich hybridization assays for DNA targets . . . . 8.3.3.2. Sandwich hybridization assays of RNA targets . . . . . 8.3.3.3. Sandwich hybridization followed by immunological capture and detection ...................... 8.3.4. Solution hybridization procedures with label probe(s) only . . . . 8.3.4.1. Selective destruction of unhybridized probes . . . . . . 8.3.4.2. Hybridization in concentrated solutions of chaotropes or urea .................................

369 370 371 372 373 375 376 376 377 381 384 385 386 386 387 388 388 391 391 393 401 401 403 406 407 410 413 415 416 416 419 421 426 427 427 427 329 329 430

X

Strand displacement assays . . . . . . . . . . . . . . . . . . Energy transfer and enzyme channeling systems . . . .

433 436

Chapter 9. Hybridization after electrophoretic fractionation of nucleic acids ............................

437

8.3.4.3. 8.3.4.4.

9.1. Electrophoretic procedures ................................ 9.1.1. Standard agarose gel electrophoresis of DNA . . . . . . . . . . . . . 9.1.2. Pulsed-field agarose gel electrophoresis of DNA . . . . . . . . . . . 9.1.3. Agarose gel electrophoresisof RNA . . . . . . . . . . . . . . . . . . . 9.2. Transfer procedures ..................................... 9.2.1. Southern transfer ................................. 9.2.1.1. Capillary transfer techniques and simple derivatives . 9.2.1.2. Pocket blotting, centrifugal transfer and vacuum or positive pressure transfer .................... 9.2.1.3. Electroblotting ........................... 9.2.2. Northern blotting ................................. 9.3. Hybridization in the gel or before electrophoresis . . . . . . . . . . . . . . . . 9.3.1. Reverse Southern analysis ........................... 9.3.2. Hybridization in gel after electrophoresis of DNA . . . . . . . . . . 9.3.3. Hybridization in gel after electrophoresis of RNA . . . . . . . . . . 9.4. Hybridization after transfer to membranes .....................

439 439

..........

475

Chapter 10. Colony and plaque lift hybridization

445

449 453 454 456 463 465 467 470 470 471 472 472

10.1. Colony and plaque hybridization: different approaches . . . . . . . . . . . . . 10.2. Colony hybridization ..................................... 10.2.1. Identification of bacterial colonies harboring specific plasmids orcosmids ...................................... 10.2.2. Colony hybridization of lower eukaryotes . . . . . . . . . . . . . . . . 10.2.3. Adaptation of colony hybridization for higher eukaryotic cells 10.2.4. Useful modifications in colony hybridization . . . . . . . . . . . . . . 10.3. Plaque hybridization ..................................... 10.3.1. Standard plaque hybridization methods . . . . . . . . . . . . . . . . . 10.3.2. Modified plaque hybridization procedures . . . . . . . . . . . . . . . 10.4. Hybridization to colony or plaque nucleic acids . . . . . . . . . . . . . . . . . . 10.4.1. Radioactive probes ................................ 10.4.2. Nonradioactive probes ............................. 10.4.3. Reduction of background ........................... 10.5. Selection, packing and purification of clones ....................

476 478

Chapter 11. In situ hybridization ......................

495

11.1. Theoretical considerations ................................. 11.1.1. Nature of probes .................................

496 498

.

478 482 483 484 485 485 486 488 489 490 491 492

xi 11.1.2. Requirements of tissue preparation .................... 11.1.3. Positive and negative hybridization controls . . . . . . . . . . . . . . 11.2. Cytological procedures ................................... 11.2.1. Permeabilization and fixation of tissues and cells . . . . . . . . . . . 11.2.2. Sectioning and mounting of cells and tissues . . . . . . . . . . . . . . 11.3. Hybridization procedures .................................. 11.3.1. Prehybridization treatments ......................... 11.3.2. Hybridization to DNA ............................. 11.3.3. Hybridization to RNA ............................. 11.3.4. Posthybridization and detection of probes . . . . . . . . . . . . . . . 11.3.5. Turbohybridizationin situ ........................... 11.4. Emerging in situ hybridization techniques ...................... 11.4.1. Quantitative analysis of mRNA ....................... 11.4.2. Combination of in situ hybridization with immunohistochemistry ........................................... 11.4.3. In situ hybridization at electron microscopic level . . . . . . . . . . 11.4.4. Gene localization ................................. 11.4.5. Troubleshooting ..................................

500 501 502 503 508 509 511 511 514 515 517 517 517 518 519 520 523

Chapter 12. Selected applications of hybridization . . . . . . . . . . 525 12.1. Aims of subtractive or suppression hybridization . . . . . . . . . . . . . . . . . 12.1.1. Subtractive hybridization ............................ 12.1.1.1. High ratio subtractive hybridization by cloning inhibition ................................... 12.1.1.2. High ratio subtractive hybridization by biotin- or poly(A)+-capture .......................... 12.1.1.3. Low ratio subtractive hybridization . . . . . . . . . . . . . 12.1.2. Suppression hybridization ........................... 12.2. Hybrid selection of mRNA ................................ 12.2.1. Procedures of hybrid selection ........................ 12.2.1.1. Membranes and hybrid selection . . . . . . . . . . . . . . 12.2.1.2. Ultracentrifugation ........................ 12.2.1.3. Hybrid selection through biotinylated DNA . . . . . . . 12.2.1.4. Hybrid selection through capture assays . . . . . . . . . 12.3. S1 analysis of mRNA with DNA probes ....................... 12.3.1. Principles and goals of S1 analysis ..................... 12.3.2. S1 analysis methods ............................... 12.3.2.1. Preparation of DNA probes for S1 analysis . . . . . . . 12.3.2.2. S1 mapping of mRNA ...................... 12.3.2.3. Quantitative S1 analysis of mRNA . . . . . . . . . . . . . 12.3.3. Critical parameters of S1 analysis ...................... 12.4. RNase protection assays .................................. 12.4.1. Advantages of RNase protection assays over S1 analysis . . . . .

525 525 526 529 531 532 533 533 533 534 535 535 535 537 539 539 540 541 542 543 544

xii

......................

545

...................................

545 546 547

...........................

548 549

............................

549 550 552

...........................................

554 554 554 555

12.4.2. RNase protection assay methods 12.4.2.1. Preparation of full length RNA probes and purifica-

tion

12.4.2.2. Hybridization of RNA probe with target . . . . . . . . . 12.4.2.3. Digestion of unhybridized target and probe RNA . . 12.4.2.4. Analysis of protected RNA by polyacrylamide gel

electrophoresis

12.4.3. SimultaneousRNA extraction and RNase protection assays . . . 12.4.4. Interpretation, critical parameters and troubleshooting in

RNase protection assays

12.5. Triple helices .......................................... 12.5.1. Sequence-specificDNA purification by triplex affinity capture . 12.5.2. Inhibition of DNA restriction at specific sites via triplex forma-

tion

12.6. Current developments and prospects ......................... 12.6.1. Screening of genetic diseases ......................... 12.6.2. Peptide nucleic acid chimerae ........................ 12.6.3. Matrix array hybridization on silicon wafers Cgenosensors on

chips')

.........................................

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Subject index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

556 557 575

Part 11: Probe labeling and hybridization techniques

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CHAPTER 7

Labeling of probes and their detection The nature and detection of the different labels, followed by the methods of their incorporation in probes, is reviewed here. Radioactive and nonradioactive labels are not distinguished with respect to the labeling method but differences in methodology are indicated when necessary.

7.1. Choice of label and labeling method A myriad of labels is available, many of which may have advantages for particular applications. Labels can be distinguished as primary and secondary labels. Primary labels yield a detectable signal directly whereas secondary labels, tagged to nucleic acid, are recognized by detection systems with primary labels. Direct primary labels, such as radioisotopes, enzymes and physicochemical reporters (fluorophors, luminophors) are covalently attached to the nucleic acid. Indirect primary labels are linked to detection systems such as streptavidin (recognizing biotin), antibodies (recognizing haptens) or sulfhydryl ligands (recognizing mercurated nucleic acids). The nucleic acid to be labeled can be divided into four types: natural; enzymatically amplified; cloned; or synthetic. The selection of an appropriate label for a given probe may depend on many factors (Table 7.1). Localization of target nucleic acid in a cell often requires high resolution. This can be obtained by low energy radioisotopes, fluorescent dyes, or enzyme detection systems that limit the spread of the generated product as much as possible. On the other hand, dot hybridization requires high detectability while resolution is not a priority. Quantification is better with radioisotope labels than with biotin systems due to a higher sensitivity, while the

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 7.1 Criteria for choice of label and type of probe A. Requirements imposed by intended use - Resolution versus detectability (i.e., nature of information required) - Size of probe (e.g., full lecgth for S, mapping, 150-750 length for filter hybridization, but shorter for in situ hybridization) - Nature of probe (DNA/RNA, ss versus ds)

B. Stability of labeled probes - Effect of label on stability of probe - Stability during storage (half-life and storage life of label (single or multiple uses)) C. Nature of label - Detectability (target concentration at which signal/noise ratio just exceeds that of blanks) and sensitivity ( = efficiency of hybridization rimes efficiency of label detection) - Ease of background reduction (natural occurrence of label in samples?) - Radioactive or nonradioactive labels: safety, stability, detectability, sensitivity, cost, effect on hybridization kinetics, intrinsic specific activity of precursor - Primary label or secondary label (section D, below) - Ease of detection and equipment required D. Overview of labels and recognition systems 1. Primary labels: a. Yield a detectable signal (radioisotopes, enzymes, fluorescent or chemiluminescent dyes or electron-dense markers) b. Can be incorporated or attached to probes directly or indirectly (via recognition systems) 2. Secondary labels: a. Haptens: can be recognized by antibodies b. Biotin: is usually recognized by (strept)avidin but can also be recognized by antibody (useful when background levels are high with streptavidin) c. Mercury: recognized by SH-ligands which may be a primary or a secondary label d. Particular hybrid structure: can be recognized by specific antibodies 3. Recognition systems for secondary labels: a. Streptavidin: is used instead of avidin for the recognition of biotin since avidin yields strong backgrounds, particularly in the presence of phosphate; streptavidin can contain primary label or be detected by biotinylated primary label b. Antibody: to particular nucleic acid structure or to haptens; antibody may be labeled or be recognized by labeled anti-IgG antibodies c. Sulfhydryl ligands: react strongly with mercurated nucleic acids

Ch. 7

PROBE LABELING AND DETECTION

271

indirect systems may have a detectability matching that of radioisotopes. One label can be shown to be superior to another just by the design of the experiment. The degree of hybridization depends on the product of C, and t. Thus in theory, C , or t can be increased, while decreasing t or C , to maintain the same Cot value, to get the same degree of hybridization (Section 2.3.2). Two additional parameters are important: (i) the extent of background is often disproportionately related to either the amount of probe added or the incubation time; (ii) the kinetics of the hybridization reaction require high concentrations of probe. Since hybrid formation depends on the product of target (TI and probe ( P ) concentrations (Section 2.3.31, it proceeds at extremely low T concentrations provided the P concentration is sufficiently high. The limitation is the number of label molecules required for detection. Commonly, a high detectability requires a = lo5 excess of probe so that signal noise should be reduced by at least the same factor (e.g., by washing). If the concentration of probe added is greater than the amount required by hybridization kinetics, the signal/noise ratio can be reduced simply by decreasing the probe concentration (e.g., for DIG-probes, concentrations should be kept below 20 ng/ml for most hybridizations). Increasing the amount of probe and shortening the time while producing the same degree of hybridization may yield a different background level. It is not surprising that detectabilities reported for the same label differ widely, rendering comparisons of labels invalid. Thus, a label with a high sensitivity does not necessarily ensure a high detectability (e.g., if noise reduction is poor) and labels with low sensitivity may have high detectability (Section 7.3.2.1). Constraints imposed by the intended application may limit the choice of labeling methods (Table 7.2). For example, when the probe should be intact, such as in S , mapping, labeling by nick translation should be avoided. In contrast, for most slot blot and in situ hybridizations, nick translation or random primer extension methods provide suitable probes with an optimum label density, often with the added benefit of ss extensions on the duplex offering the possibility of amplification through hyperpolymer formation.

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

7.2. Radioisotopic labeling and detection of nucleic acids 7.2.1. Principles of radioactivity and units

Radioactivity is the spontaneous disintegration of the nucleus of an atom with the emission of radiation, accompanied by the emission of TABLE7.2 Overview and characteristicsof labeling methods A. Uniform enzyme-mediatedincorporation into ds DNA 1. Nick translation; widely used classical method producing probes of varying lengths and labels uniformly incorporated; requires ds DNA, amount of DNA decreases during labeling 2. Random priming: often replaces nick-translation since higher specific activity can be obtained, label is used more efficiently and the reaction is better controlled; amount of DNA increases during labeling; requires ss or denatured nucleic acid, length of probes is variable and less homogeneous than with nicktranslation 3. PCR labeling: very simple and powerful labeling method, small quantities of high specific activity can be rapidly obtained, does not require cloned templates for probe synthesis, primers (and their sequence) should be known 4. Replacement synthesis, most useful for specific labeling of one of the strands of the duplex, less used than other techniques

B. Preparation of uniformly labeled single-stranded probes 1. In vitro transcription: high yields of ss RNA, high specific activity, high stability of probes with target, requires cloning in appropriate vectors or the use of ds DNA amplified with PCR using primers containing the promoter sequences, usually allows production of probes against either strand of duplex 2. Priming on M13 vectors: resembles in vitro transcription but requires gel purification of probe, stability with duplex lower than with transcripts, DNA probes more stable than RNA probes 3. PCR: several methods available but not as rigorous as PCR for ds DNA probes 4. cDNA probes: usually less specific but may be required, e.g., for subtractive hybridization, specificity can be improved using sequence-specific primers C. End-labeling 1. Kinasing: relatively low molar specific activity 2. Tailing: allows high specific activity, very useful for oligonucleotides, may require the use of homopolymers during hybridization to suppress background 3. Fill-in reactions, relatively low specific activity, may be useful for specific labeling of oligomers

Ch.7

PROBE LABELING AND DETECTlON

213

TABLE1.2 (continued) D. Chemical labeling 1. Transamination: simple introduction of reactive amino groups in cytosine on ss strands; many primary or secondary labels may be reacted with this nucleophile; useful for universal probes 2. Photohaptens: photobiotin or photodigoxigenin can be introduced by simple methods; photohaptens react with many different molecules and nucleic acid preparation should be very pure; detectability somewhat inferior 3. Cross-linking: psoralen cross-links nucleic acid under UV irradiation; specific for ds nucleic acid (purity not a necessity); 'gapped' probes (with ss in sert) possible; good detectability 4. Mercuration: simple; reagents very toxic; stability may sometimes be problematic; usually no significant advantages over alternatives 5. Fluorene labeling: simple labeling; requires specific antibody; AAF carcinogenic

a @-particle and a neutrino. This @-particle may be a negatron (electron) or a positron (orbital electron capture) and its emission changes the number of protons so that a nuclide may decay to one with a lower or higher atomic number. The total released energy is constant and is specific for the type of nuclide (e.g., 1.71 MeV for 32P but only 0.018 MeV for 3H). Its distribution over the negatron and the neutrino varies, however. The SI unit for radioactivity is Bq (Becquerel): 1 Bq equals 1 transformation per second. This unit has simple relations to other units such as disintegrations per minute (60 dpm = 1 Bq) and counts per minute (1 cpm = 1 dpm x counting efficiency). The counting efficiency in a scintillation counter is usually 0.7-1.0 for 32P.The old, still widely used, unit for radioactivity is the Curie (Ci) which equals 3.7 X 10" Bq. Specific activities are expressed as radioactivity/unit mass (mg, mol, etc.). The specific activity of commercial preparations is stated in both units, but Ci/mmol is the most commonly used. Two types of units are used in the field of radiation dosimetry. The SI unit for energy absorbed from radiation is the Gray (Gy), which equals 1 J/kg and is 100-fold greater than a radian ( = 100 erg/g). Another older unit is the Roentgen which depends on the medium (1 Gy = 114 R in air but about 105 in water or tissue). The second SI

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

unit for radiation dosage is the Sievert (Sv) and it takes the relative biological effectiveness (RBE) into account; 1 Sv = 100 Rem (Roentgen-equivalent-man), or Sv = RBE X Gy. The Sv unit of dose equivalent equals the amount of ionizing radiation of any type that produces the same damage to humans as 100 R. The RBE is close to 1 for most commonly used radionuclides. Nonexistent 'nonisotopes' are often invoked to be the key to nonradioactive assays.

7.2.2. Maximum and optimum radioactivity of probes The rate (frequency) of disintegration is an inherent property of a given radioisotope and varies widely among isotopes. The half-life (time of survival of half the initial set of radioactive atoms) may be millions of years (e.g., 237Np)to a fraction of a second (e.g., %P). Since disintegrations in a small interval of time are rare and independent of other time intervals, disintegration in equal finite intervals follows the Poisson distribution. The probability of atoms still being radioactive, P(0) (in which 0 indicates no nuclear transformations), after t intervals with a decay constant 6 (= fraction of radioactive atoms decayed per interval, 6, if seconds, 6, if days) thus equals P ( O ) = e-*'(6t)O/O!

= e-*'

When half the radioactive atoms have decayed (at half-life and P(0) = 0.5)

(1) to,,

intervals =

0.5 = em( -6t0,,) and to, = 0.693/6

(2)

Equation (21, in combination with the established half-lives, gives the maximum attainable radioactivity for a certain mass of a given radionuclide. For example, "P has a half-life of 14.3 days (= 1.24 X lo6 s) and so the fraction of radioactive atoms transformed per second is 5.61 x (= 0.693/t0,,). One mole (6.02 x atoms)

ch.7

PROBE LABELING AND DETECIlON

275

of "P would have 6.02 X X 5.61 X = 3.38 x 1017 transformations per second (Bq) which equals 9.128 x lo6 Ci. Therefore, in a commercial preparation with 1000 Ci/mmol, about 1 in 9 P is 32P and the remainder nonradioactive "P. Similarly, the maximum radioactivity of any radioisotope can be established by measuring the decay rate and letting time t = 0. Maximum specific activities can also be rapidly established using the simple formula constant maximum specific radioactivity = half-life

(3)

in which the constant equals 1.3 X lo5 (Ci - days/mmol) or 4.8 x 10l8 (Bq - days/mol) depending on which unit expresses radioactivity (half-life expressed in days). For some radionuclides only one can be incorporated per nucleotide (e.g., P), whereas for others (e.g., H) several can be incorporated. Half-life of radionuclides should not be too short (short useful life of probes) or too long (resulting in low radioactivity per reasonable interval). Long shelf-life and high radioactivity are thus conflicting requirements. The theoretical maximum specific activity (in dpm/pg) that can be obtained in a probe can be calculated. One pg nucleic acid has about 20 X 1014 nucleotides (= 3.3 X moly assuming a mass of about 330 Da/nucleotide and using Avogadro's number). If all 20 X 1014 nucleotides in the 1-pg sample contained a 32P, it would have a specific activity of 1.1 x lo9 Bq/pg ( 3 . 3 8 ~10'7X20X 1014/6.02 X loD), which is 6.6 X lo1' dpm/pg (= 30 mCi/pg). In practice, a probe prepared with a common commercial radioactive nucleotide preparation of 1000 Ci/mmol (1/9 of maximum) would have a maximum specific activity of 1.1 x 109/9 = 1.2 x 10' Bq/pg (or 7.3 X lo9 dpm/pg). In addition, about 25% incorporation is maximally obtained, since the labeled nucleotide accounts for about 1/4 of the constituent bases, thus with this precursor close to 2 X lo9 dpm/pg can be obtained. Usually an excess of radioactivity should be added to obtain this since the efficiency of labeling may be 40-75%.

More important than the maximum is the optimum radioactivity of a probe. Nuclear transformations of 32P lead to strand scissions

276

HYBRIDIZATION WITH NUCLEIC ACID PROBES

(32P+ 32S)due to the unfavorable coordination numbers of 32S and the high vibrational energy. Also, 32Ptransformation leads to 5: 10% breakage in the sister strand. Thus highly labeled probes have high initial detectabilities, but they are self-destructive and may have half-lives about 5-20 times shorter than the radionuclide. The optimum radioactivity is a compromise between maximum specific activity and long shelf-life of the probe. Labeling results in a distribution of radioisotopes over the various strands according to the Poisson distribution (with m the average and n the actual number, an integer, of radioisotopes per strand): P( n ) = e-"mn/n !

(4)

and the fraction of probes with 0, 1, 2, 3, etc., radioisotopes, is

P ( 0 ) = e-m

~ ( 2 =) e-"m2/2

P( 1) = e-"m P ( 3 ) = e-"m3/6

etc. For example, in ss probes of 250 nucleotides labeled with 32P to lo8 dpm/Fg, which is 108/6.6 X 10" = 1/660 of maximum (since the maximum is 6.6 X 10" dpm/Fg), m would be 250/660 and P(0) = 0.685, P(1) = 0.259, P(2) = 0.049 and P ( 3 ) = 0.006. These considerations imply that the intensity of radiation of probes increases linearly with the number of radioisotopes per molecule, but that molecular decay relates exponentially to the number of radioisotopes in the molecule. The severity of this decay may be attenuated if a few scissions can be tolerated. It is difficult to establish the optimal level of activity. As a rule of thumb, probes with > lo9 dpm/Fg should be used within a few days; if the detectability can be assured with lower activity probes, then a somewhat lower specific activity (e.g., lo8 dpm/pg) can be aimed at to ensure the longest shelf-life (1-2 weeks). If high detectability is required or if a rapid labeling procedure of small amounts of probes (e.g., with PCR) is feasible daily, the optimum activity may be chosen close to the maximum.

Ch.7

PROBE LABELING AND DETECTION

211

TABLE1.3 Stability of radiolabeled precursors and compounds Characteristic of decomposition Primary

- internal - external

Secondary

Chemical and physical

Cause Natural decay of radioisotope; recoil energy of radioactive decay Interaction of radiation energy with molecules of the compound Interaction of radiation-produced reactants (e.g., radicals) with molecules of the compound Thermodynamic instability of molecules of the compound; method of storage

7.2.3. Chemical stability of radiolabeled probes Labeled compounds are chemically not as stable as their unlabeled counterparts (Table 7.3). The natural decay of the radioisotope produces an impurity Le., 3H yields helium, 35S yields chlorine, 32P yields sulfur and Iz5I yields tellurium). The compound or its direct environment absorbs the emitted radiation energy and extensive self-radiolysis may occur (Tolbert et al., 1953). External primary decomposition occurs through the production of free radicals or other reactive species, such as hydrogen radicals, hydroxyl radicals or peroxides. These may all cause destruction of the molecules of the labeled compound. To counteract this effect, suppliers add, when possible, ‘scavengers’ (e.g., ethanol, cysteamine) to the solution. The effects of freezing on stability are complex, e.g., beneficial for 35Sbut not for 3H. This may be due, at least partially, to clustering of molecules in solution upon freezing, resulting in increased decomposition due to nearby radioactivity. The position of the radioactive atom in the molecule is also important. For example, 32P is directly in the backbone of the nucleic acid and a breakup will result in a scission of the strand. On the other hand, 35S is not as crucial since it replaces an 0 outside the phosphate group in the backbone.

278

HYBRIDIZATION WITH NUCLEIC ACID PROBES

7.2.4. Choice of radiolabel The choice of label depends on several factors (Table 7.1). Detectability is usually inversely related to the resolution attainable with the various radioisotopes. The fine resolution often required for in situ studies can be obtained with 3H which emits very weak &radiation and produces short path lengths and silver grains over a small area. Its low detectability requires long exposure times. The specific activity of the label and its detection efficiency both limit the detectability. For example, 1251 emits both y-radiation and low energy Auger electrons. The y-radiation penetrates the film almost without producing silver grains, whereas the low energy electrons do so more efficiently. 1251 should, therefore, be useful both for high detectability (by returning the y-radiation to the emulsion by an intensifying screen) and high resolution (without a screen). Table 7.4 summarizes the characteristics of radionuclides most often used in nucleic acid labeling. Commercial dNTP preparations usually contain 10 pCi/pl and those with higher specific activities thus have a lower concentration. For instance, if the final radioactive concentration in the mixture is 1 pCi/pl, the concentration of that radiolabeled dNTP is 1 pM if the specific activity is 1000 Ci/mmol but 0.2 pM if the specific activity is 5000 Ci/mmol. The claim that a single gene can be detected means that among the large number of copies of immobilized genomes, a unique gene can be detected. An example can be given for the detection of a unique gene in human DNA by filter hybridization. The haploid human genome has about 3 x 10’ bp; a hypothetical gene of 1000 bp (under the assumption of a gene without introns, coding for a protein of about 40 kDa) would represent 1000/3 X 10’ part of the genome. On a nitrocellulose membrane, about 80 pg of DNA can be adsorbed per cm2 or about 8 pg to a slot of 0.1 cm2. Thus, a maximum of 8 X 1000/3 X 10’ pg of the hypothetical gene, or about 2.6 pg (which corresponds to about 2.4 X lo6 copies of that gene) can be adsorbed. The detectability that is attainable with a 32P-labeled probe of 1000 nucleotides is about 0.1 pg/O.l cm2 or about lo5 target molecules (assuming a specific activity of the probe of lo8 dpm/pg and a detection limit of 100 dpm/cm2/24 h).

Ch. 7

279

PROBE LABELING AND DETECTION

TABLE 7.4 Characteristics of radioisotopes used in nucleic acid labeling Radio- Half- Radiation isolife type/max tope energy of emission (MeV) (mean)

'H

32 P

35s

1251

12 years

14 days

90 days

60 days

P

Labeling methods

25-100

Nick-translation Random primer Transcripts

0.018 (0.0055)

P

500-5000

1.71 (0.70)

P

300-1500

0.167 (0.049)

P electroncapture

Y 0.035

*

Typical SA* of nucleotides (Ci/mmol)

1000-2000

Typical

SA * of probe (dpm/pg)

5 X lo7

200

108

40

5 x lo7

200

Nick-trans- (1-5)x 108 lation (1-S)Xlo9 Random primer (1-SIX l o 9 PCR 1x lo9 Transcripts s x 106-109 End-label 108-10'0 Tailing Nick-translation Random primer Transcripts

Probe detection limit (ps/cm2)

108

0.2-1 0.02 -0.1 0.02-0.1 0.1 20-100 0.02-1 5

5x10s

0.8

lo9

1.5

Nick-translation

108

1

Random primer Direct iodination

lo9

0.1

108

1

SA, specific activity; maximum radioactivity is 28.75 Ci/mmol for 3H, 0.065 Ci/mmol for I4C, 9128 Ci/mmol for 32P, 1494 Ci/mmol for "S and 2176 Ci/mmol for '251

280

HYBRIDIZATION WITH NUCLEIC ACID PROBES

7.2.5. Detection of radioactive probes Special equipment is available to detect radioactivity on membranes (e.g., Betascope 603 Blot Analyzer from Betagen, Inc.). It is expensive and autoradiography with X-ray film remains widely used. Autoradiography provides both a photographic recording of the signal intensity and its spatial distribution. Hybridization in situ with radioactive probes often requires the use of radioisotopes emitting P-particles with low energy to limit their range and improve resolution. Here, autoradiographic emulsion is coated directly on the specimen to obtain intimate contact.

7.2.5.1. Principles of autoradiography and use of films The commonly used radioisotopes emit P-particles (electrons; 3H, 14C, 35S,32P) or gamma, ionizing radiation (X-ray) and electrons (lz1). When P-particles collide with other orbital electrons or nuclei, they are dispersed and rapidly lose energy. These paths depend on the initial energy of the particle (Table 7.4). In autoradiography, these particles collide with silver halide (bromide, chloride) grains and absorption of the energy results in the release of electrons. These electrons, when captured, transfer positively charged silver ions into atoms of metallic silver. About six atoms (which can be generated by a single interaction with P, X-ray, or y-ray photons, but requiring more than one light photon) can form a stable, latent image center that can be developed into visible images. Latent images are not stable and decay over time. Low temperature exposure or preflashing can improve the formation and stability of latent images (Hahn, 1983). Direct exposure of the film to the specimen is only effective with high energy P-particles but yields high quality images. The signal can be intensified by using (i) special films, (ii) intensifying screens, (iii) fluorography, (iv) preflashed films and (v) low temperature exposures (Fig. 7.1). Most common X-ray films have a plastic film support coated at both sides with an emulsion of silver halide grains in gelatin and covered with a protective gelatin layer. Smaller silver grains have a higher resolution but a lower sensitivity. HyperfilmTM(Amersham;

Ch. 7

PROBE LABELING AND DETECTION

281

CEA AB, Sweden), a single-coated film, comes in two versions, one lacking the protective layer (for detection of 3H) and one with an anti-scratch layer (for 14C, 35S and 1251). This film has a fine grain and a high silver content and thus a high resolution and still a high sensitivity in the detection of low energy @-particles.It is well suited for Northern and Southern blots. It is important for one-sided coated films to place the correct side toward the specimen. Phosphor-intensifying screens (e.g., Cronex Lightening Plus from du Pont or Fuji Mach 2) can be used with double-sided coated X-ray film for high energy @-particles. Since many of these particles pass directly through the twoemulsion layers, they can be absorbed by the phosphor (calcium tungstate) and produce multiple photons (blue light) that bounce back to the film to produce latent images (Swamtron and Shank, 1978). Using two screens (between which the film and the membrane are sandwiched), increases the signal if the probe is bound to a membrane that does not absorb light. All membranes used absorb light efficiently; yet, by rendering nitrocellulose membranes translucent with mineral oil, Rust et al. (1987) obtained an enhancement of signal. The @-particles in the specimen can be converted directly into light photons (fluoyography). These photons have a longer pathway and increase the signal of weak @-particles about 10-fold (Bonner and Laskey, 1974). Although initially fluors, such as 2,5-diphenyloxazole (PPO), were used, sodium salicylate is now often used to overlayer or impregnate the sample (Chamberlain, 1979). The specimen is soaked in 10 vols. of 1 M sodium salicylate (pH 6.0) for 30 min and dried before autoradiography. Commercial enhancing fluors should be used according to the manufacturer’s recommendations. Latent image formation can be initiated by preflashing X-ray film. Light photons generated by radiation (fluorography, intensifying screens) will then complete the images. Preflashed films produce higher signal densities (about two-fold) with less background (Laskey and Mills, 1977) and the density is more proportional to the intensity of radioactivity. The flash of light ( < 1 ms) is emitted with a photographic flash unit (or stroboscope) covered with an orange filter (Wratten 22A) to reduce blue light and placed perpendicular to the film (50 cm distance) in total darkness. The film should be

282

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

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Ch. 7

PROBE LABELING AND DETECTION

283

covered with a Whatman no. 1 filter paper or a diffusing screen. A series of test films is exposed for different lengths of time and the absorbance of the film measured after development in a cuvette holder of a spectrophotometer at 545 nm against a developed nonexposed film. The optimum exposure time is the one that yields an optical density of 0.15. Latent image formation is most stable at low temperature ( - 70°C) and methods based on light photons (fluorography; intensifying screens) should be done at this temperature (difference can be five-fold). Low temperatures do not improve latent image stability for direct exposure and ionizing radiation. Wet or damp specimens should never be in direct contact with the film (cannot be separated) and direct exposure requires the use of Saran Wrap (Dow Chemicals) between the film and the specimen. Saran Wrap still may block a significant fraction of low energy P-particles. Original autoradiographic images can be intensified using Kodak Chromium Intensifier (bleach and clearing baths) to darken the image (carried out in artificial room light). The autoradiogram is soaked in water for at least 10 min, immersed in the bleach bath for 5 min, rinsed briefly with water and immersed in a clearing bath for 2 min. After another rinse, the autoradiogram is redeveloped using a developer such as Kodak Dektol (diluted 1 : 3 with water). This process can be repeated several times to obtain more than 5 X intensification and make invisible bands appear. Double emulsion films produce the best results and it may be necessary to optimize preflashing to avoid high background absorbance. Table 7.5 summarizes details on film autoradiography.

Fig. 7.1. Three types of autoradiography can be distinguished: (i) direct exposure (direct contact of specimen with film, usually separated just by a Saran wrap for wet specimens); (ii) fluorography in which fluors are excited by the radiation resulting in multiple photons per /3-particle; (iii) phosphor-intensifyingscreens which can absorb radiation under the emission of photons (most stable in the cold). The proper choice of method depends on the penetration level of the radioisotope and the intensity of radiation.

284

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 7.5 Technical details of autoradiography

1. Choice of film: high speed, double-sided emulsion (Kodak XAR-5 or XAR-2 or Fuji RX) whereas Kodak XRP-1 has one-third the speed but a finer grain. Hyperfilm ( h e r s h a m ) is one-sided and has a very high resolution. All can be developed and fixed with conventional products such as Kodak GBX. For fluorography, Kodak SB is an excellent choice due to its sensitivity for light photons. 2. The specimen is placed in contact with an X-ray film in the dark and exposed for a time which is usually a compromise between a background development and maximum signal (typically 24 h for 32P). A good contact between the specimen and film is required. This can be achieved by sandwiching them between glass plates in a light-proof bag and placing weights on top or by using X-ray film cassettes (Wolf X-ray Corp.). 3. Intensifying screens are used for 32Pwhereas fluorography can be used for 35S,14C, 3H. Some highly radioactive samples may require very short exposure times or slower films without screens. 4. Films exposed at - 70°C (with intensifying screens) should be allowed to warm to room temperature to prevent condensation or be developed before condensation forms. 5. 35S ink labels are useful to locate the various specimens on the film. Alternatively, fluorescent markers or RadtapeTM are convenient.

7.2.5.2. Autoradiography with nuclear track emulsion Thin films of autoradiographic emulsion should adhere closely to the surface of cytological preparations for the detection of low energy &particles (from 3H), which have the highest resolution. Examples are the Ilford LA and the Kodak NTB-2 or NTB-3 emulsions, which are solid at room temperature but become liquid when warmed to 41-45°C. Slides are dipped in a diluted emulsion to produce a thin, even coating. Thicker coatings do not increase specific grains (due to the short pathway) but have a higher probability of nonspecific grains or grains due to extraneous causes. The emulsions should always be kept separated from radioisotopes and organic fumes. Dilute 32P solutions, present in the same refrigerator, rapidly cause an unacceptable level of grains. Quality of new batches should always be checked. Pardue (1985) suggested as upper limit 100 grains per microscopic field (using a 100 x Neofluar lens and 10 X Kpl eyepieces). Less than 50 grains per field is excellent.

Ch. 7

PROBE LABELING AND DETECTION

285

Emulsions are prepared in a light-tight darkroom under sodium vapor safelight or corresponding filter. The bottle of NTB (e.g.1 emulsion (112 ml) and a 500-ml Erlenmeyer flask with 112 ml of distilled water are warmed to 41-45°C for 30 min. Subsequently, the molten emulsion is poured very slowly down the side of the flask, which is rotated slowly under an angle to mix evenly. Diluted emulsion is then aliquotted into nylon scintillation vials (10 ml each), wrapped in aluminum foil and stored in light-tight boxes in a refrigerator. These emulsions are very stable. The evenness of the emulsion can be verified by dipping a few blank slides and viewing them near the safelight. It is useful for dipping to place the tissue preparations close to the bottom of the slide. Emulsion, warmed in the vial during 15 min at 45"C, is poured along the side into a dipping chamber, avoiding bubbles. Slides are dipped one by one slowly into the chamber and withdrawn. Excess of emulsion is allowed to drain, the slides placed in a vertical rack and dried for about 2 h (whole procedure in the dark). Dried slides are stored in a refrigerator with a small container of silica gel or Drierite, to maintain dryness, in light-tight boxes, sealed with plastic tape. Exposure time varies from a few hours to several months and it is, therefore, useful to have several replicates so that periodically a slide of each batch can be developed. For development (at 15-2OoC), the specimens are placed in 1 : 1 diluted Kodak D-19 developer (2.5 mid, water (10 s), undiluted Kodak rapid fixer (5 min) and 5 X 5-min washes in distilled water. Shaking of the baths during development can be useful. Slides are dried after autoradiography and stained immediately (Section 11.3.4). True labeling is hard to distinguish from background when labeling is weak and truly labeled structures may lack grains in the emulsion layer due to the Poisson probability of radioactive decay. For instance, when a labeled structure contains an average of 3 grains, then a fraction equaling P(0) = e-3 = 0.050 will be negative but when the average number of grains drops to 1 per labeled structure, P(0) = eC1 = 0.368. Thus the number of negative structures increases seven-fold while the average number of grains drops only three-fold. Korr and Schmidt (1988) have presented a

286

HYBRIDIZATION WITH NUCLEIC ACID PROBES

computer-aided statistical correction method that takes this binomial distribution into account.

7.2.6. Safety considerations Radiation is a major hazard and its stochastic effects should not be underestimated (Ballance et al., 1984). For instance, P-particles emitted by 32Phave a maximum range of 6 m in air, but less in glass (0.38 cm), Perspex (Plexiglas, 0.64 cm) or water (0.84 cm). The maximum permissible occupational skin dose equivalent in the United States is 30000 mRem/year (7500 mRem/3 months) but six times lower for the lens of the eye. The dose equivalent rate at the surface of a glass container with 32P(such as ‘Combi-v-vial’) can be close to 500 mRem/hmCi whereas it may be over 170000 mRem/hmCi outside a plastic syringe with a 32P solution (Zoon, 1987). Without precautions, the maximum permissible dose can thus be received in a matter of minutes or even seconds. In contrast, 35S does not penetrate the walls of typical containers or the dead layer of skin cells. The main hazard of 35S is due to inhalation. Leak-free gloves are mandatory and dosimetry badges (particularly wrist badges) should be worn for continuous monitoring. When shielding radiation, low effective atom number-material should be chosen to minimize the generation of Bremsstrahlung by the /3-particles (IAEA, 19731, particularly for high energy emitters like 32Pand lZI. Merrifield (1987) described the construction of simple shielding devices. For example, for flip-cap Eppendorf tubes, four Bijou bottles (or decontaminated ‘Duoseal’ containers, without the plastic inserts, from Amersham) are placed in a disposable Petri dish and fixed with an embedding resin (e.g., Shallowcast). Screw-cap tubes are recommended for radioactive samples. Plastic tube holders (e.g., Sarstedt 93.848.100, with splines to hold the tubes) can be made safe for &radiation by constructing a Perspex box to fit around it and adding a sliding cover (Gupta, 1985). Gilson Pipetman or similar pipettors can be fitted simply with a radiation shield (Merrifield, 1987). Cohen et al. (1990) described a ‘hybribox’ which consists of a set of three Plexiglas outer boxes (hybridization and 2 posthybridization) and a carrier box which is transferred from box to box. The

Ch. 7

PROBE LABELING AND DETECTION

287

volume is quite large (Plexiglas inserts can decrease this) and may make the procedure expensive if used for a single hybridization. The safety makes it very attractive, however. Upon receipt, packaging should be intact and radioactive material should be stored behind proper shielding (Perspex boxes with l-cm thick walls in the freezer and for transport). Labeled precursors and products should be handled behind radiation shields. We use homemade shields from Perspex (1 cm thick, 40 cm wide and 60 cm high) standing under an angle of 60" and supported for 35 cm at each side by rectangular triangles (60"; 17.5 cm at base) of Perspex. Radioactive waste is disposed of according to the guidelines of the appropriate authorities.

7.3. Nonradioactive primary labels Nonradioactive primary labels (Fig. 7.2) have been compared in several studies (e.g., Giaid et al., 1989; McQuaid et al., 1991). Their suitability depends on the nature of the hybridization experiment. For filter hybridization, enzyme labels are excellent. Several enzymes also can be used with chemiluminescent substrates that rival 32P in detectability. Acridinium esters are useful primary labels in commercial applications of solution hybridization. Most solution hybridization assays require a second step, in which the hybrid is captured selectively from the reaction mixture and then detected on a solid phase. In situ hybridization is faced with different requirements (resolution, background) and convenient markers are peroxidase and fluorescein. Both yield excellent resolution (somewhat better for fluorescein but cellular detail is less obvious) whereas electron-dense markers (gold) with silver enhancement give high background levels at the light microscope level. Gold markers, without enhancement, are excellent in electron microscopy. Alkaline phosphatase is often less suitable for in situ hybridization since (i) NBT/BCIP substrate gives a diffuse reaction within individual cells and (ii) a strong background is frequently observed. Background due to endogenous alkaline phosphatase may be reduced by the addition of 5 mM levamisole (not efficient against all alkaline phosphatases), whereas

288

b

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Ch. 7

PROBE LABELING AND DETECTION

289

background due to endogenous peroxidase (particularly in myeloid cells and erythrocytes) can be eliminated by incubation in methanolic peroxide after deparaffinization (Section 11.2.2).

7.3.1. Chemiluminescence Chemiluminescence is becoming popular for probe detection due to its high detectability and low background. It is a nonthermal emission of electromagnetic radiation (light) through a process involving three steps: (i) absorption of energy generated by chemical reaction (usually oxidation); (ii) excitation; (iii) emission of energy as radiation in the visible portion of the spectrum. Light emission can be intense, stable for long periods and detectable with luminometers, silicon photodiodes or photography with X-ray (e.g., Kodak X-Omat) or instant (e.g., Polaroid type 612 Instant black and white) films. Attention has been focused on four different chemiluminescent systems, two as substrates for enzymes and two as labels incorporated in the probes (activated after hybridization by H,O,/alkali (Table 7.6)). The latter are usually intended for diagnostic purposes and prepared by commercial suppliers, whereas those serving as a substrate for enzymes attached to the probe-hybrid complex can be readily adapted for many hybridization assays. Bioluminescence has been used in immunoassays (TCrouanne et al., 1986) but its complexity and high background, arising from ATP contamination, thus far make this system less attractive. Bioluminescent assays have also been proposed with alkaline phosphatase using luciferin phosphate as the substrate (Hauber and Geiger, 1987) and Fig. 7.2. Modified nucleotides to which primary labels can be attached (1-111) and nonradioactive primary labels (IV-VIII). Photohaptens (IX)are readily attached to nucleic acids. Particularly popular are DIG-1 1-dUTP (VI) and fluorescein-12-dUTP. Bases 1-111 contain both a linker and a reactive amino group. Usually an extra linker is included to improve further accessibility (e.g., VI and VIII). Although linkers are a necessity for biotin, they are not absolutely required for the detection by labeled antibody (BrdU (IV), AAF (VII)). Similarly, trinitrophenyl-glutathione (TNP-GSH) readily reacts with mercuricytosine (V) and the TNP can be detected using specific antibody. R, in VII can be either an H (AAF) or an I (AAIF).

290

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 7.6 Chemiluminescence systems

*

Location of Type of substrate luminescence

Substrate derivatives

Type of reaction

Dioxetane

Enzymatic hydrolysis

In medium

‘Glow’

Luminol Acridinium ester Oxalate ester

POase * / H 2 0 2 H 2 0 2/alkali

In medium In probe

‘Fast glow’ ‘Flash’

H 2 0 , /alkali

In probe

‘Flash’

Suppliers BRL, Tropix, Lumigen, Boehringer Mannheim Amersham Ciba-Corning, GenProbe American Cyanamid

Peroxidase.

glucose-6-phosphate dehydrogenase using flavin mononucleotide oxidoreductase and a luciferase (Balaguer et al., 1989).

7.3.1.1. Acridinium ester probes An acridinium ester-labeling method has been developed by Gen-

Probe, Inc.. Because of high quantum yield and flash reaction kinetics in the presence of base and H 202, chemiluminescent acridinium esters provide the possibility of designing sensitive nonradioactive probes. The detectability of these systems is 5 X mol and acridinium-labeled probes are fully compatible with hybridization. Arnold et al. (1989) discriminated hybridized from unhybridized acridinium ester-labeled DNA probes without prior separation (Fig. 7.3). In a typical experiment (Table 7.71, the ss probe is hybridized to Fig. 7.3. Principle of acridinium-ester probe hybridization in solution and detection. Acridinium-NHS is linked to ally1 nucleotides (Section 7.3.1.2) in the probe. When the probe hybridizes with its target in solution, the acridinium moiety intercalates in the duplex. Unhybridized acridinium ester probes can be hydrolyzed selectively (Table 7.7, step 21, whereas protected acridinium ester remains intact (11). After nonspecific chemiluminescence is sufficiently eliminated, specific chemiluminescence is measured (111). The whole procedure takes less than 30 min.

t ACRlDlNlUM

PROTECTED BY

UNPROTECTED ACRlDlNlUM

INTERCALATION INTO DUPLEX

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292

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE7.7 Hybridization protection assay with acridinium ester probes 1. Hybridize methyl acridinium ester probe with target nucleic acid (single-stranded or heat-denatured) for 10 min at 60°C in 0.1 M lithium succinate buffer (pH 5.2) containing 10% (w/v) lithium lauryl sulfate, 2 mM EDTA and 2 mM EGTA (total volume 100 ~1 with 0.2 pmol of probe in a 12x75 mm2 tube). 2. Hydrolyze unhybridized acridinium ester probe for 10 rnin at 60°C by adding 300 pI of 0.18 M sodium tetraborate containing 1-5% Triton X-100 at pH 7.0-8.5. 3. Measure the remaining chemiluminescence after the samples have cooled down in a luminometer with an automatic reagent injector by injecting 200 pI of 0.3% H,O, and after 1 s, 200 PI of 2 M NaOH (the ‘flash’ chemiluminescence is integrated over a 2-5 s period).

the target in solution (5-10 min at 60°C). The acridinium ester on the probe is protected (intercalated) in the duplex but not on the ss probe. In the second step, the ester bond on the unhybridized probe is hydrolyzed (5-10 min at 60°C) resulting in a rapid loss of nonspecific chemiluminescence. The remaining chemiluminescence is proportional to the amount of bound probe. The half-life (t,,J of unhybridized acridinium ester is about 45 s but about 2700 s for the hybridized label and the remaining chemiluminescence ( A ) can be calculated from

A , = A , x (0.5)1’10.5 i.e., after 15 min it would be expected that A,,, for hybridized probes is 79%, but for the unhybridized probe. This theoretical performance has not been achieved since background luminescence limits the reduction to about 0.002%. of the acridinium ester in the hybridized probe decreases The if mismatches are present. In such experiments, hydrolysis is omitted and RNA/DNA-probe hybrids are adsorbed to hydroxyapatite. Although about 80% may be hybridized, half-lives could be reduced, for some mismatches, to close to that of unhybridized probes (Arnold et al., 1989). Probes with mismatches cannot be used in solution hybridization but can be used in hybridization on solid phase where

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the unhybridized probe is removed by washing and not by hydrolysis. Discrimination remains superior in the solution hybridization technique. These methods are exquisitely reliable (Engleberg, 1991).

7.3.1.2. Acridinium labeling of probes Acridinium-NHS (N-hydroxysuccinimide) ester labeling reagent is synthesized as described by Weeks et al. (1983) and probes containing an allylamine linker arm (Fig. 7.2.1) are prepared either as described for nucleotides and for enzymatically labeled probes (Section 7.6.2.21, by transamination (Section 7.8.1) or during oligomer synthesis (Section 6.4). The acridinium-NHS ester is then reacted with the linker arm by standard methods (Section 7.4.1). 7.3.2. Fluorochrome labels Fluorochrome labels are widely used in immunohistochemistry, immunofluorometric assays and cell sorting. Fluorescein isothiocyanate (FITC) has a green fluorescence of high intensity and reacts readily with proteins (e.g., streptavidin, antibodies) or amino groups. Several alternatives (rhodamine B and tetramethylrhodamine isothiocyanates, Texas Red; all red fluorescence; Fig. 7.4) have also been used. Texas Red is bright red, resists fading and its emission spectrum is quite different from FITC, making it ideal for double staining. An important constituent of the light absorption system of red algae is a fluorescent phycobiliprotein (Rhodymenia phycoerythrin, or RPE; orange-red fluorescence) that, on a molar basis, has a 25-fold brighter fluorescence than FITC (Oi et al., 1982). A major drawback of the fluorescein labels is their stability. Direct labeling of nucleic acids with the primary fluorescent label often produces less background and higher resolution than indirect methods. The gain in resolution is counterbalanced with the gain in detectability with indirect staining through streptavidin or antibodies (or anti-antibodies, protein A, etc.). Detectability of fluoresceinated nucleic acids can be improved by using anti-FITC monoclonal antibodies (EIA Co., San Francisco).

294

HYBRIDIZATION WITH NUCLEIC ACID PROBES

I R :-OH fluorescein isothiocyanate (FITC) -N-(CHJ)2 -N-(C,H,),

I1

tetramethylrhodamine isothiocyanate (TMRITC) rhodamine B isothiocyanate (RBITC)

I11 RO‘ 4-Methylumbelliferyl-R

S0,CI

R: 8-D-galactopyranoside (for BGase) R: Phosphate (for APase)

Fig. 7.4. Fluorochromes used as labels in probes (1-11). Fluorescent dye (111) is used as a substrate, whereas Eu’+ (IV) is used in time-resolved fluorescence.

7.3.2.1. Fluorochrome labeling of nucleic acid probes Labeling of nucleic acids via N-hydroxysuccinimide (NHS) esters of fluorescein (Research Organics) or Eu3+-chelates is straightforward. Nucleic acid should then possess reactive amino groups. These can be introduced by transamination (Section 7.8.1) or by using aminohexyl-dNTP or AH-NTP as precursors during enzymatic probe synthesis (Section 7.6.2.2; Folsom et al., 1989). After AH-NTP incorporation in RNA (or AH-dNTP in DNA and heat-denaturation), the nucleic acid is diluted to 1 p g in 25 pl of H,O and added to 25 pl of freshly prepared 0.4 M sodium bicarbonate (pH about 8.2) and then to 10 ~1 of ester in DMF (1.7 mg/ml of ester final concentration). After incubation for 2 h, the reaction is stopped by adding 50 pl of 1

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M Tris-HC1 (pH 7.6) and 100 p,1 of 1 X SSC. Free fluorescein is removed by chromatography on a 6-ml Sephadex G-50 column (1 x SSC + 0.05% SDS). A fluorescein label will be present once every 30 bases. T4 RNA ligase can introduce fluorescent 3’-0-(5’-phosphoryldeoxycytidyl)S-bimane phosphorothioate into RNA or ss DNA (Cosstick et al., 1984). Other methods have been developed for fluorochrome labeling of DNA for automated sequencing, either by labeled primers or ‘dye-terminators’ (fluorescent ddNTP; Applied Biosystems) with DNA polymerase or with terminal dNTP transferase (Trainor and Jensen, 1988). We successfully used fluoresceindUTP (Boehringer Mannheim, soon available), both for automated sequencing and probe preparation. All common DNA polymerases incorporate this analogue efficiently. Labeling of nucleic acids with Eu chelates, used in time-resolved fluorescence, is described in Section 7.3.2.3. Highly fluorescent complexes have also been introduced into ds DNA via psoralen carriers (Oser et al., 1988; Section 7.8.3). A plasmid carrying an insert complementary to the target nucleic acid is cut with a convenient restriction enzyme in the insert or polylinker and partially digested with exonuclease I11 (Section 7.6.4). This renders at least part of the insert ss but leaves the remainder of the vector ds. Reactive thiols are introduced in the psoralen which are then reacted with the ds DNA. In the next step, poly-L-lysine, labeled with the metal chelator diethyleneamine pentaacetic acid (DTPA), is reacted with the DNA-bound psoralen-thiol groups. After hybridization, europium ions are attached through the chelator DTPA and detected by time-resolved fluorescence. Alternatively, highly radioactive metal isotopes can be used as well as electron-dense markers such as 0 s or U ions, or Gd3+ for NMR analysis. Although the Eu3+ detectability was excellent in the membrane hybridization method, the sensitivity was poor making quantification difficult. Oser and Valet (1988) used polystyrene microtiter plates instead of membranes, lower concentrations of Eu3+ and included different washing steps to remove nonspecifically bound Eu3+ more efficiently. The detectability was only slightly improved, but the sensitivity (signal increase/target increase) improved about = 100-fold.

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

7.3.2.2. Fluorochrome labeling of streptavidin or antibody Conjugation procedures should yield optimal fluorochrome/protein (F/P) ratios. Most economically, the desired F/P ratio is regulated by the initial weight of dye in the reaction mixture and the reaction is allowed to go to completion. Alternatively, with relatively more dye, the reaction is interrupted after a specific incubation period. The efficiency of labeling depends on the protein, the protein concentration, the specific fluorochrome and the purity of the fluorochrome (some preparations only 30%). Over- or undercoupling leads to nonspecificity or low detectability, respectively. Purified antibody (Section 7.4.0, dissolved at 3 mg/ml (4 optical density units at 278 nm/ml) in 0.1 M sodium carbonate (pH 9.0) is labeled by adding slowly (in 5-pl aliquots) FITC or tetramethylrhodamine (freshly prepared at 1 mg/ml in DMSO; solvent should be of best grade available and stored frozen to prevent oxidation) to a total of 50 pl/ml. The reaction mix is left in the dark for 8 h at 4°C. At the end, NH,C1 is added to 50 mM and incubation is continued for another 2 h at 4°C. Xylene cyanol (0.1%) and glycerol (to 5 % ) are added and the mixture is passed on a small Sephadex G-25 (fine grade) column (0.8 x 10 cm; in PBS). The conjugate elutes first and can be seen under room light. BSA (to 0.1%) and sodium azide (to 0.02%) are finally added and the conjugate is stored at 4°C. The F/P ratio for fluorescein can be determined, before adding BSA, at 495/280 nm (should be 0.3-1.0) and for rhodamine at 575/280 (should be 0.3-0.7). At low ratios, conjugation should be repeated, at high ratios labeled antibodies can be purified on DEAE-Sephacel. The column should be equilibrated with 10 mM potassium phosphate (pH 8.0) and the different antibodies eluted at increasing salt concentration. The ratios of the fractions are measured and the appropriate fractions are pooled. This method can be used for labeling with dichlorotriazinylaminofluorescein (DTAF), with the exception that the fluorescein is dissolved at 2.5 mg/ml in 1.0 M sodium carbonate (pH 9.0) and the antibody is prepared in 0.2 M of the same buffer (use 25 pg of DTAF per mg antibody). The same procedure can be used for the labeling of (strept)avidin but the F/P ratios should be about 10% lower.

Ch. 7

297

PROBE LABELING AND DETECTION

n e x t cycle

____(

\ cour ed

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

L+‘v’+ mbcc -’ Delay

Counting

pre-pulse

Fig. 7.5. Lanthanide chelates, such as Eu3+,have exceptionally long fluorescent decay times (up to over 1 ms), whereas proteins and other macromolecules may have decay times of to l o p 6 s. At every 1 ms interval, an excitation pulse is sent through the sample in time-resolved fluorescence. After a delay of 400 ps, fluorescence is measured for 400 ps (two cycles shown).

7.3.2.3. Time-resolved fluorescence In time-resolved fluorescence (TRF) (Maundrell et al., 1985), europium chelates are excited at 340 nm to emit two types of fluorescence, a shortlived background fluorescence ( < 0.1 ms) and a fluorescence due to emitted photons of Eu3+ lasting up to over 1 ms. This difference in fluorescence decay rate can be exploited by measuring fluorescence only after background fluorescence has completely decayed to obtain a very high signal to noise ratio (detectabilM) as shown in Fig. 7.5. Originally, anti-hapten ity; down to antibody was labeled with Eu3+ but in more recent procedures Eu3+ is directly attached to the nucleic acid (Sections 7.3.2.1 and 7.8.1). TRF assays require special equipment (e.g., LKB-Wallac, Arcus 1230). Dextran sulfate or polyethylene glycol, used to promote crowding and thus accelerate hybridization, cause high background levels in TRF.

298

HYBRIDIZATION WITH NUCLElC ACID PROBES

7.3.3. The use of enzymes as primary labels

Enzymes are extensively used, for example in enzyme immunoassays, to replace radioactive markers. Enzymes have the advantages that (i) they may be stable for a year instead of 1-2 weeks, (ii) the danger inherent to radiation exposure is eliminated, (iii) storage and disposal problems are avoided and (iv) detection signals can be equivalent to those from 32P. Enzymes with high turnover numbers combined with highly detectable enzyme-generated products with low background levels compare favorably with radiolabels. Enzyme detection may be automated, as in EIA (Tijssen, 19851, producing quantitated printout data. Nevertheless, 32P will continue to be widely used in research laboratories since replacing 31P by 32P will not affect the hybridization kinetics. Its predictable behavior is highly suited for nonroutine use. Similar to enzyme immunoassays, several enzymes are potential candidates as markers. Alkaline phosphatase (APase) and horseradish peroxidase (POase) are almost universally chosen although p-galactosidase and acid phosphatase are sometimes selected. Substrates should be soluble to prevent background staining, but, depending on the assay format, products should be either soluble (e.g., hybridization in microtiter plates) or precipitate immediately during conversion (e.g., in Southern hybridization) to maintain excellent resolution. Nibbering et al. (1986) showed a linear relationship in a model system between the amount of enzyme immobilized on nitrocellulose and the amount of enzyme reaction product. Two critical parameters should be considered in designing enzyme-coupled systems for probing: (i) the spacing of enzyme labels on probes; (ii) the impact of solid phase on enzyme kinetics. 7.3.3.1. Labeling density of enzymes on probes Enzymes tend to be bulky. The radius, r , of a spherical molecule can be calculated with the equation

in which M , is the relative molecular mass of the enzyme, N

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TABLE 7.8 Polymerization of alkaline phosphatase 1. Dilute enzyme (e.g., from Boehringer Mannheim cat no. 567-752) to 1 mg/ml in ice-cold 3 M NaCI, 1 mM MgCI,, 0.1 mM ZnCI, and 30 mM triethanolamine (pH 7.6) in a plastic reaction vessel (all subsequent reactions at 4°C).

2. Add twice 10 K I of a 5 mg/ml solution of disuccinimidyl suberate (Pierce Chemicals) per ml of enzyme (30-60 s apart) with gentle stirring. 3. Stir solution for about 20 min (cloudy precipitate should appear). 4. Add four times 10 p1 of cadaverine (Sigma; 0.1 mg/ml in the same buffer as in step 1) to 1 ml of the reaction mixture at 10 min intervals, then 30 pI of the cadaverine solution, followed after another 10 min by 2 pI of the undiluted cadaverine and continue stirring for 30 min. 5. Dialyze the resultant clear solution extensively against the buffer of step 1.

Avogadro’s, number, -irr3 the volume of the spherical particle and V is the partial specific volume (for proteins usually 0.74 ml/g, but for glycoproteins somewhat lower). For example, it can be calculated that the calf intestinal APase (with an M , of 115000) has a diameter of 13 nm. When attached to a nucleic acid, it would cover about a helix length (1 turn of 10 base pairs is 3.4 nm long) of almost 40 nucleotides. Thus, little can be gained by attempting to increase detectability by increasing the enzyme density beyond 2 APase molecules per 100 nucleotides. An elegant method to indirectly increase the number of enzyme molecules per length unit of nucleic acid was described by Leary et al. (1983). They created polymers of APase by crosslinking with disuccinimidyl suberate (Table 7.8). This enzyme complex retains high enzymatic activity and requires only a low degree of biotinylation (biotinylation of APase rapidly inactivates the enzyme) (Tijssen, 1985). Only one of the molecules in large complexes needs to contain a biotin to react with streptavidin on the probe. Leary et al. (1983) could thus detect single-copy genes in 7.5-pg samples of mammalian DNA with the colorimetric BCIP/NBT (Section 7.3.3.4.1) substrate. With DIG-probes, Holtke and Kessler (1990) also observed that polymeric Fab fragment-APase complexes gave superior results.

300

HYBRIDIZATION WITH NUCLEIC ACID PROBES

7.3.3.2. Impact of solid phase on enzyme kinetics The immediate vicinity of a solid phase may strongly affect the activity of the enzyme. The substrate partitions between the fluid phase and the charged polymer solid phase. This can be attributed to the ionic charges and to the limitation of diffusion of the solute to the solid phase due to an unstirred layer of about 1 km (i.e., corresponding to a double-helix length of about 3000 bp or to more than 100 times the diameter of an average protein) (Trevan, 1980). A negatively charged polyanion as solid phase attracts protons, whereas positively charged solid phases repel them, and both may change the pH in this microenvironment considerably. Many solid phases have either a positive or a negative zeta potential (e.g., Zetabind or nylon 66 membranes have a positive zeta potential). Conditions for solid phase enzymes are thus different from those in the fluid phase and the composition of the medium has to be adjusted accordingly. A shift of one or more pH units to obtain maximum activity on the solid phase is common (Goldstein, 1972). The concentration of H 3 0 + ions at pH 7-10 is much lower than the substrate concentration and an enzyme liberating or complexing protons strongly affects the pH in its microenvironment. Immobilization of the enzyme may have direct effects on its catalytic ability as conformational changes often affect the Michaelis-Menten parameters. The stability or activity of enzymes on a solid phase is often better than in the fluid phase, probably due to the local high concentration of enzyme. Certain solid phases, however, inactivate an enzyme, such as POase by polystyrene in the absence of wetting agents (Berkowitz and Webert, 1981). Inhibitors may behave differently with immobilized enzymes. Polymers may repel like-charged and attract oppositely charged inhibitors. For instance, APase has a stronger affinity for phosphate than for most substrates (Tijssen, 1985). Phosphate may be attracted by a positively charged membrane and phosphate-based buffers should not be used with APase. With product inhibition, the lack of rapid diffusion may inhibit the enzyme more than in the liquid phase. This may be serious in hybridization using APase. On the other hand, substrate inhibition, such as for peroxidase by H,O,, may be lowered by a restricted diffusion. Changes in the substrate or

Ch. 7

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301

in the polymer may affect partitioning. Substrate concentration in solid phase enzyme detection should often be higher than for soluble enzymes to counteract diffusion limitation. Diffusion effects are stronger with enzymes with high intrinsic activities. These effects on enzyme activity require the adjustment of the experimental conditions for optimal results. It is not correct to establish optimum conditions in the liquid phase and to expect that the same conditions yield optimal results with the enzyme immobilized on a membrane. Chemical modification of the support, e.g., to immobilize nucleic acid, also may affect enzyme activity. The K , , as measured in the fluid phase, is different from that in the solid phase ( K A ) . The reaction velocity is affected by both the uneven distribution of the enzyme, which requires the diffusion of the substrate from the interior of the fluid phase to the solid phase and an electrical effect, i.e., a gradient of electrical potential, grad 6, generated by charge differences between the solid phase and the interior of the fluid phase (Hornby et al., 1968). The substrate transport, due to the electrical effect, is proportional to the negative gradient of the electrical potential ( -grad 61, the concentration of substrate in the interior of the fluid phase [S], and the valence of the substrate (z). On the other hand, transport of the substrate by thermal diffusion is related by D to the substrate concentration gradient. At steady state, the net substrate transport is limiting and the reaction rate becomes vnet which can then be expressed as

where x is the distance from the solid phase, D the diffusion constant of the substrate, R the gas constant, T the absolute temperature and F the Faraday constant. This equation differs from the classical Michaelis-Menten equation only by the Michaelis-Menten constant. The second term of the product replacing the K , becomes

302

HYBRIDIZATION WITH NUCLEIC ACID PROBES

1 if either z or grad 4 is zero, so that only the diffusion term remains, whereas the second term is > 1 if z and grad 4 have the same sign. Although many enzymes used in nucleic acid probes do not follow simple Michaelis-Menten kinetics, some important conclusions can still be drawn for enzymatic activity in solid phase hybridization: (i) a higher diffusion constant of the substrate will decrease the K A of the reaction; (ii) the accumulation or depletion of ionic species (e.g., protons) influences the reaction rate. Shaking of the solid phase during the enzyme reaction may be advantageous, but for comparison care should be taken to agitate each reaction vessel equally. 7.3.3.3. Characteristics of major enzymes The most popular enzymes (POase, APase, P-galactosidase) have group specificity that allows the development of new (chromogenic) substrates with improved stability and detectability (e.g., West et al., 1990; Conyers and Kidwell, 1991). The high restricted absorptivity values and the high restricted dynamic range for many chromogens are current limitations. Besides improved chromogens, fluorometric or luminometric substrates are becoming popular. 7.3.3.3.1. Properties of alkaline phosphatase (APase) APases are found primarily in animal tissues and microorganisms. One unit of activity of APase corresponds to the hydrolysis of 1.0 pmol of p-nitrophenyl phosphate (p-NPP) per minute (in 100 mM glycine, 1 mM ZnCl,, 1 mM MgCl, and 6 mM p-NPP, pH 10.4; or in 1 M diethanolamine, 0.5 mM MgCl, and 15 mM p-NPP, pH 9.8). The bovine intestinal enzyme generally has a specific activity of 1000 and 2000 units/mg in these two buffers, respectively, at 37°C. At 25"C, activity is reduced to about half, but p-NPP suffers from spontaneous hydrolysis, particularly at temperatures above 30°C. All APases seem to be zinc metalloenzymes with at least two atoms of Zn2+ per enzyme molecule. Crude APase (Boehringer, grade 11) can be purified 20-fold and completely recovered by affinity chromatography (Mossner et al., 1980). APase transfers the phosphoryl residue via a phosphoryl-enzyme intermediate, which can be repressed by inorganic phosphate. Its

Ch. 7

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303

kinetics have been discussed by Fernley (1971) and Reid and Wilson (1971). The pH optimum of the enzyme is shifted to higher values with increasing substrate concentrations, whereas with increasing p K values of the substrate, the pH optimum usually shifts to neutrality. Addition of ions, such as Mg2+,may activate enzyme in some buffers (glycine buffers, which have insufficient buffering capacity for APase) but not in others (diethanolamine). Tris buffers produce a sharp increase in enzyme activity (Neumann et al., 1975). Inorganic phosphate is a strong competitive inhibitor of the enzyme and may be present in many biological samples and the use of PBS for the detection of APase should be avoided. Arsenate is a stronger competitive inhibitor than phosphate, whereas phosphonates are weaker. Metal chelating products (EDTA, cysteine, thioglycolic acid) are also important inhibitors. Many amino acids show a mixed competitive or uncompetitive inhibition. The detectability of APase can be improved 100-fold by a dual enzyme cascade system (Mize et al., 1989).

7.3.3.3.2. Properties of horseradish peroxidase (POase) The molecular mass of POase-C is about 44 kDa, of which about 20% is accounted for by carbohydrates (Welinder, 1979) and the enzyme has a Stokes radius of about 3 nm. A widely used practice is to give the so-called R Z (Reinheits Zahl or ‘purity number’) which is the ratio of the absorbance of POase at 403 nm (Soret band) and 275 nm as a measure of purity. Different isozymes of POase differ in R Z values, ranging between 4.19 and 3.15 or lower, although the exact range is still disputed and may vary according to the origin of the POase. ‘Pure’ commercial POase with an R Z of 3.0 contains several isozymes, the major component (isozyme C) has the highest activity and a somewhat higher R Z (Tijssen and Kurstak, 1984). The extinction coefficient of POase-C at 403 nm is 2.25 cm2/mg. POase is more sensitive to contaminants than APase, even water deionized with polystyrene resins may be toxic to POase. POase is inactivated by polystyrene in solid phase EIA if Tween 20 is omitted (Berkowitz and Webert, 1981). It is also sensitive to the presence of bacteria or bacteriostatic agents (NaN,) and is inactivated by oxygen, hypochlorous acid and aromatic chlorocarbons often found in laboratory water (degassing recommended).

304

HYBRIDIZATION WITH NUCLEIC ACID PROBES

POase is usually purchased although it is simple and fast to purify (Tijssen and Kurstak, 1984) decreasing the cost sometimes more than 10 times. This method starts with a low priced crude extract (RZ of 1.0) that is dissolved in 2.5 mM sodium phosphate buffer (pH 8.0) and passed over a DEAE-Sepharose column, previously equilibrated with the same buffer (up to 5 mg of protein per ml of gel). Impurities and less active acidic isozymes are retained whereas pure POase passes directly.

7.3.3.3.3. Properties of P-galactosidase P-Galactosidase is present in microorganisms, animals and plants and is sometimes used due to the low background, but its large size makes it less suitable. Native P-galactosidase is a tetramer and has a molecular weight of 465000 (calculated from its amino acid sequence) and a PI of 4.6 (Fowler and Zabin, 1977). The tetramer dissociates into inactive monomers at pH < 3.5 or > 11.5 or with mercurials (Loontiens et al., 1970). Aggregates are readily formed in purified preparations without SH compounds (Sund and Weber, 1963). With 100 mM 2-mercaptoethanol (2-ME) and 10 mM MgCl, (2-ME alone inactivates the enzyme) the enzyme is also much more heat-stable (at least 30 min at 40°C within pH 6-8). In some E. coli strains, about 5% of the total protein content is P-galactosidase if lactose is the source of carbon (Craven et al., 1965). The turnover number of the purified enzyme is about 3 X lo5 mol of o-nitrophenyl-P-D-galactoside (0-NPG) at 25°C in 50 mM potassium phosphate buffer (pH 6.8) containing 1 mM MgC1, and 2.6 mM substrate (the extinction coefficient of the o-nitrophenol generated is 18.5 cm2/p,mol at 405 nm). This corresponds to a specific activity of about 500 units/mg. P-Galactosidase follows Michaelis-Menten kinetics. The K , value for its natural substrate, lactose, is four times higher than for o-NPG (9.5 X M). The D-pyranoside is essential and the pH optimum of activity is 7.2-7.7. Heavy metals, organomercuric compounds and chelating agents (EDTA, citrate) counteract the inhibitory effect of 2-ME. Unfortunately, studies on these parameters in solid phase hybridization are lacking. The most widely used chromogen is X-gal (Section 7.3.3.3.0, but fluorescent substrates are valuable for high detectability. With 4-

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methylumbelliferyl-P-D-galactoside (MUG), picogram levels of target nucleic acid can be detected (Yokota et al., 1986). Coutlee et al. (1989a, b) used P-galactosidase-labeled monoclonal antibodies to RNA : DNA hybrids in solution hybridization and detected the enzyme, after selective capture of the hybrids in microtiter plates, with 0.1 mM MUG in PBS with 1 mM MgCl, and 50 Fg/ml BSA (measured in a Dynatech microfluor microtiter plate fluorometer at 450 nm (emission) and 365 nm as excitation wavelength). Detectability was about 1 pg/O.l ml. 7.3.3.4. Colorimetric detection of enzymes Highly sensitive labels do not guarantee sensitive assays. To obtain fast hybridization kinetics, an excess of input probe is required. This, in turn, may lead to background problems. Removal of free probe before detection and using labels with low background levels may overcome these problems. 7.3.3.4.1. Chromogenic detection of alkaline phosphatase APase hydrolyzes almost any phosphomonoester to give inorganic phosphate and the corresponding alcohol, phenol, sugar, etc. Many histochemical color reactions can be used (Burstone, 1960; Smith et al., 1968; Wolf et al., 1968; Stage and Avrameas, 1976). In the method of McGadey (1970) (Fig. 7.6), adapted by Leary et al. (1983) and widely used, the enzyme dephosphorylates 5-bromo-4-chloro-3-indolyl phosphate (BCIP or 'X-phosphate') after which the resulting hydroxyl group on the indoxyl moiety comes to a tautomeric equilibrium with a ketone. The ketone form of the molecule dimerizes rapidly to the insoluble 5,5'-dibromo-4,4'-dichloro-indigo molecule while releasing hydrogen. This hydrogen, in turn, can reduce nitroblue tetrazolium (NBT) resulting in diformazan that is insoluble and has an intense purple or brownish-blue color (depending on the solid phase). In proper conditions, BCIP and NBT are more stable than most alternatives and give little nonspecific staining on the nitrocellulose membrane. A strong background is observed with nylon-based membranes, although stronger blocking agents may at least partially alleviate this problem. Leary et al. (1983), using BCIP/NBT and polymerized AE'ase (Table 7.9A), were able to detect 0.5 fmol of

306 HYBRIDIZATION WITH NUCLEIC ACID PROBES

-6 *-\ /

N

"-\ / i

U Y

-==j B u

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307

TABLE 7.9 Colorimetric detection of alkaline phosphatase A. Traditional BCIP/NBT staining 1. Wash membranes rapidly with: a. 0.1 M Tris-HCI (pH 7.9, 1 M NaCI, 2 mM MgCI,, 0.05% Triton X-100 (twice) b. 0.1 M Tris-HCI (pH 9.9, 0.1 M NaCI, 50 mM MgCI, (three times). 2. Prepare staining solutions: NBT (nitroblue tetrazolium salt): 7.5% (w/v) in 70% DMF; 45 (LI of the NBT solution is added to 10 ml buffer (lb). BCIP ( = X-phosphate: 5-bromo-4-chloro-3-indolyl phosphate): 1.75 mg is dissolved in 35 (LI of 100% DMF and added dropwise with gentle mixing into the NBT solution. Final concentration: 0.33 mg/ml NBT and 0.17 mg/ ml BCIP. 3. Incubate membranes in substrate (10 m1/100 cmz in polypropylene bags) in the dark for about 4 h. 4. Terminate color development (bluish on nitrocellulose and dark purplish brown on nylon) by washing the membranes in 10 mM Tris-HCI (pH 9.9, containing 1 mM EDTA. Membranes should be stored in buffer (color fades on dry membranes) in the dark (precipitate is light-sensitive). Stain can be removed from nylon, but not from nitrocellulose, membranes by incubation in DMF (in glass container) in a heated waterbath (in hood!). B. Naphthol AS phosphates and diazonium salts 1. Prepare a 1% naphthol AS phosphate, naphthol AS-E phosphate or naphthol AS-MX phosphate in DMF or DMSO and dilute to 0.02% (w/v) in 50 mM Tris-HCI (pH 8.2). 2. Add diazonium salt (Fast Blue BB, Fast Red TR or Fast Violet B) to a final concentration of 0.1% (w/v) and filter. 3. Equilibrate the filter in 50 mM Tris-HCI (pH 8.2) and incubate in freshly prepared substrate solution for a few seconds to 30 min (depending on the amount of DNA). 4. Wash filters in 50 mM Tris-HC1 (pH 8.2) and air-dry. Best results are obtained with naphthol AS-E phosphate and Fast Violet B but the others are very useful in multicolor detection procedures.

biotinylated nucleotides on nitrocellulose. The color will fade but it can be restored by wetting the membrane as long as it has not been exposed to strong light. The detection limit can be improved about

308

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Naphthol AS phosphate (R=-CI o r - C I l 3 )

O=Y-OH OH

APase pH 8.3

i

R

H

kt

+ R2

diazonium s a l l

I

R

H

R

Rl FasC Red TR azo dye

3RJf

Rl

' R2

-Cl

Fast Violet Blue -CH

R2

-H

-CH

-.-Pa -OCH,

H

Fast Blue BB

-OCH2CH3

-y-!!e

-OCH2CH,

H

Fig. 7.7. Naphthol AS phosphate/diazonium salt colorimetric detection of APase is a very promising and cheap alternative to BCIP/NBT. Moreover, these substrates can be used to detect different probes on the same blot.

five times by rendering the nitrocellulose membrane transparent (wet with toluene). West et al. (1990) developed a powerful staining method (Fig. 7.7) by using different 2-hydroxy-3-naphthoic acid anilide (naphthol AS)

Ch. 7

formazan

NADP

309

PROBE LABELING AND DETECTION

APase

NAD+

ethanol

lactate

p - io d o n i t r o t e t r a z o l i u m violet

NADH

+ H+

acetaldehyde

pyruvate

Fig. 7.8. Amplification generated by the production of a catalytically active NAD'. The main problem with this technique is that even trace amounts of NAD', in the enzyme or NADP+ preparations, will lead to high backgrounds. Alternative amplification methods include enzyme cascades (Blake et al., 1984) and catalyzed reporter deposition (Bobrow et al., 1991).

phosphates as substrates in combination with different diazonium salts (Table 7.9B). This method not only allows the visualization of different target nucleic acids (different colors), but also produces more stable and brilliant precipitates with much lower background, faster development and better resolution than BCIP/NBT. For rehybridization, filters can be boiled in distilled water or 0.1% SDS for 5 min to remove the probe or inactivate any remaining enzyme and then hybridized with other probes. Boiling, in contrast to ethanol, does not remove the colored precipitate from nylon membranes. An additional advantage is that these substrates are much cheaper than BCIP/NBT. Amplification of the signal has been proposed for EIA by Self (1985) and seems directly applicable for probes. The principle is shown in Fig. 7.8. APase, with its group specificity, can dephosphorylate NADP+ into NAD+ and so induce the enzymes alcohol dehydrogenase and diaphorase (with all their substrates but lacking NAD') to start NAD+-specific cycling to accumulate product. With

310

HYBRIDIZATION WITH NUCLEIC ACID PROBES

each cycle, one molecule of formazan is formed from p-iodonitrotetrazolium violet. This method is only useful if the detection is limited by the low amount of indicator molecules generated, not if detectability is limited by background noise (then background would also be amplified). The second restriction is that reagents should be free of even trace amounts of NAD (reagents are available from, e.g., BRL or IQ(Bio) Ltd., Cambridge, UK). This method has proven excellent for solution systems, such as EIA (50-100 x amplification) (Clark and Price, 19861, but much less for membrane hybridization (Gatley, 1985). Chemiluminescent substrates thus seem a better choice. 7.3.3.4.2. Chrornogenic detection of horseradish peroxidase POase is widely used in enzyme immunoassays (EIA) and many suitable chromogens (which are oxidized by the enzyme in the presence of the peroxide or urea peroxide substrates) have been developed. Peroxide is the usual substrate, particularly on solid phases since its reduction results in the formation of ‘inert’ water near the solid phase (Fig. 7.9). It should be realized that POase has a very pronounced optimum concentration of H,O, substrate (Tijssen et al., 1982). Activity is low at low substrate concentrations, but inhibition is considerable at high substrate concentrations. The universally used POase, C isozyme, has an optimum in solution of 0.003% peroxide but higher concentrations are usually required on a solid phase. Although different chromogenic hydrogen donors can be deployed, for solid phase hybridization, the soluble chromogen needs to be oxidized to an insoluble, colored product. Most chromogens used in EIA remain (partially) soluble upon oxidation. Those used in immunohistochemistry should precipitate at the site of the reaction (Table 7.10). The classical chromogen, 3,3’-diaminobenzidine (DAB) is a suspected carcinogen and several alternatives have been proposed (Hanker-Yates reagent (Hanker et al., 1977), o-dianisidine (Colman et al., 1976; de Olmos, 1977)). The most often used chromogen is 4-chloro-1-naphthol (CN). This chromogen is, however, not very sensitive (although UV viewing improves the detectability 10100 X ). Sheldon et al. (1986) used the noncarcinogenic 3,3’,5,5’-tetramethylbenzidine (TMB). This colorless chromogen yields a blue

Ch. 7

I

311

PROBE LABELING AND DETECTION

Oxidrzed d o n o r

+ I

1

7 1

I

Compound I Hydrogen donor transrer

k, r

n transfer

-*-

(DH) Oxidized donor

Comoound II

Hydrogen donor

Compound 111 and IV [inactive enzyme complex)

(DH) (excess substrate)

11

Hydrogen donors:

OH

NH.

DAB

111

CN

Examples or chromogen oxidation

6 A electrophile

A phenylenedmmrne

POase

__

CN ~

[HP021

@.w

MBTll

@=NH

eleclrophde

Red Dye

Fig. 7.9. General catalytic reaction mechanism of POase. Note that excess substrate leads to enzyme inactivation. Hydrogen donors such as 3,3'-diaminobenzidine (DAB), 4-chloro-l-naphthol, 3,3',5,5'-tetramethylbenzidine (TMB) and o-dianisidine (ODA) are often used, but the color reaction (111) proposed by Conyers and Kidwell (1991) yields superior results.

product that is efficiently immobilized by dextran sulfate adsorbed to the solid phase. Membrane loading of the dextran sulfate polyanion is improved using positively charged nylon membranes.

312

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE7.10 Chromogenic detection of peroxidase on nitrocellulose or in situ (For comparative analysis, see Scopsi and Larsson, 1986; new detection systems have been developed by Conyers and Kidwell (1991)) A. Standard diaminobenzidine (DAB) at neutral pH (Graham and Karnowsky, 1966) This method yields considerable background on blots but is widely used in in situ (see also B, C). 1. Wash blots for 10 rnin at room temperature in TBS (50 mM Tris-HCI, pH 7.6, containing 0.15 M NaCl and 0.25% BSA). 2. Dissolve DAB.tetrahydrochloride (Sigma, cat. no. D-5637) at 0.5 mg/ml in 50 mM Tris-HCI (pH 7.6) and filter through Millex 0.45 pm filters. 3. Add H 2 0 2 to a final concentration of 0.003-0.1% (depending on solid phase) to the substrate solution (excess H , 0 2 will inhibit). 4. Add DAI-H,02 solution to the blots and incubate for 10 min in the dark. 5. Rinse blots with distilled water and air-dry. B. Imidazole enhancement of DAB staining (Straus, 1982) 1. Proceed with protocol as standard in A above, except for the following: a. Add 0.03 M imidazole to 0.05 M Tris prior to DAB and adjust pH to 7.4 with 1 N HCI. b. Add 0.01% H20,. C. Nickel enhancement of DAB staining (Hancock, 1982; Green et al. (1989) also described a nickel/cobalt method) 1. Wash blots in 0.1 M acetate buffer (pH 6.0) for 10 min at room temperature. 2. Add DAJ3.4HCI to 0.35 mg/ml to the same buffer containing 1.5% nickel sulfate and pass through a Millex filter (0.45 pm). 3. Add 0.007-0.1% H,02 and incubate blots in substrate for 10 rnin in the dark. D. Tetramethylbenzidine (TMB) (Mesulam, 1978; Sheldon et al. 1986) Oxidized TMB is usually soluble, but can be immobilized at the site of reaction by dextran sulfate. 1. Rinse blots for 10 rnin with TBS and 5 rnin in 0.1 M sodium citrate (pH 5.0). 2. Dissolve 5 mg of 3,3',5,5'-TMB (Sigma, cat. no. T-2885) in 2.5 ml of 99% ethanol and mix with 92.5 ml of distilled water (containing 100 mg sodium nitroferricyanide) and 5 ml of 0.2 M acetate buffer (pH 3.3). TMB must be kept in the dark to prevent photooxidation. 3. Incubate the blots for 20 rnin in this buffer (in the dark), pour off substrate and add 0.03% H 2 0 2 to the substrate and reincubate the blots in substrate (in the dark) until a good signal is obtained. 4. Rinse blots four times for 5 min at 4°C in 0.1 M sodium acetate buffer (pH 5.0). Insufficient washing results in darkening of the membrane over time. For reprobing, membranes can be decolorized in 0.18% N a 2 S 0 , and the probe can be removed by incubation for 1 h at 65°C in water containing 0.5% SDS.

Ch. 7

PROBE LABELING AND DETECTION

313

TABLE7.10 (continued) 5 . Air-dry or carry through stabilization (Rye et al., 1984): a. Dissolve 50 mg of DAB.4HCI in 100 ml of 0.1 M sodium phosphate buffer

(PB; pH 7.31, filter, add 2 ml of 1% CoCI, and chill to 4°C. b. Add H,O, to substrate to 0.01%. c. Rinse blots for 30 s in PB and incubate them for 5 min in substrate in the dark. d. Rinse blots five times for 10 min with cold PB and once with H,O.

E. 4-Chloro-1-naphthol(CN)(Conyers and Kidwell, 1991) The detectability of the Nadi (CN) reaction can be improved 20-50 times by adding DEPDA (N,N-diethylphenylenediamine),DMPDA (N,N-dimethylphenylenediaminine) or MBTH (3-methyl-2-benzothiazolinone hydrazone hydrochloride) to the CN solution. Among these substrate systems, DMPDA has very low background (the others no background) and MBTH yields a red precipitate (the others yield blue precipitates). Although DMPDA seems more sensitive than the other two, the reaction should be stopped at < 30 min due to the slight background. The others can be incubated for > 2 h. The ideal system is usually CN/DEPDA. 1. Prepare CN/enhancer solution by dissolving 0.06 M DEPDA or DMPDA or 0.1 M MBTH and 0.11 M CN in acetonitrile (adding a small amount of water may help to increase the solubility of the amine salts); can be stored in a refrigerator for several days. 2. Prepare a 0.1 M sodium citrate buffer (pH 6.0) (pH 4.0 for MBTH) and add 1 IJ.I of 30% H,O, per 10 ml of buffer (final concentration 2.9 mM). 3. Immediately before use, add acetonitrile-chromogen solution to buffer (1 :50; solution should be homogeneous). 4. When CN is used without ‘enhancer’ diamine: a. Heating of medium to 50°C before use often produces a stronger stain. b. The intensity of the bands can be increased about 10-40-fold by viewing under UV (even invisible bands become clear).

Conyers and Kidwell (1991) proposed a modified CN/‘enhancer amine’ method of POase detection (Table 7.10E) that increases its detectability to that of APase using BCIP/NBT (Fig. 7.9.111). Among the three amines proposed, the concentration of MBTH was most critical for optimal detection. It offers the possibility, due to the red precipitation, of distinguishing two probe systems on a single membrane or in situ preparations (e.g., with APase yielding a blue precipitate from BCIP/NBT). POase, oxidized by H,O,, will oxidize substituted phenylenediamines or MBTH (DH,) to cationic elec-

314

HYBRIDIZATION

wwn

NUCLEIC ACID PROBES

trophiles which in turn will react with electron-rich aromatic compounds such as CN to form colored products. Catalysis by POase is shown in Fig. 7.9. In the first reaction, the ferric ion is oxidized to the ferry1 form and the porphyrin to its 7-cation radical. Stopped-flow experiments confirmed that k , is much higher than k , but both may differ considerably according to the nature of DH, (Sakurada et al., 1990). An excess of substrate inactivates POase by forming compound 111 which is not known to participate in the usual peroxidatic cycle but is the peroxidase analog of oxymyoglobin or oxyhemoglobin. Compound I11 formation is accompanied by oxidation of aromatic amino acid groups in the protein. In contrast to H,O,, DH, chromogens protect the enzyme from the inactivating process. Thus, it can be expected that the ratio DH,/H,O, is the determining factor in the catalytic turnover of peroxidases. This was confirmed by Arnao et al. (1990) who undertook a kinetic study of the reaction of POase with the 2,2’ azino-di(3-ethyl-benzthiazoline sulfonate-6) (ABTS) system. A molar ratio of 1 yielded about three times more product than if it were 0.1. Also 5 mM ABTS yielded three times more product than 1 mM ABTS. A comparison of different DH, showed that the order of most intense absorbance is TMB > o-phenylenediamine > ABTS > 5-aminosalicylic salt > 3-(dimethylamino)benzoic acid > 3-amino-9-ethylcarbazole (Hosoda et al., 1986). H-Donors like azide and substituted hydrazines yield highly reactive radicals (in contrast to DH, like ABTS) and can be considered suicide substrates. Diffusion and potential gradients will affect the optimum ratio of substrate/chromogens for solid phase systems but studies are still lacking. 7.3.3.5. Chemiluminescent enzyme substrates 7.3.3.5.1. Dioxetane substrates for alkaline phosphatase and p-galactosidase The four-membered ring peroxides, 1,2-dioxetanes, are too unstable to be used as chemiluminescent substrates due to their low energy of activation (Adam and Cilento, 1983). Substitution of the carbonyls in the dioxetane ring has substantial effects on the rate of decomposition (Wieringa et al., 19721, i.e., half-lives from less than a second for simple dioxetanes to over 21 years for some of the

Ch. 7

315

PROBE LABELING AND DETECTION

0-D-ealactoside

1""CSPD

(&)

/

-

APase

*@"-

AMP-D

Fig. 7.10. Chemiluminescent substrates for APase and BGase. The bidadamantyl) group is quite hydrophobic and the recently introduced CSPD (with a polar CI atom) improves the detectability with these substrates even further.

bisadamantyl substituents. Stable substituted dioxetanes have been synthesized (Fig. 7.10) which emit light upon hydrolysis by APase or P-galactosidase (BGase). These asymmetrically substituted dioxetane substrates are a suitable compromise between stability in the presence and absence of enzyme (Bronstein et al., 1989; Schaap et al., 1989). They contain an adarnantyl substituent for stabilization and an aromatic phosphate or galactopyranoside substituent, which, upon hydrolysis, reduces the half-life of the dioxetane to 2-60 min (Table 7.11). Hydrolysis is proportional to the enzyme activity, but AMP-D tends to accumulate before decomposition. Decomposition, resulting in light, occurs most efficiently at pH 9.0 whereas background becomes significant only below pH 6.0 (Bronstein et al., 1989). Although for APase the reaction can be directly performed at pH 9.5 (Table 7.11B), it is necessary for BGase to digest the substrate at its

316

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE7.11 Properties and detection of chemiluminescent dioxetane substrates A. Properties of dioxetane substrates 1. Stability of AMPPD In solid form at 4°C In H,O at pH 9.5 In H 2 0 at pH 12

Indefinite 1 year at 4"C, 125 days at 30°C Years at 4"C, 1.2 years at 30°C

Stability of AMP- D at pH 9 ( = pK, of ground state of anion), depending on other solutes, equals a of 2-60 min (2 hrs on nitrocellulose and about 10 hrs on nylon) 2. Catalytic properties (at pH 9.5-9.6) K, of AMPPD

k,,, of AMPPD

0.2 mmol/l in sodium carbonate buffer 0.1 mmol/l in 2-amino-2-methyl-1-propanol buffer buffer 4100 s- in 2-amino-2-methyl-1-propanol

'

3. Light emission and relative intensity Wavelength Relative intensity 477 nm 1 Without amplifier 477 nm 2 plus 0.1% BSA 20- 100 plus Tropix Sapphire (hydroxy-coumarin) 466 nm 527,542 nm 100- 1000 plus Tropix Emerald (fluorescein) 620 nm 50- 300 plus Tropix Ruby (rhodamine) 525 nm About 400 Lumi-Phos 530 (Boehringer Mannheim, BRL)

*

*

Increase of relative intensity by activation of fluorophores by chemiluminescence (in micelles or covalently linked polymers).

B. Detection of probes with dioxetane substrates Store substrate at 4°C in the dark (stock solution 10 mg/ml = 23.5 mM). Do not use nitrocellulose membranes but Biodyne, Hyhond N or Magnagraph nylon membranes; use unpowdered gloves. Use probes at less than 20 ng/ml (Chapter 8). Signals on nitrocellulose are much weaker and require special blocking and enhancer substances (Nitro-Block; Sapphire or Emerald; from Tropix). Best results are on positively charged nylon; also PVDF with Nitro-Block. 1. Prewarm substrate (0.23 mM AMPPD (0.1 mg/ml), 750 mM 2-amino-2-methylpropanol buffer * * (pH 9.6), 0.88 mM MgCl,, with amplifier (e.g., Lumi-Phos 530: 1.13 mM CTAB and 0.035 mM fluorescein surfactant)) and use an aliquot just enough to wet the membrane. The volume of substrate can be conveniently minimized by placing the membrane (rinsed in 0.1 M Tris-HCI, pH 9.5, 0.1 M NaCl and 50 mM MgCI,) between two photocopier transparencies (acetate film, use only once), placing a few milliliters of substrate on the membrane and squeezing the liquid to cover the whole membrane.

Ch. 7

PROBE LABELING AND DETECTION

317

TABLE 7.11 (continued)

* * Instead of this buffer, the rinse buffer can also be used for dilution from the stock solution. For more economical use of substrate, it may be reused but should then be stored separately in a dark bottle. If several blots are to be processed simultaneously, 3 ml of substrate can be pipetted in the center of a large Petri dish and the membranes passed through the solution repeatedly until thoroughly wetted (always use blunt-end forceps, never touch with fingers) and enclose damp membranes in hybridization bag t o prevent drying. The volume of the substrate can also be reduced by using a type of slot-blot system which requires only small size membranes. Expose wetted membrane to X-ray film (e.g., Kodak XAR) immediately (or incubate at 37°C for 30 min to obtain steady-state light emission). Light emission remains constant for about 24 h. Multiple exposures from a single blot may thus be made to obtain an optimal signal/noise ratio. Background in substrate should be less than 25 TLU (Turner light units) over 1 h from 100 p,I of substrate, whereas lo-'' mol of APase should yield > 106 TLU. C. Detection of' probes with luminol substrates (Whitehead et al., 1983; van Gijlswijk et. al., 1992) 1. Prepare substrate solution. 5 mM luminol (0.5 M luminol (Aldrich) in DMSO, stored at -20°C) 0.006%) H 2 0 , (30% stock solution) 10 mM imidazole (stock solution 1 M imidazole, pH 8.0) 0.2 M carbonate buffer (pH 8.0 for unenhanced reaction but p H should be between 8.7 and 8.9 for the enhanced reaction (otherwise strong background)) 2. Add enhancer. Optimal is 15 mM p-iodophenol (stock solution in 0.5 M in DMSO; 150 times more signal than unenhanced) or 5 mM p-phenylphenol (stock solution of 0.1 M in DMSO; 100 times more signal). Enhancer stock solutions and substrate solutions are stored at 4°C for up to 5 days. Enhancers are available from Aldrich. 3. Soak membrane with the POase complexes with reagent and transfer rapidly (within 1 min of wetting) to filter holder, cover with transparent film and take print on instant photographic film (Polaroid Type 612 or 667; from 25 crn) o r contact print on blue-sensitive X-ray film. Note:: higher or lower concentrations of H,O, decrease the enzyme activity (depends also on the nature of the enhancer). For the unenhanced reaction, 0.03% is optimal. Imidazole increases the unenhanced reaction four-fold and the enhanced reaction two-fold) since it increases the turnover number of the enzyme (Straus,1982).

318

HYBRIDIZATION WITH NUCLEIC ACID PROBES

pH optimum (pH 7.5) and then in a second step to raise the pH to 12 to convert AMPD to AMP- D and to obtain chemiluminescence. The signal can be enhanced by so-called amplifiers, probably by an interaction of the relatively long-lived AMP-D anion with hydrophobic microdomains on the enhancer molecules. The hydrophobic domains exclude water with its protons (which could quench chemiluminescence) from the direct environment of the anion emitter. Just the addition of 0.1% BSA may amplify the signal several-fold. This may show possible problems for quantification due to unknown effects of sample components with hydrophobic domains. Amplification with an independent system can be achieved by adding commercially available fluorescent micelles (Schaap et al., 19891, such as those prepared from CTAB (Section 3.1.2.5) and 5-N-tetradecanoylaminofluorescein, which sequester the more hydrophobic AMP-D. Chemiluminescence transfers enough energy to the co-micellized fluorescer to provide for a = 100-fold increase in photon output. Beside solution assays, AMPPD and AMPGD can be used for detection of nucleic acids on membranes since the AMP-D anion is hydrophobic and will attach to the membrane in some fashion in the direct vicinity upon formation. This allows its application in assays where resolution is required such as Northern and Southern hybridization. A potential problem with APase, rarely mentioned, is poor quality of water. Since extremely small amounts of APase (such as produced by contaminating bacteria) can be detected, poor quality water will lead to high background levels. Nonintestinal APase can be specifically inhibited by adding 2 mM levamisole (Sigma) to the substrate. Aggregation of AMPPD or AMP-D causes a time lag before steady-state light emission whereas resolution of imaged bands degrades over time. Martin et al. (1991) modified the hydrophobic adamantyl group (Fig. 7.10) to prevent these potential problems. Moreover, the light intensity in the first few hours was about twice as high with this modified substrate (CSPD) than with AMPPD.

7.3.3.5.2. Luminol substrates for peroxidase Cyclic diacylhydrazides, such as luminol (Fig. 7.111, can be oxidized by POase in the presence of H,O, (Matthews et al., 1985). Luminol is then converted via an

Ch. 7

I

319

PROBE LABELING AND DETECTION

0

N"* 3-AMINOPHTHALATE

LUMINOL

I1

PRIMER I N P C R - PRODUCT

0

I11

BIOTIN STREPTAWDINE CONJUGATE

POase

//////////////,

MEMBRANE

///////////////

Fig. 7.1 1. Luminol produces fairly rapid chemiluminescence with good detectability (I). It can be used conveniently, e.g., for the rapid detection of PCR products (I1 and 111).

endoperoxide into an excited-state 3-aminophthalate dianion, which decays under the emission of light. The reaction signal can be improved considerably by adding certain phenol, naphthol and amino derivatives as enhancers. These enhancers do not increase the quantum efficiency of the reaction but regulate the reaction among POase, oxidant and luminol. A kit containing all the necessary ingredients is supplied by Amersham CECL Gene detection system'). In Southern blots, Pollard-Knight et al. (1990) could detect attomole (6 X lo5 molecules or about 2 p,g of human genomic DNA) amounts of target nucleic acid with probes of approximately 0.5-3.0 kb (one enzyme molecule per 50-100 nucleotides). This corresponds to a low picogram range of target DNA. A cooled charged coupled device (CCD) camera can be used to quantify the light output directly from blots over a wide range instead of blue sensitive film that does not respond proportionally (although improved by preflashing).

320

HYBRlDIZATlON WITH NUCLEIC ACID PROBES

The original Amersham system was based on labeling the probe with POase before hybridization. Prior labeling may have several consequences: (i) the enzyme is not stable in stringent conditions (Amersham suggests the use of 6 M urea, changing the stringency by changing the SSC concentration and avoiding temperatures above 42°C); (ii) hybridization kinetics and hybrid stability, particularly for short probes, may be affected. Pollard-Knight et al. (1990) observed no change in T,,, for long probes. A recent modification of the Amersham system allows the introduction of POase after hybridization. A possible problem with the luminol system may be the nonuniform variation in band patterns as recently noted in Western blotting (Harper and Murphy, 1991). The time required to obtain maximal luminescence varies since shortly after the reaction strong bands are preferentially detected while blotting the membrane later after the reaction preferentially detects faint bands. This phenomenon has not yet been described for nucleic acid probes.

7.3.3.6. Labeling of nucleic acid with enzymes Enzymes can be linked to nucleic acid via haptenation of nucleic acid or crosslinking with labeled basic polymers (Section 7.8.4). Enzymes can also be linked to oligomers using bifunctional reagents (Section 6.5). A simple procedure is the crosslinking of protein with nucleic acid using UV irradiation. Conjugation of protein to DNA by UV irradiation at low pH is efficient, fast and simple (Czichos et al., 1989). The major drawbacks are the size of the label (may interfere in hybridization), the stability of the enzyme during labeling and the relatively harsh conditions in the hybridization steps. Protein A, which binds certain (sub)classes of IgG (e.g., rabbit antibodies) and is stable at high temperatures in the range pH 1-12 and in denaturing solutions such as aqueous formamide, is a most useful intermediate. Plasmid DNA, 0.5 p g and 3 p g of the protein in 50 pl of 50 mM sodium citrate (pH 2) in an Eppendorf tube is maintained on ice and irradiated for 10 min from a low pressure mercury lamp (8 W, 254 nm) at a distance of 7 cm above the opened tube (resulting light intensity about 2 mW/cm2). After neutralization with 7 p1 of 1 N NaOH, an aliquot (5 pl) is analyzed by elec-

Ch. 7

PROBE LABELING AND DETECTION

321

trophoresis to confirm a mobility decrease of the conjugate as compared to the control DNA. Circumstantial evidence indicates that covalent bonds are formed between the DNA and the protein. Protein A in the complex will capture IgG-enzyme (IgG can have any specificity but should react with protein A). However, detectability is improved by detecting protein A with anti-protein A chicken antibodies and rabbit anti-chicken IgG antibody conjugates. The detectability by these colorimetric tests is about 20 pg of target DNA.

7.3.3.7. Conjugation of antibodies or streptavidin with enzymes Many conjugation procedures have been described for each of the enzymes used in indirect probe detection (most common are described in Table 7.12). It is important to adhere to the relative amounts of the recognition protein :enzyme since this will determine to a large degree the detectability and the background levels in the assays. It is useful to compare the performance of the conjugates obtained with commercial preparations. 7.3.4. Electron-dense markers Electron-dense markers can be distinguished into three categories: (i) heavy metal (gold/silver/osmium/cobalt/nickel) enhancement of product generated by enzyme (e.g., Table 7.10); (ii) heavy metal introduced into the complex by chelators (Section 7.3.2.1); (iii) the use of colloidal-gold labeled antibodies or streptavidin for electron microscopic studies. Silver enhancement of colloidal-gold stained nucleic acids in situ (light microscopic level) leads to strong background and poor definition (Giaid et al., 1989; McQuaid et al., 1991).

7.4. Recognition systems Central to secondary label systems is the strategy chosen to introduce the primary label into the hybrid complex. Most approaches rely on the introduction of small modifications in the probe to avoid interference during hybridization. The two major secondary label/detection systems are biotin/streptavidin and hapten/antibody, whereas

322

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 7.12 Preparation of antibodies and conjugation of alkaline phosphatase or peroxidase (Note: conjugates should not be frozen) A. Purification of antibodies from antisera Principle: antibodies used in a sandwich assay do not need to be purified but those to be conjugated need to be as pure as possible. Many different procedures are possible (Tijssen, 1985); only a widely used convenient method of salting-out of IgG and subsequent purification on ion exchangers is presented. 1. Prepare a saturated (NH412S04 solution and adjust p H to 7.0 (check pH on a 20 x diluted solution since the saturated solution is essentially ‘dry’ which would yield erroneous p H values) and add slowly, while mixing, 0.8 vol. to the antiserum. Stir solution for 30 min at room temperature and collect IgG by centrifugation (15 min, 4000X g ) . Dissolve pellet and dialyse against 17.5 mM phosphate buffer (pH 6.5) to remove ammonium salts. 2. Pre-equilibrate DE-52 or a DEAE-Sephacel column with the same buffer. Pass IgG fraction over the column (contaminants are retained but can later be removed with 0.2 M phosphate buffer, p H 8.0). 3. Establish the concentration and quality of IgG. IgGs have an absorbance ratio at 278/251 nm of 2.5 to about 3.0 in contrast to contaminating proteins which have about 1-1.5. The extinction coefficient of IgG is about 1.4 (per mg/ml, at 278 nm and using 1 cm cuvettes). B. Coiugation of alkaline phosphatase (method from Boehringer Mannheim) Principle: a homobifunctional agent (glutaraldehyde = pentanedialdehyde) is allowed to react with amino groups on alkaline phosphatase and the activated enzyme is allowed to react with IgG or streptavidin. 1. Combine in a test tube 1.0 ml of 50 mM potassium phosphate buffer (pH 7.2), 0.3 ml APase (e.g., Boehringer Mannheim; cat. no. 567744) and 10 pl of glutaraldehyde (25%, EM-grade) and incubate for 1 h at room temperature. 2. Add 0.3 ml (IgG at 5 mg/ml or streptavidin at 2 mg/ml in same phosphate buffer) and incubate for 90 min at room temperature. 3. Pass the reaction mixture through a Sephacryl S-300 column (125 ml in phosphate buffer; 4°C) and elute with 50 mM Tris-HCI (pH 8.0) containing 0.1 M NaCI, 1 mM MgCI, and 0.1% sodium azide. 4. Collect conjugate (first fractions up to 15 ml contain protein as detected by absorbance at 280 nm; free enzyme, antibody or streptavidin follow in later fractions). 5. Assay for both antibody (or streptavidin) and enzyme activity, add BSA to combined fractions (3 mg/ml) and store at 4°C. Freezing is detrimental to the enzyme. About 2000 units of enzyme are recovered and most of the antibody is conjugated. For use, the preparation is usually diluted approximately 1 :5000 (depending on the titer of antibody).

Ch. 7

PROBE LABELING AND DETECTION

323

TABLE 7.12 (continued) ~~~

~~~

C. Conjugation of horseradish peroxidase (POase) to antibodies or streptavidin (Tijssen and Kurstak, 1984) Principle: the carbohydrate shell of POase is lightly oxidized, i.e., vicinal groups in sugar t o aldehyde groups (over oxidation yields -COOH groups) by NaIO,. The aldehyde groups then react with the amino groups on IgG or streptavidin (POase groups). itself contains very few free amino . 1. Oxidize POase- by adding NaIO, to 12 rnM to a 0.1 M sodium bicarbonate solution containing 10 rng/ml enzyme (RZ 3.0; type C or VI) and incubate for 2 h in the dark at room temperature (color change should be at most minor). 2. Add 3 vols. of IgG (10 mg/ml and dialyzed against 0.1 M sodium bicarbonate) and 0.12 vol. of 0.1 M sodium carbonate and incubate for 3 h in the dark at room temperature. 3. Stabilize the Schiff bases by adding, while mixing, 1/20 vol. of freshly (!) prepared NaBH, twice with a 30 min interval. 4. Precipitate conjugate with an equal volume of saturated ammonium sulfate, collect by centrifugation (free POase remains in supernatant) and store at 4°C as precipitate (stable for at least a year).

the mercurated nucleic acid/SH-ligand (Fig. 7.2.V) is also used occasionally. 7.4.I. Streptavidin and biotin-labeling of enzymes or linker proteins Avidin is a very basic glycoprotein with a p l of 10.5 (Fraenkel-Conrat et al., 1952) and adsorbs nonspecifically to nucleic acids. Its analogue from Streptomyces avidinii (ATCC 27419; purified as described by Hofmann et al., 1980) has a much lower PI and is widely used for nucleic acid probes. It is a tetramer with four binding sites situated in two pairs; i.e., if one site is occupied then its neighbor is hardly accessible. Moreover, the binding sites are deep, almost 1 nm, beneath the van der Waals surface of the molecule. Therefore, biotin on probes or proteins should be linked via spacer molecules to allow efficient binding of biotin to streptavidin. Yehle et al. (1987) observed a better detectability with avidin than with streptavidin (requiring the succinylation of avidin to prevent nonspecific adsorption). McNeil et al. (1991) confirmed this superiority of avidin for in situ

324

HYBRIDIZATION WITH NUCLEIC ACID PROBES

hybridization, but avoided nonspecific staining by avidin by using citrate instead of phosphate buffers. The binding of avidin with M) is undisturbed by extremes of pH or chaotropic biotin agents, such as guanidineeHC1 (up to 3 M). They are commonly employed in either of three configurations: (i) labeled avidin with biotinylated probe; (ii) biotinylated probe and labels with avidin as a bridge; or (iii) a labeled probe with a preincubated avidin-biotin-label complex. Proteins (IgG, enzymes) can be biotinylated with activated biotin (e.g., photobiotin, NHS-biotin). Particularly useful are the watersoluble sulfo-NHS esters synthesized by Pierce Co. which eliminates the necessity of potentially harmful organic solvents. The reaction occurs via a nucleophilic attack of an amine (usually lysine €-amino groups) towards the ester, yielding a stable amide bond and the release of NHS as a by-product. This reaction is notably effective at higher pH (primary amine unprotonated). Protein should be dissolved at 10 mg/ml and the ester at 0.1 M (fresh solution) in 0.1 M sodium carbonate buffer (pH 8.5-9.0). Ester solution is then added to the protein solution (60 pl/ml for IgG; 5 pl/ml for POase; 20 pl/ml for BGase). It is not recommended for APase (inactivation of the enzyme; BSA can be biotinylated at 20 pl/ml and then co-polymerized with APase to obtain highly active large complexes). The reaction mixture is incubated for at least 4 h and then desalted on Sephadex G-25. Commercially available complexes are expensive. The biotin-streptavidin system is widely used in nonradioactive probes. However, this system may occasionally lead to nonspecific staining, particularly for clinical specimens. For example, biotinylated probes may be excellent for the detection of virus produced in the laboratory but give strong background levels on clinical samples (Verbeek et al., 1990). In some cases (e.g., Kennedy et al., 1989) the background is caused by avidin-binding proteins, such as the ompF protein in E. coli. This can be remedied by using relatively high probe concentrations with concomitant short hybridization times (respectively 200 ng/ml and 30 m i d and color development (5-10 m i d . Others (Bialkowska-Hobrzanska, 1987; Haas and Fleming, 1988) subjected the blots to extensive protease or organic solvent treatments, often with limited success (Huovinen et al., 1988). In our

Ch. 7

PROBE LABELING AND DETECTION

325

experience, an alternative labeling method is the best solution (e.g., DIG-labeled probes; Fig. 7.2.VI) although the use of anti-biotin antibodies instead of streptavidin can eliminate the problem if the nonspecific staining is due to nonspecific binding of avidin. It may, however, also be due to nonspecific biotin-binding (Verbeek et al., 1990). 7.4.2. Antibodies 7.4.2.1, Use of anti-nucleic acid antibodies Patients with systemic lupus erythematosus produce antibodies that react with duplex DNA. These antibodies hardly distinguish native from denatured DNA. Experimentally produced antibodies to RNA helices or RNA : DNA hybrids, triple helices, or left-handed Z-DNA can be highly specific (Stollar, 1975, 1985). Purified nucleic acids are poor immunogens but can serve as high molecular weight haptens. Synthetic RNA : DNA hybrids, such as poly(A) :poly(dT), can serve as immunogens (Stollar and Rashtchian, 1987) and the antiserum obtained can be absorbed with ds RNA or denatured DNA. The remaining antibodies may specifically capture hybrids from solution or in competitive ELISA test. Boguslawski et al. (1986) prepared a monoclonal antibody to DNA :RNA (using a duplex prepared by transcripts from 4x174 DNA with genomic DNA as immunogen) and applied it to immunodetection of hybrids. Stuart et al. (1981) and Bauman et al. (1984) obtained monoclonal antibodies to poly(A) :poly(dT) immunogens. The affinity of antibody prepared against the heteropolymer duplex was, however, significantly higher (8.5 X 10" M-'). 7.4.2.2. Hapten antibodies Activated haptens (e.g., NHS-haptens, photohaptens (biotin, digoxigenin; Fig. 7.12)) can be linked to nucleic acid (e.g., after transamination) and to amino groups on proteins. Haptenated proteins can then be used to generate antibodies in order to have a suitable recognition system. Soluble carriers such as BSA, keyhole limpet hemocyanin (KLH) or ovalbumin are excellent and 1 mo1/5 kDa is a reasonable coupling ratio. KLH has a high molecular mass and is

I 0

-

N-hydroxysuccinirnide ester

OF

'I

+

R-NHZ

pH 9-10

NO,

Nitroaryl

I11

halides

NH

+

NH R-NH,

-C:

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pH 9-10

OCH

+

CH,OH

y-R H

Imidoesters

IV

+

-N=C=S

H H -Ib-E-t!J-R S

R-NH,

Isothiocyanate

0

yx

H

____)

illumination Azidophenyl (nitrene; nonspecific binding

VI

~

in UV)

R

B-NHS

(BI

NHS

N

w

+ CDI +

c

o

+

4 %N

N=7 &

CDI

o

H c-arninocaproic

NHS

N=7 B10-7-NHS

II

Biotin

I

acid

Ch. 7

PROBE LABELING AND DETECTION

327

foreign to mammalian immune systems and is thus highly immunogenic. However, it suffers from poor solubility, particularly after crosslinking. Nondenatured, highly purified KLH should be blue (binding copper) and an excellent preparation is available from Pierce Co. (cat. no. 77100). BSA is useful as a carrier (30 lysine groups per molecule are available for conjugation) but the frequent use of BSA in EIA or hybridization makes it a less suitable choice. For immunization of rabbits, 0.05-1.0 mg should be used, e.g., for subcutaneous injection (10 sites/rabbit, maximum of about 0.5 ml per site), once as an emulsion with an equal volume of complete Freund's adjuvant and once with incomplete adjuvant (after 4 weeks) and bleeding of the rabbit after 6-8 weeks. Animals should be fasted for 12 h before bleeding but should be provided with water. After coagulation of the cells, the antiserum is collected and purified as described in Table 7.12A. These antibodies can be used to detect the probe and then be recognized by anti-IgG antibody (or protein A) enzyme conjugates. Alternatively, these antibodies can be conjugated directly. Haptens are detected with antibodies as described in Table 7.13.

7.4.3. Sulfiydryl ligands The hybridization potential of a probe remains intact after mercuration. Sulfhydryl compounds bind strongly to mercurated nucleic acids and a variety of sulfhydryl-(primary label) ligands can be introduced in the complex after hybridization. Simpson (1961) observed a typical association constant of mercury for sulfhydryl compounds of 10l6 M - but the mercury-sulfhydryl bond in polynucleotide complexes may be less stable (Hopman et al., 1986a). An interesting application

'

Fig. 7.12. Coupling mechanisms of haptens to probes (I-V). As an example (VI), the preparation of BIO-7-NHS is shown. First NHS is coupled to biotin (a) by carbonyldiimidazole (CDI). B-NHS then becomes reactive towards a linker. After the linker has been attached (b), an NHS is again introduced to obtain a reactive BIO-7-NHS (Costello et al., 1979). These intermediates are commercially available.

328

HYBRlDIZATlON WITH NUCLEIC ACID PROBES

TABLE 7.13 Hapten detection by antibodies 1. Equilibrate membrane after hybridization and posthybridization washes for 1 min with a filtered buffer (0.45 km) containing 1 M Tris-HCI (pH 7.5) and 0.15 M NaCI. 2. Drain liquid from membrane (without drying) and incubate in BM blocking buffer (2% blocking reagent (Boehringer Mannheim cat. no. 1096176) added to buffer from step 1) with gentle shaking. 3. Add antibody (or conjugate) to a final dilution of 1 : 1000 to 1 : 10000 to buffer as in step 2 and transfer the membrane to this solution (incubation for 30-60 min). 4. Transfer the membrane to another container with the same buffer as in step 1 (twice 15 m i d . 5. Equilibrate the washed membrane for 2 min in the same buffer as used later for enzyme detection.

of this principle is the immobilization of the mercurated probe (after hybridization in solution; Section 8.3) on thiol-containing solid phases (e.g., thiol/CPG-550 glassbeads resins from Pierce Chem. Co. or sulfhydryl-Sepharose 6B as prepared by Cuatrecasas (1970)). Hopman et al. (1986a) described the synthesis of several sulfhydryl ligands. For instance, trinitrophenyl-glutathione (TNP-GS) was prepared by adding equimolar amounts of 2,4,6-trinitrobenzenesulfonic acid to oxidized glutathione dissolved in 10 mM EDTA, 0.5 M carbonate buffer (pH 9.5) (Bauman et al., 1983). After a 1-h reaction at room temperature, while passing nitrogen through the reaction mixture, TNP-GS was isolated by repeated precipitation with HCI and dissolving in carbonate buffer (all steps in the dark). Hopman et al. (1986b) observed that the negative charge on TNP-GS could have unexpected consequences. This ligand gave excellent results in a Sephadex model system but not in in situ hybridization. By reversing the net charge (reacting aliphatic diamines to the carboxyl groups of TPN-GS using a water-soluble carbodiimide), this method attained a detectability comparable to other nonradiographic methods. TPN-GS and anti-TPN antisera are commercially available (Euro-Diagnostics BV, Box 2870, Apeldoorn, The Netherlands).

Ch. 7

PROBE LABELING AND DETECTION

329

7.5. Overview of enzymatic incorporation of labels into

probes Different enzymatic methods exist for the labeling of nucleic acids, both for DNA and RNA (Table 7.21, yielding probes with different characteristics. These methods can be subdivided into two groups: (i) those resulting in more or less uniform labeling (nick translation, random or specific primer extension, PCR, transcription, replacement synthesis); (ii) those resulting in end labeling. End labeling, PCR, specific primer extension, transcription and replacement labeling methods, yield intact probes, whereas random primer extension and nick translation methods yield fragmented probes. Usually less label is introduced with 5’-end labeling and the detectability will be less, but 3’-end labeling can yield probes with high detectabilities. Haptenated precursors (e.g., biotin, digoxigenin) or base analogues (e.g., BrdU, recognized by commercially available antibodies) can often be incorporated by standard methods. The K , , i.e., the concentration of substrate at which DNA synthesis occurs at half the maximal rate, is significantly different for each of the four dNTPs and for the different enzymes. A high proportion of the label is incorporated only if it represents a high proportion of the precursor. If the concentration of the labeled dNTP, e.g., dCTP*, is low, then it may become considerably less than K , in the reaction mixture. This cannot be compensated by adding cold dCTP without affecting the proportion of dCTP */dCTP. Therefore, the rate of the reaction may be far from maximal, particularly if labeled dNTPs are chosen with high K , values. Thus dATP is a suitable labeled substrate whereas the other dNTPs should be used at concentrations exceeding their K,. The product size will be small when one of the precursors is present in concentrations below K , unless the template concentration is low (e.g., 0.1 pmol or = 50 ng). However, at low template concentrations, the hybrid formation with primer will be slower unless the primer concentration is increased (Section 2.2.4). A 10-fold excess of primers over template should then be used.

330

HYBRIDIZATION WITH NUCLEIC ACID PROBES

7.6. Uniform incorporation of labels in nucleic acids 7.6.1. Nick translation Rigby et al. (1977) introduced a powerful, uniform labeling method, nick translation, which has been widely used and still remains the most common means of labeling. In this approach (Fig. 4.11, DNase I is used to create single-stranded nicks randomly in ds DNA (hydrolysis of a phosphodiester bond producing free 3’ hydroxyl and free S’phosphate groups). A second enzyme, E. coli polymerase I (DNA pol I), which has both 5’ + 3’ exonuclease and polymerase activity (and 3’ + 5’ exonuclease activity which is inactive in nick translation conditions), is then used to progressively remove nucleotides in the nicked strand from the nick towards the 3’ end and simultaneously to add nucleotides (polymerize) to the free 3‘ hydroxyl end in the nick using the complementary strand as a template. The net result is the movement of the nick in a 5’ -+ 3’ direction (‘nick translation’) and newly synthesized stretches in the nicked strand. If among the dNTPs added (required for polymerase activity), labeled nucleotides are present (e.g., [ w3’P]dNTP or biotinylated dNTP), then labeled nucleic acid is obtained. The concentration of the DNase I creating the nicks will affect the uniformity of the labeling and the length of the probes. Standard procedures are presented in Table 7.14. The DNase I activity in nick translation protocols is critical to obtain probes of suitable length. Usually, a mean of about 500 bases is optimal, but shorter probes are advantageous for in situ hybridization. DNase I stock solutions are stored at -20°C (1 mg/ml) in 5-111 aliquots (each aliquot is used once). Variation in the activity of DNase I preparations is often observed and the exact amount needed to introduce the desired number of nicks should be determined for each enzyme batch. Sometimes template switches occur which will result in ‘snap-back’ structures (zero-binding nucleic acid), which remain S nuclease-resistant upon denaturation. Rigby et al. (1977) suggested that this effect was due to a differential loss of 5’ + 3‘ exonuclease activity upon storage leading to a displacement of the nicked strand and a template switch from the complementary

Ch. 7

PROBE LABELING AND DETECTION

33 1

TABLE 7.14 Nick-translation A. Prepare in advance 1. DNase aliquots (e.g., from Worthington Biochemicals). DNase I resuspended at 1 mg/ml in 0.15 M NaCI, 5 mM CaCI, and 1 mM MgCI,; 5 pl aliquots stored at -20°C 2. DNA polymerase I stored at 5 units/kl (usually supplied in 50% glycerol). Units as defined by Richardson et al., 1964 3. Nick-translation (NT) buffer ( l o x , stored i n 0.5 ml aliquots at -20°C): 0.5 M Tris-HCI (pH 7.4) 0.1 M MgSO, 1.0 mM dithiothreitol 0.5 mg/ml BSA, fraction V 4. dNTP mix (200 pM of each, except of the labeled species; i.e., dGTP, dATP and d l T P if [a-32P]dCTP is used) is stored in 10 )LI aliquots at -20°C.

B. Conventional (simultaneous) method 1. Prepare DNA to be labeled; it is important to remove agarose as agarose impurities strongly inhibit DNase I 2. Add the following chilled reactants to the wall of a 1.5 ml microfuge tube: a. 1 pg of DNA plus H,O (total of 14 ~ 1 at) the bottom b. 2 pl of DNase I, optimally diluted immediately before use (e.g., 1OOOO times in 10 mM Tris-HCI (pH 7.4), 5 mM MgCl, and 1 mg/ml BSA, Pentax fraction V c. 8 kl of unlabeled dNTP mix d. 5 pl of 1OX NT buffer e. 1 pI of DNA polymerase I f. 20 kl of labeled dNTP (final concentration about 2 pM) 3. Mix reactants and incubate for 30 min at 14-16°C (at higher temperatures, undesired snap-back DNA may be generated). 4. Stop reaction by adding 2 F I of 0.5 M EDTA (pH 8.0). 5. Remove unincorporated dNTPs by spin chromatography or selective ethanol precipitation and determine incorporation as detailed in Section 7.4.1.1. C. Quick, sequential method (Koch et al, 1986) 1. Titrate DNase I activity from a new batch by mixing 0.5 pg of DNA (1 kI), 1 kl of 3 x N T buffer and 0.05-2.5 ng of DNase I ( 1 kl) and incubate for 15 min at 37°C. Estimate the size of the DNA fragments generated by electrophoresis through alkaline agarose gels (optimal size 300-500 bases, except for small DNA (or in situ hybridization) where smaller fragments may be optimal. 2. Incubate 1 pI of DNA (0.5 kg), mixed with 1 pl of 3 x N T buffer and 1 kI of DNase for 15 min at 37°C. 3. Add dNTPs (10 kl of unlabeled and 20 pI of labeled dNTPs), polymerase (2.5 kl) and 2 X NT buffer to a total of 60 PI and incubate at 14°C until polymerization is complete (several hours or even overnight).

332

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE7.14 (continued) D. Large scale, sequential method 1. Mix 3 pg of DNA in 6 pI of 1.16X NT buffer and 1 p1 of DNase (dilution-titrated for incubations of 16 h) and incubate for 16 h at 37°C. 2. Add 7 pI of unlabeled dNTF's, 14 p1 of labeled dNTP, 2.5 pl of lOxNT buffer and 2.5 p1 of polymerase and incubate at 14°C until polymerization is complete. Note: this method is excellent for the preparation of DIG- or BIO-probes, using 100 p M dNTPs of which d'ITP is partially replaced by BIO-11-dUTP (50%) or DIG-dUTP (35%).

strand to the displaced strand; it was observed later (e.g., see Maniatis et al., 1982) that incubation temperatures over 14-16°C were responsible. Nick translation should be arrested once maximum incorporation is obtained (0.5-5 h; faster when more enzyme is used) since both exonuclease and DNase I will become relatively more important in time, resulting in a loss of incorporated label. SDS and EDTA are preferred over heat denaturation for enzyme inactivation. Labeled dNTP is usually supplied at 10 pCi/pl and the concentration (in pM) is estimated as 9100/specific activity (Ci/mmol). In the classical nick translation procedure (Fig. 7.131, or in kits, DNase and E. cofi DNA pol I are mixed. The stability of these enzymes is not equal and fairly variable degrees of labeling may be obtained. The suggestion by Koch et al. (1986) to separate the enzymatic steps constitutes a significant improvement since nick translation is better controlled with respect to fragment size obtained, it yields higher specific activity and almost 100% incorporation (classical procedure about 35%) and large amounts of probes can be obtained. In their approach, DNA is digested with DNase at 3TC, where the enzyme has 8-fold higher activity than at 14"C, to obtain the optimum distribution of nicks. The DNase/DNA mixture is then diluted and incubated with DNA pol I and dNTPs at 14°C. In the second step, the DNase activity decreases to nonsignificant levels since dilution greatly affects the enzyme activity. They devised two methods (Table 7.14C, D): (i) a quick procedure with relatively high DNase concentrations followed by a 20-fold dilution in the second

Ch. 7

PROBE LABELING AND DETEaION

5' 3. 5' 3'

33

__

4

~

E.co1i pol I

5'

3'

~

E.coli pol I

5' 3'

E.co/i pol I

+->

3'

5'

I

*

Intact duplex DNA

DNasel

--4

(5'-

3' 5'

NickedDNA with3' OH

3' 5-

Gaps of oneto several bases

- 33

Synthesis D N A with label

3'exonuclease) 3' 5'-

5'

3'

5,

53

333

i 1

(5'-

3'exonuclease)

5,

(5'3'exonuclease t polymerase)

3,

1

5'

Movement of nicks to 3'-end ("translation")

Denaturation

* *>+I*->

Short, partially re-synthesized DNA New DNA contains labeled precursors

Fig. 7.13. Preparation of labeled probes by nick translation. Note that a ds molecule is needed and that the total amount of labeled DNA does not exceed the original amount of template DNA.

step; (ii) a larger scale procedure with lower DNase concentrations (64 X longer incubation period) followed by a three-fold dilution in the second step. DNase (over a 1 to 1000 pg range) is titrated for the intended procedure to a concentration giving desired fragment lengths (usually about 300-500 bases) as judged by alkaline agarose gel electrophoresis (Section 9.1.1). Since nicking activity in the second step is very low, polymerization is conducted until optimum incorporation (even up to 2 days). Variation in DNA quality is the main difficulty, e.g., DNA obtained by minipreps will require more DNase than DNA obtained by more rigorous purification methods and calibration should be carried out accordingly. Nick translation on a solid phase (e.g., nylon membranes) offers important advantages such as (i) easy removal of nonincorporated precursors and (ii) labeling of specific fragments after electrophoretic transfer from agarose gels to the membrane (Section 9.2.1) and cutting of the membrane to obtain the desired fragment (Kainz and Kainz, 1989). The main difference between nick transla-

334

HYBRIDIZATION WITH NUCLEIC ACID PROBES

tion of filter-bound DNA and of DNA in solution is the increased amount of DNase required. Nick translation of controls shows whether a failure is due to the quality of the sample DNA or of the other components. The enzymes used are sensitive to agarose contamination of DNA. 7.6.1.1. Radiolabeling by nick translation High specific activities of the probe can be achieved by changing to labeled nucleotide preparations of higher specific activity and reducing the labeled dNTP precursor concentration to 1-2 pM. Consequently, if the specific activity of the precursor is 1000 Ci/mmol, then 1-2 mCi should be added per ml (i.e., 25-50 pCi/25 pl). With higher specific activities of the precursor, proportionally more radioactivity should be used to maintain the same precursor concentration (e.g., 3-6 mCi/ml if the specific activity is 3000 Ci/mmol). This can be expensive if the volume of the mixture is not scaled down, but it can be advantageous when low concentrations of probe with high specific activity are required (e.g., for certain Southern blots). The efficiency of incorporation is lower when two or more labeled nucleotides are used as precursors. Although [a-32P]dNTP is generally used as a radiolabel in nick translation, [ a-3sS]dNTP can be used and may have some advantages where detectability is not the primary concern: (i) it has a longer half-life (6 X ); (ii) shows less radiolysis; (iii) tighter bands are obtained (Radford, 1983). The standard nick translation protocol should then be modified since [a- 35S]dNTP incorporation is much slower than its 32 P counterpart (in Radford’s method, overnight incubation with DNA pol I). Moreover, autoradiography of 35S-blots may take days instead of hours, even if sprayed with scintillants, and the label is quite expensive. Probes radiolabeled by nick translation are usually stable up to 1-2 weeks (depending on specific activity, Section 7.2.2). After nick translation, enzymes are inactivated by adding EDTA to 20 mM; DNA can be selectively precipitated with ethanol (Section 3.1.4.1) and, if necessary, using 2.5 pg of carrier tRNA. The precipitate can be dissolved in 1 ml of water. To determine the incorporation of radiolabel, 2 pl of the sample is placed on a glass filter (Whatman

Ch. 7

PROBE LABELING AND DETECTION

335

GF/C). When the filter is dry, it is swirled using blunt-end forceps in a beaker containing 200 ml of ice-cold 5% trichloroacetic acid (TCA) and 20 mM sodium pyrophosphate for 2 min (repeated three times in a new solution). The filter is then dipped for 2 min in 70% ethanol and dried. The incorporated radioactivity can now be measured with a scintillation counter. If 1 ng of DNA was spotted, then 0.5-10 X lo5 cpm should be expected. By comparing with the cpm obtained from the original sample, directly spotted and not washed, the efficiency of incorporation can be calculated (Table 7.15).

7.6.1.2. Nick translation to incorporate secondary labels Secondary, nonradioactive labels are usually bulky nucleotide analogues which are less good substrates for most polymerases. For instance, BIO-4-dUTP (biotin attached with a 4-atom spacer arm to C-5 of dUTP; Fig. 7.10) is a much better substrate than BIO-16dUTP. On the other hand, the biotin binding sites are deeply buried in two pairs in the tetrameric avidin molecule. This explains why BIO-4-dUMP residues in DNA, in contrast to those with longer spacer arms, bind poorly to avidin or streptavidin (Leary et al., 1983) whereas antibodies to biotin bind to all BIO-nucleotide analogues equally well. Optimum binding is achieved when about 30 base analogues are introduced per kilobase, otherwise either decreased specific activity or decreased duplex stability is observed. Nick translation is the most common procedure for incorporation of biotinylated nucleotides. Commercial kits follow the classical procedure, but the alternative procedure of Koch et al. (1986; Table 7.14D) yields superior results. Biotinylated probes should not be too long since this tends to increase background staining. Nick translation is not as often used for digoxigenin-dUTP (DIG-11-dUTP) but can be used similarly (25% of the dTTP replaced by DIG-11-dUTP). Although the supplier of DIG-11-dUTP (Boehringer Mannheim) advises random priming, nick translation yields a more uniform size of probes. In contrast to biotin, digoxigenin is not widespread (only in digitalis plants) and is becoming a favorite hapten. Phenol extraction of biotinylated or DIG-probes should always be avoided due to the possible partitioning of the probe into the phenol layer. Similarly, NACSTMresins should not be used for the purifica-

336

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE7.15 Example of calculation of specific activity and amount of probe synthesized Proportion of precursor incorporated? A 20 pl reaction mix, containing 30 pCi (i.e., 3 p1 of 10 mCi/ml) is assumed. After incorporation of precursor, the mix is diluted twice and 2 pl is diluted 100-fold with TE containing 10 pg of carrier nucleic acid. Half of the TE-diluted sample (100 pl) is directly counted in a scintillation counter whereas 2 ml of cold 5% TCA (containing 20 mM pyrophosphate) is added to the other half, then mixed and incubated on ice for 5 min and the precipitate collected on GF/C glass-fiber filters. The tube should be washed once with 5% TCA and twice with 95% ethanol and the washes also passed through the filter (alternatively, the TE-diluted sample can be applied to the filter and then rinsed with the TCA solution; Table 8.11A). The radioactivity counted on the filter indicates the amount incorporated. For example, 1 X 106/2X lo6 or 50% is incorporated.

How much of the precursor was present in the reaction? If 30 pCi of [CZ-~*P]~CTP was added and the specific activity was 1500 Ci/mmol, then 30 pCix(1 Ci/106 pCi)X(l mmo1/1500 Ci)=2XlO-' mmol or 2X10-* nmol was added. If 5 p M cold dCTP was also included in the reaction mix of 20 pl, then it contained 0.1 nmol cold d(3TP. The total amount of dCTP precursor is therefore 0.12 nmol. How much of probe was synthesized? Since 50% of radiolabel was incorporated, 50% of 0.12 nmol or 0.06 nmol was incorporated. If we assume that all four nucleotides are equally represented in the probe, then 0.24 nmol of probe was synthesized. Assuming a molecular mass of 330 Da per base (i.e., 1 nmol= 330 ng), a total of 0.24X330 ng or about 80 ng was synthesized. What is the specific activity of the probe? If, in step 1, 1 X lo6 is precipitable from 1 pl, then a total of 2 X lo7 cpm would be precipitable from 20 pl. Since 80 ng nucleic acid was produced, the specific acitivity would be 2 X lo7 cpm/80 ng = 2.5 x lo8 cpm/ pg.

tion of biotinylated probes. It is necessary to include 0.1% SDS when Sephadex G-50 spin columns are used to avoid nonspecific sticking of the probes to the column. The shelf-life of these nonradioactive probes is at least 1-2 years if nuclease activity is avoided. Often, probes can be reused several times if stored at 4°C although they should be heat-denatured prior to use.

Ch. 7

337

PROBE LABELING AND DETECTION

5' 3'

3'

4

5'

~

Intact duplex DNA

Denaturation

5'

~39

+ 5'

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R a n d o m 1 primers (hexamer) 5'

3'

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5-

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-5'

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-5'

'5

4

fragment

5-

-5

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----

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-5'

-5'

-5

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'

5' 33

Extension

5'

primers

-5'

5-

of

Denaturation

- - - i _ L = o

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Fig. 7.14. Preparation of labeled probes by random priming. Both ss and ds DNA can serve as a template.

7.6.2. Random-primed synthesis of labeled DNA High specific activities can be obtained by the random-priming technique (Feinberg and Vogelstein, 1983, 1984). In this procedure (Fig. 7.14), hexanucleotides with random sequences (or, alternatively, specific primers) are annealed to denatured or single-stranded DNA and elongated using the large Klenow fragment of E. coli DNA pol I and four dNTPs, one of which is labeled. The Klenow fragment can be used so that virtually only the polymerase is active, since it lacks the 5' + 3' exonuclease activity and can be used at pH 6.6 where the 3' + 5' exonuclease is much reduced. DNA synthesis is initiated at several sites along the template and the length of the nascent probe molecules will be an inverse function of the primer concentration (Hodgson and Fisk, 1987). Standard protocols yield fragments of about 200-400 bases with usually up to about 75% incorporation of the labeled dNTP. The method is also rapid, does not depend on the activity of a second enzyme and usually yields higher specific activities than nick translation.

338

HYBRIDIZATION WITH NUCLElC ACID PROBES

A brief heat-denaturation step is essential for double-stranded DNA templates and linear molecules are preferable to avoid rapid renaturation. The purity of the template is not as critical as in nick translation and DNA can be used in low temperature gelling agarose or directly from minilysates (Section 4.3). Subcloning is not necessary as electrophoretically resolved restriction fragments can be easily labeled. A convenient random-priming method is labeling on a nylon membrane. Template can be spotted on the membrane, but it is also possible to use DNA restriction fragments transferred after electrophoretic separation onto nylon membranes (Chapter 9). DNA fixed on nylon membranes can serve as a template and the unincorporated precursors can be removed by simple washing for 1-2 min. The probe is then eluted from the membrane in formamide or in water. These membrane-bound DNAs can be reused. The probes synthesized by this method are as efficient in detecting nucleic acid as those synthesized in solution (Bhat, 1990). Similar methods have been proposed earlier for the synthesis of ss DNA probes from M13 templates (Ashley and MacDonald, 1984; Hansen et al., 1987). 7.62.1. Radiolabeling by random primer extension The labeled nucleotide is the limiting factor in the reaction since the K , often exceeds its concentration. The standard method (Table 7.16) proceeds in solution but the immobilized-DNA template method has some interesting advantages (e.g., simple purification). For instance, adding 3 p1 of [a-32PldATP (specific activity 3000 Ci/mmol; 10 pCi/p.l) per 10 p.1 of reaction volume represents 30 p.Ci/lO p.1 or about 1 p.M dATP and during synthesis this concentration falls even further below the K,. Usually 50-200 ng of template is used which will be sufficient for at most a few hybridizations. Increasing the template concentration does not appreciably change the efficiency of incorporation, but decreases the specific activity due to an excess of nonlabeled template. Similarly, adding cold dNTP leads to more DNA synthesis (and longer fragments) but with lower specific activity. The probe length is thus determined by the concentration ratio of the labeled nucleotide versus template. The variation in fragment sizes is wider than observed with nick

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TABLE 7.16 Enzymatic extension from random primers A. Incorporation of radiolabels Add to a 0.5 ml tube on ice: 2.5 pl of 0.5 mM dNTF' mix (except dATP) 2.5 pl of 10XRP-buffer (0.9 M HEPES, adjusted to pH 6.6 with 4 N NaOH and containing 100 mM MgCI,) 4 p1 of [ C X - ~ ~ P I ~(800-3000 ATP Ci/mmol; 10 pCi/pI) 1 pl of Klenow fragment (3 units). Combine linearized DNA (50-200 ng) with hexanucleotides (10-fold excess with respect to mass) in 15 pl, boil for 4 min and place on ice/NaCl (except when DNA is added in a molten gel slice; in that case it is rapidly cooled to 37°C). A somewhat higher specific activity is obtained if the DNA is alkali-denatured (0.2 N NaOH+0.2 M EDTA (final concentration) for 5 mid, neutralized by adding 0.1 vol. of 2 M ammonium acetate and ethanol-precipitated. Immediately combine the two solutions and incubate for 3 h at room temperature. The reaction is stopped by adding 1 pl of 0.5 M EDTA, 3 pl of 10 mg/ml tRNA and 100 pI of T E buffer. Extract the solution twice with phenol/chloroform and remove unincorporated label by spin chromatography.

B. Incorporation of nonradioactive labels Prepare reaction mixture as in A, except: a. The four final dNTP concentrations should be 100 pM. b. The label partially (depending on efficiency of incorporation) replaces its unlabeled counterpart (e.g., 35 p M digoxigenin-dUTP+65 pM dTTP or 50 pM bio-7-dUTP+50 pM d'M'P). c. The amount of template DNA can be increased 10-25 times without significant loss in specific activity. However, the relative amount of labeled DNA drops with more DNA substrate (e.g., for DIG-dUTP 30 ng is synthesized from 30 ng template, but 60 ng from 100 ng, 260 ng from 1 pg and 530 ng from 3 pg). C. PCR-labeling by random priming 1. Place in a 0.5 ml microcentrifuge tube: 10 ng of template DNA 5 kg of nonamer primers 200 p M of 3 dNTP (final concentration) 50 WCi of [cI-~*P]~NTP 5 pl of Taq polymerase buffer (Section 5.3.2.1) 2 units of Tuq polymerase Complete to 50 pl with distilled water 2. Cover with 75 pI of mineral oil and program DNA cycler for 1.5 cycles (or transfer in appropriate baths). A cycle consists of 2 min at 24°C 1 min at 72°C and 1 min at 94°C. The last cycle finishes with 7 min at 72°C.

340

HYBRIDIZATION WITH NUCLEIC ACID PROBES

translation. Probes with high specific activities should be used immediately. The efficiency of incorporation can be determined as discussed in Section 7.6.1.1. The convenient solid phase method of Bhat (1990) starts with the denaturation of template (plasmid) DNA in 0.5 N KOH at room temperature for 5 min. Samples of 1-2 pl (100-200 ng) are then spotted on 3 X 3 mm2 pieces of nylon membrane. The DNA membrane can be neutralized by soaking in 2 M Tris-HC1 (pH 7.4) or used directly for DNA binding, e.g., by UV-binding for 3 min on a transilluminator or by incubation at 80°C for 1-2 h. A random priming mix (50 pl) with all the reagents (except enzyme) is placed into an Eppendorf tube and one nylon membrane square with DNA is added. After 5 min, enzyme is added and the reaction left to proceed for 4 h. The filter bit is then removed and left immersed in 2 X SSC (Table 8.4) for 10 s (5 times, each in a fresh 100 ml). The final wash was in 1 ml of H,O in an Eppendorf tube and heated for 5 min at 90°C (no more than 2% of probe remains bound and the filter is removed for reuse at this point). About 60% of the precursor is chased into the probe. 7.6.2.2. Incorporation of secondary labels by random primer extension The protocol for the incorporation of secondary, nonradioactive labels differs in several respects from that for radiolabels (Table 7.16B) since (i) tagged precursor dNTPs can be bulky and less suitable as a substrate and (ii) the label density usually should be less (Section 7.6.1.2). In contrast to the protocol of radiolabeling, the concentration of each dNTP largely exceeds its K , although one of the dNTPs is partially replaced by labeled precursor. Consequently, it is easier to make large quantities without decreasing the specific activity. N6-(6-Aminohexyl)dATP (AHdATP), which is virtually as efficiently incorporated as dATP, via its primary amine group on the carbon chain, provides a reactive group for an N-hydroxysuccinimide (NHS) ester of any hapten (including biotin, various haptens for which antibodies are available, fluorescent dyes, etc.). Although this procedure has been described for nick translation (Gebeyehu et al., 1987), it is more useful in random priming. In this procedure, the

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modification of AHdATP depends on the nature of the hapten added and the efficiency of the NHS-mediated reaction (usually 60-100% replacement). The labeled DNA is separated from nucleotide precursors by spin chromatography and precipitated by ethanol. The precipitate is taken up in 0.1 M sodium borate buffer (pH 8.5) to 20 ng/p,I and 1 vol. of DNA suspension is mixed with 0.4 vol. containing 50 mg/ml of the NHS ester of a convenient hapten molecule, freshly prepared in anhydrous DMF (note that some esters, e.g., the sulfo-NHS esters, are water-soluble). After incubating for 2 h at room temperature, DNA is purified by spin chromatography and precipitated as before. Note that antibodies to the same hapten molecules can be easily prepared by immunizing rabbits with proteins modified with the NHS-esters of these haptens. BIO- 11-dUTP or DIG-11-dUTP are incorporated efficiently by random priming by replacing 25% of the d l T P in the reaction mix with the analogue (Cherif et al., 1989).

7.4.2.3. Random-primed DNA synthesis by PCR The random-primer extension method has been adapted for PCR conditions. The company Bios devised a kit containing random nonamers (5’-N,(G/C),) by which the target nucleic acid can be amplified and labeled without any sequence knowledge (otherwise specific primers should be used) (Table 7.16C). In 15 cycles, from 10 ng of template a 10-fold amplification is achieved with a typical specific activity of 108-9 dpm/p,g. The limiting factor is the amount of [a-32 PIdNTP. It is important to realize that a random primer may anneal anywhere along the template so that on average the template size is halved after each duplication. Consequently, the probes would be very short if (G/C), is closely spaced as in GC-rich templates. 7.6.3. Defined primer extension on single-stranded templates M13 and phagemid ss DNA is a convenient template for the preparation of probes. Two systems are usually distinguished with respect to the location of the insert: (i) upstream priming; (ii) downstream priming. It is important to realize that the insert in the phage should

342

HYBRIDIZATION WITH NUCLEIC ACID PROBES

+

have ‘ -’ polarity for upstream priming and ‘ ’ polarity for downstream priming in order to be complementary to mRNA (Fig. 7.15).

7.6.3.1. Upstream priming on single-stranded templates The upstream primer (5’-CACAATTCCACACAAC)binds upstream of the insert and is extended by DNA polymerase (Klenow) away from the insert. Any labeled dNTP, as in random priming, can be used to obtain a probe with reasonably high activity. Under the experimental conditions (Brown et al., 1982; Hu and Messing, 1982), the synthesis is not allowed to reach completion to leave the insert region ss. After phenol extraction and spin chromatography on Sephadex G-50, the probe is ready for use (no heat-denaturation!). This method is extremely simple and yields strand-specific probes. 7.6.3.2. Downstream priming on single-stranded templates In this approach (Barker et al., 1989, a universal sequencing primer (e.g., 5 ‘-TCCCAGTCACGACGT) is hybridized just downstream of the insert and extended in the presence of label through the insert and the polylinker with a suitable restriction site (same conditions as above). The partially ds molecule is then digested on the 5’-side of the cloned insert and denatured to purify the ss strand-specific probe. Bruening et al. (1982) heat-denatured the reaction mixture (3 min at lOOOC) in 35% formamide and electrophoresed the sample for 75 min at 30 mA on a sequencing gel (5% polyacrylamide in TBE containing 7 M urea). In the case of 32P-labeled probes, a 2-min autoradiography was sufficient. Specific activities over lo9 dpm/p,g can be obtained. 7.6.4. Labeling through sequential exonuclease-polymerase activities (‘replacement synthesis’)

The elegant replacement method can be achieved by a single enzyme possessing both exonuclease and polymerase activities (Fig. 7.16) or Fig. 7.15. Use of MI3 for probes. Clones with antisense inserts (I) are labeled using an upstream primer whereas those with a sense insert (11) are labeled using a downstream primer followed by gel purification.

Ch. 7 PROBE LABELING AND DETECTION

/

I

343

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

I

;:

3' 5'

T4 DNA p o l y m e r a s e 1 without dNTPs 5'

3' 3'

Addition of dNTPs - ' 5

c

5'

(+labeled precursor)

Fig. 7.16. T4 DNA polymerase (I) without dNTPs behaves as an exonuclease. After a convenient time span, dNTPs (with label) can be added to obtain partly labeled probes. Restriction and purification yields strand-specific probes. A similar approach can be taken by exonuclease and fill-in reactions.

by two different enzymes sequentially. T4 DNA polymerase (T4 DNA pol) or native T7 polymerase have both 3’ .+ 5‘ exonuclease activity (in the absence of dNTPs) and 5 ’ + 3’ polymerase activity. One of the strands of the double-stranded target DNA can thus be digested partially from the 3’-end in the absence of dNTPs and then be repaired towards the 3’-end simply by adding dNTPs (including the labeled precursor; Table 7.17). Figure 7.16 demonstrates that once the digestion proceeds beyond the midpoint, the molecules can no longer serve as a primer:template. It is thus necessary to cali-

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brate the degree of digestion (e.g., estimated by electrophoresis after phenol/chloroform extraction and S1 digestion; optimal when about 35% is removed from each end). The average number of nucleotides removed per minute from each 3'-end in standard conditions is TABLE7.17 Replacement synthesis after digestion of single strands from duplex by exonuclease ~

~~

A. T4 polymerase method Prepare in an Eppendorf tube on ice: 1 pg of DNA 5 pI of 10XT4 buffer (330 mM Tris-acetate, 660 mM potassium acetate, 100 mM Mg-acetate, pH 7.9) 5 units of T4 polymerase water to a final volume of 50 PI; if restriction is necessary, it can be usually carried out in this buffer before adding T4 enzyme Incubate at 37°C for the digestion step (digestion rate should be about 60 nucleotides/min from each end). Add 3 vols. of dNTP mix for repair step (1 XT4 buffer containing 200 p M 3 dNTPs and labeled dNTP which should in mass exceed at least the mass of excised DNA (i.e., > 5 pM)) and incubate for 30-120 min at 37°C. Stop the reaction by adding EDTA to 20 mM. Purify by phenol/chloroform extraction and spin chromatography. ~ . . Nofe:BIO-11-dUTP and BIO-16-dUTP, but not BIO-7-dATP, are efficiently incorporated by T4 polymerase. B. Exonuclease 111-Klenow polymerase method Reverse transcriptase is especially effective for small fill-in reactions. 1. Prepare in an Eppendorf tube on ice: 1 pmol of DNA ends (0.35 pg/kb), free of nicks 4 pI of 10 X exo 111 buffer (0.66 M Tris-HC1, pH 8.0, and 66 mM MgCl2) (NaCI inhibits the reaction) 10 units of exo 111 enzyme (will also act on nicks!) Water to a final volume of 40 pI 2. Incubate for 1-30 min at 37°C until the desired digestion (remarkably uniform, 200-400 bases per min) and stop the reaction by heating to 75°C for 10 min. 3. Repair with Klenow fragment by adding 10 pl of Klenow mixture (as prepared in Table 7.16A, step 1) to 10 pI of digested DNA. Proceed with the reaction and extraction as described in Table 7.16A, steps 3 and 4. Nonradioactive labels can he incorporated as described in Table 7.16B. Note: BIO-1 1-dUTP, BIO-16-dUTP and BIO-7-dATP are efficiently incorporated by Klenow.

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 7.17 (continued) C. Lambda exonuclease-oolrmerase method - 1. Prepare DNA so that one 5‘-end is phosphorylated and the other bears a hydroxyl group ( e g , after kinasing of one of the primers for PCR; digestion, dephosphorylation and another digestion (blunt-end)). 2. Prepare in an Eppendorf tube on ice: 5 pmol of DNA (1.75 pg/kb) 10 p1 of lox LE buffer (0.67 M glycine-KOH buffer, pH 9.4, 25 mM MgCI,) 25 units lambda exonuclease Water to a final volume of 100 pI 3. Incubate for 1-30 min at 37°C (depending on the extent of digestion required) and stop the reaction by adding 2 pl of 0.5 M EDTA. 4. Phenol/chloroform extract and change buffer by spin chromatography 5. Add 50 pmol of primer, heat to 65T, cool slowly and proceed with Klenow reaction as in B, step 3.

about 16 X the ratio of enzyme (units/pg DNA) up to a ratio of about 2.5 (at which point 40 nucleotides are removed per minute per end). Digestion is usually rather uniform but replacement synthesis yields molecules that are progressively more labeled towards their 3’-ends. Low concentrations of labeled dNTP risk rapid exhaustion of that dNTP and exonuclease may take over again. Cold dNTP can be used to chase the molecules into full length. The central part of the molecule will not be labeled whereas one strand is labeled at one end and the other strand is labeled at the other end. Digestion with a restriction enzyme and fragment purification by electrophoresis (Section 9.1.1) yields specific probes for opposite strands. Bio-11-dUTP can be used instead of [cx-~*P]~TTP with almost the same efficiency. A useful alternative to the T4 DNA pol method is offered by the sequential 3’ + 5’ digestion with exonuclease I11 (ex0 111) and repair by Klenow polymerase (Table 7.17B). Although exo I11 can be used in a similar way as the exonuclease of T4 DNA pol, it has the interesting property that 3’-protruding ends (at least three nucleotides long) generated by certain restriction enzymes are resistant to this exonuclease (the PstI overhang is leaky) while blunt or 5’ overhangs are sensitive. Fill-in 5‘ protrusions with a-phosphothioate dNTP (Pharmacia), using Klenow polymerase, also renders them

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resistant to exo 111. It is thus possible to selectively label one of the two strands by selecting the appropriate restriction sites. The exo I11 activity is dependent on the base composition as the rate of degradation is C > > A = T > G. One unit of exo I11 removes about 10 nucleotides from each end of 1 p,g of 5 kb double-stranded DNA (in 10 PI) in 10 min at 37°C. Digestion is remarkably uniform although exo I11 will act upon nicks in the duplex. The easiest method to obtain nick-free DNA is by acid-phenol extraction. Phenol (500 g) is equilibrated several times with 500 ml of 50 mM sodium acetate (pH 4.0) since it is essential that this pH is reached; at pH 4.2 no selective removal of nicked plasmid DNA is achieved. The ionic strength is also important because at higher salt concentrations DNA may partition into the phenol. DNA from a maxiprep is taken up in water and 0.025 vol. of 2 M sodium acetate (pH 4.0, always measure pH in diluted solutions!) and NaCl to 75 mM. This suspension is extracted with the equilibrated phenol (2 or 3 times may be needed to remove all nicked DNA) and ethanol-precipitated. After ex0 I11 digestion of the restricted plasmid, Klenow polymerase (or modified T7 polymerase) is used for repair as discussed in Section 7.6.2. A different approach to generate strand-specific probes is offered by lambda exonuclease (lambda exo; Table 7 . 1 7 0 It has 5' + 3' exonuclease activity but only from 5 '-phosphoryl termini (5'-hydroxyl termini are resistant) (Little, 1981). One of the two strands can selectively bear a 5 '-phosphoryl group by different approaches, such as phosphorylation of one of the two primers prior to PCR or successive restriction, dephosphorylation and restriction steps. In the first approach, the primers are removed after PCR cycling and the strand with the 5'-phosphoryl group digested from the duplex. The phosphorylated or its 5 '-P-less equivalent can then be used to generate a strand-specific probe. Synthesis is achieved as described above.

7.6.5. Preparation of nonradioactive probes by PCR Two different approaches can be distinguished: (i) labeling of primers and subsequent elongation and amplification by PCR; or (ii) amplification of target nucleic acid with unlabeled primers but using labeled nucleotides. Radiolabels are incorporated by substitution of the

348

HYBRIDIZATION WITH NUCLEIC ACID PROBES

appropriate dNTP using standard methods (Section 5.3) (Verbeek and Tijssen, 1991). Single-stranded probes can be synthesized by simple modifications of the PCR method (Section 5.3.3.5) (Espelund et al., 1990). A variety of nonradioactive labels can be introduced into primers (Section 6.4) and then used in conventional PCR to incorporate these labels into probes. These labels should be introduced to the 5’ end of the primer since for most labels, their attachment to the 3’ termini would preclude PCR amplification. The most common modified dNTPs used in PCR are biotinylated dUTP (e.g., BIO-11-dUTP) and DIG-11-dUTP (Boehringer Mannheim)). Solid BIO-11-dUTP from Sigma should be dissolved to 0.3 mM in 100 mM M Tris-HCI (pH 7.5) containing 0.1 mM EDTA. In a typical reaction, 1/4 of dTTP is replaced by BIO-11-dUTP, or 1/3 of dTTP by DIG-11-dUTP, in the conventional PCR (e.g., Lion and Haas, 1990, Chapter 5). In PCR, DIG-11-dUTP and dTTP can be decreased to 20 pM and 60 pM, respectively (while the other dNTPs are used at 200 pM) without loss of amplification efficiency (An et al., 1992). Some modified nucleotides are not efficiently used by Taq polymerase. For example, Dye-terminator (trade mark of Applied Biosystems; a dNTP with a large fluorophor used in sequencing) is not incorporated efficiendy unless the extension temperature is lowered from 72°C to 60°C. Others, such as BrdUTP (Tabibzadeh et al., 19911, are efficiently incorporated by equimolar substitution of dTTP. After PCR, 1 pI (20 pg) of glycogen (Boehringer Mannheim), 20 p1 of 2.5 M sodium acetate (pH 5.2) and 220 pl of ethanol are added to 100 pl of the reaction mixture and precipitated overnight. The probe is collected by centrifugation and dissolved in 100 p1 of 1 X TE. This is sufficient for 20 ml of the hybridization mix. The usual criteria for maintaining probe specificity during PCR amplification apply, such as low concentration of target nucleic acid ( = 0.5 ng of a plasmid), limitation of the number of cycles (an excess of cycles rapidly increases the amount of nonspecific probes), ‘hotstart’ of PCR to avoid nonspecific annealing and extension of primers to nucleic acid at low temperature (i.e., nucleic acid or primer or enzyme are added at high temperature).

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7.6.6. In vitro synthesis and labeling of RNA by transcription In vitro synthesis of RNA by bacteriophage RNA polymerases from double stranded DNA containing the proper promoters has become a simple and powerful method of generating large amounts of strand-specific RNA with defined sequences (‘riboprobes’). Since it is easy to include labeled NTP precursors, highly labeled, specific hybridization probes can thus be obtained. Another advantage is that RNA: DNA is more stable than DNA :DNA duplexes. A major disadvantage is the susceptibility of RNA probes to degradation. This method is based on the observation that some bacteriophage RNA polymerases are extremely specific for their own promoter sequences and for a specific strand (less than 1/500 from opposite strand) (Melton et al., 1984). Moreover, these polymerases do not initiate from nicks in the double-stranded DNA. Three bacteriophage RNA polymerases commonly used are from SP6, T3 and T7 (Chamberlin, 1982; Davanloo et al., 1984; Tabor and Richardson, 1985; Morris et al., 1986). Some low level ‘cross-talk‘ between T3 and T7 has been decreased even further for the cloned enzymes. The SP6 enzyme is much more expensive than the other two. Sequences recognized by these polymerases (‘promoters’) can be placed at either side of the polylinker (Fig. 7.17) so that sequences complementary to either the ‘ + ’ or the ‘ - ’ strand of the insert or amplified product can be generated (pSP, pTZ, pBluescript, pUCl18 vectors). The bacteriophage RNA polymerase transcribes efficiently downstream from its promoter, even through homopolymeric tails, provided that the NTP concentrations are sufficiently high. Although plasmid vectors have been generally used as substrates, PCR products can be easily obtained with the appropriate promoters. These promoters are relatively short (Fig. 7.17) and can thus be appended to one or both of the primers used in PCR. Since the phage promoter sequence can be appended directly to one of the two primers in a PCR setup and a portion of the amplified product can be transcribed by standard methods (Weier and Rosette, 19881, transcription can start and finish at any point in the sequence. The procedure given in Table 7.18 is essentially the same for all three polymerases. The only difference is that spermidine is included

350

1

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Bacteriophage RNA polymerase recognition s e q u e n c e s

SP6

ATTTAGGTGACACTATAGaa

t t

*

-..-___-

T3

ATTAACCCTCACTAAAG

T,

AATACGACTCACTATAG

*

___.-..-

1-. -. ... -*

I1

Iv

Transcription in vitro

* * *--..--*-.

T3 RNA polymerase

or

T7 RNA polymerase

..___ ___..._____... .___ .._____

. . . . ..__

*--*

.-*

_._. .._._ .....__. .._ ...

*.~~.------.--.-.---*.-

.._.______...__._.._...

a

b

Fig. 7.17. RNA can be generated in vitro from any DNA containing the proper promoter sequence such as those in frame I. Plasmid vectors in which the promoter sequences have been inserted (Table 4.4) or PCR products generated with primers to which the promoters were appended (Fig. 5.5), are readily amplified (frame 11). Depending on the side of the promoter, sense or antisense RNA can be obtained.

if SP6 polymerase is used. Since stock solutions of spermidine (10 mM) will precipitate DNA at 0-4"C, care should be taken that the various solutions are at 20°C. Obviously, it is essential to use RNase-

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free components and substrate. DNA substrate may be purified with almost any method as long as RNase is removed. In standard conditions, 1 mol of DNA yields 10 mol of RNA; after 30-60 min another batch of enzyme can be added to double the yield (if enzyme

TABLE 7.18 In vitro transcription A. Preparation of [3SS1UTP-labeledtranscripts 1. Dry 250 pmol of [35S]UTP( = 25 p,I of nucleotide at 1000 Ci/mmol; 10 pCi/pI) in SpeedVac or N, stream (use GTP for SP6 polymerase). 2. Mix: 2 p1 of 5 X transcription buffer (0.2 M Tris-HCI, pH 7.5, 10 mM spermidine (only if SP6 is used), 50 mM NaCl and 30 mM MgCI,) 1.5 pl of water 1 pI of 0.1 M dithiothreitol 0.5 p1 of RNasin (20 units) 2 pI of ATP, CTP and GTP (2.5 mM each) 2 pI of linearized DNA template (0.2 pmol = 0.4 pg of 3 kb) with the appropriate promoter (Table 4.4) and transfer to the [35S]UTP tube and add 1 pI of RNA polymerase (15 units) 3. Incubate for 1 h at 40°C (for SP6 polymerase) or at 37°C (for T7 or T3 polymerase). 4. Add 1 pl of unlabeled UTP (5 mM) and 1 p1 of RNA polymerase and incubate for an additional 30 min (optional). 5. If necessary (e.g., RNase protection assays), add 40 units of RNasin, 6 ~1 of DNase I(100 Fg/ml) and 11 pl of H,O and incubate for 10 min at 37°C. 6. Purify labeled transcripts: a. Add 2 pI of 10 mg/ml tRNA and water to 50 pl b. Save 1 pI for step h and extract the rest with phenol/chloroform c. Add 200 pl of 2.5 M NH,OAc to the aqueous phase and 750 pl of ethanol d. Mix and precipitate RNA by incubating for 15 min on ice and centrifuge for 15 min at 4°C e. Redissolve the pellet in 50 pl of H,O and repeat steps c and d f. Repeat step e g. Rinse pellet with 75% ethanol/25% 3 M NaOAc (pH 5.2) h. Redissolve in 100 p1 of hybridization buffer and count 1-5 pI in a scintillation counter and compare with the cpm from unprecipitated sample (1 pI from step b) to establish the degree of incorporation.

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE7.18 (continued) B. Preparation of [32P1NTP-labeledtranscripts 1. Mix: 2 p1 of 5 X transcription buffer (section A above) 1 p1 of 0.1 M dithiothreitol 0.5 p1 of RNasin (20 units) 2 p1 of NTPs, e.g., ATP, CTP and UTP (2.5 rnM each) 3 p1 of labeled precursor (e.g. GTP; 30 pCi) 2 p,I of linearized DNA template (0.2 pmol= 0.4 pg of 3 kb) with the appropriate promoter (Table 4.4) 0.5 pl of BSA, fraction V (2 rng/ml) 1 pl of RNA polymerase (15 units) 2. Continue as above, steps 3-6. Note: if RNA pellets become dry, they are difficult to redissolve in formamide-containing buffers.

is limiting component). About 60 p,g of transcripts (of 1 kb length) can thus be generated from 3 p,g of template DNA. Tracer amounts of labeled N l P precursor can be added for analysis of RNA transcripts. Sometimes, 3' overhangs cause problems by serving as a template for nonspecific initiation. Another enzyme or fill-in reaction (Table 7.17B) usually solves this problem (Krieg and Melton, 1987). T7 RNA polymerase incorporates biotinylated nucleotide analogues efficiently (Fenn and Herman, 1990). They noted that this enzyme did not discriminate significantly between UTP and BIO-4UTP, but its turnover number was reduced (V,, from 130 to 28 pmol/min and the K , from 32 to 77 p,M for UTP and BIO-4-UTP, respectively). T3 RNA polymerase incorporates BIO-11-UTP most efficiently (3 x SP6 and 2 X T7 RNA polymerase) (D'Alessio, 1985). Fenn and Herman (1990) separated the nucleotides obtained after alkaline hydrolysis of the transcripts by reverse-phase HPLC. Gel retardation assays (Theissen et al., 1989) are simpler alternatives (change in mobility of transcripts on polyacrylamide if streptavidin is bound to biotin moieties) although RNA with secondary structures may not allow the streptavidin to bind under native conditions. DIG-UTP has been shown to be an excellent substrate (30% efficiency of UTP for SP6 or T7 RNA polymerases (Holtke and

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Kessler, 1990). They prepared an alkaline-resistant spacer arm which might be advantageous for in situ hybridization to reduce probe size by alkali treatment (Cox et al., 1984). The transcription conditions of Nielsen and Shapiro (1986) gave better results for DIG-UTP than those of Melton et al. (1984). Moreover, increasing the NTP concentration to 1 mM almost doubled the yield and the optimum incubation time was 2 h (at 35% replacement of UTP by DIG-UTP). The reaction mix is usually 20 p,l, but for very large-scale labeling, 0.1 mg of linear DNA, 0.2 ml of DIG-NTP labeling mix, 0.2 ml of 10 X transcription buffer and 4000 units of T7 RNA polymerase in a final volume of 2 ml gave 1.3 mg of DIG-RNA within 2 h. 7.6.7. Reverse transcription of RNA Reverse transcription of RNA is sometimes used, e.g., for clone selection by colony hybridization (Verbeek and Tijssen, 1988). Although both avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MMLV) reverse transcriptase can be used, we prefer the AMV enzyme if there are no strong secondary structures in the RNA (MMLV enzyme can be used at higher temperatures, even up to 55°C). The optimal pH range for the AMV enzyme is very narrow (should be 8.3) whereas the MMLV enzyme is active in a range of 7.6-8.3. Superscript reverse transcriptase from BRL is a cloned MMLV enzyme from which the RNase H activity has been deleted. The procedure is similar as described in Section 4.4, the only difference being the amount of labeled precursor added. Either oligo(dT), or random or specific primers can be used. After firststrand synthesis, the RNA can be removed by alkaline hydrolysis (0.2 N NaOH 2 mM EDTA) and spin chromatography through Sephadex G-50. Single-stranded DNA probes are obtained.

+

7.7. End-labeling of probes 7.7.1. Labeling of 5'-ends of DNA or RNA T4 polynucleotide kinase (T4 PNKase) transfers the terminal phosphate (i.e., gamma position) of ATP to 5'-OH termini of DNA or

354

HYBRIDIZATION WITH NUCLEIC ACID PROBES

RNA. Using ATP, of which the P in the gamma position is radioactive ([y-32P]ATP),the 5’-end of the DNA or RNA molecule can be labeled. This kinasing reaction is particularly efficient with 5 ’-OH in single-stranded polynucleotides or in 5‘ overhangs in ds molecules. T4 PNKase can also transfer the 5’-phosphate group from a DNA or RNA strand to ADP if the latter is present in excess. Therefore, it is also possible to exchange the nonradioactive 5’-phosphate group for a radioactive group. This exchange reaction is less efficient than the forward reaction. Alkaline phosphatase can be used to remove the 5’-phosphate group prior to kinasing if high activity is required. Synthetic oligonucleotides have, unless they are phosphorylated, a 5 ’-OH group, whereas restriction fragments generated with most endonucleases have a 5 ‘-phosphate group. In the case of recessed 5’-phosphate groups, labeling can be improved by denaturing the DNA and immediately carrying out the reaction or by macromolecule crowding (Harrison and Zimmerman, 1986), e.g., with PEG 8000. The reactions are represented schematically in Fig. 7.18. Experimental details of the kinasing reactions, both forward and exchange, are presented in Table 7.19. The purity of the strand to be labeled is critical in the kinasing reactions. Even a small contamination with respect to weight may be very significant in the relative amount of 5’-ends if the contaminating molecules are very small. The best enzyme preparations are from overproducing E. coli strains into which the gene coding for this enzyme has been cloned (Midgley and Murray, 1985). Care should be taken in the preparation of the nucleic acids since phosphate and ammonium salts are strong inhibitors of this enzyme.

7.7.2. Labeling of 3‘ ends of DNA or RNA Both ds and ss DNA (e.g., oligomers) can be efficiently tailed at the 3‘-end using terminal deoxynucleotidyl transferase (TdTase). This enzyme has a broad specificity (Deibel et al., 1985) but the efficiency of incorporation of the various nucleotides differs considerably. TdTase, like T4 PNKase, is quite sensitive to contaminants such as Na+ or protein and does not label as efficiently recessed ends as protruding ends. In case a single nucleotide is to be added, it is

Ch. 7

355

PROBE LABELING AND DETECTION

I

I11 5'HO 3'

5 ' 'P

OH 3'

c

5' T, PNKase [ T ~ ~ATP P ] 3'

3'

5'

5' P 3'

+

3'

3' - ' 5 5,

5'

3' P

5

'

M

A

3'

+

3'

5'

RTase or Sequennse

'-'3 5' 'P

3'

+ +

3-'

OH 3' 5'

5'

Restriction

5'

' 5'

3'

-3

5'

Denaturation

IV

I1 5'

OH 3'

3'

5'

4

TdTase dNTP

5'

(NL.3'

3'

5'

5'

OH

3'

3'

5'

5'

(N),3'

3'

5'

3'

+ 3'

5'

3'

5'

Hybridize

5'

4

3'

-5'

c

3' 5'

with ; l ~ u ) c l e o t i d e

3' 5-

5' 3'

5'

Restriction

3'

3' 5'

RTase or Sequenase 5'-3'5-

3'

-35'-

+

3' 5'

Denaturation

Fig. 7.18. End-labeling can be achieved by different approaches. Kinasing (I) is most effective with 5'-OH overhangs. In the presence of excess ADP, radioactive phosphate can also be exchanged with a 5' phosphate. Whereas kinasing introduces at most one label per DNA end, tailing (11) allows multiple labels to be introduced (Co2+ is necessary with recessed ends). Fill-in of restriction sites (111) also yields 3'-end labeling. RTase or sequenase should then be used. Oligomers are required if the 5'-end is recessed (IV).

possible to use [a-"P]cordycepin (a dATP but instead of the usual 2'-deoxy it is 3'-deoxy) which, due to the lack of a 3'-hydroxyl group, prevents further addition. For most applications, however, a large number of labeled nucleotides are added.

356

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Collins and Hunsaker (1985) observed that the detectability of the 3’-end 32P-labeledoligomer probes was similar to that of nick-translated probes and about 24-50 times better than 5’-end labeled probes. The thermal stability, and thus specificity, of tailed oligomers (radiolabeled or labeled with digoxigenin) is virtually identical although the hybridization efficiency can be significantly affected by the tails. This may have a direct effect on the hybridization pattern as probes with shorter tails than the average will be more likely to hybridize (hybridization selects the more mobile probes with shorter tails). Although eukaryotic genomes contain large tracts of poly(dT1 (Lustig and Petes, 19841, background is absent since the T, of poly(dT):poly(dA) is sufficiently low. In contrast, if dC or dG is chosen for tailing, strong background levels can be expected. The useful lifetime of 32P(dA)-tailed oligomer probes is about 1 week (30% decrease in radioactivity is hardly detectable), whereas after 2 weeks, the signal decreases about three-fold with a much higher background (spin chromatography prior to hybridization can decrease this background). 35S-labeled probes have about the same detectability as 5’-end 32P-labeled probes, but the useful lifetime is 1-2 months. An alternative to tailing, but yielding a specific activity similar to that of 5’-end labeled probes, is the fill-in reaction (to extend the 3’-end) of endonuclease digested DNA. This requires 5’ overhangs at the restricted sites. Discriminatory 3’-end labeling of restriction endonuclease co-digested DNA (leaving 5 ’ overhangs) is usually achieved by a fill-in reaction with Klenow fragment. For instance, an EcoRI and BamHI digested DNA can be specifically labelled at the BamHI sites with [ ( Y - ~ ~ P I ~ C and T Pat the EcoRI sites with [a32PldTTP. However, it has been observed that Klenow fragment often labels nonspecifically (Theveny and Revet, 1987). This is probably due to 3’ + 5‘ exonuclease and 5‘ + 3’ polymerase activities. Reverse transcriptase does not possess 3’ + 5’ exodeoxyribonuclease activity and has been proposed to obtain specific labeling (LeBlond and Ts’o, 1989). They observed that the proper choice of endonucleases, labeled dNTP and incubation temperatures are critical. Even with these precautions, prolonged hybridization results in background staining. Sequenase is a very valuable alternative for labeling.

Ch. 7

PROBE LABELING AND DETECTION

357

TMLE 7.19 End-labeling by kinasing reactions A. Kinasing of 5’-OH groups 1. Mix: 1-50 pmol of dephosphorylated DNA or oligomers (1 pmol of 5’-termini equals 1 pg of linear DNA of 3 kbp) 5 pI of 10xkinase buffer (0.5 M Tris-HCI, pH 7.6, 0.1 M MgCI,, 50 mM dithiothreitol, 1 mM spermidine-HCI and 1 mM EDTA) 50 pmol of [Y-~*P]ATP (specific activity 3000 Ci/mmol); should be at least 1 p M at final concentration 20 units (1 pl) of T4 polynucleotide kinase Add water to 50 pI 2. Incubate for 30 min at 37°C 3. Add 2 pl of 0.5 M EDTA, extract with phenol/chloroform and precipitate with ethanol (Table 3.1). 4. Redissolve DNA in 50 p1 of TE and remove ATP by spin chromatography (polynucleotides) or polyacrylamide gels (oligomers; Section 6.3.2). Note: ammonium salts inhibit kinase enzyme; DNA should be pure, particularly small fragments present in small amounts can become predominant with respect to 5’-termini. Kinasing of oligomers is sometimes inhibited by the presence of residual chemicals from their synthesis. Increasing the MgSO, concentration to 10 mM and precipitation with 5 vols. of ethanol can then improve kinasing. Kinasing of recessed or blunt 5’-OH groups Mix in this order: 1-50 pmol of dephosphorylated DNA 4 pI of 1Oximidazole buffer (0.5 M imidazole-HCI, pH 6.4, 180 mM MgCI, and 50 mM dithiothreitol) H,O to 15 pl 10 pl of PEG 8000 (25% w/v) TP activity 3000 Ci/mmol) in a volume of Add 50-75 pmol of [ Y - ~ ~ P I A (specific 15 pl. Add 1 pl (20 units) of T4 polynucleotide kinase and mix. Continue as in steps A2-4. Exchange reaction of 5‘-P group with 32P 1. Mix in this order: 1-50 pmol of 5‘-termini of DNA 5 IJ.I of ]Oxexchange buffer (0.5 M imidazole-HCI, pH 6.4, 180 mM MgCI,, 50 mM dithiothreitol, 1 mM spermidine-HCl and 1 mM EDTA). 5 pl of 1 mM ADP 1 p1 of 50 nM ATP 50 pmol of [ Y - ~ ~ P I A (specific TP activity 3000 Ci/mmol) H,O to 40 p1 10 pI of PEG 8000 (25% w/v) 1 p1 (20 units) of T4 polynucleotide kinase 2. Continue as in steps A2-4.

358

HYBRIDIZATION WITH NUCLEIC ACID PROBES

An elegant approach of 3'-end labeling of oligomers (Fig. 6.3) is the use of two partially complementary oligomers (Studencki and Wallace, 1984; Dattagupta et al., 1987). When two oligomers are synthesized so that upon hybridization they contain one or two 5' overhangs, a fill-in reaction with one or more (labeled) dNTPs can be obtained, as described above. The primers can also be designed so that one overhang lacks the base complementary to the labeled nucleotide while the other overhang contains all bases. Fill-in with complementary dNTPs will lead to labeling of only one of the two strands. Since these oligomers will have different lengths, they can be easily purified as described in Section 7.6.3.2. Klenow should be avoided since it contains also exonuclease activity (Rigaud et al., 1991). A typical 30 pl reaction mix contains 15 pmol of primers in 40 mM Tris-HC1 (pH 7 3 , 20 mM MgCI, and 50 mM NaCI. It is incubated for 2 min at 75°C followed by cooling to 20°C within 20 min. Then 3 pI of 100 mM DTT and 1 pI of each of 10 mM dTTP, dGTP and dCTP and 20 pl of label ([CY-~'P]~ATP at 10 pCi/pI and a specific actixfityof 3000 Ci/mmol or equivalent) and 0.5 pl(5 units) of sequenase are added and incubated for 20 min at 20°C. The labeled oligomer(s) is purified on a 15% polyacrylamide gel. The amount of oligomers and label are given for the incorporation of four dATP residues, but the ratio should be modified with different oligomers. The specific activity can be very high.

7.7.2.1. Radiolabeling of 3'-ends of DNA In a typical radiolabeling reaction, the appropriate amount of probe (0.5 ng per base, i.e., a 20-mer requires 10 ng) in 0.1 M potassium cacodylate, 1 mM CoCl,, 1 mM 2-ME, 200 pCi of a-labeled [32P]dATP(5000 Ci/mmol), 100 pg/ml BSA and 20 units of TdTase in a total volume of 10 p1 is incubated for 60 min at 37°C. This may yield a specific activity of > 10'' dpm/pg (mass of original oligomer). Similarly, [a-3sS]dATP can also be incorporated (reaction volume 40 pI) but unlabeled dTTP (5 ng) should also be added, to reduce background, while maintaining the same concentrations of the other components. Tailed probes can be purified after phenol extraction and by spin chromatography over 10-15 vols. of Sephadex G-25 equilibrated with 10 mM EDTA (pH 7.0). SDS (0.2%) is added to

Ch. 7

PROBE LABELlNG AND DETECTION

359

the ',P-probes which are stored at 4"C, while 10 mM D l T is added to 35S-probeswhich are stored at -60°C (DTT is also added to the hybridization steps). 7.7.2.2. Nonradioactive labeling by tailing of 3'-ends of DNA TdTase is an excellent enzyme to incorporate the often bulky nonradioactive labels. Here, we discuss the use of the cardenolipid steroid DIG and the vitamin biotin, but similar experiments can be set up with other labels. Schmitz et al. (1991) used two different DIG labels (DIG-dUTP and DIG-ddUTP). The use of DIG-ddUTP leads to the addition of a single DIG hapten. The labeling reaction contained (assembled on ice): 100 pmol of oligonucleotide, 4 pl of 5 X tailing buffer (0.125 M Tris-HC1, pH 7.6; 1 M potassium cacodylate; 1.25 mg/ml BSA), 2 ~1 of 0.05 M CoCl,, 1 p1 of the labeling mixture and completed to 19 p1 with H,O. The labeling mixture contains (all in 10 mM Tris-HC1, pH 7.5) either DIG-dUTP (1 mM DIG-dUTP + 9 mM dATP) or 10 mM DIG-ddUTP or BIO-dUTP (1 mM BIO-16dUTP + 1 mM dTTP). One pl (55 units) of TdTase is then added and the mix is incubated for 15 min at 37°C. Co2+ concentrations for dA/dT are at an optimum at 1.5 mM whereas for dG/dC it should be 0.75 mM. The optimum ratio of 3'-OH termini to dNTP is 1 : 100 (yielding dA-DIG-dU tails of about 40-50 bases with one label per 10-12 bases). The most efficient way to stop the reaction is by the addition of EDTA to 0.2 M with a carrier like glycogen (Boehringer Mannheim). Phenol extraction should be avoided. Optimum hybridization was achieved with DIG-probes at concentrations of about 150 pmol/ml. When BrdU is used for tailing, it can be substituted directly for dTTP (Jirikowski et al., 1989). 7.7.2.3. Labeling of 3'-ends of RNA T4 RNA ligase (Uhlenbeck and Gumport, 1982) can join the 3'-end of an RNA (or ss DNA) to a ss 5'-phosphoryl end of DNA or RNA or to the short [5'-32P]nucleoside 3'3' bidphosphate) if ATP is provided as an energy source:

RNA-OH + 5'-32P-N-P-3'+ ATP + RNA-',P-N-P

+ AMP + PPi

360

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 7.20 Transamination A. Transamination with ethylene diamine (Draper, 1984) 1. Prepare fresh transamination solution (1 M sodium bisulfite, 3 M ethylenediamine (pK 7.61, 3 mM hydroquinone) by slowly adding 1 ml of concentrated HCI to 1 ml of water, 1 ml ethylenediamine (0.9 g; caustic!) and 0.475 g of sodium metabisulfite on ice. Adjust the pH to 6.0 by adding concentrated HCI and bring the volume to 5 ml with distilled water. Add 100 JLI of ethanol containing 5 mg of hydroquinone (a scavenger of free radicals). 2. Heat-denature DNA (10 pg/40 JLI in 1 x T E ) for 10 min at 98°C and cool rapidly on ice. 3. Heat transamination solution to 42”C, add 9 vols. of this solution to 1 vol. of DNA suspension and incubate for 2 h at 42°C (ss DNA) or 7 min at 98°C ( d s DNA, at 42°C DNA may reanneal and thus limit the transamination). 4. Purify the probe intermediate by dialysis or centrifugation on Centricon (Section 3.1.4.2). Note: dCTP can be transaminated with 1,6-diaminohexane by slight modifications of this method (Gillam and Tener, 1986) and then be incorporated by the usual enzymatic methods.

B. Biotin-labeling of transaminated DNA (Viscidi et al., 1986) 1. Dilute modified nucleic acid to 10 pg/lOO pl in 0.1 M sodium phosphate (pH 8.5); heat-denature ds DNA. 2. Prepare a 2 M stock solution of NHS-biotin in DMF and add 5 pI to the reaction mixture. 3. Incubate the reaction mixture for 1 h and then dialyze three exchanges of 0.15 M NaCI/10 mM sodium phosphate buffer (pH 7.0) and 1 mM EDTA. The probe can be stored at 4°C. C. Eu-labeling of transaminated DNA (Hurskainen et al., 1991) (Other fluorophores are labeled in a similar way: e.g., 5-carboxyfluorescein-NHS ester from Research Organics) (Folsom et al., 1989). 1. Dialyze transaminated DNA against four exchanges of 5 mM sodium phosphate (pH 7.5). Exchange to 0.1 M sodium carbonate buffer (pH 9.8) containing 0.1 mM EDTA (note that ethanol precipitation results in precipitation of phosphates). 2. Add 2 mg of Eu-chelate (Fig. 7.4) to 100 pl of DNA and let the reaction proceed overnight at room temperature. 3. Remove reagents by passage through Sephacryl S-400 column ( 5 0 x 0 3 cm2) using 10 mM Tris-HCI (pH 7.5) containing 0.1 M NaCI. EDTA concentrations should be kept low ( < 12-fold over Eu) to prevent Eu-dissociation.

Ch. 7

PROBE LABELING AND DETECTION

361

TABLE7.20 (continued) D. Biotin hydrazide (Reisfeld et al., 1987) 1. Heat-denature linear DNA and cool rapidly on ice/NaCl. 2. Dissolve 10 mg/ml of biotin hydrazide (not very soluble) and add to an equal volume of DNA (50 pg) in 0.1 M acetate buffer (pH 4.5) and add bisulfite to 1 M (total volume 0.5-1.0 ml). 3. Incubate at 37°C for 24 h. Transamination can be checked by measuring hyperchromicity at 260 nm: 20 pI of sample in 1 ml 0.2 N NaOH should yield an increase of 0.1-0.2 over 24 h. 4. Dialyze against water (several changes) for 24 h at 4°C. 5. Precipitate DNA, resuspend in 50 pl of T E buffer and store at 4°C until used.

In the usual reaction, 2 Fg of RNA is suspended in 50 pl containing 50 mM HEPES (pH 8.3), 10 mM MgCl,, 5 mM DTT, 2 mM ATP, 50 pg/ml BSA and a 10-fold excess of label. Since the RNA may circularize, it should not be too diluted. Similarly, if the label has a certain minimum length (e.g., labeled ss DNA), it can also circularize.

7.8. Chemical modification of probes to introduce labels Both aromatic amino and hydroxyl groups present on nucleotidic bases are rather unreactive. One of the best choices is to introduce a primary aliphatic group which has high nucleophilicity. Four different labeling strategies are widely used: (i) transamination; (ii) photolabeling; (iii) crosslinking; (iv) direct adducts. A controlled transamination of the cytidine residues allows the introduction of a reactive diamine or the direct introduction of a label (e.g., hydrazide biotin, sulfonation). Photoreactive molecules react via free radicals and are thus nonspecific and the extent of the reaction is sensitive to contaminants. Both biotin and haptens can be introduced by this method. Molecules which can be brought into close contact with nucleic acids (basic molecules, intercalating agents, nucleic acid-binding proteins) can be crosslinked with nucleic acid, e.g., by UV-irradiation. Products which readily form adducts with nucleic acids, e.g., N-acetoxy-

362

HYBRIDIZATION WITH NUCLEIC ACID PROBES

kq

NHCHzCH2NHZ

0 f

I

R

!

<-"'and

bok9

NHCHzCH z N H y N H - L i g a n d S

\NHS-iigand

R

Fig. 7.19. Transamination of unpaired cytosine with a bifunctional amine (ethylene diamine) is catalyzed by sodium bisulfite. The primary amino groups on the cytosine derivatives can then be coupled with, e.g., SCN- or NHS-ligands.

N-acetylaminofluorene, are usually very toxic or carcinogenic and require safety and disposal measures.

7.8.1. Introduction of reactive groups in nucleic acid by transamination Hydrazine, semicarbazide, hydrazide derivatives and bifunctional linkers can replace amino groups of cytidine. This reaction Ctransamination') can be catalyzed with bisulfite (about 100 times faster) and can be followed by measuring the hyperchromicity at 260 nm (Table 7.20D, step 3). Bisulfite-catalyzed transamination (Shapiro and Weisgrass, 1970) of cytidine residues in ss nucleic acid is a simple, cheap and practical means of preparing nonradioactive probes. Viscidi et al. (1986) described a method involving transamination of unpaired cytosine residues in nucleic acid with NaHSO, and ethylenediamine. This yields a versatile intermediate with side chains bearing reactive side groups (Fig. 7.19). Two competitive reactions occur in this process, transamination and deamination (converting cytidine into uracil). A lower amine p K is associated with faster transamination since in this process the pK of the nucleophile affects the concentration of the reactive amine at a given pH. Transamination has a higher activation enthalpy than deamina-

Ch. 7

PROBE LABELING AND DETECTION

363

tion and is thus favored by higher temperatures. The adduct formation (Fig. 7.19) is affected by the pH with both the sulfite-bisulfite (pK = 6.2) and cytidine N 3 protonation equilibria favoring the adduct at low pH. Transamination and deamination should proceed by the same mechanism. However, transamination also depends on the nucleophile (-RNH,) concentration which increases with higher pH. It has been observed that deamination is optimal near pH 5 and transamination is optimal near pH 6 (Draper, 1984). This method also works well with RNA, but it is essential to include hydroquinone to prevent RNA degradation. In the standard protocol (Table 7.20A), about 33% of the cytosine is usually derivatized. In the second step, the purified, transaminated DNA is brought to a higher pH (8.5-10) in order to decrease the protonation of the reactive amino group. Viscidi et al. (1986) labeled the N4-substituted cytosine residue with biotin using the NHS-biotin ester, whereas, e.g., Hurskainen et al. (1991) labeled the same intermediate with a europium chelate (for time-resolved fluorescence). The Cot,, increases about 1 log but this is compensated by the high probe concentration. This method yields probes with a similar detectability (lo5 molecules) as enzymatically labeled biotin probes, but the reagents are inexpensive and easily available. An interesting approach to selectively label regions outside the probe is to tail ds molecules (e.g., inserts from plasmids or PCR-produced duplices) with C-homopolymers and to transaminate these tails without heat-denaturation of the duplex. Subsequent restriction of the duplex and purification of the fragments yields strand-specific probes. Reisfeld et al. (1987) described a hydrazide biotin labeling method (Table 7.20D) by which they could detect 1 pg of target DNA. Hydrazide compounds can be used similarly for a large number of other labels (enzymes, chromo- or fluorophores, electron-dense markers, etc.). Since the reaction is specific for ss nucleic acid, ss loops in duplices can be specifically labeled.

7.8.2. Photohaptens (biotin, digoxigenin) Forster et al. (1985) synthesized a derivative of biotin containing a photoactivable azido group. This product is commercially available

364

HYBRIDIZATION WITH NUCLEIC ACID PROBES

(BRL) as is photodigoxigenin (Boehringer Mannheim). The main drawbacks of these products (Fig. 7.2.IX) are that: (i) aryl nitrenes may react with a wide range of functional groups which can react as scavenger agents (thus probe preparations should be clean and free of protein); (ii) the number of label moieties rarely exceeds 1 per 100 bp (more often 0.2-0.5); (iii) the detectability is not as good as that of most alternatives. However, the method is extremely simple, can be readily applied to both DNA and RNA and leads to the preparation of probe up to at least milligram quantities. Photobiotin or photodigoxigenin can be photo-activated in visible light to yield highly reactive aryl nitrenes which react with the aromatic bases. The spacer should be hydrophilic to ensure a high solubility in water. The nucleic acid should be free of organic buffers such as Tris and can be ss or ds (McInnis et al., 1987). Photoreactive reagents should, when possible, be kept out of light. NO,-groups on the phenyl group (Fig 7.2.IX) shifts the optimum wavelength range towards visible light. Such photohaptens are easier activated but demand also handling under very dim light or photographic safelight. Photobiotin acetate (1 pg/pl) is added to an equal volume of nucleic acid (1 pg/kl) in water. Alternatively, 1 p,l of photodigoxigenin stock solution (1 pg/pl in DMF) is added to 1 pg of nucleic acid in 9 p1 of water. The open Eppendorf tubes containing the samples are placed on an ice/water bath, 10 cm below a sun lamp (e.g., Philips HPLR or Osram MB/U 300 or 400 W lamps) for 15 min (RNA, 10 mid. After adding 9 vols. of 10 X TE, the sample is extracted twice with an equal volume of 2-butanol and the probe is ethanol-precipitated. The biotinylated nucleic acid yields an orange-brown pellet. Store DNA in TE buffer, containing 0.1% SDS. When the photobiotin/DNA mass ratio in the reaction is too high, a precipitate may form and the ratio should be reduced to 2. Weak signals may be due to too stringent hybridization conditions but also to insufficient labeling (presence of scavengers, organic buffers) or adherence of DNA to the storage tube. This can be prevented by siliconizing tubes or adding 0.1% SDS. Similarly, many different photoactivable haptens can be synthesized. Keller et al. (1989) prepared a 2,4-dinitrophenyl (DNPIlinker-aryl azide compound by a simple method from commercially

Ch. 7

PROBE LABELING AND DETECTION

365

available chemicals and produced anti-DNP antibodies by immunizing rabbits with 6-DNP-aminohexyl BSA. The photohapten is prepared by dissolving 8 mg of sulfosuccinimidyl 6-(4’-azido-2’nitropheny1amino)hexanoate (sold as sulfo-SANPAH by Pierce) in 5 ml of 0.1 sodium carbonate (pH 9.0) in a polypropylene tube and adding 2 ml of 13 mM 6-(2,4-dinitrophenylamino)-l-aminohexanein methanol. After an incubation for 1 h at room temperature, the product (2,4 dinitrophenyl (DNP) attached via a linker to aryl azide) is collected by centrifugation (2000 X g for 10 min). The red-brown precipitate is dissolved in 3 ml of CHCl, and extracted in a polypropylene tube five times with water. Purity can be checked by TLC chromatography (silica gel; chloroform/methanol, 85 : 15). Photo-DNP moves with the front. CHCl, is removed in vacuo and the dried material redissolved in DMSO. The extinction coefficient of photo-DNP is about 1.74 X lo4 cm-’ M-’ at 360 nm.

7.8.3. Psoralen crosslinkers Psoralens (furocoumarins used by some plants in defence from insects and fungi) photoreact with ds DNA to form interstrand crosslinks in the presence of near-UV light by two steps: intercalation in the dark and formation of monoadducts or interstrand crosslinks (Song and Tapley, 1979). This reaction has the advantage that psoralen binds, in contrast to photobiotin, only to nucleic acid. Saffran et al. (1988) described the preparation of a biotinylated psoralen. First, diamine psoralen is prepared from chloromethyl trioxsalen and N,N’-dimethylethylenediamineas described by Goldenberg et al. (1988). Twelve milligrams of diamine psoralen is then dissolved in 0.4 ml of dimethylformamide (DMF), 15 mg of solid NHS-biotin (Pierce) is added and the reaction is allowed to proceed for 1 h at room temperature (covered with aluminium foil when possible). The product is purified by flash chromatography (silica gel TLC) (Still et al., 1978) after taking up the yellow oil in 0.1 ml of methanol and then adding 1.5 ml of CH,CI,. The product gives a single spot with blue fluorescence and an R , value of 0.28 on TLC plates. Two kg of linear DNA in 10 p,I of TE is mixed with biotin

366

HYBRIDIZATION WlTH NUCLElC ACID PROBES

I

I1

Psorslen

Psoralen

uv

+ drr DNA

Psoralen t ss DNA + 0P

A

2

C

H

z

R

111

)3-y

H

-

-

- C-(CH&

H

H

R2

-A- (cH,),-NH

unmodified

CHz

CH3

R, = - Y - C H 2 - C H * - ( O C H 2 C H z

uv

photocycloaddition xlth thymidine

f

(cH,)$-N-cH

(cH~),-sH

GH

Detector

IV&

-I I I I I I I I I I I I I I I I I I I I I

Fig. 7.20. Psoralen (I) reacts only with ds DNA (II), at a rate of 5-10 groups/lOO bp, and can be derivatized with different side-groups (111). These side-groups can then be detected. M13 or phagemid clones, hybridized with M13 or phagemids without inserts, are convenient. This leaves only the insert of the clone single-stranded, which will be the only region not labeled.

psoralen and irradiated with near-UV light (about 350 nm). The biotin retains the affinity for streptavidin. The property of crosslinking ds DNA was used by Sheldon et al. (1986) who prepared ‘gapped circle’ M13 probes. The probe sequence is a ss DNA (‘gap’) whereas the rest is double-stranded and is effectively crosslinked (Fig. 7.20). They prepared gapped circle M13 probes by in vitro hybridization, as described by Courage-Tebbe and Kemper (1982), using ss M13 DNA with the appropriate insert and complementary DNA from linearized R F M13 without an insert. With the crosslinking method described above, about 3-5 psoralen moieties should be obtained per 100 base pairs allowing the detection of single-copy genes in about 1 p,g of total human DNA. The commercial HLA probes from Cetus are based on this principle.

7.8.4. Crosslinking of haptenated or enzyme-labeled basic polymers Basic polymers readily bind to nucleic acids and this interaction can be made permanent by crosslinking (Renz, 1983; Renz and Kurz,

Ch. 7

PROBE LABELING AND DETECTION

367

1984; Syvanen et al., 1985; Al-Hakim and Hull, 1986). Bulky side groups on the nucleic acid may interfere with hybridization. Direct coupling of enzymes (Renz and Kurz, 1984) requires the enzymes to be stable under the hybridization conditions. This means that the enzyme often limits the stringency of hybridization. In this labeling procedure, POase (20 mg) is dissolved in 220 pl of 90 mM sodium phosphate (pH 6.0) to which 60 pl of a solution containing 30 ng p-benzoquinone in 1 ml of ethanol is added and incubated for 1 h at 37°C in the dark. The free benzoquinone molecules are then separated from the derivatized POase by chromatography on a small column of Sephadex G-100 (7 ml in 0.14 M NaCI). The colored POase fractions are combined and 1/10 vol. of 1 M NaHCO, and 2.7 p l of 5% Polymin G35 (BASF) are added. The reaction mixture is kept at 37°C in the dark for 20 h, subsequently dialyzed against 5 mM sodium phosphate (pH 6.8) and stored at 4°C. This approach has been taken by Amersham for their commercial probes (direct coupling of peroxidase to basic polymer), but results have been variable in our hands. The Amersham probe technology has been combined with a sensitive detection system (Section 7.3.3.5.2). Syvanen et al. (1985) used E. coli single-stranded binding protein, and crosslinked by glutaraldehyde and subsequently biotinylated, but the probes had a disappointing detectability. The method of Al-Hakim and Hull (1986) has been the most successful among these approaches. Histone H1, cytochrome c or polyethyleneimine was biotinylated with NHS-biotin (500 p g of NHS-biotin in, if necessary, 10 p1 of DMF (those with soluble spacers in water) per mg protein or 2 mg of NHS-biotin per mg of polyethyleneimine in 250 p1 of 50 mM NaHCO,) during 1 h at room temperature and then dialyzed overnight against 5 mM sodium phosphate. Polyetheyleneimine with a molecular weight of 1400 (‘Polymin G35’)detected about 25 times less target nucleic acid than polyethyleneimine with a molecular weight of 60 000, cytochrome c or histone H1 (probably due to steric hindrance of the larger molecules). Heat-denatured or single-stranded DNA in 100 pl of 5 mM sodium phosphate (pH 6.8) was crosslinked with the biotinylated basic polymer (4 p1) by adding 20 p l of 2.5% of the crosslinking agent (Fig. 7.21) and incubating for 20 min at 37°C. The probe can be

368

HYBRIDIZATION WITH NUCLEIC ACID PROBES

I

0

pH 6 . 0

I

0

OH

bH

p~ 8 . 0

i-

+ polyethyleneimine (PEI)

PEI-(NH j), polycation)

POase Q( \

glutaraldehyde O=F-(CH,), -F=O H H

7

denatured DNA (polyanion)

OH

OH

I1

BIO-n-NHS

+

protein or PEI

I

BIO-basic polymer (BIO-BP) H2C-CH-(CH z),-CH-CHz ‘0’

\o/

denatwedDNA

1.2,7.8 diepoxyoctane BIO-BP-(CH,),

-DNA

Ch. 7

PROBE LABELING AND DETECTION

369

ins e r t

0 pUCf1

cloning d

M13K07

fl

pUCRfl

secondary probe (universal probe)

Fig. 7.22. Iodination of pyrimidines in RNA or DNA.

purified by chromatography through a small (1 ml) Sephadex G-75 column (5 pl of Orange G at 25 pg/ml added as a marker). The probe elutes just ahead of the Orange G and can be added directly to the hybridization solution. 7.8.5. Radioactive labels Radioiodination of nucleic acid (Fig. 7.22) is one of the earliest, convenient methods to obtain high specificity (Commerford, 1971) but its use has been declining considerably. It might still have some usefulness for in situ hybridization and, in the case of RNA, in two-dimensional fingerprint analysis. One should be aware of the volatile nature of lZI. The reaction is carried out in two stages: (i) nucleic acid (10-1000 p M as cytosine) is heated to 60°C for about 10 min in the presence of 30-100 p M radioiodide, TlCl, at 5-10 times Fig. 7.21. Enzyme coupling via p-benzoquinone (pBQ) activation (pBQ also reacts with carbohydrates) and coupling to a basic polymer (I). At pH 6, only one of two potential sites of pBQ reacts, whereas at pH 8 the second site is activated. The complex is conjugated to denatured DNA using glutaraldehyde. Alternatively, haptenated basic polymers can be linked by 1,2,7,8-diepoxyoctane (carcinogenic?) to DNA (11).

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 7.21 Mercuration of nucleic acid 1. Incubate RNA or DNA (5 pg/50 pl) in 5 mM mercury acetate and 10 mM NaOAc (pH 5.5-6) overnight at 50°C. The 15 mM mercury acetate stock solution is freshly prepared by dissolving 45 mg mercury acetate in 500 p1 of 0.2 M NaOAc and diluting to 10 ml with distilled water. 2. Stop mercuration by adding an equal volume of a buffer containing 0.12 M KCN, 40 mM NaCl and 2 mM EDTA in 20 mM Tris-HCI (pH 7.2). 3. Add 400 pg of carrier DNA (Table 8.4) and purify probes by chromatography over Sephadex G-50 (8X0.5 cm2), equilibrated in 10 mM sodium phosphate and containing 1 mM KCN (pH 7.2). 4. Concentrate the nucleic acid solution by 2-butanol extraction.

the iodide concentration and 0.1 M sodium acetate buffer (pH 4.7); (ii) the pH of the mixture is raised with NaOH to 7-8 and heated again for 20 min at 60°C to stabilize the 5-iodocytosine derivative. The efficiency of incorporation is about 25% when equimolar amounts of cytosine and '''I are used. Since RNA is not as stable as DNA, it is more difficult to label but several methods have been described (Heiniger et al., 1973; Lee and Fowlks, 1982).

7.8.6. Mercurated nucleic acids for sul'ydryl-hapten

ligands

Cytosine and uracil are readily mercurated (7.2.V) at the 5' position in a warm, buffered solution containing mercuric acetate (Dale et al., 1975; Hopman et al., 1986a) (Table 7.21). Although this technique yields excellent results, the chemicals needed are very toxic. There is little or no reaction with adenine, thymine or guanine bases. Polymer mercuration is, in contrast to mononucleotides, influenced by the ionic strength of the reaction mixture: the reaction rate is higher at a lower ionic strength. Residues in ss or ds polymers react at essentially the same rate. Two simultaneous reactions occur: a noncovalent complexing reaction and a slower covalent introduction of mercury at the 5' position. It is important to remove the complex-bound mercury ions from the nucleic acids because they interfere with the annealing. Both cyanide and iodide are effective in

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371

TABLE7.22 N-Acetoxy-N-2-acetylaminofluorene (AAF or AAIF) labeling Dissolve nucleic acid (fragments of about 1000 nucleotides) in 2 mM sodium citrate buffer (pH 7) at a concentration of 0.5 kg/kl. Heat-denature (lOO°C, 5 min) and add 1/10 vol. of a three-fold excess of AAF or AAIF and incubate the reaction mixture for 2 h in the dark at 37°C. Eliminate cross-linking by treatment with alkali (50 mM sodium borate buffer, pH 9, 1Oo"C, 3 min) followed by neutralization (0.1 M Tris-HCI buffer, pH 7.0). Remove unreacted fluorene by five extractions with cold ether; ethanol-precipitate DNA and redissolve in citrate buffer. About 5% of the bases should be modified.

removing complex-bound mercury but decomplexing with iodide also removes covalently bound mercury. Moreover, removal of Hg with iodide results in dimerization of pyrimidine bases which then interfere with hybridization. In the case of cyanide, no such problems were observed if the p H of the mixture did not deviate from about 7.2. Mercaptoethanol may also lead to demercurization.

7.8.7. N-Acetoxy-N-2 acetylaminofluorene ( M F ) labeling Guanine residues in sequence (RNA or DNA) can be tagged chemically with AAF or its 7-iOdO derivative which can, in turn, be recognized by specific antibodies (Tchen et al., 1984). The fluorene derivatives are linked mainly at the C-8 position (Fig. 7.2.VII). AAF-modification of DNA (Table 7.22) can be followed by the absorbance at 305 nm (extinction coefficient of dG-AAF is 18000 M-' cm-'. The T, of long probes is only slightly affected (about l.l°C/l% of bases modified) (Tchen et al., 1985) but significantly more so for oligonucleotides (Sanford and Krugh, 1985). In Z-form DNA, the C-8 is located at the exterior of the helix and base-pairing need not be disrupted by the AAF adduct. About 0.5 pg/ml of ds or 0.04 pg/ml of ss probe is used in the hybridization mix and about 1 pg of target nucleic acid can be detected with APase conjugates and about 8 pg with POase conjugates. These probes remain stable for at least 2 years at 4°C.

372

HYBRIDIZATION WITH NUCLEIC ACID PROBES

n R Fig. 7.23. Universal probes can be prepared from phagemids (Table 4.4). The insert of interest is cloned into a phagemid and ss DNA is produced using the M13K07 helper phage. A phagemid with the f l in the opposite orientation (allowing the production of the complementary ss DNA) permits the production of a secondary (universal) probe which is hybridized to the primary probe with any insert. This hybrid serves to detect the specific target. AAIF, an iodinated derivative, gives better results than AAF, probably due to higher thermal stability of the duplex and/or greater accessibility to the modified site by the antibodies. AAIF tends to adopt an outside conformation because of the steric hindrance of the iodine atom (Fuchs et al., 1976). Tchen et al. (1984) observed optimal results when hybridizing at 65°C without formamide or dextran sulfate and using glycine instead of albumin in Denhardt's solution. Highest detectabilities are obtained with indirect immunological detection systems (e.g., hapten : antibody : anti-antibody-enzyme conjugate) (Tijssen 1985). These probes have been used in all hybridization techniques with excellent results. Their main drawback is that AAF is carcinogenic.

7.9. Universal probes Universal probes (Fig. 7.23) which are also specific require two components: (i) a tag which is recognizable after hybridization by a

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313

universal means; (ii) the specific sequence which recognizes the target. Such a system can be simply devised by a two-stage sandwich hybridization method (Boyer, 1986). For instance, the replicative form of a phage (or a phagemid or plasmid) without inserts can be labeled with any of the methods described above. The ss M13 phage containing the specific insert is allowed to react with the target sequence and the phage sequences form a target for the subsequent hybridization with labeled R F M13 DNA. It should be noted that in order to obtain hyperpolymers, the R F should be fragmented (e.g., by nick translation or random priming, or alternatively by a limited DNase I digestion followed by heat denaturation. Many variants of this method can be devised (e.g., Dunn and Hassell, 1977; Ranki et al., 1983; Leser et al., 1986). It is particularly useful when many different probes are to be used. Nakagami et al. (1991) proposed a different universal probe system. In their procedure, phagemids are used to obtain strandspecific ss DNA molecules, one with the insert complementary to the target sequence and a second without the insert and with the opposite polarity (Fig. 7.23). The ss DNA without insert is labeled with any label (usually a secondary label) and this labeled DNA is hybridized with the complementary strand containing the insert. This leaves only the probe sequence ss whereas the tag sequence is ds. This method has several advantages: (i) like the sandwich method it leaves the probe sequence single-stranded; (ii) hybridization of the tag sequences in solution instead of on the solid phase as in a sandwich assay is much faster; (iii) no fragmentation is required.

7.10. Biological probes Hartley et al. (1986) devised a nonradioactive probe, called probevector, which can, after hybridization, transform competent E. coli cells and, depending on the transformation efficiency, detect as little as 0.1 pg of target nucleic acid. The probe-vector molecules are linear, partially ss DNA prepared by hybridizing individually prepared DNA strands. The ds region of the probe-vector encodes a phenotypic marker and origin of replication. The two terminal ss

314

HYBRIDIZATION WITH NUCLEIC ACID PROBES

regions (A’ and B’) are complementary to regions A and B of the DNA to be detected. Hybridization results in a circularization and an increase in transformation efficiency of at least lo5. The number of transformants is directly linear to the amount of target DNA.

CHAPTER 8

Mixed phase and solution hybridization formats Solid phase hybridization methods have largely overtaken the traditional techniques of DNA renaturation in solution. Hybridization is much faster and much more efficient in solution than on solid phase, but the signal from hybridized probe in solution is often hard to distinguish from that of free probe. The inherent superiority of solution hybridization has led to efforts to improve the specificity of its signal. Three different (semi-)solution hybridization approaches can be distinguished: (i) capture hybridization, in which a capture probe, anchored on a solid phase, hybridizes with the label probetarget hybrid; (ii) sandwich hybridization, in which both hapten and label probe hybridize to the target in solution (the target is ‘sandwiched’ in solution) followed by collection of ternary complex via the hapten molecule (e.g., by biotin and immobilized avidin); (iii) true solution hybridization, in which a single (label) probe is hybridized to the target and then selectively detected. Both the capture and the sandwich assays allow the release of captured target nucleic acid for further studies or for background reduction. The merits of these formats are summarized in Table 8.1. Solid phase hybridization is in most cases achieved on membranes. Target nucleic acid is immobilized and subsequently detected by a probe. This approach forms the basis of slot/dot blot hybridization, Northern and Southern hybridization and colony or plaque hybridization. Dot/slot blot hybridization (Kafatos et al., 1979) demonstrates the presence of target sequences but not their size. Although solid phase hybridization is convenient for hybrid/free probe separation, it has the disadvantages that nucleic acid is most often bound noncovalently and that targets are immobilized at fre-

376

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 8.1 Merits and disadvantages of different hybridization formats Format Mixed phase

Advantages Simple Rigorous

Less optimization required Widely applicable Capture

Sandwich

Solution

Efficient use of binding sites on solid phase Higher annealing efficiency Faster than mixed phase Crude extracts Flexibility, due to use of two different probes Faster than capture Up to 100% hybridization Crude extracts Free probe can be removed efficiently Very fast Large-scale screening

Disadvantages Slow hybridization Competition of other molecules for membrane binding sites Lower hybridization efficiency Loss of target nucleic acid from membrane Two probes required (1 labeled) Increased background Stringent optimization

Stringent optimization

2 probes required (2 labeled) Stringent optimization Detectability usually limited by efficiency of hybrid separation from free probe

quent intervals on the chains (every 10-100 bases). Each of these short stretches then act as an independent target.

8.1. Materials and solutions 8.1.1. Solid phase systems

Solid phase hybridization is convenient and popular but some limitations should be noted. Background due to nonspecific adsorption of the probe to the solid phase can be considerable without the proper precautions. Both charged and hydrophobic surfaces can yield non-

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specific signals. Saturation with nonspecific macromolecules prior to adding probe and the proper salt conditions to suppress nonspecificity are important. Second, target nucleic acid on the solid phase is often poorly retained during hybridization (often only 10% of the original amount) (Gamper et al., 1987; Gingeras et al., 1987). In capture hybridization, the impact of this leaching can be more pronounced. Since hybridization in solution is far more efficient upon release of even a small fraction of capture probe (which is present at 103-106 times higher concentrations than the target), it will compete efficiently with the immobilized capture probe for target nucleic acid. The capture probe surface or affinity matrix should thus be prepared so that leakage of capture molecules would be minimal. In formats where strand displacement of the probe by a longer nucleic acid (form more stable hybrids than short probes) is possible, it will eventually occur. It is then not uncommon to have a decrease of signal after a few hours, independent of the retention of target nucleic acid. This phenomenon may be particularly important in capture assays with an oligomer as capture probe and ds target nucleic acid. 8.1.1,1, Membranes

Essentially, six distinct membranes are used for immobilization of nucleic acid: nitrocellulose; reinforced nitrocellulose; diazotized membranes (paper or nylon); nylon; charged nylon; or polyvinylidene difluoride (PVDF) membranes. Major suppliers are listed in Table 8.2. DEAE-anion exchange membranes permit reversible transfer elution and are used primarily for isolation procedures for RNA and DNA. The most commonly used membranes are (reinforced) nitrocellulose and nylon (Table 8.3). Nitrocellulose has a lower background but also a lower binding capacity than nylon and is chosen primarily when background, but not detectability, is the main concern. Nylon, in contrast, is ideal for lower copy number sequences, short target sequences (down to oligomers) or for reprobing. Membranes are available with different pore sizes. For DNA blots, membranes with a pore size of 0.45 Fm are usually chosen for large fragments, but 0.22 p m for fragments of <500 bases. For RNA blots, membranes with a pore size of 0.1 or 0.22 km are most

w 4 m

TABLE 8.2 Major suppliers of membranes Suppliers

Amersham Bio-Rad Dupont NEN Gelman Sciences Micron Separations Millipore * Pall Corp. Schleicher & Schuell Stratagene

Nitrocellulose Basic Reinforced Hybond-C Nitrocellulose

3

Nylon Amphoteric

Hybond-C-Super

Hybond-N

NitroScreenWest

GeneScreen

Biotrace NT Nitrocellulose HAW, HATF

Nitroplus 2000

Magna

S&S NC BAS5

S&S BA S85 Duralose-UV

4

m Charged

E

Hybond-N +. Zeta-Probe GeneScreen Plus Biotrace RP Magnagraph

P

Biodyne B Nytran Duralon-UV

A survey on molecular biology products (Ausubel et al., 1991) indicated that for nitrocellulose about 70% of respondents preferred S&S NC (other manufacturers < 10%) but that the preference for nylon membranes differed little among Schleicher & Schuell, Dupont NEN and Amersham. In the same survey, respondents had no clear preference for either nylon or nitrocellulose for Southern hybridization but a clear preference for nitrocellulose in the case of colony/plaque hybridization (where concentration of target is sufficient highly but background should be suppressed). * Millipore supplies Immobilon (PVDF) membranes.

z z E

7

2

z

C

P

m0

b

6

2 G1

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TABLE8.3 Advantages and disadvantages of membranes Membranes Nitrocellulose

Advantages No activation Low background

Supported nitrocellulose

Nylon

DPM/DPT paper

Low background No activation Strong reprobing High capacity ( = 450 pg/cm2) Target of all sizes Multiple probing Binding in low salt Covalent binding Reprobing

Disadvantages Inefficient binding of small nucleic acid Fragile Reprobing difficult Low capacity ( < 100 pg/cm2) Low capacity Not for small target

Higher background Variation in binding No activation required

Require activation Very low capacity

efficient. Membranes are available in different size specifications: sheets; rolls; circles for colony or plaque lifts; small rectangles for transfer from agarose gels. Membranes should never be handled with bare hands. Pure nitrocellulose binds only about 80 pg nucleic acid/cm2 but gives low background levels (varies with the different lots; mixed nitrocellulose esters bind poorly). Nitrocellulose membranes are fragile and should be stored in a dark and cool place which should be devoid of solvent vapors to prevent drying out or making them brittle. The wettability of the membrane is important since it should be uniformly wet for transfer from gels or blotting to obtain acceptable results. The binding of nucleic acid depends directly on the salt concentration (optimal when wetted first with water without trapping air bubbles and then with 20 X SSC, 20 X SSPE or 1 M NH,OAc). Alkaline transfers are not possible with nitrocellulose. Nitrocellulose is quite flammable and it is necessary to use a relative low tempera-

380

HYBRIDIZATION WITH NUCLEIC ACID PROBES

ture with a vacuum when baked to fix the nucleic acid. After baking, the membranes become even more brittle. The efficiency of UV crosslinking of nucleic acid on damp (important for safety!), UVgrade nitrocellulose membranes depends on their lot. Reinforced nitrocellulose (e.g., Hybond-C Super, Duralose-UV) has a tensile strength which may be 500 times more than for ordinary nitrocellulose. It has about the same binding capacity and background level as nitrocellulose. As with ordinary nitrocellulose, alkaline transfers are not possible but one or two reprobings may be possible. Nitrocellulose membranes are versatile and are preferred for colony/plaque lifts. Diazotized paper (Alwine et al., 1979) or membranes were initially used to bind short target sequences. The binding capacity is generally quite low (about 1 pg/cm2) and the preparation is involved. There are now more useful alternatives (nylon, even for oligomers) and diazotized membranes are rarely used (sometimes for the enrichment of specific RNA sequences by hybrid selection). Schleicher and Schuell markets Transa-bind APT (aminophenylthioether) paper which is an arylamide-substituted cellulose paper to which both RNA and DNA can be covalently bound. It is different from the aminobenzyloxymethyl cellulose paper described by Alwine et al. (19791, has a binding capacity of about 40 pg/cm2 and requires a different activation procedure. The surface of the paper is sensitive to light (a light yellow color indicates oxidation, but will not interfere) and it should be stored at -20°C. Alternatively, cyanuric chloride-activated (CCA) paper has been used in the detection of subpicogram quantities of specific DNA in Southern transfer hybridization of genomic DNA (RFLP analysis) (Hunger et al., 1987). Amphoteric (‘uncharged’) nylon 66 membranes have exceptional strength, thermal stability and a binding capacity of about 400-500 p,g nucleic acid/cm2. Nucleic acids are fixed by baking or UV crosslinking. Although nylon can be dry for crosslinking, it takes 10 times more time. These membranes can be used for alkaline transfer (but then lose the capacity for reprobing). Their high capacity also increases the background and blocking becomes very important. However, blocking strategies have recently become quite efficient. Charged nylon membranes (Nytran, Zeta-Probe, Magnagraph;

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active surface amines) retain small fragments ( z 40 bases) particularly well in low or high ionic strength conditions. Some brands require a higher ionic strength for optimal immobilization than others. For instance, nucleic acid immobilization in NaOH on Nytran is superior when the NaCl concentration is > 1 M whereas for Genescreen Plus, a lower ionic strength is optimal. The positive charges permit electrostatic interaction or covalent binding. The high mechanical strength and chemical resistance of these membranes permits highly stringent assay conditions and repeated reprobing of the blots. The charge on the solid phase can, however, have a strong effect on enzyme activity if these primary labels are used (Section 7.3.3.2). Enzyme assay conditions may thus have to be adjusted for the particular type of membrane. PVDF membranes have been used occasionally (Hicks and Vecoli, 1987; Williams, 1990). They have binding characteristics similar to charged nylon although the binding is hydrophobic, with possible electrostatic interactions since PVDF polymers have a strong dipole character. This may be the reason why PVDF functions very well in alkaline conditions. 8.1.1.2. Paramagnetic beads Paramagnetic particles, such as ferric oxide particles ( < 1 pm) from Gene-Trak Systems (Framingham, MA), do not act as magnets (do not cling to each other) but are drawn to a magnetic source. The highly convoluted surfaces are coated with silicon hydrides to which capture molecules such as oligo(dT1 ‘hairs’ can be covalently linked. These beads can be used for mRNA purification (Table 3.16) as well as hybridization. The use of ss dT-polymers offers the advantage of low complexity, hence rapid hybridization and selective denaturation at low temperatures (e.g., for reverse target capture; Section 8.3.2). Superparamagnetic beads, with covalently linked streptavidin, are also commercially available (Dynal, Inc., Oslo, Norway, or Great Neck, NY; Advanced Magnetics, Inc., Cambridge, M A Promega). Hornes and Korsnes (1990) found that hybridization is complete within a few minutes and that it is not necessary to have a large excess of capture probe (even at 1 : 1, the efficiency is close to 100%). A typical hybridization procedure, excluding detection step, takes

w N OD

I

I1

Sense oligomer 5’

3’ I

1 5‘

1

3’ 5’ I

TdTase

I

4

dA& AAAAAAAA Hybridization (0.5M NaC1)

TTTTTTTT 3’ AAAAAA

TdTase *

dATP AAAAAAAA

AAAAAAAA

1

0

Hybridization (0.5M NaC1)

TTTTTT

5’

1

0

ds DNA

5’ 3’

Primer extension

TTTTTTTT

3’

AAAAAAAA

5’

I

Strand

1

separat’on

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TTTTTTTT AAAAAAAA

V

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n

3’

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Strand

1

TTTTTTTT Antisense or s e n s e ss DNA

AAAAAA

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SOLID PHASE AND SOLUTION HYBRIDlZATION

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less than 15 min (Table 3.16). Carboxylated, paramagnetic latex particles (Seragen, Bangs Lab.) have been used to attach avidin instead of the more expensive and less sensitive streptavidin (Yehle et al., 1987). It was then necessary to succinylate the avidin to avoid nonspecific adsorption of the probe although the proper choice of buffer will also reduce background (Section 11.1.1). Albretsen et al. (1990b) synthesized oligomers on paramagnetic beads using a urethane type of linker (Sproat and Brown, 1985). After capping the remaining NH, or OH groups on the beads, these paramagnetic beads could be used in hybridization in the presence of guanidine thiocyanate. The efficiency varied directly with the oligomer length and inversely with the concentration of the capture probe. A simple method to convert oligo(dT1 cellulose beads into beads containing a specific ss DNA has been described by Goldkorn and Prockop (1986) and can also be used for paramagnetic beads. Double-stranded DNA, containing the strand to be linked, is (dA)-tailed as described in Section 7.7.2.1. Following tailing, restriction and fragment purification can be used to select for the fragment with the specific strand tailed. Alternatively, (dA)-tailed oligomers can be used (Fig. 8.1). The tailed ds DNA is denatured and snap-cooled; 3 p g DNA and 1 mg of oligo(dT) beads are added to 0.2 ml of 0.5 M NaCl in a 10 mM Tris-HC1 (pH 7.5) at 65°C. Hybridization is maintained during 30 min at 65°C. After gradually cooling to room temperature, the beads are washed with the same buffer and then with 0.1 M NaCl in 10 mM Tris-HC1 buffer and resuspended in 0.1 M NaCl, 10 mM MgSO,, 0.1 mM DTT and 50 pg/ml BSA in 50 mM Tris-HCl (pH 7.2). The polymerase reaction is carried out by adding 1 unit of Klenow fragment and 80 pM of each dNTP to 50 p1 of this sample. In the case of a polynucleotide linkage, this step is followed Fig. 8.1. Oligo-dT beads (cellulose, paramagnetic) can be modified to obtain specific probes on these beads. Both ss (I) and ds (11) DNA can be used. In the case of ds DNA, both strands can be linked to the beads. Unless the DNA is digested with a restriction enzyme prior to hybridization, complementary strands may be coupled to the beads. Purification of one of the two poly(dA)-containingends after restriction but before hybridization to the beads will yield strand-specificprobes.

384

HYBRIDIZATION WITH NUCLEIC ACID PROBES

by ligation (Section 4.5) for 2 h by adding 0.15 ml of 10 mM MgCl, and 20 pl of 10 x ligation buffer (supplied with enzyme) and 2 units of T4 DNA ligase. To separate the strands, the sample is heated for 5 min at 95°C in 0.8 ml of 10 mM Tris-HC1 (pH 7 . 3 , snap-cooled and immediately centrifuged (repeated twice). For oligomers, formamide may have to be added due to the rapid renaturation.

8.1.1.3. Particulate solid phase Oligomer-coated nylon beads allow rapid hybridization with very low affinity for protein or nonspecific nucleic acid and are stable to all commonly used hybridization formats (Van Ness et al., 1991). Poly(ethy1eneimine) (PEI) coating of nylon 6/6 (2.4 mm) beads permits the immobilization of large amounts of oligomers. The nylon bead is ethylated with triethyloxonium tetrafluoroborate, to obtain amine-reactive imidate esters on the surface, and is then reacted with PEI to provide an extended amine surface for the attachment of oligomers and improve hybridization. Van Ness et al. (1991) observed that oligomers with a 5’-amine tail (Section 6.4) could be immobilized exclusively through this linker to PEI after activation with an excess of cyanuric chloride. Succinic anhydride is used as a capping reagent. The detectability is about 15-75 amol per hybridization. Wolf et al. (1987) attached DNA to submicrometer hydrazidefunctionalized latex beads and observed that hybridization rates of these conjugates were comparable to those in true solution hybridization. These beads (0.19 pm or 0.038 p m diameter) can be collected and washed in Centricon 10 devices. Diazotization to Sephacryl S-500 was found by Langdale and Malcolm (1985) to be more efficient than to Sephadex G-50, cellulose or magnetizable cellulose (Langdale and Malcolm, 1984). Moreover, Sephacryl s-500 adsorbs very little nonspecific nucleic acid and DNA bound to these particles hybridize efficiently. Gingeras et al. (1987) achieved 5 ’-end specific coupling of aminohexylphosphoramidate-derivatized oligomers through EDC (Section 6.4) to Sephacryl 500 dextran supports (coupling time 1 h). The support is stored in TE at 4°C and 50 mg (wet weight) is used for each hybridization (250

Ch. 8

SOLID PHASE AND SOLUTION HYBRIDIZATION

385

~ 1 ) . The hybridization efficiency on these supports is five times higher than on nitrocellulose membranes. Maskos and Southern (1992) designed a linker for the synthesis of oligomers on glass supports which is resistant to ammonia treatment. The system appears to be highly specific in hybridization and sensitive to single mismatches. Their predictable behavior should make this system very useful for hybridization and for the isolation of DNA-binding proteins.

8.1.1.4. Microtiter plates The popularity of microtiter plates in diagnostics (e.g., enzyme immunoassays) and the development of systems for washing, measurement of enzyme activity or chemiluminescence, etc., has led to scattered attempts of using these plates for hybridization. Microtiter plates are not suited for immobilization of target nucleic acid but are promising for capture and sandwich assays, particularly for large numbers of samples. Microtiter plates are also an excellent choice as the affinity matrix for the capture of hapten probe-target-label probe ternary complexes (Syvanen et al., 1986). The detectability of this technique is about 20 amol. The original methods of immobilizing capture probes resulted in ill-defined DNA-plastic bonds. DNA was passively adsorbed and immobilized with UV irradiation (Nagata et al., 1985; DahlCn et al., 1987). Immobilization of DNA was improved by covalent binding of base-modified DNA (Keller et al., 19881, by covalent binding to poly(Lys-HBr, Phe) (Running and Urdea, 1990) adsorbed to microwells or by carbodiimide-mediated condensation of DNA onto CovaLink NH microtiter plates (Nunc) (Rasmussen et al., 1991). The method of Keller et al. (1988) is suitable for long DNA capture probes, such as M13 probes, whereas the method of Rasmussen et al. (1991) is recommended for oligomers. In the last method, DNA molecules are attached only at one end which should improve hybridization characteristics. The detection limit, however, is about 40 amol. The covalent linking of DNA permits stringent hybridization conditions and this method is promising for capture assays (probably repeated use possible).

386

HYBRIDIZATION WITH NUCLElC ACID PROBES

8.1.1.5. Affinity columns Hydroxyapatite (e.g., Bio-Rad) has been used extensively in renaturation studies (Britten et al., 1974). Both ss and ds DNA are adsorbed to hydroxyapatite at sodium phosphate concentrations less than 20 mM. At higher concentrations (130 mM sodium phosphate buffer), ss DNA is eluted but ds DNA is retained. The ds DNA can be eluted at 0.5 M sodium phosphate buffer. The affinity columns are usually water-jacketed to allow control of renaturation and discrimination among various sequences. Both optical density and radioactive tracers have been used to establish the reannealing rate. This method is useful for reassociation kinetics studies but other methods are usually preferred for hybridization. In solution hybridization systems, probe can be hybridized to target against a large background of nonspecific nucleic acid and hydroxyapatite may be used to distinguish hybridized from unhybridized probes (Steffan and Atlas, 1990). 8.1.1.6. Alternative solid phases Corti et al. (1991) adapted Porex absorbent filters for the detection of DNA using DIG-labeled probes. This flow-through assay is rapid (although for detectabilities below 50 pg it is necessary to increase hybridization times to 6 h) and seems readily adaptable if detectability is not a major concern. Poly(ethy1eneimine) (PEI) cellophane membranes have been used in nonradioactive hybridization assays (Vener et al., 1991). These authors used a stained latex-streptavidin conjugate (diameter 1.8 p.m) to detect captured biotin groups. Since the cellophane pores are only 0.4 p,m, accessibility was limited, but the detectability could be improved by modifying cellophane with PEL Cellophane membranes are boiled in 200 ml of 1 M Na,CO, for 30 min, washed three times in distilled water and treated with SO ml of 0.1 M NaIO, for 30 min at 22°C. The membrane is again washed in distilled water and incubated with 50 ml2% PEI in 0.1 M NaHCO, for 2 h with shaking and the Schiff bases were stabilized by rinsing the membrane with fresh 0.1% NaBH, and incubating for 1 h in the borohydride solution (constant shaking). After rinsing with distilled water, the membrane can be stored wet. DNA is immobilized on the membrane

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placed on a filter prewetted in distilled water and subsequently dried by IR irradiation. Hybridization is as for nylon, except that the biotinylated hybrids are detected by adding 0.1% gelatin to the signal buffer. Conway de Macario et al. (1990) adapted the slide immunoenzymatic assay (SIA) for DNA hybridization. This may be of interest for laboratories using the modular SIA system or instrumentation. Slides with sample circles are coated with polylysine or polystyrene, dissolved in toluene, to anchor the DNA.

8.1.2. Equipment Spot blotting is usually achieved in specifically designed vacuum filtration manifolds (e.g., Schleicher and Schuell Minifold I and 11, Bio-Rad, BRL). Two principal formats can be distinguished, each with its particular advantages. The dot format has the same dimensions as a microtiter plate and has almost twice as many samples/cm2 as the slot format. On the other hand, the slot format is easily adapted for scanning with a densitometer. Scanning of dot signals requires a more expensive scanner (e.g., USB SciScan 5000) which counts the density and total surface of the signal dot. Defined dots or slots can be excised and counted for both formats. Nucleic acids are traditionally fixed to membranes in a vacuum oven (a conventional oven can be used for nylon membranes). The house vacuum system is usually adequate and baking of most membranes is at 80°C. UV crosslinking is an attractive alternative although it is only recommended for specific membranes. Special equipment is available for sophisticated, well-controlled UV irradiation (e.g., Stratalinker from Stratagene). However, conventional transilluminators available in the laboratory perform equally well. Special hybridization ovens or convenient acrylic cassettes to be placed in a waterbath are commercially available. The membrane is placed in the hybridization cassette (e.g., from Bios Corp., New Haven, CT 06511; Fax (203) 773-0017), covered with (prelhybridization solution, the top closed over a cover and incubated at the desired temperature. ‘Hybriboxes’ can also be constructed as reported by Cohen et al. (1990; Section 7.2.6). The hybridization

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solution is retrieved through stopcocks and the membrane is washed. For reusable containers, it is essential that the removal of probe is complete to prevent a buildup of nonspecific label. More convenient, but also ten times more expensive, are hybridization incubators. Essentially, these temperature-controlled incubators have a built-in turning device (about 4 rev./min) to which firmly closed roller tubes, containing the membranes, are clamped. Since the tubes turn continuously, little hybridization solution is required. The various commercial models may differ in several aspects: (i) nature of tube (plastic or borosilicate glass; this is important for nonspecific adherence of probes and expected life-time); (ii) rate by which temperature is adjusted in the oven after door openings; (iii) whether the turning device has, in addition to rotation, also a lengthwise rocking (to prevent the solution staying at one end of the tube). In the case of frequent hybridization, this may be a wise investment. This special equipment can be economically replaced by inexpensive polyethylene bags. However, not all freezer bags are suitable. Sears Seal-N-Save, Layflat or Decosonic bags are widely used but KAPAK/Scotch Pak heat-sealable bags should be avoided for nonradioisotopic probes. After the bag is sealed along three sides near the membrane (with any electrical heat-sealer), buffer is added and the bag is examined for possible leaks. A minimal volume of buffer is added to the membrane (several membranes may be placed in the same container if they can move freely). Then, with as little air as possible, the fourth side is sealed off. The remaining air bubbles are forced out, after snipping one corner, by running a ruler along the bag to the opening and the bag is sealed. If the bag is agitated during (prelhybridization, a few air bubbles will not harm. To facilitate this procedure, it is useful to have at least one rounded corner of the membrane. This corner can be snipped off (removing air bubbles, removing prehybridization solution and/or introduction of hybridization solution). It is important to prevent radioactive spills (always on a piece of Benchkote (Whatman) in a tray). 8.1.3. Solutions and buffers used in hybridization 8.1.3.1. Blocking reagents Initially, the membrane-filter technique (Gillespie and Spiegelman,

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1965) constituted of hybridizing RNA to DNA bound to nitrocellulose. Nygaard and Hall (1963) observed that denatured DNA, but not free RNA, sticks to the membrane. Hybridization implied the intermediate step of making RNA complementary to the DNA (Green, 1963). Denhardt (1966) discovered that, after immobilization of the target sequence, remaining binding sites could be efficiently blocked with a cocktail of macromolecules (Denhardt’s solution; Table 8.4) without affecting the hybridization potential of the target. Tchen et al. (1984) obtained a better result in immunodetection of probes on nitrocellulose when albumin in Denhardt’s solution was replaced by glycine (same concentration). Nonfat dry milk can serve as a blocking agent, particularly in protein blots (Johnson et al., 1984). This ‘BLOTTO’ (‘bovine lacto transfer technique optimizer’) solution is easier to handle and a cheaper alternative to Denhardt’s solution, even in Northern blots after treatment with diethylpyrocarbonate (DEPC) (Siege1 and Bresnick, 1987). The sodium citrate in SSC causes dispersion of the white milk emulsion and the solution becomes clear. BLOTTO may contain considerable amounts of RNase and is not recommended for RNA hybridization unless this enzyme is inactivated. Denhardt’s solution is widely used and is excellent for all types of hybridization involving a solid phase. BLOTTO hybridization cocktails may occasionally yield inconsistent results and high background levels, particularly on nylon membranes. This may reflect variation in components of different lots or brands of dried skim milk (Westneat et al., 1988) and this blocking reagent is not recommended for the detection of small amounts of target. BM blocking reagent from Boehringer Mannheim (cat. no. 1096176) is a proteolytic digest of gelatine (contaminating enzymes are equally digested). BM blocking reagent is more efficient than BSA or BLOTTO. Autoclaving is not possible (precipitates will form and a strong background is observed with some detection systems). For RNA probes, a treatment of BM blocking reagent with DEPC to inactivate RNases is essential. However, treatment of this blocking reagent with DEPC for 30 min and heating for 2 h at 60°C leads to increased background on nylon. Holtke and Kessler (1990) obtained excellent results by dissolving BM blocking reagent at 5% in 0.1 M

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HYBRlDlZATlON WITH NUCLEIC ACID PROBES

TABLE 8.4 Hybridization stock solutions a. 20 x SSC 0.3 M sodium citrate + 3 M NaCl (pH 7.0). Sterilize by autoclaving. b. 2 M ammonium acetate (NH,OAc) (pH 7.0) C. 2 M NH,CI d. 3 M sodium acetate (NaOAc) (pH 5.7) e. Carrier DNA: 2 mg/ml (herring or salmon sperm DNA). Dissolve in water at 60°C, overnight, or cut in pieces with scissors, suspend in water and heat repeatedly in a microwave oven (a few minutes each time). Sonicate for 10 min and store at 4°C or - 20°C f. 100x Denhardt's solution: 2% Ficoll (av. MW 400000), 2% polyvinylpyrrolidone (av. MW 360000) and 2% BSA. Store at - 20°C after filtration g. 20xSSPE: 3.6 M NaCI, 0.2 M sodium phosphate (pH 8.0) and 0.02 M EDTA. Sterilize by autoclaving. h. 20% SDS (may be necessary to heat to 60°C to get in solution; store at room temperature) 1. Formamide (teratogen): deionized just before use with a mixed-bed ion exchange resin such as Bio-Rad AG501-X8 (1 g/100 ml and mixing for 1 h, followed by filtration through coarse paper); pH of formamide-containing solutions should be verified j. 50% dextran sulfate (store at 4°C): 50% dextran sulfate and 0.2% sodium azide. To prepare this suspension, 50 g DeSO, (close to 100 ml) is poured into a graduated bottle (previously marked at the level of 100 ml water plus a magnetic bar), containing the magnetic bar and a small amount of water is added. The DeSO, will dissolve slowly (add water to the 100 ml level). The suspension should be clear and, before use, should be taken out of the refrigerator and mixed for some time. Sodium azide should not be added when enzymes such as peroxidase are used. k. Glyoxal (7 M) should be stored in the presence of mixed-bed resin Amberlite MB-3. Glyoxal(10 ml) is mixed with 1 ml of resin and swirled on ice for 5 min. The phases are separated by filtration through Whatman GFC filters (pH of glyoxal solution should rise from about 3 to about 5.5 and the solution should turn less yellow. The deionized glyoxal is stored in 100 FI fractions at - 70°C. 1. Trichloroacetic acid (TCA): add 227 ml of water to a bottle containing 500 g of TCA, this yields 100% (w/v) TCA.

sodium maleate which had been previously treated with DEPC for 30 min at 25°C and then sterilized by autoclaving. This stock solution could be diluted up to five times with a sterile solution of 0.1 M sodium maleate for use on nylon and subsequent detection of RNA probes by the chemiluminescent system.

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Heparin (50 mg in 4 X SSPE or 4 X SSC) is also used as blocking agent at 50 p,g/ml or at 500 pg/ml if dextran sulfate is present (Singh and Jones, 1984). Lebacq et al. (1988) observed that heparin (a polyanionic mucopolysaccharide) lowered the general background significantly on positively charged nylon when used at 3.5 mg/ml. 8.1.3.2. (Pre)hybridization solutions

Formulations of different hybridization solutions and buffers are given in Table 8.4. Hybridization solutions can be prepared by combining the reagents, except carrier DNA (usually from salmon or herring sperm), and warming to 50"C, denaturing the sheared carrier DNA by boiling for 5 min, combining the suspensions and using the warmed solution immediately. Alternatively, all reagents can be combined and boiled for 5 min while mixing. The solution is then quickly chilled on ice to 50°C and used immediately. SSPE is often used, instead of SSC, when formamide is present to improve the buffering capacity. All hybridization solutions should be filtered through cellulose acetate filters (e.g., MSI Cameo 11s 0.22 p,m or S&S Uniflo 0.45 p m filters). Carrier DNA should be sheared with a sonicator to decrease the viscosity. Large quantities can be prepared at a time and a sonicator is needed infrequently. Alternatively, sheared carrier DNA can be purchased for a minimal extra charge.

8.2. Slot /dot blot hybridization on membranes Five steps can be distinguished in membrane hybridization: (i) immobilization of target nucleic acid; (ii) prehybridization to saturate the remaining binding sites which would otherwise adsorb probe nonspecifically; (iii) hybridization in low stringent conditions to adsorb probe as efficiently as possible; (iv) posthybridization washes to define the stringency of the hybridization and thus the specificity of the reaction; (v) the detection step (Fig. 8.2). In addition, the hybridized probes can sometimes be stripped from the blots to expose the targets to other probes.

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I

9 1 9

C

-w

I11

‘E Fig. 8.2. The detection of membrane-bound sequences (shown for a tailed probe in I) can be modified to considerably enhance the signal. One system (11) consists of target-specific primary probes and labeled, target-independent secondary probes. The secondary probes are specific for vector sequences of the primary probe. It is possible to have multiple primary probes, each recognizing a different region of the target. If the primary probes are M13, then all M13-specific secondary probes serve as universal probes. This patented system is marketed by ImClone Systems as AmpliProbe. Primary probes, labeled by random priming or nick-translation, also yield hyperpolymers (111) and hence a signal amplification. The current development of branched DNA could lead to a replacement of the hyperpolymer systems. Probes with partial overlaps, e.g., obtained with nick-translation, may also form hyperpolymers.

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Although there are adaptations for specific nucleic acids (Chapter 3) and a number of shortcuts (Section 8.2.41, traditional procedures to obtain DNA or RNA from cells are commonly used. For DNA, cells are pelleted and resuspended (to about 3 X lo6 cells/ml) in 10 mM Tris-HC1 (pH 7.4). An equal volume of 0.4 M Tris-HC1 (pH 8.01, containing 0.1 M EDTA, 0.1% SDS and 200 pg/ml proteinase K is added, mixed and incubated for 1 h at 55°C. The mixture is then extracted with phenol/chloroform (Section 3.1.2.1). For RNA, about 3 x lo6 cells are washed with cold PBS and pelleted in a microcentrifuge tube (15 s at 14000 X g ) . The cells are first resuspended in 40 p.1 of cold, sterile TE, containing 30 mM vanadyl-ribonucleoside complex and then mixed with 5 p1 of 5% Nonidet P-40 and placed on ice for 5 min. The nuclei are then pelleted and the supernatant is transferred to a fresh tube. RNA should be immediately denatured (Table 8.5; step C2) and immobilized or stored at -70°C. Alternatively, guanidine thiocyanate (GuSCN) allows a direct extraction of RNA or DNA and simultaneous inhibition of RNase (Sections 8.3.2 and 8.3.4.1) (Hopp et al., 1991). Usually, cells are dissolved in a solution with a GuSCN concentration between 2.5 and 5 M, and 0.5% sarcosine, 0.3 M Tris-HC1 (pH 7.5) and 10 mM EDTA. DTT at 0.5% may improve the solubility of proteins in GuSCN (Morissey and Collins, 1989). Elu-Quik bits (S&S) are convenient.

8.2.1. Immobilization of target nucleic acid Immobilization and retention of nucleic acid on solid phase is a critical step (Table 8.5). Baking of nitrocellulose membranes, to which nucleic acid has been adsorbed, is believed to create hydrophobic bonds between nitrocellulose and the nucleic acid. UV crosslinking (Church and Gilbert, 1984) results in a covalent bond between the NH, groups of the nylon membrane and the UVactivated thymine groups of the nucleic acid. UV has also been reported to be beneficial for nitrocellulose (Khandjian, 1987). In order to achieve uniform blotting, it is necessary to prewet the membranes with the blotting (transfer) buffer (e.g., 6 X SSPE, 20 X SSC, etc.) before placing in the blotting manifold. Although for nucleic acid immobilization, a high ionic strength yields the best results, we observed (Verbeek and Tijssen, 1988) that for immobiliza-

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 8.5 Standard procedures of nucleic acid immobilization A. DNA blotting on nitrocellulose 1. Denature DNA. Add 0.1 vol. of 3 M NaOH and 0.1 M EDTA and heat the sample to 95°C for 5 min. Neutralize by adding an equal volume of cold 2 M ammonium acetate (pH 6.5). Concentrated DNA should be treated before use (restriction enzymes, sonication) to decrease viscosity and improve binding. DNA can also be microwaved for 0.5-5 min and rapidly cooled. 2. Prewet the membrane. Float on water taking care not to trap air bubbles (otherwise boil briefly), immerse when completely wet, blot lightly on Whatman 3MM paper or S&S GB003 (cheaper) and transfer to a dish with 20 x SSC or 1 M ammonium acetate and assemble in the slot or dot apparatus (always wear gloves when handling membranes) according to the manufacturer’s recommendations. Without a manifold, high salt concentrations improve DNA binding and keep dots small. 3. Wash membrane with 1 M ammonium acetate or 6XSSC, apply a gentle vacuum, turn vacuum off when dry and add 50-500 pl of denatured sample (smaller volumes of sample give better results; < 10 p g of DNA) and turn on vacuum. 4. Add 500 pl of 2 X SSC after the sample has filtered through. 5. When the wells are empty, disassemble the apparatus, rinse the membrane in 2 X SSC and allow the membrane to dry. 6. Bake the membrane for up to 2 h at 80°C in a vacuum (for S&S membranes 30 min to 2 h, depending on the number of blots) or fix DNA with UV as in B4 (for those cellulose brands for which W irradiation is recommended by the suppliers; e.g., Duralose W). Note that the nitrocellulose should be damp for best binding and prevention of ignition. 7. Store membrane between filter papers in a plastic bag at room temperature.

B. DNA blotting on nylon or Immobilon-P 1. Denature DNA as in Al. Alkali-denatured DNA can be applied directly to a Zeta-Probe membrane (but 0.2 vol. of 5 M NaCl should be added to the sample prior to applying to Nytran), but can then not be reprobed (otherwise neutralize before blotting). 2. Proceed as in A2-3, except for wetting the membrane with water. Immobilon is first rinsed for a few seconds with methanol. If alkaline DNA is blotted, it is preferable to wet the membrane also with 0.2 N NaOH. 3. Add 500 pl of denaturation solution to each well and apply a vacuum (optional). 4. Disassemble the apparatus when wells are empty and UV irradiate the samples (better and more convenient than baking). Baking or UV irradiation of alkaline-immobilized DNA on charged nylon membranes may be counterproductive. When using UV irradiation for the first time, it is recommended to optimize the time required (usually 1-5 min) for best binding to the damp membranes (e.g., uncovering a successive dot every 30 s); always use the same distance from the source. Store as in A7.

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TABLE8.5 (continued) C. RNA blotting, 1. Prepare membrane as in A2 (20 X SSC for nitrocellulose and 5 X SSC for nylon). 2. Denature RNA; although it is usually ss, it may contain considerable secondary structure. Several methods are used (prevent RNase activity): a. Weak alkaline denaturation; RNA is dissolved in 500 pI of ice-cold 10 mM NaOH, 1 mM EDTA and immediately applied to a nylon membrane. The membrane is then washed with the same buffer. b. Formaldehyde denaturation; 30 pI of 2OXSSC and 20 IJ.I of 37% formaldehyde is added to 50 p1 of RNA and incubated for 15 min at 60°C and applied immediately to nitrocellulose or a nylon membrane. c. Glyoxal denaturation; dry down 10 pg of RNA in a microfuge tube and dissolve in 5 pl of RNase-free water. Complete the solution to a final concentration of 50% DMSO, 10 mM sodium phosphate (pH 7.0) and 1 M glyoxal and incubate for 1 h at 50°C (avoid evaporation). Spin briefly and apply immediately to a nylon membrane and wash with 500 pl of TE. Remove glyoxal adducts (after baking) by pouring 20 mM Tris-HCI (pH 8.0) and 1 mM EDTA, heated to 9 5 T , onto the membrane and shake until cooled down (alternatively use nitrocellulose as in Section 8.2.1). d. Metbylmercuric hydroxide (Section 9.1.3). 3. Proceed as in A3-5, but use 2OXSSC for rinse before and after the application of RNA. 4. Bake nitrocellulose membranes as in A6 whereas nylon membranes are air-dried. Both types of membranes can be stored as in A7.

tion of viral nucleic acid without prior RNA extraction from the virus (to prevent RNA degradation), low ionic strength buffers are best suited. For instance, spotting of virus with 1 X SSC, followed by baking produced superior immobilization of the virus and subsequent liberation and fixing of the viral RNA during baking. Loss of extracted RNA target during solid phase hybridization due to RNase activity in the samples can be considerable (Rotbart et al., 1987) and the signal depends directly on the quantity of intact virions in the specimen. RNase inhibitors are essential for reliable RNA hybridization assays. The amount of DNA required per dot depends on the number of target sequences present. For single-copy genes in mammalian chromosomes, detection is achieved with 1-10 k g of total target nucleic acid. Proportionately less can be used when multiple copies are

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present. On the other hand, for high detectabilities, as much as possible of a sample is loaded in a dot. Supercoiled DNA should be linearized before denaturation. This can be achieved by restriction digestion (followed by phenol extraction and ethanol precipitation; Section 3.1.4.1) or by boiling (in 100 p,l of 0.02 M Tris-HCI, pH 7.4, 1 mM EDTA) for 10 min, resulting in partial depurination, and subsequent alkali treatment during blotting which results in the breaking of phosphodiester bonds at the sites of depurination. Genomic DNA is very viscous and its size should be reduced by restriction endonuclease digestion or by sonication (optimum size about 2-3 kb; check by agarose gel electrophoresis). The DNA concentration can be determined by measuring its optical density (1 OD,,, = 50 pg/ml). Nucleic acid should be ss for immobilization although ds can be blotted onto nylon. Blotting of ss nucleic acid is achieved by either heat-denaturation in low ionic strength buffers, by alkali treatment or microwave denaturation. Alkali denaturation eliminates RNA and the base-catalyzed fixation of DNA on charged nylon obviates the need for a separate fixation step. A separate UV fixation can even be counterproductive since it often diminishes the ability of fixed DNA to form hybrids (Reed and Mann, 1985). Microwave denaturation (0.5-5 min in water) and snap-cooling is superior to heat denaturation and leaves nucleic acid intact, in contrast to heat-denaturation (Stroop and Schaefer, 1989). Samples are then diluted in 500 p,l of high ionic strength buffer (6 X SSPE, 20 X SSC, etc.) in the case of nitrocellulose membranes (and sometimes also for nylon) and applied to the membranes in the blotting manifold under vacuum. Among the transfer buffers, Allefs et al. (1990) found 1 M NH,OAc or NH,C1 to yield slightly superior results (signal twice as strong), both for nylon and for nitrocellulose membranes. Other nylon membranes (e.g., Genescreen) require low ionic strength buffers for efficient binding. If a vacuum manifold is not available, membranes can be dried to prevent large dots, after they have been prewetted and soaked in high salt solutions and stamped with a rubber stamp and ink pad with an array of 5 mm circles. The dry membrane is placed on a plastic box without a cover so that only the edges of the box touch

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the membrane. Small samples can then be blotted, if necessary with repeated applications allowing time after each application to dry. Denature DNA by floating the nitrocellulose membrane (application side up) on a Whatman 3MM paper saturated with 1.5 M NaCl and 0.5 N NaOH for 5 min. The membrane is then transferred to another filter paper saturated with 0.5 M Tris-HC1 (pH 7.4, 30 s> and a second one saturated with 1.5 M NaCl and 0.5 M Tris-HC1, pH 7.4 for 5 min. The membrane is then dried before baking. Tchen et al. (1985) squashed complete etherized Drosophilu flies on nylon or nitrocellulose membranes. The membranes were placed on a filter which was sandwiched between two plastic sheets cut from heat-sealable plastic bags. The flies were squashed one by one using a spatula (photocopies after squashing for further localization of specific flies). The plastic sheets were then discarded and the membrane layered successively for 2 min on Whatman 3MM paper (soaked with 10% SDS or H,O), twice for 5 min on 0.5 M NaOH (or KOH)/2.5 M NaCl and finally three times 2 min on NaOAc (pH 5.0). After neutralization, large fragments (wings, etc.) were removed with forceps and the blots dried for 30 min at room temperature and baked in a vacuum at 80°C for at least 1 h. Here, best results were obtained with Pall ‘Posidyne’ membranes. Although RNA is ss, it usually does not bind to membranes (Nygaard and Hall, 1963) due to the often considerable secondary structure. Alkali treatment is hardly possible (hydrolysis RNA) and DMSO dissolves nitrocellulose membranes. Glyoxal has been proposed by Thomas (1980) and has been widely used. Glyoxal binds to guanidine residues and the complex binds efficiently to nitrocellulose after which the glyoxal is removed by incubation at 100°C at pH 8.0. However, RNA should be free of protein which may react with glyoxal. Baking of nitrocellulose or nylon is often optimum for 2 h in vacuo (vacuum not necessary for nylon). Shorter baking periods lead to inferior detectabilities for some brands or lots but not for others. On the other hand, it is necessary to bake for as short a time as possible since the nitrocellulose membranes become progressively more brittle. UV crosslinking in a StratalinkerTMis optimal at about 100-120 mJ/cm2 (at 254 nm) and takes less than a minute. UV

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fixation was found to give superior results, especially for amphoteric nylon (Holtke and Kessler, 1990). It is necessary to calibrate the transilluminator used in the laboratory for optimum results (intensity of UV irradiation decreases over time). Usually, it will take about 3 min at 300 nm. Allefs et al. (1990) found an optimum time of 1 min using a Philips TUV 30 W tube. DNA immobilized by baking will leach off the surface, particularly if incubated for prolonged periods at high temperatures. If the probe covers the entire length of the target sequence, the hybrid could dissociate into the solution (Haas et al., 1972). Although there are some counterclaims about the improving effect of UV crosslinking for nitrocellulose or charged nylon (Nierzwicki-Bauer and Gebhart, 19901, the simplicity and rapidity of the procedure are attractive. Nevertheless, there is a difference in lots of membranes of the same brand with respect to their performance in baking and UV crosslinking. UV immobilized nucleic acid seems not as well retained during reprobings as that after baking. The blotting on diazotized transfer media is different. The diazonium groups on the surface of the membrane are very unstable and will be inactivated at high pH and temperature (hydrolysis). Maximum reactivity is maintained at pH 4.0. Just before use, the paper is activated (explained here for APT paper). Ice-cold 1.2 N HCl is prepared (120 ml for a 120 cm2 sheet) and 3.4 ml of fresh 10 mg/ml NaNO, is added. It is essential that the nitrite solution is fresh. The APT paper is immersed and left to react for 30 min or longer at 0°C (paper will turn into a deep yellow color). Just prior to transfer, the paper is washed twice for 1 rnin in ice-cold 20 mM NaOAc (pH 4.0). Low temperatures are essential to preserve the activity of the surface groups. The nucleic acid to be blotted should not be in a solution with a primary amine group (e.g., Tris) since these will inactivate the diazo groups. Low ionic strength of the transfer medium gives best results (e.g., 20 mM NaOAc, pH 4.0). The APT paper will change color from canary yellow to a reddish-brown as immobilization occurs. The unoccupied diazonium groups remaining on the membrane can be completely inactivated by prehybridization with the hybridization solution (Table 8.6) containing glycine (1.0%) or NaI (0.1 M).

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TABLE 8.6 (Pre)hybridization solutions and conditions Abbreviations: 1 X salt ( = 1 X SSPE or 1 X SSC + 10 mM sodium phosphate (pH 6.8), + 1 mM EDTA; EDTA is omitted if no DNA is involved); DHS, Denhardt’s solution; carDNA, carrier DNA, BAHS, basic aqueous hybridization solution; BFSH, basic formamide hybridization solution; NC, nitrocellulose; N, nylon; CN, charged nylon; prefiv D indicates DNA target (e.g., D-NC is nitrocellulose with DNA target) and R is RNA target; PH, prehybridization; HYB, hybridization; DHS, SDS, carDNA, BM blocking reagent or BLOTTO are all intended to avoid nonspecific sticking of the probe to the membrane, albeit with different efficiencies and different side-effects; for single-copy genes or RNA, 5 X DHS + 0.5% SDS + 100 p.g/ml carDNA is often optimum, whereas BLOTTO instead of DHS is often a good starting point for abundant target DNA; DHS tends to give higher background on most brands of nylon. When DeSO, is used, carDNA can often be omitted from hybridization solutions but not from prehybridizations. Recommendations for blocking agents: 5 X DHS, all applications; 10 X DHS, only for colony/plaque lifts; BLOTTO, primarily for colony/plaque lifts; yeast tRNA, primarily for RNA blots but not recommended for colony/plaque lifts; homopolymer DNA, for AT- or GC-rich target DNA. Hybridization solutions

BAHS

BFHS

Salt DHS (decrease to 1-3 X for nylon) SDS DeSO, or PEG (optimal) carDNA (250 pg/ml for RNA) Tetrasodium pyrophosphate (optional) Formamide Sodium phosphate (pH 6.8) (optional)

5x 1-5 X 0.5% 10% 100 kg/ml 0.3%

5X 1-5 x 0.5% 10% 100 pg/ml 50% 50 mM

Note: DHS should be replaced by other blocking agents as noted above and in the protocols below; SDS concentrations should be increased to at least 1% for nylon membranes although for nonradioactive probes it may have to be lowered. For oligomers, 1.8 M NaC1, 0.24 M Tris-HCI (pH 7.81, 12 mM EDTA, 1 0 x DHS and 0.1% can also be used. When oligomers bind poorly to membranes, e.g., nitrocellulose, blocking agents are less necessary for oligomer probes.

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TABLE8.6 (continued) Al. Nitrocellulose, radioprobes a. DNA probes D-NC: BFHS; PH for 1-6 h at 42°C and HYB for 15-24 h at 42°C. D-NC: BAHS; PH for 2-6 h at 68°C and HYB for 15-24 h at 68°C. R-NC: BFHS; PH for 8-16 h at 50°C and HYB for 5-24 h at 50°C. R-NC: BAHS not recommended. b. RNA probes D-NC BFHS; PH for 1-6 h at 50-55°C and HYB for 15-24 h at 50-55°C. D-NC: BAHS not recommended. R-NC BFHS; PH for 4 h at 55°C and HYB overnight at 55°C R-NC BAHS not recommended c. Oligonucleotide probes (the simplest method is usually adequate) D-NC: 2 PHs; PH1: BAHS without DeSO, but with 0.1 mg/ml proteinase K at 65°C for at least 2 h; PH2 (8°C below T , ) in BAHS containing 100 p,g/ml poly(A) (if oligomer probes used are tailed with ["PI- or [35S]dATPwith TdTase) and 0.1 mg/ml proteinase K. HYB: same solution as PH2 and same temperature, overnight. R-NC: BAHS containing 200 p,g/ml poly(A); PH: Td -8°C for 4 h; HYB: same. R,D-NC: 5xsalt, 5xDHS; PH for 0.25 h at Td -8°C and HYB for 1 h at same temperature. A2. Nitrocellulose, nonradioactive probes As in Al, except for an increase in blocking reagent, e.g., 6 x D H S and 300 p,g/ml carDNA. Higher probe concentrations allow shorter hybridization periods. A convenient method is: D,R-NC: 5 xsalt, 0.5% BM blocking reagent (dissolve by heating for 1 h at 60°C; Boehringer Mannheim), 0.02% SDS, 0.1% sarkosine (solution remains turbid); PH for 2-6 h at 68°C and HYB for > 6 h at 68°C. If 50% formamide is added, increase BM blocking reagent concentration to 5% and (pre)hybridize at 42°C (DNA probe), 55°C (DNA target, RNA probe) or 68°C (if both target and probe are RNA). B1. Nylon, radiolabeled probes a. DNA probes: as in Al; the carDNA concentration can usually be decreased to 50 pg/ml. b. RNA probes: 0.25 M sodium phosphate (pH 6.9, 0.25 M NaCI, 50% formamide, 7% SDS, 1 mM EDTA, 15% PEG;; PH for 2-6 h at 42°C and HYB for 15-24 h at 42°C. This method gave optimal detectability and specificity in papilloma virus typing (Auvinen et al., 1989). c. Oligonucleotide probes: as for nitrocellulose, but with increased blocking agent and carDNA concentrations since nylon binds oligomers efficiently. Alternatively: PH in: 6xsalt, 0.5% SDS, 100 p,g/ml carDNA, 0.1% BLOTTO for 2-6 h at Td -8°C; HYB in 3.0 M TMAC (or 2.4 M TEAC or 2.4 M TEAB), 10 mM sodium phosphate (pH 6.81, 0.5% SDS, 100 p,g/rnl carDNA, 0.1% BLOTTO for 2-6 h at Td -8°C.

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TABLE 8.6 (continued) B2. Nylon, nonradioactive probes a. RNA or DNA probes: as in A2 (convenient method) with 50% formamide and 5% BM blocking reagent (can be replaced by 5 X DHS, although with increased background). b. Oligonucleotide probes: D,R-N: PH: 6Xsalt, lOxDHS, 0.1% SDS (or 0.02% SDS and 0.1% sarkosine), 100 pg/ml carDNA, for 1-3 h at T, -6°C; remove PH solution and add probe in PH solution (without DHS and carDNA) and incubate for up to 4 h at the same temperature as PH. C1. Charged nylon, radiolabeled probes It may be necessary to use 10 X DHS or 0.5% BLOTTO a. DNA probes: D-CN: 0.5 M sodium phosphate (pH 7.21, 7% SDS, 1 mM EDTA, PH for 0.1 h at 65°C and HYB for 5-24 h at 65°C. R-CN: PH: BAHS for 12-24 h at 42°C; remove PH solution completely and proceed with HYB: BFHS (1% SDS, SO pg/ml carDNA (10% DeSO, optional)) for 24 h at 42°C. Include 100 pg/ml tRNA in both PH and HYB. b. RNA probes: as for DNA probes to RNA targets, except for adding 100 pg/ml tRNA to both PH and HYB solutions and incubations at 50°C. In the case of RNA targets, the incubation temperature is raised to 55°C. c. Oligonucleotide probes D-CN: PH in BFHS for 3 h at Td -6°C; remove PH solution completely and proceed with HYB (6xsalt/l% SDS) at T, -6°C for up to 4 h. C2. Charged nylon, nonradioactive probes As for radiolabeled probes (Cl) D. APT paper (RNA or DNA target) PH: in BFHS+ 1% glycine (or 0.1 M NaI) for 30 min at 42°C and HYB (without glycine) overnight at 42°C.

8.2.2. Hybridization conditions

Hybridization conditions vary with the nature of the target (RNA, DNA), of the probe (polynucleotide versus oligomer; radiolabel versus nonradioactive labels; RNA versus DNA; ds versus ss) and of the membrane. The impact of the different parameters will be reviewed to facilitate the optimization of hybridization. 8.2.2.1. Prehybridization

Prehybridization serves two purposes: (i) it blocks the binding sites on the solid phase to which probe may nonspecifically adhere; (ii) it

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equilibrates the solid phase with the hybridization solution and conditions (e.g., temperature). In some cases of capture hybridization on particulate solid phase, prehybridization has little effect on the background levels but promotes hybridization efficiency (Gingeras et al., 1987). The composition of the prehybridization solution buffer may have to be adjusted for optimal detectability. In particular, the nature and incubation of the heterologous nucleic acid can be essential to prevent adherence of the probe to either the heterologous nucleic acid or unoccupied binding sites on the solid phase. The hybridization to nontarget nucleic acid can, if it is a problem, be eliminated or decreased by adding the same heterologous nucleic acid in excess to the hybridization solution. The addition of poly(A) can be useful if the membrane-bound sequences are rich in A,T residues. Similarly, poly(C) is useful for GC-rich targets or when GC homopolymer tailing has been used in the construction of target nucleic acid. Before prehybridization, nitrocellulose should be wetted with 1 x SSPE (or 1 X SSC), containing 0.1% SDS, whereas nylon membranes can be used directly. Prehybridization and hybridization is performed in polythene bags, plastic boxes (hybridization cassette) or hybridization incubators (Section 8.1.2). The cassettes and incubators are economical in the use of hybridization solutions. For plastic bags, use 0.25 ml/cm2 for nylon membranes and 0.1 ml/cm2 for nitrocellulose membranes of prefiltered (important!) prehybridization solutions. The bags should be massaged to completely distribute the solution and incubated with agitation. As a general rule, prehybridization conditions (buffer and temperature) are, except for oligonucleotide probes and charged nylon, identical to those of hybridization although the time is shorter (usually fast but prolonged prehybridization does not harm). Most (prelhybridization buffers are variants of two basic buffers, one with formamide and one without. Formamide allows the use of lower incubation temperatures and an easy adjustment of the stringency and is generally used, or even essential, for RNA hybridization or for nonradioactive probes. It is recommended, particularly when formamide is used, to include a buffer, such as sodium/potassium phosphate, Tris-HCI, or PIPES-NaOH, or to use SSPE instead of

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SSC. EDTA is added to the buffers to protect DNA although SSC is slightly chelating. Carrier DNA is added to high concentrations in the case of RNA probes and relatively low concentrations on charged nylon membranes. Instead of (or in addition to) carrier DNA, tRNA is sometimes chosen for RNA probes. High SDS concentrations improve the signal/noise ratio for hybridization on nylon membranes, i.e., > 0.5% SDS for radioprobes although nonradioactive probes may require the use of lower concentrations (e.g., 0.02% SDS + 0.1% sarcosine). SDS at > 0.5% inhibits RNase and is useful whenever RNA is involved. Nylon membranes require an efficient blocking due to their high binding capacity. For this purpose, the Denhardt’s solution is often increased to about 10 X in prehybridization solutions but is omitted from the hybridization solution on charged nylon since at > 5 X it may interfere with the hybridization. RNA and oligomer probes have less tendency to stick to nitrocellulose than DNA probes and lower concentrations of Denhardt’s solution can be used. Tchen et al. (1984) observed that for nonradioactive probes, glycine instead of BSA in Denhardt’s solution yields better results. The incubation temperature during prehybridization depends on the probe used and the formamide concentration. Usually 40-50°C for DNA probes (in 50% formamide) or 55-65°C for RNA probes (in 50% formamide) and 20°C higher in aqueous solutions. For each % of mismatch, the incubation temperature can be lowered by 0.8-1°C or the formamide concentration decreased by 1.5%.

8.2.2.2. Hybridization Prehybridization solution is removed from the bag and the hybridization solution is added but probe may be added directly to prehybridization solution (Section 8.1.2). Hybridization buffers should match those used for prehybridization in the case of radiolabeled probes and nitrocellulose membranes. The volume of the hybridization solution should be kept as small as possible. The probe is evenly distributed by massaging the bag and incubating at the proper temperature with agitation. The probe should be clean (spin column, Cameo IIS or Uniflo cellulose acetate filters for radioprobes) and denatured, if necessary, by boiling in TE for 5 min (radioprobes,

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nonradioactive probes) or incubating with 0.1 vol. of 0.1 M NaOH for 5 min at 37°C (radioprobes; many nonradioactive probes are not stable in alkaline conditions). Probes denatured by microwaves (0.5-5 min and snap cooling) yield about 20 times stronger signals than heat-denatured probes (Stroop and Schaefer, 1989). For single-copy genes 1-5 x 106 cpm/ml (10 ng of 1-5 X 108 cpm/p,g per ml) may be required, but > 10 ng/ml radiolabeled probe should be avoided. The optimum concentration for ss and for ds probes can differ more than 10-fold (Tchen et al., 1984) due to reannealing of the latter. Hybridization conditions are usually chosen so that the annealing rate is optimal although with increased cross-hybridization it is advantageous to use more stringent conditions. Relaxed conditions (T, - 25°C) for optimum hybridization also lead to an increased formation of nonspecific hybrids which can usually be removed by subsequent washing at the desired stringency. Table 8.6 lists several protocols, which may serve as a starting point, but optimization of hybridization conditions and incubation time may be necessary for a particular target/solid phase/probe combination. Salt conditions are usually the least critical, provided they are sufficiently high. Blocking reagents each have particular efficiencies and (disladvantages. An optimum balance among Denhardt’s (or BLOTTO with high concentrations of target) solution, detergents (SDS, sarkosyl) and carrier nucleic acid should be determined empirically. This is particularly important when the carrier nucleic acid has a tendency to anneal with the probe (otherwise quite effective). When accelerators are used (Section 8.2.2.31, it is necessary to include them when optimizing the blocking mixture (Auvinen et al., 1989). Although the optimal incubation period can be predicted to some degree (Section 2.3.5.11, it is necessary to establish empirically which combination of probe concentration and incubation time yield best signal/noise ratios. Probe reassociates (if denatured ds), target nucleic acid leaches off and probe is gradually degraded during hybridization. A probe with a complexity of 5 kb and at 10 ng/ml will obtain its Cot,, in about 20 h in typical hybridization conditions, whereas a purified 1.5 kb insert (also at 10 ng/ml) will reach it in about 6 h. A simple rule of thumb (probe in excess) is T , (h)

=

[complexity of probe]/ [ 25( ng/ml of probe)]

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Nonradioactive probes, which can be used at 10-40 times higher concentrations than radiolabeled probes, thus have a shorter incubation period. However, this period is only 3-8 times shorter since (i) labeling of these probes decreases the T, and the hybridization rate compared to radioprobes (hybridization often at 45% formamide at 42°C); (ii) degradation of probe and desorption of target are less important; (iii) accelerators, such as DeSO,, are usually avoided to prevent background; (iv) less background problems, particularly for nitrocellulose membranes, allow longer incubations. Although nonradioisotopic probes are used at high concentrations (e.g., 100 ng/ml), they may be reused if stored at 4°C (after heat-denaturation). DeSO, is often included in formamide-containing buffers for radioprobes since formamide slows hybridization. In some protocols, hybridization solutions are prepared by adding 0.25 vol. of 50% DeSO, to the prehybridization buffer (decreasing the concentration of the other components to 0.8 of the original) whereas in other cases a separate hybridization buffer is made (similar to the original plus DeSO,). The addition of DeSO, to nonradioactive or oligomer probe solutions would be at most marginally beneficial but is often counterproductive (e.g., Holtke and Kessler, 1990) due to increased background. In contrast, van Gijlswijk et al. (1992) noted that DeSO, increases hybridization about 10-fold while background could be reduced by washing and blocking at higher temperatures. Moreover, casein used in the immunodetection and Tween-20 in the washing buffer, reduce background staining. RNA (ss) is nonenzymatically hydrolysed in aqueous solutions at elevated temperatures (Tenhunen, 1989; Kaga et al., 1992). At 80"C, degradation occurs within 2 h, whereas at 60°C ss RNA is partially destroyed within 6 h. Conditions generally used for DNA hybridization should therefore be modified, especially for quantitative studies of RNA. Since the length of hybridization affects the final results, this could explain why the degree of mismatch necessary to form a stable hybrid in RNA: RNA hybridization is much less than that predicted by calculations. The addition of formamide allows hybridization to be carried out at lower temperatures without changing the stringency. Immediately before use, RNA probes are precipitated and 0.1 ml

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of yeast tRNA (20 mg/ml), 0.5 ml of carrier DNA and 0.6 ml of deionized RNA are added, mixed thoroughly and heated at 70°C for 5 min. RNA probes tend to give high background signals on nylon and stringent conditions may be required. In reverse hybridization, unlabeled probes are immobilized on nitrocellulose membranes (0.5 pg per spot), the test sample is photochemically labeled (Section 7.8.2) and hybridized simultaneously to the panel of probes (Dattagupta et al., 1989). It is not necessary to purify sample DNA before or after labeling and a 300 pl sample is added to 1.1 ml of the prewarmed hybridization solution (3 X SSC, 20 mM pyrophosphate, 5% BLOTTO, 10% PEG, 1 mg/ml carDNA, prehybridization 30 min, hybridization 2 h). The haptens are then detected with labeled antibody or streptavidin. Labeling of oligonucleotides to high specific activity using TdTase is essential to achieve high signal/noise ratios in solid phase hybridization (Henderson et al., 1991). Tailing does not affect the hybridization rate at the same oligomer concentration, since the complexity changes very little if, e.g., only As are added.

8.2.2.3. Acceleration of hybridization by polymers Some polymers can be used as accelerators of nucleic acid hybridization. They sometimes produce background staining and are only recommended when the hybridization rate is low (small amounts of probe or target). Dextran sulfate is often used as an accelerator but polyethylene glycol (PEG 6000) and sodium polyacrylate have been shown to be valuable alternatives. These polymers probably act by exclusion of probe molecules from the volume occupied by the hydrated polymer which results in an effective increase in the probe concentration (Wetmur, 1975). The optimal probe concentration in membrane hybridization is considered to be about 106 cpm of probe/ml (1-10 ng/ml), but Amasino (1986) found a 2- to 10-fold reduction in the probe concentration to be optimal when 10% PEG was included (note that these effects may depend on the specific activity of the probe). These polymers do not act in a similar way and have their own characteristics. In hybrids with ds probes, DeSO, is often found to give stronger signals, probably due to the enhanced networking effect of this

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polymer (Wahl et al., 1979). However, hybridization of ss probes in the presence of PEG is significantly faster than with DeSO, (Amasino, 1986). In addition, Parkkinen et al. (1989) observed that PEG yielded also a higher signal. Moreover, PEG is cheaper and less viscous. The optimal PEG concentration is from 10 to 15%. SDS at a concentration of 7% in combination with 15% PEG also gives optimal specificity (Auvinen et al., 1989). These authors also compared the suitability of DeSO, and PEG for membrane hybridization with RNA probes for typing of papilloma virus strains and observed that the use of PEG and high concentrations of SDS provides specific and sensitive conditions for differential typing. Yehle et al. (1987) used 2% sodium polyacrylate instead of 10% DeSO, since it is less viscous and easier to pipette. Hybridization can also be accelerated by the addition of certain DNA-binding proteins (e.g., those involved in natural recombination processes). Alberts and Frey (1970) observed that DNA hybridization kinetics was comparable in low salt (Mg*+-containing)buffers, but in the presence of T4 gene 32 protein, to that seen at an ionic strength of 1 at 70°C. Similar results were observed with, e.g., single-strand binding (SBB) protein (Christiansen and Baldwin, 19771, RecA protein (Bryant et al., 1989), helicases (Kodadek and Alberts, 1987; Roman and Kowalczykowski, 1989), or SV40 large T-antigen (Schiedner et al., 1990). Similar effects exist for RNA-binding proteins, e.g., nuclear riboprotein A1 increases the local concentration of complementary strands for rapid molecular assembly ( k , increased 300-fold) (Kumar and Wilson, 1990; Pontius and Berg, 1990). Although these proteins are as yet not used on a large scale in hybridization, they have promising potential. 8.2.2.4. Posthybridization washes The temperature and salt concentration during washes are usually more stringent than during hybridization since the overall stringency of the hybridization procedure is generally determined at this stage. The conditions are commonly chosen so that the incubation temperature is constant, e.g., 20°C below T, (at 5 X SSC), with increasing stringency of the washes (e.g., from 2 X SSC to 0.1 X SSC (with

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SDS)). The optimal conditions are established empirically although those given in Table 8.7 can serve as a guideline. Well-matched hybrids ( > 100 bp) can be washed in stringent conditions (0.1 X SSC, at the same temperature), whereas poorly matched hybrids are washed in high salt conditions (2-6 X SSC). An important consideration is the criterion which establishes the lower limit of fidelity of base pairing. Hybridization at an open (relaxed) criterion followed by a wash at a stringent criterion may not yield the same result as hybridization and wash at a stringent criterion. Polynucleotides usually contain AT-rich and GC-rich regions and the T,, reflects that of the total hybrid. However, the AT-rich regions will melt first and the difference in T, of these distinct regions can be as much as 20°C in the same duplex. Nonspecific hybrids, still existing through GC-rich regions below the criterion temperature, are very slowly removed. Whenever this is a problem, washing solutions which negate the difference in stability of A/T and G/C base pairs can be used. TEAC, or its bromide counterpart (TEAB), allow the differential elimination of hybrids differing as little as 1°C in their T, (TEAC purified as in Section 8.3.1). Although theoretically the T,, then becomes concentration-dependent (Tdr; Section 2.2.4.3), washing periods are short compared to the progressive release of probe and this effect will be negligible. Nitrocellulose membranes are not stable when submerged over prolonged periods in solutions containing TMAC, TEAC or TEAB; hence, washing periods with these salts should be kept short. Washed nylon membranes can be kept damp (e.g., sealed in a bag) so that more stringent washes can be added, if necessary. Moreover, damp membranes allow stripping and reprobing. The formulation of the washing buffer is important for nonradioactive probe detection where a blocking step to avoid nonspecific detection is required. This is possible with 5% BM blocking reagent (Section 8.1.3.1). For the detection of digoxigenin, Fab fragments gave considerably less background than conjugates of complete antibodies (Holtke and Kessler, 1990). Biotinylated probes are detected after a blocking step by vigorous shaking in 0.1 M NaCI, 0.1 M Tris-HC1 (pH 7.9, 0.5% Tween 20 and 3 mM MgCl, for 90 min at 22°C. Streptavidin is added at 2 pg/ml and the membrane is shaken

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TABLE8.7 Posthybridization washes and probe detection Use 0.5 ml/cm2 and a stringency which yields optimum signal/noise ratios while maintaining the desired detectability. The stringency can be adjusted by the ionic strength, the temperature of the wash and the concentration of SDS. For oligonucleotides and nonradioactive probes, 0.5% Tween 20 can be used instead of SDS. The hybridization solution is first collected (can often be reused), followed for radioprobes by a ‘hot rinse’, to remove the bulk of radioactivity, at room temperature. A. Radiolabeled probes

Low stringency: Twice 15 min in 1 XSSPE, 0.1% SDS at 22°C Twice 15 min in 1 XSSPE, 0.1% SDS at 37°C Medium stringency: Twice 15 min in 1 XSSPE, 0.1% SDS at 37°C Once 15 min in 1 X SSPE, 1% SDS at 37°C Once 15 min in 0.1 xSSPE, 1% SDS at 37°C High stringency: Once 15 min in 0.1 XSSPE, 1% SDS at 37°C Three times 15 min in 0.1 X SSPE, 1% SDS at 65°C Autoradiography: rinse membrane in 0.1 X SSPE and wrap membrane in Saran wrap and expose to film as described in Section 7.2.5.1.

B. Nonradioactive probes Low stringency: Twice 3 min in 2XSSPE, 0.1% SDS at 22°C Once 3 min in 1 x SSPE, 0.1% SDS at 37°C Medium stringency: Twice 3 min in 1 xSSPE, 0.1% SDS at 37°C Once 3 min in 0.2XSSPE, 0.1% SDS at 37°C High stringency: Once 3 min in 1 XSSPE, 0.1% SDS at 37°C Twice 15 min in 0.16XSSPE, 0.1% SDS at 50°C (nitrocellulose) or 65°C for nylon-bound hybrids Probe detection: a. Rinse membrane twice in 2 X SSPE (SSC if enzymes have been immobilized) at room temperature (3 m i d . b. Incubate membrane with NSSB buffer (Section 8.2.3, formulation dependent on membrane and nonradioactive label) to prevent adsorption of streptavidin, antibodies, etc., to solid phase. c. Incubate membrane with conjugate in NSSB buffer for 30-90 min and rinse three times with same buffer.

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TABLE8.7 (continued) Note: van Gijlswijk et al. (1992) observed (using positively charged nylon membranes) that incubation of antibodies (polyclonal) at high ionic strength (0.6 M NaC1) combined with a wash at low ionic strength (0.15 M NaCI) gave much better results than having both steps at either low or high ionic strength. d. Incubate membrane with developing buffer for the detection of enzymes (Sections 8.2.3, 7.3.3.4, and 7.3.3.5). C. Oligonucleotide probes Wash several times (e.g., 4 X 10 min) in 5 xsalt (Table 8.6)+0.1% SDS at T, - 6°C to T, 6°C to obtain desired stringency.

+

for 30 min at 22°C (4 m1/100 cm2). After washing with development buffer, the enzyme is detected as described in Section 7.3.3.4.1. BLOTTO at 5%, added to the streptavidin conjugate to prevent nonspecific staining, may inhibit the streptavidin-biotin interaction (Hoffman and Jump, 1990). Alternatively, BLOTTO at 1% is usually as effective in preventing nonspecific staining but does not interfere with their specific interaction. Dialysis of the 5% BLOTTO-streptavidin suspension prior to use will also eliminate the inhibiting factor. For RNA probes, background can be reduced by an RNase treatment (e.g., 30 min 25 pg/ml RNase A and 10 units/ml RNase T1 in 2 X SSC). 8.2.3. Probe detection and analysis

The use of primary tissue preparations and plasmid probes may lead to nonspecific hybridization (Howell and Kaplan, 1987). Xenogeneic tissues are frequently cultivated in immunodeficient animals and may contain bacterial DNA (episomes, plasmids) and lead to erroneous conclusions. In addition to athymic mouse xenografts, bacterial contamination may occur in tissues from other sources such as patients immunocompromised by disease or chemotherapy (opportunistic infections) or even tissues from healthy individuals from organs rich in bacterial flora (e.g., the gut). Spurious hybridization is easily identified with vector probes and can be avoided using probes generated

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from the insert only. This nonspecific hybridization is common (Ambinder et al., 1986). Several problems may occur with autoradiography (Table 7.5). First, the nitrocellulose membrane may be brittle (disintegrating), particularly due to improper neutralization after alkali treatment (yellowish membrane) or after prolonged baking. The lack of signal can be attributed to: (i) low specific activity of the probe, low concentration of the probe or short hybridization time; (ii) short exposure time or improper setup during autoradiography; (iii) incorrect immobilization of target nucleic acid (control signal?); (iv) degradation of the probe or lack of its denaturation; (v) too high stringency of hybridization or posthybridization conditions. A negative image (dark background and clear spots) is obtained when the concentration of the 32P probe is too high. The film may also be uniformly dark or black (during hybridization, or washing, membranes have become dry, thus immobilizing probe anywhere, or probe has not been filtered or the stringency was far too low), black in parts (membrane partly dried during hybridization or touched with bare hands), or has dark spots at random (air bubbles on membrane during hybridization, dust on membrane, unincorporated precursors in hybridization mixture). Nonradioactive probes require adaptations of detection to solid phase format. For instance, nonspecific binding of antibody or enzyme may differ greatly between nitrocellulose and nylon or the conditions may have to be adjusted to permit the reaction product to precipitate in situ (e.g. for TMB). For the detection of biotinylated probes, membranes are immersed in nonspecific signal blocking (NSSB) buffer ( = TBS (0.15 M NaCl, 0.1 M Tris-HCI, pH 7.5) containing 3 mM MgCl, and 0.5% (v/v) Tween 20 (or Triton X-100)) for 90 min at 22°C (shaking). Membranes are then washed briefly with reaction buffer (as NSSB buffer but 0.05% Tween or Triton) and incubated with 2 pg/ml of streptavidin-alkaline phosphatase conjugate in reaction buffer (30 min at 22DC). The enzyme is detected after washing three times for 5 min with developing buffer (0.1 M NaCl, 0.1 M Tris-HC1, p H 9.6 and 10 mM MgCI,) and adding substrate as described in Section 7.3.3.4.1. Clinical or bacterial samples often give high background

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levels (Kennedy et al., 1989; Verbeek et al., 1990). Streptavidin was found to bind specifically to a limited number of polypeptides which are difficult to remove. The best alternative is then the use of antibodies (anti-biotin or against other haptens). For other enzymes, the developing buffer is adjusted accordingly (Sections 7.3.3.3 and 7.3.3.4). Haptens are detected, after blocking nonspecific sites by a 20 rnin incubation with TBS/lO% normal serum at 37"C, by adding specific antibody (dilution, e.g., 1:lOOO in TBS/lO% normal serum) or enzyme-conjugated antibody (diluted, e.g., 1: 100 (polyclonal) or 1: 100 to 5000 (monoclonal), depending on affinity and background) to the blots and incubated for 1 h at 37°C. Unreacted antibodies are removed with three 5 rnin washes in TBS (to prevent background staining). In the indirect method (higher detectability), enzyme-conjugated anti-IgG antibody is diluted 1 : l O O in TBS/lO% normal serum and incubated for 1 h at 37°C. The enzyme is detected, after another three washes, as described in Section 7.3.3.4.1. One should be aware of the possibility of interference with heterologous antibodies (Tijssen and Adam, 1991) and change the normal serum component for BM blocking reagent if necessary. The popular DIG-monoclonal antibody-alkaline phosphatase complex is detected after a 1 rnin wash in TBS, 30 rnin incubation in TBS containing 0.5% BM blocking reagent and 1 min in TBS. The conjugate is diluted 1:5000 in TBS and added to the membrane (0.2 ml/cm2). After incubation for 30 min, unbound conjugate is removed and the enzyme detected as described above. Lebacq et al. (1988) detected sulfonated probes (Section 7.8.1) on charged nylon membranes after blocking with heparin (0.35% in 0.3% Tween 20, 3% BSA, 3% BLOTTO and TBS). An elegant and powerful method for the detection and screening of M13 libraries has been reported by Donegan et al. (1989). Target nucleic acid is immobilized on a solid phase and 0.1 ml of M13 clones (supernatant from cultures), containing an array of inserts, is added to tubes containing 0.4 ml of a solution 5 x SSC, 1.25 x Denhardt, 0.31% SDS and 250 pg/ml sonicated heterologous DNA and hybridized overnight at 65°C. After washing in 2 X SSC/O.l% SDS (for 30 min at 6 5 0 , labeled wildtype M13 (RF; in 0.8 X

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dilution buffer of clones) is added and serves as a universal probe for the detection of hybridized clones. This method has important advantages: (i) DNA need not be isolated from the clones since it is released by the SDS in the hybridization solutions; (ii) the relative degree of hybridization on two or more target blots with related sequences allows the recognition of common and specific clones; (iii) ss clones prevent self-annealing and facilitate detectability by a universal probe. 8.2.4. ‘Fast’ blots of nucleic acid from cells Fast blot methods to minimize nucleic acid extraction and immobilization steps have been developed. Those with nylon as a solid phase can take advantage of the ability of NaOH to dissociate cells, denature DNA and immobilize DNA. Nitrocellulose membranes have a lower binding capacity and co-immobilization of nucleic acid and protein from neutral solutions can be a problem. Bresser et al. (1983) used hot concentrated NaI to inhibit protein immobilization, to denature DNA and to irreversibly bind the nucleic acid to nitrocellulose (no baking required). This method can also be used for RNA. About lo5 cells are minimally required for a unique DNA sequence, whereas > 0.01% of total mRNA can be detected by the NaI methods. Methods for detecting DNA from whole cells on nylon, without DNA purification and processing of the samples in individual vials (Brandsma and Miller, 1980), have recently been developed (McIntyre and Stark, 1989; Reed and Matthaei, 1990; Hammermueller et al., 1991). These procedures avoid enzymatic dispersion of cells, RNase and pronase treatments to hydrolyze cellular macromolecules, etc. These alternative methods are based on the capacity of hot alkali to disperse and solubilize cells and hydrolyze macromolecules including RNA and protein, but not DNA. Positively charged modified nylon membranes then irreversibly bind nucleic acid (Reed and Mann, 1985) while remaining suitable for hybridization. Critical parameters in this procedure are the temperature (80”C), the length of the incubation period (20 min) and the NaOH

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concentration (0.4 N). For example in the method of Hammermueller et al. (1991), virus-infected cell monolayers in flat-bottom 96-well microtiter plates are solubilized with 200 p1 of 0.4 NaOH, sealed in a plastic bag and incubated at 80°C in a water bath for 20 min (65°C yielded insufficient solubilization, whereas incubation above 80°C increased the risk of hydrolysis of DNA). After incubation, the DNA suspension can be transferred to a dot-blot vacuum filtration manifold (nylon membrane is previously soaked in hot water and then rinsed in 0.4 N NaOH). A very low vacuum (filtration rate about 10 min) facilitates an even distribution of DNA over the membrane. After a rinse with 200 p1 of 0.4 N NaOH, the membranes are washed twice with 2 X SSC followed by fixation of the DNA (UV crosslinking of damp membrane or air drying and baking at 80°C). In the usual DNA extraction methods (proteinase K-SDS, Sections 3.3.1 and 8.21, peptides are generated which are extracted to prevent co-immobilization on nitrocellulose. Bresser et al. (1983) added 1 vol. of hot NaI (10 M; 75 g NaI, completed to 50 ml with water, in a 50 ml screw-capped tube and dissolved by placing in boiling water and intermittent vigorous shaking) directly to the digest (no extraction) and denatured DNA by incubation for 10 min at 95°C. If required, dilutions can be made with hot 5 M NaI. The hot suspension is then filtered through the nitrocellulose membrane (prewet and soaked in SSC; Table 8.5) and the membrane washed in three changes of 70% ethanol (5 min at room temperature each) and, for crude DNA, once for 10 min in fresh acetic anhydride solution (0.25 ml acetic anhydride in 100 ml of 0.1 M triethanolamine). After air drying, the membrane is ready for hybridization. The ‘quick blot’ procedure, used for mRNA (not efficient for rRNA or tRNA), is based on the same principle (Costanzi and Gillespie, 1987). A cell pellet is resuspended in Hanks’ balanced salt solution (Gibco), containing 50 pg/ml of cycloheximide and 10 mM vanadyl-ribonucleoside complexes, to about 106-107 cells/ml. The cells are incubated for 10 min at 37°C after adding 1 vol. of proteinase K stock solution. Then, 0.05 vol. of 10% Brij 35 is added and mixed, followed by mixing with 0.05 vol. of sodium deoxycholate. The subsequent steps with fresh hot NaI are as for DNA, except that 5 M NaI containing 0.5% Brij is used for dilution.

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415

TABLE 8.8 Probe stripping for rehybridization A. Reinforced nitrocellulose 1. Heat solution of 0 . l x S S C plus 10 mM EDTA to boiling, remove from heat source and add SDS to 0.5%. Immerse membrane in the hot solution (about 1 ml/cm2) for about 15 min. 2. Repeat step 1 (do not allow the membrane to dry). 3. Rinse the membrane with 0.01 X SSC at room temperature and place the membrane on a pad of paper towels. 4. Check the removal of the probe. The membrane can be dried if the probe was successfully removed (e.g., in aluminum foil under vacuum at room temperature).

B. Nylon membranes Place the membrane for 2 h in buffer (1 mM Tris-HCI, 1 mM EDTA, 0.1 X Denhardt’s solution and 0.5% pyrophosphate, pH 8.0) at 75°C or immerse the membrane for 1 h in 2 X SSPE plus 50% formamide at 65°C or wash for 1 h in 0.5xDenhardt’s solution, containing 25 mM Tris-HCI (pH 7.5) and 0.1% SDS at 95°C or simply for 15 min i n distilled water at 65°C. Rinse the membrane with 0.1 XSSPE at room temperature and place on a pad of paper towels. Continue as in A4.

8.2.5. Reprobing after stripping original probe from blots Sometimes, it is useful to reuse the template(s) on the blot for another probe. All original probe should then be removed unless a different detection system is used. The membranes should not have become dry (because this would irreversibly immobilize the probes) and the membranes should be durable enough to withstand the stripping (Table 8.8). Several potential problems remain. The gradual loss of target DNA from the membrane, unless covalently linked, leads to a loss of sensitivity (Section 8.1.1). Incomplete removal of a plasmid probe, even if detection with a different label, may lead to its recognition by a second probe (nonspecific reaction with plasmid sequences). For reprobing, excised inserts are recommended. Repeated stripping and heterologous reprobing results in a faster loss of target DNA from

416

HYBRIDIZATION WITH NUCLEIC ACID PROBES

nylon after UV immobilization than after baking (Nierzwicki-Bauer et al., 1990). On nitrocellulose, this effect is even more pronounced. 8.2.6. Hybridization to solid phase other than membranes A wide variety of hybridization conditions can be expected for this heterogeneous group of solid phases. The hybridization time depends on the nature of the solid phase and complexity of the strand in excess and may range from that comparable to membrane hybridization (Section 8.2; e.g., microtiter plates and polynucleotide probes), to a few minutes (small particulate solid phase will display a hybridization rate which approaches that of solution hybridization; Table 3.16). Many target nucleic acids are covalently linked to a solid phase with a lower tendency to stick probes. The hybridization solution containing the probe often consists of a high salt (e.g., 0.8 M NaCl), a buffer (e.g., 10 mM sodium phosphate), EDTA (5 mM), formamide (50%), carrier DNA (100 pg/ml) and a wetting agent (0.1% Tween 20). Prehybridization is usually not more than a rinse in this solution. Oligonucleotide radioprobes or capture probes on paramagnetic beads will hybridize in a few minutes (low complexity, high efficiency due to dispersion in solution) in 6 X SSPE, 0.1% SDS at room temperature (Hornes and Korsnes, 1990).

8.3. (Semi)-solution hybridization Solution hybridization assays are superior to mixed phase hybridization formats since (i) hybridization is much faster (Flavell et al., 1974a; Bunemann, 1982), particularly when the target nucleic acid is the rate-limiting component, (ii) there are no transfer or fixation steps, (iii) they can be performed in small volumes, (iv) they can be easily manipulated and (v) the efficiency of hybridization can be 100%. In contrast, signals after nitrocellulose membrane hybridization are estimated to be only 1-3% of maximum (Gamper et al., 1987) due to secondary structure remaining in the target nucleic acid and loss of these target molecules from the membrane. Moreover,

Ch. 8

417

SOLID PHASE AND SOLUTION HYBRIDIZATION

I

-

+ +

Hapten probe Target

P Hybrid Probe

I -<

4

P Hybrid

4

*

Detectable complex

Fig. 8.3. Sandwich hybridization consists of two steps: hybridization in solution with nonoverlapping labeled and 'haptenated' probes (I) and the trapping of the hybrid (via the haptens) on an affinity matrix and washing so that labeled probe is only retained when the target is present (11).

tethering of target nucleic acid at several points along the chain to the solid phase reduces the effective of the probe, and the diffusion, electrical gradient and steric interference factors influence both hybridization and detection. Sensor-based systems (Fig. 8.4) are becoming popular in diagnostics and may become also useful for quantitation in hybridization (Olson et al., 1991). The main drawback of solution hybridization, i.e., distinguishing signals from hybridized and nonhybridized probes, is compounded by the large excess of probe. Separation must then be very efficient to achieve a satisfactory detectability. A simple way to improve detectability without affecting sensitivity is to decrease the probe concentration (Section 8.3.4.1). Separation systems include: (i) selective enzymatic or chemical destruction of free probe (Saiki et al., 1985); (ii) selective capture of sandwiched hybrids (hapten probe-targetlabel probe) by affinity matrix, e.g., by biotin (Delius et al., 198.51, mercurated probes (Banfalvi et al., 1985), or ligands (Fig. 8.3); (iii) by hybridization of target-label probe complex to capture probes immobilized on a solid phase (high capture probe concentration ensures efficient trapping of target-label probe hybrids); (iv) specific recogni-

418

HYBRIDIZATION WITH NUCLEIC ACID PROBES

I

I

rea +H20 CO2

-

primer probe target

-

wmmz

0

biotin

+ ZNH,

streptavidin

0

fluorescein

0 urease

Fig. 8.4. Biosensors may become interesting for probe detection. In this example (from Olson et al., 1990, a biotinylated and a fluoresceinated probe are hybridized in solution to the target (I). The primer sequences should be located outside this region for PCR-product detection. Avidin acts as a bridge between the biotinylated probe and the biotinylated surface of the biosensor (11). Fluorescein is detected with a urease-antibody conjugate. The rise in pH, after the addition of urea, is detected with the pH-sensitive biosensor. The total assay time is less than 2 h with a detectability of 30 amol and a coefficient of variation (standard deviation/mean) of less than 10%.

tion of the hybrid by antibodies (e.g., in the case of RNA:DNA hybrids); (v) strand displacement assays. The main difference between the capture and the sandwich assays is how the label probe-target complex is immobilized on a solid phase. In capture assays, hybridization with the immobilized capture probe determines the kinetics and specificity/detectability/sensitivity characteristics of the assay. In sandwich assays, an immobilized molecule, which is not a nucleic acid (e.g., streptavidin, antibodies), serves to capture the hapten probe-target-label probe ternary complex (thus both probes need to be modified). The affinity matrixhapten interaction thus determines the kinetics (usually 3-10 times faster than solid phase hybridization), the sensitivity and detectability

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419

and contributes to the specificity. The equilibrium constant for the hapten-affinity matrix interaction seems unfavorable compared to hybridization and limits detectability. However, the presence of several haptens on the same molecule and multiple interactions of the complex with the affinity matrix result in an affinity bonus (‘avidity’) (Tijssen, 1985, p. 132). Capture and sandwich assays can be designed so that the target-label probe complex can be released and recaptured to decrease noise and thus improve detectability. In both the capture and sandwich assays, the hvo probes should not cross-react and be located fairly close to each other on the target (especially RNA targets). If two targets that differ by a restriction site are to be distinguished, then the hapten or capture probe should be located at one side and the label probe at the other side of the restriction sequence. Restriction of the target, prior to denaturation and hybridization, will prevent the binding of the labeled probe to the matrix if the restriction site is present. This method has been applied for the detection of sickle gene (not cut by DdeI, but normal DNA is).

8.3.1. Capture hybridization procedures A compromise between the mixed phase and solution hybridization formats is the capture hybridization method originally described by Dunn and Hassell (1977) and adapted by Ranki et al. (1983). Target immobilization in standard solid phase hybridization has the disadvantage that many nonspecific molecules can be present and outcompete the target molecules for the binding sites available on the solid phase and thus influence the amount of target nucleic acid which can be detected. Although in some cases these competing macromolecules can be selectively removed (Verbeek et al., 1990), it makes the method laborious. Moreover, hybridization can be very slow at lower target nucleic acid concentrations on the membrane. These problems often can be avoided by using a capture probe immobilized on the solid phase. The label probe hybridizes with high efficiency and rapidly to the target in solution and the hybrid is efficiently captured on the solid phase due to the high concentration of capture probe. This method (Table 8.9) is also interesting for

420

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE8.9 Capture hybridization

Microtiter plate format (DahlBn et al., 1987). 1. Denature the capture probe (e.g., 3 min in 0.3 N NaOH, then chilled on ice and neutralize). 2. Attach the capture probe to the solid phase: a. In the case of UV binding: dilute the capture probe to 500 ng/ml in PBSM ( = PBS: 8 mM Na,HPO,, 1.5 mM KH,PO,, 140 mM NaCI, 3 mM KCI, pH 7.2, plus 100 mM MgCI,) and incubate 100 ng/well overnight. (Morissey and in 1.5 M NaCI, 0.3 M Tris-HCI Collins (1989) used 100 ng/ml poly(dT),, (pH 8.0) and 0.5 M MgCI,). Wash wells once with PBS and UV irradiate the remaining DNA at 254 nm (250 pW cm-’) for 5 min (Nagata et al., 1985). c. Covalent linkage to CovaLink NH (Nunc) plates (Rasmussen et al., 1991): to ss DNA (8 ng/ml), 0.1 vol. of ice-cold 0.1 M 1-methylimidazole (pH 7.0) is added and 75 K I is dispensed per well (plate on ice). Twenty-five microliters of EDC (Section 6.4.2; 0.2 M in 10 mM 1-methylimidazole) are added and the plates are incubated for 5 h at 50°C. The wells are then washed with Nunc immunowash (5 x 5 min) and finally with 0.4 M NaOH/0.2% SDS. 3. Carry out prehybridization for 1 h at 42°C (solution m, Table 8.3). The binding capacity for target or binder probe can be determined at this point (wells without capture probes as controls). The amount bound equals cpm/specific activity, where the specific activity is expressed in cpm/p,g. 4. Hybridize overnight at 42°C in the same solution containing 400 ng/ml signal probe (optimize for specific cases) and heat-denatured target probes in the wells (signal probe hybridizes much faster with the target than the capture probe; samples prepared as described in Section 8.2). 5. Wash wells twice for 20 min at 50°C with stringent buffer (Dahl6n et al. (1987) used 0.1 XSSC plus O.l%SDS, but less stringent conditions may be required, particularly for oligomers (2 X SSC plus 0.1 % SDS at room temperature). 6. Detect label according to methods presented in Chapter 7.

screening related targets (e.g., capture probe to conserved sequence and label probe to target-specific sequence can be used and vice versa). The use of two probes makes it essential to optimize both probe concentrations. The amount of capture probe is established as the one with optimal detectability and the widest range of linearity. Too much capture probe causes high backgrounds and thus limited detectability. Not enough capture probe limits the binding capacity

Ch. 8

SOLID PHASE AND SOLUTION HYBRIDIZATION

42 1

and the signal is then not proportional at the high end of the concentration scale. A direct and an indirect capture probe assay can be distinguished. In both assays, the capture probe is immobilized. In the direct assay, the capture probe recognizes the target whereas in the indirect assay the capture probe hybridizes with an intermediate, the binder probe. The binder probe contains two domains, one of which is complementary to the target and the other to the solid phase bound capture probe (e.g., oligo(dT) sequences). Optimization of the relative amounts of probes is important. As a rule of thumb, the label probe should have the lowest concentration (but in large excess of the target), e.g., lo9 molecules per assay, the binder probe at least 100 X more and the capture probe another 100 X more. This approach is particularly useful (i) when the binder probe is an oligomer (which can be (dA)-tailed), (ii) for reverse target capture assays (Section 8.3.2) and (iii) for the detection of different targets with an identical capture matrix. In addition, capture hybridization procedures can be simultaneous or successive (label probe followed by capture probe hybridization). The simultaneous hybridization yields signals which better reflect the target nucleic acid concentration, although there is a nonlinearity at the high end of the concentration range or a higher background, depending on the probe concentration. The successive hybridization method allows the use of less label probe and tends to display lower background levels than the simultaneous methods and requires somewhat less optimization, but is more time-consuming, particularly at low target concentrations. This format tends to yield nonlinear responses at the low end of the concentration range.

8.3.2. Reverse target capture hybridization Reverse target capture hybridization assays (Morissey et al., 1989; Thompson et al., 1989) are powerful in decreasing background levels (Fig. 8.5). The binder probe provides the link between the targetlabel probe complex and the capture probe. It consists of oligonucleotides of about 40 bases which recognize target molecules and is tailed with poly(dA), 100-200 bases long. The avidity of the binder

422

HYBRIDIZATION WITH NUCLElC ACID PROBES

I

7

-

4

ohgo 2

Ollgo 1

Labeling

L

den y lation

+

I1

DNA Sample

Hybridization

5'

3'

t

5'

~

-8

I

in solution

I1

I

-

binding (noise)

Target capture

IV

r

t

3'.

I I

2nd target capture

5'

+ 3'-

Fig. 8.5. Reverse capture assays allow an improvement of detectability by a decrease in background staining. Two oligomers (labeled and polyadenylated) are hybridized with the target (I, 11) and captured with paramagnetic beads (PMB)covered with oligo-dT hairs (111). Nonspecifically adsorbed oligomers are usually tighter bound than hybrids to dT-hairs, allowing a selective desorption of specific hybrids (IV). These can be recaptured (V), followed by the determination of the amount of label bound.

Ch. 8

SOLID PHASE AND SOLUTION HYBRIDIZATION

423

probe ( > 50% GC in the target-recognizing domain) and the label probe for the target are chosen so that the capture probe-target complex can be denatured without affecting the binder probetarget-label probe complex. Repeated releasing and hybridizing of the complex to fresh capture probe decreases rapidly the fraction of nonspecific nucleic acid captured. The background reduction capabilities of this method allow the reversible capturing of target out of crude samples, as well as the use of a vast molar excess of label and binder probe over target to drive hybridization to completion in less than 15 min. This is particularly interesting for nonradioactive probes which often require the purification of target. Examples are given in Table 8.10. The detectability of these methods is about 0.02 amol. When this powerful technique is combined with the use of paramagnetic beads, it also becomes also very fast (this detectability in under 2 h). In the virtually identical methods described by Lomeli et al. (1989) and Thompson et al. (1989), GuSCN was used to rapidly isolate the target (Thompson and Gillespie, 1987) and to change the stability of the capture probe-target complex in the various steps (Table 8.10A). Free label probe, which initially adsorbed nonspecifically to the beads, should be absent after two cycles. Compared to poly(dA) tails, poly(A)-containing RNA was found to bind inefficiently to the oligo(dT) hairs on the paramagnetic beads (Morissey et al., 1989; Thompson et al., 1989). The efficiency of capture can be improved by using multiple binder probes (Gillespie et al., 1989). Morissey et al. (1989) and Hunsaker et al. (1989) attempted to reduce background even more efficiently by using different capture solid phases, different elution strategies and background capture steps (Table 8.10B). During the first cycle, about 0.01-0.1% of the input label probe can bind nonspecifically (‘nonspecifically bound’ or NSB) to the beads (measured in the absence of target) and often is in excess of the specifically bound complex. Although during the first wash conditions are chosen so that only dA-dT hybrids dissociate, but some NSB will dissociate also. After recapture and washing, > 99% of the NSB adsorbed during the first cycle has been removed. Progressively less NSB is removed in subsequent cycles (3.6, 2.1, 1.3 log reduction of NSB in the lst, 2nd and 3rd cycle reported by

424

HYBRIDIZATION

wrw

NUCLEIC ACID PROBES

TABLE8.10 Reverse target capture hybridization A. Differential GuSCN concentration-based assays Examples are given for radiolabeled probes and for QP-replicatable probes for HIV 1. Prepare buffers and solutions: a. Cell extraction solution: 5 M GuSCN/O.l M EDTA/10% DeSO, b. BBH buffer: 0.375 M Na,HPO,, 0.375 M NaH,PO,, 0.2% acetylated BSA (Sigma), 35 mg/ml degraded herring sperm DNA and 1% SDS c. Wash buffer: 30% 5 M GuSCN and 70% BBH without herring sperm DNA d. Release solution: 3.25 M GuSCN e. Labeled, capture probe, ss, with two domains (40-mer recognizing the target, tailed with dA-polymers which can interact with oligddT) on paramagnetic beads) 2. Pellet cells, wash and dissolve them at 10' cells/ml in cell extraction solution. 3. Incubate extract (or, e.g., 103-10' HIV target RNA molecules) with 2X lo9 label probe molecules (radiolabeled probes or QP replicatable probes with HIV-specific inserts) and 10" binder probes (oligo(dA)-tailed oligonucleotides) in 70 p1 of 2.5 M GuSCN overnight at 37°C. 4. Add to each tube, 50 ~1 of suspension of paramagnetic particles with oligddT)-hairs (Section 8.1.1.2; prehybridized and diluted to 1 mg/ml with BBH buffer as described in B2). 5. Incubate for 10 min at 37°C (concentration of GuSCN becomes 1.46 M). 6. Draw particles to the side of the tubes using a magnet, aspirate the liquid and wash twice with 400 p1 of wash buffer. 7. Dissociate oligddA): oligddT) hybrid but not the capture probe/target/label probe complex by adding 50 pl of release solution (containing 10" capture probe molecules) and incubating the suspension during 5 min at 37°C. 8. Aspirate supernatants (as in A6) and transfer them to another tube, add another 50 pl of suspension of prehybridized beads to supernatants (resulting in 1.62 M GuSCN) and incubate during 10 min at 37°C. 9. Wash beads twice and release complex as before (no capture probe added to release solution). 10. Detect complexed label probe: a. For QP probes: in the last cycle, remove GuSCN and wash beads twice with 0.1 M KCI (200 pl) and resuspend them in 50 )LI of TE. Dissociate complexes completely by incubating for 10 min at 37°C and measure the amount of label probe by the usual QP amplification method (Section 5.6). b. For radiolabeled RNA probes, add 50 pI of released hybrids to 500 )LI of fresh RNase solution (Section 8.3.4.1; 20 pg/ml RNase A, 20 units/ml RNase T1 in 2xSSC/O.1 M EDTA), mix and incubate for 1 h at 37°C. Precipitate hybridized label probe by adding 210 p1 of 30% TCA/0.23 M sodium pyrophosphate. Vortex the tubes and collect the precipitate on nitrocellulose membranes ( e g , in Minifold). Wash the membrane once with 70% ethanol and proceed with autoradiography or cutting out the dots for scintillation counting.

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425

TABLE8.10 (continued)

B. Differential solid phase and elution assays Instead of multiple elution and capture through the modification of the GuSCN concentration, these methods use different solid phases or different elution methods (chemical, thermal) to reduce background levels. 1. Prepare buffers and materials: a. standard bead solution (2XSBS): 8% fraction V BSA, 1% sodium lauroyl sarcosine (SLS) and 0.1% bronopol (optional). b. 100X T: 1 M Tris-HCI (pH 7.5); 100 x E: 0.1 M EDTA (pH 8.0) c. Sample dilution buffer: 2.5 M GuSCN, 4 0 x T , 40XE, 0.5% SLS, 10% DeSO,. d. Bead elution solution (2 X BES): 0.2% acetylated BSA (fraction V), 1% SLS and 2 pg/ml denatured E. coli DNA. e. Prehybridization solution: 1 X SBS plus 10 xT, 10 x E. f. Bead storage: 1 X SBS plus 10xT, l o x E, 0.02% sodium azide. g. Bead wash solution: 0.5 M GuSCN, 4 X T, 1 X SBS and 0.8 X E. h. Chemical elution solution: 2.5 M GuSCN, 20 X T, 4 X E, 1X BES. i. Thermal elution solution: 0.5 M NaCI, 1 0 x T , 1 0 x E , 1xBES. 2. Pretreat paramagnetic beads with oligddT),, hairs by washing them several times with water, washing and diluting beads to 1 mg/ml with prehybridization solution, heating to 68°C in a Pyrex bottle (occasional vigorous shaking) for 4 h, cooling overnight to 22°C and washing with sterile water ( 3 x 1 and prehybridization solution. 3. Extract cells or bacteria as described in A2, add an equal volume of 40 X T, 1 % SLS and 10% DeSO,, dilute if necessary with sample dilution buffer and incubate 0.25 ml with 10" molecules of label probe and 5 X 10" molecules of binder probe for 15 min at 37°C. Controls do not contain either binder probe or target. 4. Add 0.5 ml of prehybridized beads (3X loi3 molecules of capture probe), vortex vigorously for 10 s and incubate for 5 min at 37°C (GuSCN concentration is then 0.83 M). 5. Draw particles to the side of the tubes using a magnet, aspirate the liquid and wash twice (vortex for 10 s each time) with 0.6 ml of bead wash solution. 6. Remove as much as possible of the remaining liquid, add 0.25 ml of prewarmed (37°C) chemical elution solution to the beads, vortex vigorously and incubate for 2 min at 37°C. 7. Draw the beads to the side of the tube and transfer the supernatant to fresh tubes containing 0.5 ml of beads (3 X loi3 molecules of capture probe) and incubate for 5 min at 37°C. 8. Repeat step 5, remove the remaining liquid and add 0.5 ml of prewarmed (69°C) thermal elution solution to the washed beads, vortex and incubate for 2 min at 69°C. 9. Prefilter the sample through preblocked (two 0.2 ml aliquots of prehybridization solution) Teflon acrodiscs (Gelman, 0.2 pm) and filter slowly (50 pl/min) through a preblocked poly(dT),,,, nylon membrane (prepared by immobilizing poly(dT), filtered trough an acrodisc, as described in Table 8.5). Determine (radio)activity on membranes.

426

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE8.10 (continued) C. Optimization of reverse target capture assays 1. Optimize probes: label probe is preferably an RNA probe or a ss DNA probe of at least 100 bases ( > 50% GC) with "P or biotin labels whereas the binder probe should be an oligomer of 40 bases ( > 50% GC) with a dA-tail length of 100-200 bases (tailed overnight with 1000 units of TdTase, 0.01 mM oligomer and 2 mM dATP as described in Section 7.7.2). 2. Prepare or purchase beads with oligddT) of optimal length (14 in method B). 3. Determine the optimum temperature and salt conditions for label and binder probe, e.g., using 2.5 M GuSCN and hybridization between 20 and 50°C (3-5°C intervals) for 5 min (fixed target concentration (lo9) and label probe copies (10") and binder probe copies (10") and five-fold bead-oligddT) excess). 4. Set up two hybridization reactions (0.1 ml): variable binder probe ((0.1-5)X 10" copies) with fixed target (0 and lo9 copies) and 10" label probes ( a10' cpm/kg) and hybridize for 15 min at the optimum salt and temperature conditions. 5. Capture and wash twice as in method A or B and establish cpm bound. 6. Optimize for label probe as in steps 4 and 5 with the optimum binder probe concentration.

Morissey et al. (1989)). Using a different solid phase in the third cycle (poly(dT)-nylon) allowed Morissey et al. a background reduction of 1.9 log,, and more for stringent washes. Hunsaker et al. (1989) also introduced an NSB reduction step by background capture (unhybridized label probe is specifically captured and removed producing a 4-8-fold gain in signal/noise ratio). 8.3.3. Sandwich hybridization assays Sandwich hybridization, using affinity-based hybrid collection, is based on two nonoverlapping nucleic acid probes (one is labeled, the other can be collected by the affinity matrix) (Syvanen et al., 1986; Jalava et al., 1990). The principles are shown in Fig. 8.3. Target nucleic acid thus mediates binding of labeled probe to the matrix. The detectability is about lo5 molecules with a linear range to at least. lo8.' molecules with radioisotopes as labels. In contrast to capture hybridization assays, the immobilization of the complex is at 22-37°C (leaching is then usually less important).

Ch. 8

SOLID PHASE AND SOLUTION HYBRIDIZATION

427

8.3.3.1. Sandwich hybridization assays for DNA targets Syvanen et al. ( 1986) described sandwich assays with radiolabeled and nonradioisotopic label probes (Table 8.11). Collection is the crucial step and can be achieved on a variety of solid phases (microtiter plates, agarose or paramagnetic beads, membranes, etc.). Syvanen et al. (1986) observed that, for streptavidin-agarose, the batch procedure (15 min at 37°C) was superior to the column procedure. High salt washing and stringent washings were both necessary to reduce background. The highest signal/noise ratio is obtained when 2.5% PEG is included. High concentrations of PEG (10%) caused precipitation or nonspecific binding of the probe DNA to agarose. The microtiter-based procedure, using nonradioisotopic detection, was reported to have a detectability of about 15 amol as compared to 1 amol for the radiolabeled procedure. 8.3.3.2. Sandwich hybridization assays of RNA targets For the detection of mRNA (extracted with proteinase K/SDS or SDS), the signal/noise ratio improves 50-100 times when one of the probes is cRNA (particularly label probe) and another 3-5 times when both probes are RNA (transcripts from the proper inserts in vectors) (Tenhunen et al., 1990). They have also determined optimum concentrations (with respect to signal/noise ratio) of DeSO, and formamide as 2% and 20%, respectively, for cRNA probes. 8.3.3.3. Sandwich hybridization followed by immunological capture and detection Antibodies to DNA :RNA, DNA : DNA and RNA :RNA hybrids have been described (Section 7.4.2) (Stollar and Stollar, 1970; Rudkin and Stollar, 1976; Pisetsky and Coster, 1982). Monoclonal antibodies to unusual nucleic acids have also been reported (Lee et al., 1989) as well as against RNA :DNA hybrids (Bogulawski et al., 1986). In capture assays, with DNA capture probe linked to nylon beads, hybrids resulting from annealing of the RNA target with the capture probe can be detected with the specific antibody. Yehle et al. (1987) immobilized avidin to carboxyl-modified paramagnetic beads and blocked the amino groups on the avidin with succinic anhydride. A biotin-labeled probe is hybridized to its target

428

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 8.11 Sandwich hybridization assays A. DNA targets 1. Prepare: a. Affinity matrix: streptavidin-agarose (BRL) or streptavidin-paramagnetic beads (Promega) can be purchased; alternatively, coat microtiter plates, add 10 kg/ml antibody or 5 pg/ml streptavidin in 10 mM sodium carbonate (pH 9.6) to microtiter plates (150 pl/well) and incubate overnight at 4°C. After washing three times with PBS containing 0.1% Tween 20, block the remaining sites by incubation with PBS containing 0.2 mg/ml denatured herring sperm DNA. b. 1OX SPE: 100 mM sodium phosphate (pH 7.5) and 10 mM EDTA 2. Incubate a 40 pI solution containing target DNA and phage probes (either 5 x lo9 biotinylated hapten probes and 2 X 10' radiolabeled probes or lo9 sulfone-labeled hapten probes and 10'' biotin-label probes) in 0.6 M NaCI, 2 x S P E and 0.1% SDS at 65°C. 3. Collect probe: a. Biotinylated hapten probe complex: dilute sample to 100 k1 with 1 M NaCl and 1 X SPE and add 100 ~1 of a 50% suspension of streptavidin-agarose (in the same buffer) for 15 min at 37°C. Wash agarose first for 5 min in the 1 M NaCl solution and then twice with 1 ml of 0.1 X SSC/O.2% SDS (1 min 55°C and 5 min 37"C, each). b. Sulfone-probe complex: dilute hybridization mixture with 120 pl so that a final concentration of 1% BSA, 0.15 M NaCI, 1 X SPE and 0.1% Tween 20 is obtained and transfer to the wells coated with the antibody. After incubation for 3 h at 37°C and washing three times as in 3a, horseradish peroxidasestreptavidin conjugate (e.g., diluted 1:2000 in 1% BSA, 0.1% Tween 20 in PBS) is added and incubated for 45 min at 37°C. After washing, detect enzyme with a substrate, e.g., 0.46 mg/ml o-phenylenediamine, 0.01% H,O, in 0.1 M sodium acetate for 15 min at 15-22°C and stop the reaction by adding 50 pl of 2 N H,SO, (reading at 492 nm).

B. RNA targets 1. Incubate sample for 4 h at 60°C in 120 pl containing 0.5% SDS (inhibits RNase) (Tenhunen, 1989),1 X Denhardt's solution, 20% formamide, 2% DeSO,, 100 pg/ml tRNA, lo9 molecules of capture probe and 7.5 X lo5 cpm (about lo9 molecules) in SSCP buffer (40 mM sodium phosphate, 4 X SSC, pH 7.0). 2. Collect hybrids in microtiter plates coated with streptavidin (2 h, 37°C; see Ala) and wash six times with 0.01 X SSC/O.2% SDS (at 60°C). 3. Detect the labeled probe: a. Radioactive probe: elute probe by a 5 min treatment with 0.2 M NaOH at 22°C and count in a scintillation counter. b. Enzyme probe: as in A3a.

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(here rRNA) in solution (10 min at 80°C) and Fab’-P-galactosidase conjugate (antibody against RNA : DNA hybrids) and avidin beads were added and allowed to react for 1 h at room temperature. After washing, the enzymatic activity was measured. These authors used succinylated avidin rather than the less efficient streptavidin. Similarly, Coutlee et al. (1989a) developed a sandwich assay in which (i) hybrids formed in solution between the biotinylated hapten probe and the target were collected by anti-biotin antibodies immobilized in wells of a microtiter plate and (ii) detected by P-galactosidase-Fab’ fragment (of monoclonal antibody to RNA :DNA) by conversion of a substrate to a fluorescent product. Optimum conditions included hybridization for 16 h at 75°C in 2 X SSC, 10 mM HEPES, using 0.1 pg/ml probe with 7% biotinylation. DNA targets can also be detected by this method by producing an ss biotinylated RNA hapten probe by in vitro transcription (Coutlee et al., 1989b). This method is ten times faster than the reverse method in which a ds hapten probe was denatured before use. Moreover, ten times less probe was found optimal (otherwise high background; the background level with DNA hapten probe was low). High background levels may be due to the reactivity of the antibody to the secondary structure in nonhybridized RNA (which is also immobilized since it contains biotin). 8.3.4. Solution hybridization procedures with label probe(s) only True solution hybridization has been carried out with labeled probes using several approaches: (i) selective destruction of unhybridized probes or their label (Section 7.3.1); (ii) strand displacement of a label probe; (iii) energy transfer or enzyme channeling after two specific probes are brought together on the target. Particularly the first two approaches seem promising. 8.3.4.1. Selective destruction of unhybridized probes Solution hybridization is carried out and followed by hydroxyapatite chromatography (Section 8.1.1.5) or by S1 digestion of unhybridized probe and subsequent precipitation of hybrids and counting incorporated radioactivity (Durnam and Palmiter, 1983).

430

HYBRIDIZATION WITH NUCLEIC ACID PROBES

In the S1 procedure, ss label probe (DNA; 35 cpm/pl) is hybridized overnight at 68°C to the target (30 p1 of 0.6 M NaCI, 4 mM EDTA, 10 mM Tris-HC1, pH 7.5, 40% formamide containing 10 pg total nucleic acid and covered with paraffin oil to avoid evaporation in a 1.5 microfuge tube). Following hybridization, samples are treated with S1 nuclease by adding 1 ml of 0.3 M NaCI, 30 mM NaOAc, 100 pg/ml of herring sperm DNA and 8 units of S1 enzyme for 1 h at 55°C. The reaction is terminated by adding 0.1 ml of 6 M TCA, precipitated on glass fiber filters (Table 7.15) and counted in a liquid scintillation counter. Logical extensions of this method are the S1 nuclease protection assay (Reyes and Wallace, 1987) and the simultaneous quantification of several mRNA species by solution hybridization with oligonucleotides (Section 12.3) (O’Donovan et al., 1991).

8.3.4.2. Hybridization in concentrated solutions of chaotropes or urea Chaotropic anions at high concentrations promote dissolution of macromolecular superstructures such as cells, probably due to disordering the water lattice and powering the 27, of hybrids (Section 2.2.3). The optimum temperature of hybridization is also reduced. The T, and optimum hybridization temperature will probably vary with each chaotrope (Hamaguchi and Geiduschek, 1962). Thompson and Gillespie (1987) observed that the hybridization rate with RNA probes in GuSCN (3-6 M) was greater than that in 50% formamide at 42°C. Moreover, cells were readily dissolved in concentrated GuSCN allowing hybridization without prior nucleic acid purification. The issue of specificity of hybridization in GuSCN remains incompletely resolved. Thompson and Gillespie (1987) developed two solution hybridization assays in the presence of GuSCN, the RNase/TCA assay and the SSC/NC assay (Table 8.12). RNA probes are preferred since (i> they can be readily synthesized in high concentrations and at high specific activities, (ii) they are more stable in GuSCN and (iii) the removal of nonhybridized probes is easier. The probe should then be free of DNA (DNA digestion with RNase-free DNase; Section 3.1.3.4) and, in control hybridizations (RNase/TCA assay), more than 50% should hybridize and backgrounds should be low.

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SOLID PHASE AND SOLUTION HYBRIDIZATION

43 1

TABLE8.12 Hybridization in concentrated guanidine isothiocyanate (GuSCN) solution A. RNase/TCA assay 1. Dilute sample in 5 M GuSCN/O.l M EDTA (pH 7.0) (10 pl aliquots in duplicate) and cap tubes. 2. Incubate for 5 min at 60°C and cool rapidly to room temperature. 3. Add quickly RNA probe (300 ng/ml), mix and hybridize for 2 h. 4. Add 200 pI of RNase solution (20 p,g/ml RNase A (DNase-inactivated) and 20 units/ml RNase T, in 2 X SSC and 0.5 mM EDTA, containing 1 pl (per 200 bl) of a stock solution of 10 mg/ml poly(A) and 0.1 M EDTA (RNase-free) and incubate for 30 min at 37°C. 5. Chill solution and make 10% with respect to trichloroacetic acid (TCA). 6. Filter through ME25 membrane (Schleicher & Schuell) and wash with 10% TCA/2% PP,, turn over the membrane and wash again. Rinse with 70% ethanol, dry and count in a scintillation counter.

B. 2 X SSC/NC assay 1. Proceed as in Al-3 and mix then with 200 pl of the same solution as in A4 but lacking the RNases. 2. Filter the hybridization mixture at a rate of 1 ml/min through nitrocellulose BA85 (previously wetted with H,O and soaked in 2 X SSC). 3. Rinse the membrane strip (1 x 5 cm; otherwise adjust volumes) in 25 ml of 2 X SSC/EDTA at room temperature. 4. Soak strip for 30 min at 37°C in 50 ml of 2XSSC containing 0.1 ml of RNase stock solution (50 mg RNase in 5 ml 2XSSC, heated for 10 min at 95"C, cooled slowly to room temperature, after which 50000 units of RNase T, are added) and rinse again in 25 ml of 2 X SSC/EDTA. 5. Measure radioactivity by autoradiography or liquid scintillation counting. C. Dissolving cells in GuSCN and hybridization 1. Centrifuge cells and dissolve in 5 M GuSCN/O.l M EDTA (10' cells per ml). Dissolution will take about 2 min at room temperature with agitation. Usually the suspension is slightly viscous (can be reduced by a few freeze-thaw cycles). 2. Gently heat cells to 60-70°C to denature DNA, add probe and incubate at the hybridization temperature (usually room temperature). 3. Trap RNA: DNA hybrids on nitrocellulose as described in B.

With probe excess, about 0.1 pg can be detected after hybridization for 2 h. Although with high probe concentrations (300 ng/ml or more) faster hybridization rates can be expected, detectability will not necessarily increase (Fig. 8.6). With a modest probe excess,

432

HYBRIDIZATION WITH NUCLEIC AClD PROBES

100

-2

-

10

w

2

1.0

n

2

0.1

E

2

...0.01 1

8

Fig. 8.6. Sensitivity and detectability depend on various factors. In example I (Thompson and Gillespie, 1987), the sensitivity is 1. Increasing the probe concentration does not improve sensitivity but deteriorates the detectability. In another example (van Gijlswijk et al., 1992), the sensitivity in a hybridization assay, using POase-catalyzed luminol reaction, was similar in the one-step and three-step methods but the detectability improved for the latter. Similarly, in reverse capture assays detectability improves after one or a few cycles while sensitivity decreases only slightly. In example I1 (Oser and Valet, 1988), simple adjustments in the (time-resolved fluorescence) procedure improved the detectability somewhat but the sensitivity increased about 100-fold for the well-strip method.

apparent hybridization signals increase with higher incubation temperatures and higher GuSCN concentrations, whereas with lower concentrations of probe (7 ng/ml) hybridization is less efficient. Kinetics of hybridization accelerate about 100 X in GuSCN as compared to 50% formamide (Thompson and Gillespie, 1987). The stability of DNA :DNA, DNA : RNA or RNA :RNA duplexes is also dramatically decreased. Including 1 M NaCl slightly decreased the T, (particularly with increasing GuSCN concentrations and for DNA : DNA). Urea, in solution hybridization, has several advantages over formamide (Dutton and Chovnick, 1987): (i) mild temperatures (5055°C) provide stringent conditions for both RNA : DNA and DNA : DNA hybridization; (ii) the solubility of nucleic acid (particularly at high concentrations) is greater in urea than in formamide; (iii) urea is more stable. In their procedure, Dutton and Chovnick (1987) hybridized the target and probe in solution, digested the free ss probe with S1 nuclease and collected the hybrids on glass fiber

Ch. 8

SOLID PHASE AND SOLUTION HYBRIDIZATION

433

filters. However, background is usually higher than 1% (depending on the probe; those with fold-back configurations show higher background). This method can be used for rare sequences, but with higher abundance it is necessary to separate free probe from hybrid by other methods (e.g., gel electrophoresis). Annealing between the target and probe (ss, originally M13, but ss PCR probes may be more useful) is carried out in 8.0 M urea, 0.05% sarcosine, 1 mM EDTA, 0.35 M NaCl and 0.15 M HEPES, pH 7.2, for 10 min at 85°C and then 5 h at 50°C (at least 10-fold excess of probe; usually between 0.1 and 10 ng/ml). The suspension is then diluted in 9 vols. of S1 buffer (30 mM NaOAc, pH 4.6,50 mM NaCl, 3 mM ZnSO, and 10 pg/ml tRNA) and 25 units of S1 enzyme is added followed by an incubation for 1 h at 42°C. The surviving hybrids are retained on glass fiber filters (e.g., Whatman GF/C) by standard methods (Table 8.12; see also Table 7.15) and the radioactivity determined by liquid scintillation counting.

8.3.4.3. Strand dkplacement assays Among the solution hybridization methods, strand displacement assays have great potential. In this approach, the ability of a strand to displace another in a hybrid is measured. Well-established processes of D-loop and R-loop formation are based on strand displacement. In the D-loop formation, a ss DNA displaces its homologous sequence in superhelical DNA through the energy of the negative supercoils during transient denaturation (Beattie et al., 1977). R-loops can be formed with linear molecules since the stability of the RNA : DNA hybrid, obtained through strand displacement, is greater than that of ds DNA (Thomas et al., 1976). In the strand displacement assays (Ellwood et al., 1986; Vary et al., 1986), a signal DNA (same sequence and polarity as the nucleic acid to be detected, but shorter), homologous to part of the target DNA sequence, is hybridized before adding the sample. The nucleic acid in the sample, since it is longer and will form hybrids that are more stable with the target, will displace the signal DNA very rapidly (Fig. 8.7). The rate-limiting step is the initial hybridization with the portion of the target that is ss while branch migration is extremely rapid (20 ksc/bp) (Green and Tibbets, 1981).

434

HYBRIDIZATION WITH NUCLEIC ACID PROBES

A

Immobilizbd probe complexes

Sample

Hybridization

\

D

E

F

4

S'

Detection Strand separation Branch migration Fig. 8.7. In strand-displacement assays, two regions are distinguished in an analyte (S' and T'). The T' reacts with the T (target; C) and displaces a short signal sequence from this complex by branch migration (D). The liberated signal sequence ( S ' ) is removed from the well (E) and detected (F).

In the strand displacement assay of Vary (19871, an RNA strand is hybridized with a longer DNA strand ('probe') which in turn is complementary to the target DNA. The remaining free RNA is removed. Hybridization of the target with the probe results in the displacement of the RNA. The RNA becomes ss and thus a substrate for polynucleotide phosphorylase (EC 2.7.7.8) and yields nucleoside diphosphates such as ADP which in turn is converted to ATP by pyruvate kinase (EC 2.7.1.40). The concentration of ATP generated can be measured by bioluminescence (luciferase enzyme). Although this method is elegant, some of the enzymes used are not very stable, RNase may interfere and background bioluminescence is

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435

often present. Labeled displaced strands can probably be detected better with alternative (capture) methods. The detectability in a model system was 15 amol. Strand displacement can also be used in an isothermal in vitro DNA amplification method (strand displacement amplification). In the original method (Walker et al., 1992a1, cleavage by restriction enzymes was necessary to obtain defined overhangs. This limited the applicability (target must be ds DNA, convenient restriction sites should flank target). In a subsequent modification (Walker et al., 1992b), exo- Klenow (USB) was used in conjunction with HincII to nick the unmodified strand of a hemiphosphorothioate of the recognition site. This technique is generally applicable but requires four primers. An amplification of lo7 to 10’ was obtained after 2 h at 37°C. Also promising are the branch capture reactions (BCR) (Weinstock and Wetmur, 1990; Wong et al., 1991). This approach takes advantage of the higher stability conferred by some base analogues

520 n m

(520. 550 n m

-

575 n m )

11111111111111111 ’ 5-7 n m (optimal)

Fig. 8.8. Energy transfer assays are exemplified here by a probe containing a 5’4erminal fluorescein (I) and a probe containing a 3’-end rhodamine. Upon excitation at 490 nm, fluorescein emits radiation (green fluorescence) at 520 nm. While rhodamine can be excited at 520 nm, the nonhybridized probes are not close enough for energy transfer. Probes are designed so that hybridization brings the two labels to optimal proximity. Excitation of fluorescein yields fluorescence which also serves to excite rhodamine. Rhodamine fluorescence can be specifically determined at 575 nm.

436

HYBRIDIZATION WITH NUCLEIC ACID PROBES

in the displacer (Sections 2.2 and 5.5.4) to obtain (at least temporarily) D-loop formation with linear DNA, even when oligomer displacers are used. The recipient can be tagged permanently with the displacer complex if ligase is present. Duplexes with 4-base overhangs, as generated with restriction enzymes, can act as a recipient, particularly at higher temperatures which cause duplexes to ‘breathe’.

8.3.4.4. Energy transfer and enzyme channeling systems Probes can be designed so that when they come in close proximity, a signal is emitted (Fig. 8.8). These probes may contain two different enzymes one of which yields a product which is a substrate for the other. When the probes are hybridized to the target, the enzymes would be in close vicinity and the local concentration of the substrate for the second enzyme substantially higher than in the solution as a whole. Different combinations have been proposed for homogeneous enzyme immunoassays (Tijssen, 1985) and an example for probes can be found in the patent literature (Albarella et al., 1989). Similarly for energy transfer, two probes (e.g., chemiluminescent donor with rhodamine absorbant/emitter, respectively) can be brought together on a target molecule, resulting in light emission (Heller and Morrison, 1985). Despite their obvious theoretical advantages, very few of these systems have proven practical.

CHAPTER 9

Hybridization after electrophoretic fractionation of nucleic acids Gel electrophoresis is a powerful and versatile method to resolve mixtures of different nucleic acid molecules and allows the fractionated molecules (i) to be viewed directly, (ii) to be recovered in pure form or (iii) to be characterized directly by hybridization. Hybridization of the probes to fractionated DNA (‘Southern technique’) (Southern, 1975) or fractionated RNA (‘Northern technique’) (Alwine et al., 1979) can be achieved after the transfer of the resolved molecules to a membrane, but in some cases also directly in the gel using oligonucleotide probes (‘unblot’) (Purrello and Balazs, 1983; Tsao et al., 1983). The steps in these protocols are summarized in Table 9.1. Simultaneous extraction of DNA and RNA (Section 3.4.3) (Chan et al., 1988) may be advantageous when the mass of tissue available is small. Although for small DNA fragments electrophoresis is sometimes performed in polyacrylamide, generally agarose gels are preferred. Transfer of small fragments (0.5-5 kbp) from agarose gels to hybridization membranes is rather efficient, but larger fragments are poorly transferred with standard methods. Therefore, a partial hydrolysis step is included (acid treatment to depurinate a small fraction of bases and exposure to alkali to cleave phosphodiester bonds at depurinated sites), to obtain fragments of about 1 kb. The denaturation step also serves to render DNA single-stranded, which is required for the binding of the nucleic acid to the membrane and for the subsequent hybridization. Different membranes can be used for the transfer (Chapter 8) and this has direct consequences for the detectability, background and procedures for fixation. In the last step, the probe is hybridized and detected. In the case of Northern

438

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 9.1 Southern, Northern transfer and “unblot” hybridization.

B. Northern transfer

A. Southern transfer

RNA 1 denaturing agarose gel electrophoresis 1 stain with acridine

DNA (digest)

1 fractionation by electrophoresis

stain with ethidium bromide 1 depurinate with 0.25 N HCI

1 wash and denature

I 1 transfer to membrane by I

capillary blotting

-I

vacium or positive pressure

fix by baking

eleciroblotting I

I

C. Hybridization in Agarose gel electrophoresis, staining 1 denaturation, wash

1 dry (not completely)

1 prehybridization hybridization

(pre)hybridization .1 wash and dry again

1 detect label

detection of label

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hybridization, RNA is fractionated in denaturing agarose gels by electrophoresis and subsequently detected much the same way as in case of Southern hybridization. Early problems with this technique were due to insufficient denaturation of the RNA. These hybridization methods have found wide application in both diagnostic (e.g., RFLPs) and recombinant DNA research and have become indispensible. As an alternative to transfer methods, gels in which nucleic acid is fractionated can, after drying, be submitted directly to denaturation and hybridization within the gel. Finally, fractionation by electrophoresis after a solution hybridization step (Section 12.4) or after PCR and in-gel detection is also convenient. A tracer amount of labeled precursor can be added to the PCR mix (Verbeek and Tijssen, 1991) or a 5‘-labeled internal primer can be added at the end, followed by 1 PCR cycle (Parker and Burmer, 1991) and the products analyzed in the gel. Drying of the gel enhances the signal.

9.1. Electrophoretic procedures 9.1.1. Standard agarose gel electrophoresis of DNA Around pH 7.0, nucleic acids are negatively charged and will move in an electric field towards the positive pole (anode). The agarose or polyacrylamide gels have pores of varying sizes depending on their concentration. The smallest molecules are least impeded but larger molecules have to squeeze through to an increasing degree until, at a certain molecular size, they are too big for even the largest pores. Molecules at and above this size all migrate at the same rate (‘limiting mobility’). Decreasing the gel concentration will result in an average larger pore size and a larger fraction of molecules of a particular size will then, during a determined small time span, migrate uninhibited. The larger the fraction of these molecules migrating uninhibited in that time span (‘sieving’), the larger their mobility. Since all molecules have their turn of uninhibited migration and sieving is a statistical parameter, molecules of a certain size will migrate as a single band. The gel concentration thus determines the

440

HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 9.2 Range of separation of double-stranded DNA and estimated migration of tracking dye Medium

Concentration

Range

(%I Agarose

SeaKemTMSd 1X T A E 1 XTBE NuSieveTMSd 1 X TAE 1 XTBE

Size of fragments co-migrating with

a

Bromophenol blue

Xylene cyanol FF

0.5 0.8 1.2 1.6

1-25 0.6-0.8 0.4-6 0.2-0.5

0.5 ’, 0.3

5b,4C

0.5 0.75

1-25 1-25

0.7 0.5

6.5 4.5

4.0 4.0

0.01-1 0.01-1

0.07 0.03

-

Polyacrylamide

5 6 8 10 20

100-700 80-500 60-400 40-200 6-100

100 65 45 20 12

460 260 160 70 45

DNA size in kbp for agarose and in bp for polyacrylamide. In 1xTBE. In 0.5 X TBE. The agarose concentrations given for NuSieve and SeaKem are optimum; NuSieve gels with less than 4% agarose are fragile and may need the addition of 0.5% of SeaKem. In denaturing polyacrylamide, dye markers move with fragments about half the size of those in neutral gels (e.g., 35 and 130 nucleotides in 6% denaturing gel). a

size range of molecules that can be effectively fractionated (above a certain size, there is (virtually) no uninhibited migration). In general (Table 9.21, polyacrylamide gels are used for small molecules ( < 700 bp) and agarose gels for larger molecules (100-25 000 bp), whereas pulsed-field gel electrophoresis may be used to resolve even larger molecules (up to about 5-10 000 kbp). The charge to mass ratio (i.e., number of negatively charged phosphate groups per nucleotide) of DNA is constant for molecules

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of different lengths. The DNA size therefore determines the rate of migration and fractionation (Helling et al., 1974). As in SDS-polyacrylamide electrophoresis of proteins, the migration of linear molecules is inversely proportional to the log of their molecular weight. The mobility of a given DNA can thus be expressed by the equation (Tijssen et al., 1976)

where 1is the electrophoretic mobility with respect to a standard at a given gel concentration, k0 the mobility extrapolated to 0% gel, G the gel concentration and K R the retardation coefficient (Fig. 9.1). Some minor exceptions have been described, but in general these are of little consequence. Circular or supercoiled molecules have a different mobility pattern, e.g., in the absence of ethidium bromide, supercoiled DNA migrates faster than its linear counterpart (smaller hydrodynamic radius of supercoiled DNA), but circular DNA (e.g., nicked) migrates slower. The concentration of ethidium bromide present during electrophoresis determines the number of superhelical turns and thus the mobility. Increasing the ethidium bromide concentration lowers the number of superhelical turns up to a critical concentration of about 0.4 pg/ml (then circular DNA), above which negative superhelical turns are introduced and mobility increases again. Sometimes, ethidium bromide is included at 0.5 pg/ml in the gel and running buffer; it decreases the mobility of linear DNA by about 15%. Either xylene cyanol or bromophenol blue, or both, are included in the sample as tracking dye. They migrate not according to size but are indicators of migration (Table 9.2). For size markers, suitable restriction digests (e.g., EcoRI plus Hind111 digest of lambda DNA) or ligation ladders can be run on a separate lane. Details of DNA electrophoresis methods are given in Table 9.3. The most widely used buffers in gel electrophoresis of nucleic acids are Tris/acetate/EDTA (TAE) and Tris/borate/EDTA (TBE). TBE has the best buffering capacity but the use of TAE tends to result in somewhat sharper bands. For some purposes, such as DNA extraction with glassmilk, TBE should be avoided unless sorbitol is

442

HYBRIDIZATION WITH NUCLEIC ACID PROBES

I1

I

4

4

-

a e - 3 M

0.75: 1.50:

2

T

I

I

,

2

4

6

8

TAE

TBE

2

2

4

6

8

Mobility ( c m )

Mobility ( c m )

IV

111

4

4

I

a

-

p

3

M

2

2

(EtBr) I

2

I

,

,

4

6

8

,

1

2

1

4

1

6

8

Mobility ( c m )

Mobility(cm)

VH

KI to

7 4

0.4

0.0

1.2

G e l (%)

1.6

0.04

0.08 0.12 KR

0.16

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added during the extraction (Section 5.3.2.7). Glassmilk is crushed glass, which is useful for the extraction of small DNA fragments but tends to shear large DNAs (less problems with uniform glass beads). Although the mobility of nucleic acid is proportional to the applied voltage, there is less resolution of large fragments at high voltages (e.g., 5 V/cm), perhaps due to insufficient diffusion of salts in the sample. Therefore, large DNA molecules should be fractionated at low agarose concentrations and low voltages (e.g., 0.5-1 V/cm, overnight). In most gel boxes, the voltage drop over the gel is about 80% of that between the electrodes; consequently, applying 1 V/cm for a gel of 16 cm would mean a setting of (1 X 16)/0.8 or 20 V. The maximum concentration of nucleic acid, before overloading produces a smear, is about 15 ng/mm2 in the cross section for each band (i.e., about 200 ng/5 mm slot) and the minimum that can be detected with ethidium bromide is about 0.3 ng/mm2 (Sharp et al., 1973). The amount of DNA loaded per well should increase with the number of bands up to about 10 pg for restriction digests of genomic DNA. Commercially available agarose is not well defined and may differ from batch to batch. Some highly purified or chemically modified agarose preparations, such as NuSieve, SeaPlaque and SeaKem, are quite expensive and used only for special purposes. For instance, agarose gelling at low temperature is excellent for the fractionation of small DNA fragments, whereas agarose melting at low temperature can be used to liquify agarose at moderate temperatures and thus allow the extraction of DNA or enzymatic reactions. NuSieve GTG (which has a superior resolution of small DNA (10-1000 base

Fig. 9.1. The mobility of DNA depends on several factors: concentration of agarose (I); type of buffer (11); the field strength (111; particularly for large fragments); the presence of ethidium bromide (IV); the form of DNA (linear, ds or ss, circular (supercoiled or nicked)). The length of the DNA can be determined by comparison with co-migrated DNA fragments of known lengths (e.g., HindIII/EcoRI digests of lambda DNA). Alternatively, the mobility (with respect to a reference) can be determined in gels with different agarose concentrations (V). The retardation coefficients, K , , allow the construction of Ferguson plots (VI) to determine the size of the sample fragments (X and Y).

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

TABLE 9.3 Electrophoretic separation of DNA fragments in agarose gel 1. Prepare electrophoresis buffer: a. TAE: ( l o x : 48.4 g of Tris, 11.4 ml of glacial acetic acid and 20 ml of 0.5 M EDTA per liter). b. TBE: (10 X : 108 g of Tris, 55 g of boric acid, 40 ml of 0.5 M EDTA per liter). This standard solution develops a precipitate upon prolonged storage. A higher pH (up to 8.9) avoids this problem without affecting electrophoretic separation. c. Alkaline ‘buffer’: (1 X : 50 mM NaOH and 1 mM EDTA). 2. Dissolve agarose in electrophoresis buffer (1 XTAE or 0.5xTBE). For most purposes 0.8% agarose is adequate. Heating in a microwave oven is convenient (avoid sticking of agarose grains to wall of container). The container (glass bottle or Erlenmeyer) should never be closed during or after heating otherwise the bottle may explode. Prolonged heating can cause superheating and sudden violent boiling. The solution is cooled to 60°C before pouring. Ethidium bromide can be added to a final concentration of 0.5 pg/ml. 3. Seal the gel mold with a tape or place the mold in a closely fitting box (should be level) and position the sample comb 0.5-1.0 m m above the plate. Seal the edges with a small quantity of the agarose solution using a Pasteur pipette. Pour warm agarose into the mold until a 3-4 m m gel layer is obtained. In the case of low concentration agarose gels ( < 0.5%), it is convenient to first pour a thin supporting gel (l%, without comb). The gel sets within 30-60 min. 4. Remove the tape or mold box and place the mold in an electrophoresis tank and add electrophoresis buffer until the gel is submerged (a few mm). 5. Add samples (in 5% glycerol with 0.05% bromophenol blue and/or xylene cyanol FF; usually added as 6 X concentrate to 5 vols. of sample). Although for ordinary electrophoresis the same pipette tip can be used (rinsing in between), a separate tip is recommended for each sample for Southern hybridization. Usually 10-30 pI can be introduced per well. 6. Close electrophoresis tank and run samples towards the ‘ ’ pole. Although 5 V/cm is usually used (or even more for minigels), superior results are obtained at 0.5-1.0 V/cm (overnight).

+

pairs) and is suited for in-gel ligation and transformation), Seaplaque GTG (for in-gel cloning and labeling of large DNA (> 1 kbp)) and SeaKem GTG (for large DNA fragments (> 1 kbp) and digestion and ligation of recovered DNA) are some special purpose agaroses from FMC which are essentially DNase- and RNase-free. Sometimes, e.g., to calibrate nick translation reagents or to analyze the size of cDNA in S1 resistant DNA:RNA hybrids, it is

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necessary to separate ss DNA in an alkaline agarose gel. Since agarose cannot be melted in the presence of NaOH, concentrated NaOH/EDTA is added after melting the agarose and cooling to about 55°C. 9.1.2. Pulsed-field agarose gel electrophoresis of DNA Standard agarose gel electrophoresis does not fractionate long DNA molecules. Field-inversion gel electrophoresis (FIGE) is a simple periodic inversion of the electric field, with a slightly longer forward pulse (Carle et al., 1986). A simple version of the pulsed-field gel electrophoresis was first described by Schwartz and Cantor (1984) and applied for the fractionation of large molecules. This method is based on the principle that DNA will only move when it is aligned with the axis of the electric field and that this alignment takes more time for longer molecules. FIGE can be performed in standard agarose gel boxes for DNA fragments up to 800 kbp and is optimal when the reverse time interval is roughly equal to the time required for the conformation change of the molecule (0.01 s for 10 kbp, 0.1 s for 25 kbp, 1 s for 100 kbp, 10 s for 500 kbp and 30 s for 1500 kbp; in 0.5 X TBE plus 0.1 M glycine, 0.8% agarose, 8 V/cm and at 4°C) and using a ratio of forward to reverse time of 3: 1. FIGE requires only standard gel boxes and a pulse controller. A small, self-contained microcomputer sold by MJ Research, Inc (Kendall Square, Box 363, Cambridge, MA 02142, USA) under the name of PPI-200 (programmable power inverter) provides a flexible, accurate and affordable electronic interface for FIGE. FIGE controllers are also available from IBI, Bio-Rad, Consort, Hoefer and EC Apparatus. Higher resolution (up to 10-fold) can be obtained with alternating angle gel electrophoresis systems, such as contour-clamped homogenous field electrophoresis (CHEF) (Chu et al., 1986b). The design of orthogonal electrophoresis boxes and the need for cooling make this equipment quite expensive. The FIGE system will work for many purposes and the expense of other systems may not be worthwhile, although the design of alternating angle systems is still rapidly evolving (Eby, 1990). Although in FIGE a fractionation of molecules up to about 800 kbp is achieved, it also considerably improves the

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resolution in the 10-50 kbp range by using different switching times. This PPI-200 device has a built-in library of standard programs and allows both linear and quadratic time ramps. Among other companies, Bio-Rad offers a satisfactory albeit expensive CHEF-DR I1 system (up to 5-10000 kbp resolution). Power supplies should have a constant voltage output and not a pulsed direct current (the latter power supplies have usually a fixed or stepwise, rather than continuously variable, output). Alternative designs include the transverse alternating field electrophoresis (TAFE) system, rotating gel electrophoresis (RGE) and its variants (e.g., RAGE) and the rotating field electrophoresis (RFE). In the TAFE system (Gardiner et al., 19861, a small gel is placed vertically and two sets of electrodes are placed at the front and back of the gel so that the DNA zigzags through the gel. In the RGE system (Southern et al., 1987), the gel platform rotates between two angles when the electrodes are off, whereas in RFE the electrodes are rotated (Ziegler et al., 1987). Special gel media (larger and more uniform pores) improve resolution and cut running times. Fast Lane agarose (FMC BioProducts) and CGA agarose (Bio-Rad) are widely used and synthetic media are in development. Running times increase more rapidly than the fragment size and a week may be necessary for long fragments. Lower ionic strength buffers and better computer algorithms reduce running times and often a large number of gels can be stacked. It is necessary to prepare undegraded DNA to take full advantage of pulsed-field gel electrophoresis. This can usually not be achieved by the standard methods presented in Chapter 3. Techniques for the preparation of very high molecular weight DNA are given in Table 9.4. After electrophoresis of this DNA (Table 9.51, the gel is stained by immersion in a solution containing 0.5 p.g/ml of ethidium bromide for about 60 min and destaining in water for about 1 day. Southern hybridization can then be performed after the exposure to acid and base to achieve partial hydrolysis and complete denaturation prior to transfer. Possible problems and remedies are summarized in Table 9.6.

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TABLE9.4 Isolation of DNA for pulsed-field gel electrophoresis 1. Resuspend cells at a concentration of 5 X lo7 cells/ml in ice-cold lysis buffer (10 mM Tris-HCI, pH 7.6, containing 20 mM NaCl and 100 mM EDTA). Cells may be obtained from: (i) cultured cells washed several times in PBS; (ii) fresh tissues (homogenized in ice-cold PBS in chilled glass homogenizer and filtered through a cheesecloth); or (iii) frozen tissues (- 70°C) ground to a fine powder using a chilled mortar and pestle, resuspended in PBS and filtered through a cheesecloth). Washing adequately with PBS is essential to prevent inhibition of proteinase K in the lysis step. 2. Prepare 1%low melting temperature agarose in the same buffer and cool to 45°C while warming the cell suspension to 45°C. Then add an equal volume of agarose to the cell suspension and mix well. In the case of yeast, add 1/100 vol. of zymolase (ICN Immunobiologicals; 2 mg/ml in 10 mM sodium phosphate and 50% glycerol) or lyticase (Sigma; 900 units/ml, 7 pg/ml) to the liquid mixture. 3. Transfer the liquid mixture to Plexiglass block molds (containing wells which have the same size and shape as the comb used for making the gel but are open at both the top and the bottom; the bottom is sealed with tape); alternatively, the mixture can be drawn into Tygon tubing (2.4 m m internal diameter) or deposited as drops on the bottom of a Petri dish. Let gel set at 4°C. One pI should contain about 2.5 X lo4 cells or about 100-200 ng DNA (i.e., 5-10 k g / l cm of Tygon tubing). 4. Transfer hardened blocks (or 1 cm tubing cylinders, or blocks cut from agarose drops in dishes) to conical tubes containing 50 vols. of buffer containing 10 mM Tris-HCI (pH 8.0). 0.1 M EDTA, 1% Sarkosyl and 100-400 pg/ml of proteinase K (enzyme added to stock solution just before use). Incubate for 24 h at 3TC, replace with fresh digestion solution and incubate for another 16-24 h at 37°C. Replace with 50 vols. of TE buffer and incubate overnight at 4°C. 5. Inactivate protease prior to digestion with restriction enzymes. For this purpose, add phenylmethylsulfonylfluoride (PMSF, a covalent protease inhibitor, usually stored as a 100 mM stock solution(l7.4 mg/ml in isopropanol) at -20°C) to TE (at 25°C) just before use to a final concentration of 80 bg/ml. PMSF solutions have a short effective half-life in water and should be replaced twice with a fresh solution (each incubation 1 h). Moreover, PMSF is not stable in alkaline conditions or at higher temperatures. This dangerous chemical (handled in the hood) can be inactivated by rendering the used solution alkaline and leaving at room temperature. 6. Wash blocks in at least three times 20 vols. of 10 mM Tris-HCI (pH 8) for 30 min at room temperature and twice with 10 vols. of restriction buffer (1 X ) for 30 min. 7. Drain excess buffer and add 2 vols. of restriction buffer containing the restriction enzyme (e.g., 50 units, but titration may be necessary to find optimum concentration) and incubate for as long as enzyme is active (up to 15 h). 8. Soak blocks for 1 h in 50 vols. of TE (4°C) and transfer to wells of agarose gel. If it is difficult to insert the blocks, they can be melted at 65°C and poured in the well by taking care to avoid shearing (wide-hole pipettes, e.g., by cutting 5 mm off the end).

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TABLE9.5 Pulsed-field agarose gel electrophoresis 1. Prepare a lambda-DNA or T2-DNA ladder to be used as size markers. The method of Vollrath and Davis (1987) and Sambrook et al. (1989) using lambda-DNA yields a nice set of concatemers: a. Dissolve 10 pg of lambda-DNA in 177.5 pI of TE (pH 7.61, heat for 5 min at 56"C, cool to 37°C and add rapidly 62.5 kl of 8% PEG 8000, 5 &I of 0.1 M ATP, of 0.1 M DTT, 0.5 Weiss units of T4 ligase and 250 pI of 1% molten low 5 melting temperature agarose solution (in 20 mM MgCI,, 0.1 M Tris-HCI, pH 7.6 and at 37°C). b. Transfer to blocks (as in Table 9.4, step 31, allow to solidify and transfer the gel to 5-10 vols. of ligation buffer. Incubate for at least 24 h. c. Transfer the block to 10 vols. of 20 mM EDTA, incubate for 1 h and transfer to another EDTA solution in which it can be stored at 4°C. 2. Prepare 1% agarose gel (0.5XTBE or 0.5XTBE plus 0.1 M glycine). The gel should be as thin as possible for better dissipation of heat and cover gel with buffer (few mm). Insert samples in wells as described in Table 9.4, step 8. 3. Connect programmable power inverter between the gel box and the power supply and place a peristaltic pump between the buffer tanks (flow 10 ml for minigels and 40 ml for large gels). 4. Apply direct current until bromophenol blue has migrated about 1 cm into the gel, then activate the power switching device program.

TABLE9.6 Possible problems and remedies in pulsed-field agarose gel electrophoresis 1. Excessive 'smile' or bands disappearing from top of gel (temperature too high): - decrease temperature - reduce gel thickness for better heat dissipation - increase buffer recirculation 2. Smeared bands: - use lower voltages - run samples into gel for longer time before starting the switching device 3. Compressed bands at top of gels: - increase time intervals, voltage, temperature, or all - increase buffer concentration - use lower agarose concentration 4. Compressed bands at bottom of gels: - measures the opposite of 3.

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9.1.3. Agarose gel electrophoresis of RNA Analysis of RNA is more difficult than that of DNA since (i) RNases are ubiquitous and can rapidly degrade RNA resulting in smears upon electrophoresis, (ii) RNA is, generally, at least partially singlestranded and (iii) complex secondary structures influence the mobility unpredictably. Exceptions in this respect, such as the reoviruses with their segmented double-stranded RNA genome, are easier to analyze and are handled as DNA. Single-stranded RNA molecules are fractionated on denaturing agarose gels since complete denaturation of RNA renders electrophoretic mobility proportional to molecular weight (or length). Moreover, this denaturation is required for elution from the gel (or enhances) and for immobilization on the membrane in the Northern transfer procedure (Thomas, 1980). Three different denaturants are currently employed: formaldehyde; glyoxal/dimethylsulfoxide (DMSO); or methylmercuric hydroxide (in order of decreasing popularity). It is also possible to electrophorese native RNA and denature RNA before transfer (3 gel volumes of 10% formaldehyde at 65°C for 30 min) (Khandjian and MCric, 1986). The sensitivity equals that obtained with denatured RNA. This approach can be useful for studying nicked RNA or to compare native with denatured RNA. Irrespective of the electrophoresis and transfer procedure to nylon, UV fixation was found to be beneficial. Methylmercuric hydroxide is very toxic and should not be used if it can be avoided. This denaturant reacts with the RNA, before electrophoretic fractionation, primarily with the imino groups of uridine and guanosine (Gruenwedel and Davidson, 1966; Bailey and Davidson, 1976) and electrophoresis is carried out in the presence of methylmercuric hydroxide. Sulfhydryl compounds (Zmercaptoethan01, DTT) can be used to displace methylmercuric hydroxide from imino groups. Methylmercuric hydroxide is not only extremely toxic but also volatile and should be handled in chemical hoods. All waste should be disposed of as toxic material (Junghans, 1983). The choice between glyoxal/DMSO and formaldehyde is dictated by minor differences since the resolving power does not differ much (Miller, 1987); the glyoxal/DMSO method is somewhat more diffi-

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cult to perform and takes more time but may yield sharper bands. Rapid recirculation of the buffer during glyoxal/DMSO gel electrophoresis is required to maintain a constant pH, otherwise the pH may rise above 8.0 where the glyoxal adducts dissociate from the RNA. Formaldehyde is also toxic and should be handled in a chemical hood. For most sizes, RNA migrates faster than the corresponding DNA. The concentration of agarose in the denaturing gels (Table 9.7) is usually 1% for molecules larger than 1 kb and 1.4% for smaller molecules. RNA mobility is also determined by sieving and a logarithmic relation exists between migration speed and RNA size. Commonly, rRNA is included as standards (or still present) and as indicators of the integrity of RNA (assuming that degradation would be equal over all RNA species). Mouse 28s rRNA contains 4712 nucleotides (Hassouna et al., 1984) and mouse 18s rRNA contains 1869 nucleotides (Raynal et al., 1984). Other eukaryotic rRNAs have similar sizes. Markers are also available from commercial suppliers such as BRL. Two indicators are useful to estimate the integrity of RNA (i) the 28s RNA should be twice as fluorescent than the 18s RNA; (ii) the absence of smearing (i.e., the rRNA bands should stand out if the gel is not overloaded). The amount of RNA that needs to be loaded in a gel well depends on the abundance of the species to be detected. Whereas for Southern blotting a single-copy gene (i.e., about 1/106 of the total genome) can be detected in about 5 pg DNA, if the detectability is 5 pg (i.e., detectability of lo6 molecules), such a gene can be transcribed into numerous copies per cell (typically 100-500 pg per 5 p,g of mRNA). Using 5 pg of mRNA per well enables the detection of rare mRNAs comprising no more than 0.001% of the total mRNA (50 pg). In turn, mRNA consists of about 5% of the total RNA content of the cell. Thus mRNA is often easier detected than the corresponding gene and purification of mRNA from total RNA is often not necessary. It is a sine qua non to avoid any contamination with RNases. Sterile, disposable plasticware is essentially RNase-free and should be used whenever possible. General laboratory glassware, however, should be treated by baking at 180°C overnight, or, in the case of

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TABLE 9.7 Denaturing agarose gel electrophoresis for the fractionation of RNAs A.Preparation of electrophoresis tanks Use dedicated (preferably) o r cleaned gel box and combs. RNases are removed by thorough washing with detergents, rinsing with water, drying with ethanol, filled and incubated with 3% hydrogen peroxide for 10 min and rinsed with diethylpyrocarbonate (DEPCbtreated water.

B. Formaldehyde agarose gel electrophoresis (Seed, 1982; Miller, 1987) 1. Prepare running and sample buffers (use DEPC-treated warer): Running buffer ( 5 X ): 20.9 g 3-(N-morpholino)propane sulfonic acid (MOPS, free acid; from Sigma) in 800 ml of water containing SO mM sodium acetate; adjust pH to 7.0 by adding NaOH (or acetic acid). Add 10 ml of DEPC-treated 0.5 M EDTA and adjust the volume to 1 I with water. Sterilize by filtration through a 0.2 p m Millipore filter and store in the dark (when buffer becomes strongly colored, it should be discarded). See Table 9.11 for an alternative to MOPS buffer. Sample buffer: Mix 10 pI of 5 x running buffer, 8.75 pI of 37% formaldehyde and 25 p1 of formamide. RNA up to about 6 pl (maximum of 30 pg) can be added just before use, before or after heating (15 min at 55-65"C) and chilling o n ice. Loading buffer: DEPC-treated solution, containing 50% glycerol, 1 mM EDTA, 0.25% bromophenol blue and 0.25% xylene cyanol FF. Note: The DH of the formaldehvde solution (37% or 12.3 M) should be at least 4.0 and the formamide solution should not be yellowish; otherwise deionize, e.g., by mixing with Dowex XG8 and filtration through Whatman filters or over an AG-501-X8 column (Bio-Rad). The formaldehyde concentration in the gel depends on the length of the electrophoretic run: short runs (high voltage) require less formaldehyde (1.2%), whereas overnight runs may require 7% denaturant. It has even been reported that formaldehyde can be eliminated from the gel without affecting electrophoretic separation or subsequent transfer if runs are shorter than 3 h (only formaldehyde/formamide in loading buffer) (Liu and Chou, 1990). We generally use 1.2% ( = 0.41 M) formaldehyde, to decrease the level of toxic vapors in the gel. Melt agarose in water and cool to 6O"C; then add concentrated running buffer and formaldehyde so that the appropriate concentrations of agarose, 1 X running buffer and formaldehyde (see note) are obtained. Pour gel and allow gel to solidify. After the gel is set, submerge with 1 Xrunning buffer and prerun gel for 5 min at constant voltage (rapid electrophoresis: 5 V/cm or for most systems a setting of (6.25Xgel length) V) and run sample. Samples may be applied in duplicate so that half the gel can be stained and the other half can be used for hybridization. Recirculation is not necessary, but buffer may be remixed after 2 h. The run can be stopped when the bromophenol blue is about half way through the gel. Stain the duplicate half of the gel with ethidium bromide (0.5 pg/ml in 0.1 M NH,OAc) for 45 min.

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TABLE9.7 (continued) C. Glyoxal/DMSO gel electrophoresis (McMaster and Carmichael, 1977; Thomas, 1980, 1983) Note: (i) These gels should be run slower than formaldehyde gels and recirculation is necessary to avoid pH gradients during electrophoresis. (ii) Glyoxal (40% ( = 6 M) ethanediol in aqueous solution) should have a pH of at least 5.0; otherwise it contains too much glyoxalic acid and should be passed over a mixed-bed ion exchange resin (e.g., Bio-Rad: AG 501-X8) and stored at -20°C in tightly capped tubes. (iii) DMSO aliquots of high quality should also be frozen when purchased. (iv) Formamide is purified as in the previous protocol (section B). (v) Glyoxal reacts with ethidium bromide (no dye should be used). (vi) Glyoxal also reacts with proteins; samples should, therefore, be well deproteinized. (vii) Both glyoxal and DMSO are necessary for complete denaturation (Thomas, 1980). 1. Prepare agarose gel (1.5% for small RNAs; 1% for others) with 10 mM sodium phosphate (pH 7) running buffer. 2. Mix 15 pI of DMSO, 4.4 ~1 of 6 M ( = 40%) glyoxal and 3 FI of 0.1 M sodium phosphate, add RNA and complete with water to 30 ~ 1 Incubate . for 1 h at 50°C. 3. Cool the sample on ice and add 8 ml of loading buffer (50% glycerol, 10 mM sodium phosphate, pH 7 and 0.4% bromophenol blue). Load samples and run gel at 3-4 V/cm until the bromophenol blue is midway. It is essential to recirculate the buffer, using a peristaltic pump, from the cathode to the anode compartment or to change the buffer every 20-30 min. Magnetic stirring of the buffer in the compartments is also recommended.

plasticware, rinsing with chloroform. Diethylpyrocarbonate (DEPC; 0.1% in water; Section 3.1.4.3) (Fedorcsak and Ehrenberg, 1966) is a strong inhibitor of RNase and can be used to incubate glass- and plasticware with (2 h at 37°C) and subsequently inactivated by autoclaving or boiling at 100°C for 15 min. Otherwise carboxymethylation of the purines in RNA may occur although to a low degree this would hardly interfere with the hybridization. Thomas (1983) found that staining of RNA with ethidium bromide prior to transfer decreased the hybridization signal significantly. Consequently, it has been general practice to avoid staining (or, in

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some cases, to stain with acridinium orange) or stain only the duplicates for Northern hybridization. Kroczek and Siebert (19901, found that ethidium bromide added to the RNA in the loading sample, before heating (in their case 15 min at 55"C), produced very bright fluorescence of both high and low molecular weight RNA. Upon electrophoresis, this signal permitted an immediate assessment of the quality of RNA (particularly if rRNA is present), which can be documented by photography, as well as the evaluation of the degree of transfer achieved, i.e., fluorescent bands should then be present on the membrane. Whereas in standard Northern protocols (e.g., Wahl et al., 198%; Ausubel et al., 1987-1990; Perbal, 1988) the outcome is only available at the end of the experiment, in this adaptation each step can be easily assessed, thus saving time and expense. The decrease in signal due to staining in the method of Kroczek and Siebert (1990) is only about 10-20% (page 469, B).

9.2. Transfer procedures Initial transfer procedures involved nitrocellulose membranes that have the drawbacks of being brittle, of displaying diminished binding at lower ionic strength and inefficient binding of small fragments. Moreover, retention of nucleic acid during hybridization is poor. Nevertheless, the convenience, low background with nonradioactive probes and considerable experience with these membranes make them still a frequent choice. Several modifications have been made to retain the small fragments more efficiently. Most often, however, nylon is used for this purpose since it binds nucleic acid tightly (ss or ds, neutral or alkaline conditions, long or short fragments (> 40 bases), high or low ionic strength) and the membrane is strong, resistant to heat or solvents (reprobing is possible) and does not require prewetting. Separation of small fragments is best achieved with acrylamide or NuSieve agarose gel electrophoresis. Capillary transfer has been the classic procedure but simplified modifications and different versions of vacuum blotting are becoming popular. Large fragments are not efficiently transferred and DNA is clipped into = 1 kb fragments between electrophoresis and transfer.

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An alternative approach is to hybridize in dried agarose gels. This is

most efficient with short probes. Short target fragments are usually lost in this procedure during the drying in a gel dryer (pulled out with the gel liquid). Placing a nylon membrane between the gel and the vacuum source may trap small fragments.

9.2.1. Southern transfer Southern (1975) demonstrated that ss DNA could be transferred by capillary blotting from an agarose gel to a nitrocellulose membrane in high salt buffers (e.g., 20 X SSC or 1 M NH,OAc), producing a replica of the separated nucleic acid on the membrane which can then be analyzed by hybridization. The original technique is still widely used although it suffers from several drawbacks. Small fragments tend to diffuse laterally during blotting and are poorly bound to nitrocellulose. Lateral diffusion can be reduced by faster blotting methods such as those with vacuum or positive pressure transfer, whereas binding is improved by using nylon membranes. The transfer of large fragments tends to be inefficient. However, Wahl et al. (1979) improved the transfer of such fragments by limited depurination and fragmentation of the DNA after electrophoresis but before transfer. After transfer to the membrane, DNA is fixed (baking for nitrocellulose, baking or UV irradiation for nylon) and the target detected by the same techniques as for dot/slot blot hybridization (Section 8.2). The amount of DNA required to obtain a signal depends on several factors: (i) the complexity of the genome; (ii) the efficiency of transfer of the DNA to the membrane; (iii) the potency of the detection system. If the detectability is 5 amol, then 15 pg of a single plasmid insert band of 4500 bp would be needed but 5 amol of a single-copy gene in mammalian chromosomes (3 X lo9 bp) corresponds to 10 kg of total DNA. Restriction fragments of a 10 kg digest can be separated without problems on an agarose gel. Lower activity probes will require proportionately more DNA per lane. Restriction of high molecular weight DNA can be a problem since this DNA often aggregates and the inside of such clumps remains poorly accessible to enzymes. Prior to digestion, DNA is diluted to

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50 ng/p1 with restriction enzyme buffer (usually supplied as a 10 X concentrate with the enzyme) and incubated for several hours at 4°C (gently stirring periodically). The suspension is also gently mixed for a few minutes after the enzyme is added just before the suspension is brought to the appropriate incubation temperature. Spermidine can be added to 1-2.5 mM after the sample reaches the incubation temperature (spermidine precipitates at 4°C). This polycation often facilitates the digestion of genomic DNA by binding negatively charged contaminants. More enzyme is added after 30 min of incubation. Controls to which 10 and 1 pg of plasmid have been added serve as indicators of the completeness of digestion. After digestion, the diluted DNA is precipitated with ethanol (Table 3.1; not dried because large DNA resuspends only slowly) and resuspended in loading buffer (heated for 10 min at 70°C for dispersion of DNA and for evaporation of remaining ethanol). Nicked DNA gives inferior hybridization results and ligation of DNA prior to digestion improves the genomic blots (Koch et al., 1988; Verma, 1989). As a rule of thumb, 10 p,g of restricted mammalian DNA should be used if the specific activity exceeds lo9 cpm/p,g (i.e., at least about 1 in 25-50 P atoms is radioactive) and the probe length exceeds 500 bases (i.e,, containing at least ten 32Pper probe molecule; Section 7.2.2). Oligonucleotide probes will contain less radioactivity per attomole (usually 1 atom after kinasing) although the specific activity as expressed in cpm/p,g may be as high or even higher. When oligomer probes are used, more target should be loaded per lane (up to 50 Fg) or several, nonoverlapping probes to the same target, or more radioactivity introduced (e.g., by tailing). For DNA of lower complexity, proportionately less DNA can be used. Particularly with large amounts of DNA per lane, it may be better to stain the gel after electrophoresis instead of including the stain in the gel. It is also advantageous with genomic digests to run the gels at low potentials ( < 1 V/cm). After electrophoresis and staining, the gel should be photographed with a transparent ruler (sticking Scotch tape to the back and removing renders the partitions on the ruler fluorescent) so that migration of unlabeled markers in outside wells is documented.

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9.2.1.1. Capillary transfer techniques and simple derivatives Conventional capillary transfer, which has changed little since its introduction (Southern, 1975), is widely used. Recent modifications offer a much faster and more efficient transfer and are recommended (see paragraph below). The classical setup is simple (Fig. 9.2) and the basic method using nitrocellulose gives reasonable results for DNA fragments from 0.5 to 10-15 kbp (Table 9.8A). Small fragments (0.5-1.5 kb) are rapidly transferred upward in a few hours whereas the larger fragments (8-15 kb) require at least an overnight transfer. The efficiency of transfer decreases during the blotting since the gel becomes increasingly dehydrated (‘rubbery’) as more liquid is drawn by capillary force from the gel than is taken up from the buffer reservoir and the gel becomes compressed due to the weight. Prolonged blotting is therefore rarely beneficial. The efficiency of transfer of large fragments can be improved by fragmentation. A limited depurination of DNA by acid renders it susceptible to scission at depurination sites, by exposing to a strong alkali, producing smaller fragments (Section 6.1). The exposure to alkali at the same time serves to denature the nucleic acid. It is essential to prevent excessive depurination since this would produce too small DNA fragments (optimum = 1000 bases). Smaller fragments would diffuse laterally during the transfer and produce fuzzy bands. Moreover, binding of small DNA fragments is inefficient with nitrocellulose membranes. Downward capilllary transfer (Lichtenstein et al., 1990; Chomczynski, 1992) is very simple, does not require a depurination step, is efficient (1 h), does not cause flattening of the gel which is the main reason for inefficiency in the classical method, and produces higher hybridization signals since less nucleic acid is lost from the membrane due to prolonged transfers. The setup is shown in Fig. 9.2 and the transfer, according to Chomczynski (19921, is presented in Table 9.8B. This method is the most convenient among all transfer methods, both for DNA and RNA; however, for larger gels we found it necessary to use several trays with transfer solution to maintain uniformity of nucleic acid transfer. A bidirectional capillary transfer (Smith and Summers, 1980) (Table 9.8C) is feasible for Southern analysis of DNA present in high

I1

I

Glass plate

I

I

1

I I

II

I

Platform

h

II

-

Cover

r7v

111

Weight Transfer solution

-

Gel Membrane

Support

U

U

Whatmen 3 M M

~

Paper towels

Fig. 9.2. In conventional blotting systems (I), transfer solution flows by capillary force from the tray through the gel and the membrane to the dry paper towels. DNA in the gel co-migrates with the transfer solution to the membrane. In downward capillary transfer (I1 and III), a weight-induced gel flattening is avoided so that blotting is both faster and more efficient. In the initial descending system (11), transfer solution is placed in a frame above the gel. In the simplified system (III), transfer solution is brought to the top of the gel by a Whatman 3MM filter bridge. For minigels, one bridge is sufficient.

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TABLE 9.8 Capillary transfer of DNA A.Capillary transfer of DNA from agarose to nitrocellulose 1. Resolve DNA by agarose gel electrophoresis. Take into account the amount of DNA required for detection, the optimum voltage (0.7-1.0 V/cm for genomic DNA, otherwise higher) and whether or not ethidium bromide should be included (stain after run for genomic DNA). Photograph the gel after the run along a fluorescent ruler to establish the mobility (trim away unused areas of the gel with a razor blade and a corner for future orientation). 2. Depurinate DNA if fragments are longer than 10 kb by soaking the gel for 10 min in 500 ml of 0.2 N HCI with gentle agitation to ensure exposure to HCI (bromophenol blue will turn yellow). If the gel floats, it should be forced down (e.g., with glass pipettes). Rinse subsequently with water. 3. Incubate the gel in 500 ml of 1.5 M NaCI, 0.5 M NaOH (3X 15 min) to ensure denaturation and to fragment acid-treated DNA. 4. Rinse gel with deionized water and neutralize by incubation in 1 M Tris-HCI (pH 7.4) and 2 M NaOH (2 X 15 rnin). 5. Cut a nitrocellulose membrane to a size slightly smaller (a few mm) than the gel and float on deionized water (Table 8.5). If the membrane does not wet evenly from underneath within a few minutes, another membrane should be taken (unsatisfactory membranes can be autoclaved between 3MM filter papers soaked in 5 XSSC and stored wet in sealed bag). The wetted membrane is then immersed in the transfer buffer (e.g., 10 min 20 X SSC or 1 M NH,OAc). 6. Remove the gel from the neutralization solution and place on wet 3MM paper wick (Fig. 9.2) which is about 2 cm wider and 8 cm longer than the gel. The support for the wick and the gel can be a simple box which fits in an electrophoresis tray (e.g., from BRL; simplifying handling) or a glass plate resting on the edges of the tank with the overhangs of the wicks dipping in the transfer solution. The gel should be handled with gloves. Air bubbles between the support, the wick and the gel should be avoided. 7. Surround the gel with plastic film to prevent a short circuit (i.e., flow bypassing the gel producing uneven or no transfer) and place wet nitrocellulose membrane over the gel. Place one edge of the membrane just over the edge of the sample wells and carefully proceed downward. Do not move back once the membrane has touched the gel, particularly for blots with substantial amounts of target DNA. The membrane should not overhang the gel and no air bubbles should be trapped. 8. Place a 3MM paper (same size as membrane) in 2 X SSC, remove excess liquid by blotting and cover the membrane. Place three dry 3MM paper filters of the same size on the damp paper and place on a stack of dry paper towels (5 cm high; just smaller than the 3MM filter papers). 9. Place a glass plate on top of the towels for even pressure and weigh it down with 300-500 g.

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TABLE 9.8 (continued) 10. Blot overnight (short fragments for a few hours), remove towels and mark slots and edges of the gel on the membrane. Soak the gel for 5 min in 6 x SSC to remove agarose fragments. Remove the membrane with blunt forceps, allow to dry for 30 min, bake between two 3MM sheets in a vacuum at 80°C (Section 8.2.1). Staining of the gel to assess the efficiency of transfer is usually not very informative as ss DNA does not stain well.

B. Downward alkaline capillary transfer to nitrocellulose, nylon or PVDF This method is suited for nitrocellulose, nylon and Immobilon ( = PVDF); however, positively charged nylon permits direct transfer of DNA in strong base (shorten denaturation step 1 to 30 min and omit step 2). 1. Denature DNA, following electrophoresis, by incubating the gel for 1 h in a solution of 0.4 M NaOH and 1.5 M NaCI. 2. Wash the gel for 15 min in alkaline transfer solution (8 mM NaOH in 3 M NaCl (nitrocellulose) or in 1.5 M NaCl (nylon)). 3. Place a stack of 3 cm dry paper towels on a base (2 cm wider and longer than the gel) and cover with five sheets of blotting paper, wetted hybridization membrane and agarose gel (cut along the slot line to prevent leakage), as shown in Fig. 9.2. Cover the gel with three moist (in transfer solution) blotting papers (same size as the gel) and three moist blotting papers forming the bridge to the tray with the transfer solution. For minigels, one tray suffices; however for large gels, a tray at either side, each with three moist blotting papers are recommended (the level of transfer solution in these trays should be the same; achieved by adding a tube between these trays and pulling the solution through with a syringe). Finally, the bridge papers are covered with a plastic cover ( e g , from a microtiter plate). 4. Transfer during 1 h, disassemble and neutralize the gel by immersion for 10 min in 0.2 M sodium phosphate buffer (pH 6.8) (1 ml/cm2). 5. Dry the membrane and fix DNA by incubation for 15 min at 80°C (vacuum for nitrocellulose). C. Bidirectional transfer of DNA to nitrocellulose 1. Resolve DNA by agarose electrophoresis (gel not too thin!), depurinate if necessary and denature DNA as described in A1-4. 2. Soak the gel for 30 min in 1OXSSC (one corner of the gel cut off for future orientation). 3. Cut two nitrocellulose membranes (also with one corner removed) and wet in water and 10 X SSC, respectively (see A 9 4. Place one wet nitrocellulose membrane on wetted (excess blotted off) 3MM filter paper, place the gel on top and then a second nitrocellulose membrane (cut corners aligned; no air bubbles; precautions as in A6) and another damp 3MM filter paper. 5. Place the sandwich between two stacks of paper towels, put a glass plate on top and weigh down with 300-500 g. 6. Remove membranes after 2 h of blotting as described in A10.

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TABLE9.8 (continued) D. Neutral capillary transfer of DNA from agarose to nylon 1. Resolve DNA as described in A1-4. 2. Cut positively charged nylon membrane to the size of the gel and float on water until completely wetted from underneath and immerse in 1OXSSC. 3. Assemble transfer pyramid as in A6-9. Note that some manufacturers recommend a particular side of the nylon to be used. 4. Remove the membrane as in A10 and fix DNA by drying (hair dryer, conventional oven, vacuum oven) or expose the side of the membrane with the DNA to an optimal dose of W (Section 8.2.1). UV irradiation yields superior results for several nylon brands.

E. Alkaline capillary transfer of DNA from agarose to nylon 1. Resolve DNA molecules and if necessary depurinate as described in A1-2. 2. Soak the gel for 2X 15 rnin in transfer buffer (0.4 NaOH and 1 M NaCI) to denature the DNA. Gel can be weighed down with glass pipettes if it floats. 3. Prepare nylon as in D2, but use transfer buffer instead of 10 X SSC, assemble transfer pyramid and blot as in A6-10. 4. Neutralize the membrane for 15 rnin in 0.5 M Tris-HCI (pH 7.2) and 1 M NaCI. 5. Blot fluid from the membrane, dry the membrane for 30 rnin and fix DNA as in C4. F. Alkaline capillary transfer of DNA from polyacrylamide gels to nylon membranes 1. Treat one glass plate with Bind-Silane (Pharmacia) and the other with silicon (Pledge, PAM). The polyacrylamide gel will bind to the Bind-Silane-treated plate. Polyacrylamide at 4% (20 X 20 cm2, 1 mm thick; 30% acrylamide stock contains 29 g of acrylamide and 1 g of bisacrylamide; TBE buffer) allows the separation of 2 kb down to at least 0.2 kb (higher polyacrylamide concentrations allows other size windows, Table 9.2). 2. After electrophoresis (850 V, 15 W, 3 h), separate the glass plates and place the plate with the gel in depurination solution (0.25 M HCI) for 20 min and then transfer it to a solution of 0.5 M NaOH (20 min). 3. Remove excess liquid and place wetted charged nylon membrane (as in D3) on gel followed by 3MM paper and a blotting stack. As in bidirectional transfer, the solution in the gel is the only transfer medium. 4. Disassemble the transfer pyramid after 4 h (soaking of gel with membrane for 10 min in distilled water facilitates separation). Continue as in E4-5.

concentrations in the gel (e.g., from plasmids, phages or viruses). In this setup, the gel is sandwiched between two membranes and two stacks of paper towels. The liquid in the gel is drawn to both sides

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and the gel becomes fast dehydrated. Only a small amount is transferred to the two membranes and this method is not usable for the analysis of genomic DNA. However, it is fast (2 h) and two replicas are obtained from the gel. In the unidirectional modification described by Nakano et al. (19901, the gel is blotted after step 1 (Table 9.8C) to remove excess liquid and then covered with charged nylon membrane, two sheets of Whatman 3MM paper, a stack of absorbent papers (5 cm) and a glass plate (transfer takes 5 h). This unidirectional method can be combined with in-gel hybridization of DNA left behind during the transfer, as described in Section 9.3, to obtain duplicates with a single transfer (Rao et al., 1987). This method is better suited than the bidirectional method for genomic blots. In a comparison of the effect of different salts on the transfer of DNA to nylon (Hybond-N and Genescreen) and nitrocellulose (BA45) membranes, Allefs et al. (1990) observed that NH,CI or NH,OAc yielded twice as much signal as 10 X SSC, 1 M NaCl or 1 M NaOAc. Small DNA fragments bind poorly to nitrocellulose and covalent linking to diazotized membranes has been used to circumvent this problem (Reiser et al., 1978; Stellwag and Dahlberg, 1980). Alternatively, glyoxal/DMSO may promote the transfer and retention of small DNA fragments to nitrocellulose (Thomas, 1983) (Section 9.2.2). However, more recently nylon membranes have been used to capture small fragments which are then immobilized by UV irradiation (Church and Gilbert, 1984). For transfer to diazotized membranes (Wahl et al., 1987a,b), DNA is fractionated by electrophoresis as for other Southern procedures. However, after electrophoresis, the gel is rinsed with water and soaked in 1 M NaOAc (pH 4.0, 30 min) followed by rinsing in water and soaking in 20 mM NaOAc for 30 min. Transfer to diazotized membranes is as for nitrocellulose except that 20 mM NaOAc is used as a blotting buffer. Since DNA becomes covalently linked, it is not necessary to bake the membranes. Nylon membranes are generally preferred over these membranes for hybridization. Two types of nylon membranes (amphoteric and positively charged) can be used although differences among brands can be

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considerable (Section 8.1.1.1). These membranes offer the advantages of (i) easy handling, (ii) efficient binding of small fragments, (iii) binding of ds DNA, (iv) high detectability (retention of target) and (v) possibility of reprobing. They tend, however, to yield higher levels of background, particularly for nonradioactive probes. The blocking step is then particularly important. The basic method for capillary transfer of DNA to nylon membranes under neutral conditions is described in Table 9.8D. Positively charged nylon membranes also bind denatured DNA under alkaline conditions (Table 9.8E). This can be useful, e.g., for the immobilization of palindromic DNA. The background tends to increase with the time of exposure of the membrane to alkali and higher concentrations of blocking agents will be necessary. Rigaud et al. (1987) confirmed that baking after an alkaline transfer (Reed and Mann, 1985) does not increase the signal, in contrast to that after an NH,OAc transfer, and that short alkaline transfers (4 h) are beneficial in contrast to long transfers. Overall, transfer in NH,OAc yields a 10-fold higher signal than in NaOH. Conventional Southern transfer from agarose gels is unable to distinguish fragments with small size differences and polyacrylamide, NuSieve GTG agarose or HydrolinkTMgels are used instead. Although polyacrylamide gels offer very high resolution, the transfer from these gels is difficult (we have not tried downward capillary transfer) and electroblotting is usually required (Section 9.2.1.3). Martinson and Clegg (1990) reported a simple procedure (Table 9.8F) in which the gel is bound to one of the glass plates and the restriction fragments transferred under alkaline conditions to a charged membrane. The glass plate simplifies the handling of the gel and prevents distortion of the gel during the preparation of the blot. It should be noted that NaOH depolymerizes acrylamide which may then trap DNA. NuSieve agarose offers the possibility of rapid alkaline, capillary transfer of small fragments to nylon (Hatcher et al., 1990). However, it is then necessary to depurinate DNA for longer than the standard 3 min for fragments > 0.5 kb. It is not clear whether the irreproducible results reported by Kawasaki et al. (1988), using the same technique, are due to the membrane (Zetaprobe instead of Genatran).

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9.2.1.2. Pocket blotting, centrifugal transfer and vacuum or positive pressure transfer Vacuum blotting, in which DNA or RNA is drawn to the membrane by low pressure, is convenient, easy and rapid (Olszewska and Jones, 1988). Moreover, this transfer gives a high band resolution. However, it requires special equipment (e.g., Pharmacia LKE? 2016 VacuGene XL) although simple, attractive alternatives can be used (‘pocket blotting’). In the commercial system, the gel to be transferred is placed on the transfer membrane held within a window in a waterproof mask on a porous screen. This setup is locked to the top of the vacuum unit, a slight vacuum is applied and the gel sequentially flooded with the different solutions (Table 9.9A and B indicate Pharmacia’s recommendations). The transfer efficiency is highly dependent on the pressure and optimum transfers are obtained in a small range of 30-50 mbar. Transfer of DNA can be as efficient as 95% but RNA transfers can be uneven or nonreproducible at high concentrations of formaldehyde. Vacuum blot units should be installed and used according to the manufacturer’s recommendations. Although vacuum blotting is usually done with agarose gels, it is also applicable to polyacrylamide gels (Rosenberg and Amrani, 1989) and takes only 1-2 h. In the PosiblotTM pressure blotter (Braman and Dycaico, 1989) (Stratagene), transfer is achieved by positive pressure. The positive pressure above the buffer drives it through the gel and reduces the problem of gel collapse which may occur in vacuum blotting if the vacuum is too high. Therefore, a considerably higher pressure can be applied to the gel and the efficiency or speed may be higher. Wilkins and Snell (1987) used a microtitre tray lid in an IEC rack rotor, at 600 rev./min, to centrifuge fractionated DNA from the agarose gel onto the membrane. For this purpose, gels of up to 8 x 12 cm2 are sandwiched between a nylon membrane and two Whatman paper sheets, all soaked in transfer buffer (e.g., 20 X SSC) and centrifuged for 20 min. Pocket blotting (Cuny et al., 1991) is a simple, low cost alternative to vacuum blotting and does not require special equipment. It is slightly slower, but demands less attention and allows a large number of gels to be blotted simultaneously (Table 9.9C).

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TABLE9.9 Vacuum blotting A. Vacuum blotting - of DNA 1. Set up a vacuum blotting unit according to the manufacturer’s recommendations, pretreat the nylon membrane (Table 9.8.D2) and place on a porous screen. Place a mask, with gel window, on the screen so that it overlaps the membrane in the window by about 5 mm on each side. 2. Place the gel carefully on the membrane (no air bubbles) so that the gel and mask overlap by at least 2 mm and fit the top frame. Small cracks in the gel should be repaired with molten agarose. Switch on the vacuum pump (trap between the blotting unit and the pump to avoid solutions reaching the pump) and adjust to 50 mbar. Note that all solutions should be ready since the gel is covered with solutions whenever the vacuum is on. 3. For genomic DNA, depurinate by pouring 25-50 ml of 0.2 N HCI onto the gel (with a pipette; if poured beside the gel, it may lift from the membrane) until the bromophenol blue turns yellow (usually about 20 mid. Excess liquid can then be removed. Alternatively, DNA can be nicked by exposure to UV light at 302 nm for about 10 min (UV table should be calibrated) or half the time at 254 nm. 4. Pour denaturation solution (0.5 M NaOH and 0.5 M NaCI; about same volume as required for depurination). Neutralize with 1 M Tris-HCI (pH 7.5) and 1.5 M NaCl for about the same time as depurination. 5. Add transfer buffer (20XSSC) to the upper chamber and transfer for three times the time reauired for depurination. Note: for alkaline transfer, steps 4 and 5 are replaced by a single step in which 1 M NaOH is poured onto the gel and enough is added to the upper chamber for a transfer of 1-1.5 h. 6. Turn off the pump, mark the wells and remove the gel. Wash the membrane with 2 X SSC (alkaline transfer) or 20 X SSC to remove agarose.

B. Vacuum blotting of RNA 1. Proceed as in A1,2; use low concentrations of formaldehyde in resolving gel and nylon membranes. 2. For formaldehyde gels: wash gel by vacuum for 5 min with water, add 50 mM NaOH/10 mM NaCl also for 5 min and neutralize the gel with 0.1 M Tris-HCI (pH 7.4) for 5 min. 3. Transfer RNA by adding transfer buffer (20 X SSC or 0.1 M sodium phosphate buffer, pH 6.5) for at least 30 min. 4. Turn off the pump, mark the wells and remove the gel. Wash the membrane rapidly in transfer buffer.

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TABLE 9.9 (continued) C. Pocket blotting 1. Resolve DNA fragments by electrophoresis in 0.8% agarose. Cuny et al. (1991) observed that prolonged depurination (2X 20 min) and denaturation (2X 25 min in 0.4 M NaOH) produced a superior transfer without loss of small fragments. 2. Combine, using a glass support, a stack of wiping cloths (four times the thickness of the gel), two 3MM filter sheets wetted in denaturation buffer, a wetted nylon membrane cut to the size of the gel and the agarose gel. Nitrocellulose membranes are not recommended for this procedure. 3. Place the stack in a hybridization bag, heat-seal while leaving a small opening in one corner and create a vacuum within the bag with a water-driven pump (a few seconds) and heat-seal the open corner. 4. Allow the transfer to proceed for 1.5-2 h at room temperature until the gel is dry. 5. Peel the membrane from the gel and rinse twice for 2 min in 2XSSPE and dry or proceed with hybridization.

9.2.1.3. Electroblotting Electroblotting (Bittner et al., 1980) is used only if capillary or vacuum blotting fail, such as in some cases for the transfer of DNA fragments from polyacrylamide. It requires low ionic strength buffers to prevent an excessive heat buildup and to increase the mobility of the DNA. Membranes should be used which have superior binding in such buffers (e.g., Genescreen or diazotized membranes). Blotting will be complete in at most 3 h. Electrolysis may rapidly weaken the buffering capacity and large volumes of buffer will be needed. Other problems include distortion of bands by air pockets, heat buildup between gel and supporting 3MM papers, or the creation of pH gradients during transfer. Fang (1991) investigated a large number of parameters for electroblotting to positively charged nylon membranes (lower voltage, longer transfer, better cooling, different transfer buffers) none of which improved the transfer. It was observed, however, that soaking of 3MM paper for > 2 h prior to transfer improved the blotting, as did the use of neutral instead of charged nylon membranes. Both ss and ds DNA fragments can be electroblotted on nylon. After nondenaturing electrophoresis, ds can be transferred first to

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TABLE 9.10 Electrophoretic transfer of DNA from polyacrylamide gels 1. Separate DNA fragments on nondenaturing (for ds DNA) or denaturing (for ss DNA) polyacrylamide gel. A small amount of labeled markers are included in a separate track to assess the efficiency of transfer. Staining of gel is generally only useful for ds DNA at about > 25 ng/band. 2. Cut (preferably neutral) nylon membrane to the size of the gel and mark with a pencil the side that will be in contact with the gel and hydrate the membrane for 30 min in 50 mM TBE. 3. Pry the glass plate from the gel (which remains on other plate) and cover the gel carefully with filter paper (no air bubbles). This facilitates the handling of the gel (sticks to the paper). Lift the gel carefully by the paper from the glass plate and place it (gel side up) on a Scotch-Brite pad with a 3MM filter paper. 4. Wet the gel with a small amount of TBE and place the nylon membrane carefully on the gel. Place successively two 3MM filter papers (soaked for at least 2 h in TBE) on top of the membrane and cover with a second Scotch-Brite pad. Air bubbles should be avoided during each addition of membrane or filter paper (otherwise remove by rolling pipette). 5. Place the blot sandwich in a tank with 50 mM TBE and electrophorese for 3 h at 40 V at 4-15°C. 6. Disassemble the sandwich and in the case of ds DNA, place the membrane on filter papers saturated with 0.4 M NaOH for 10 min. Rinse the membrane with 2 X SSPE and proceed with fixation and hybridization (Section 8.2).

the membrane (faster than ss DNA) before denaturation (Table 9.10). Denaturation before transfer is also common. If problems are experienced by the depolymerization of acrylamide resulting in the trapping of the DNA, then, alternatively, the gel can be tightly sealed in a bag and incubated for 10 min in boiling water (dish must be covered) and 10 min in icy water. DNA can then be electroblotted without problems (Prtat, 1990). Nitrocellulose does not retain DNA efficiently at low salt concentrations although the incorporation of 4,5’,8-trimethylpsoralen(TMP or Trioxsalen; Sigma) at 10 Fg/ml in the gel improves both the transfer of small fragments to nitrocellulose and their detectability (Frossard et al., 1983). In this case, electrophoresis is carried out in ambient light, denaturation and neutralization in the dark and the gel is submitted to ambient light for 1 min before electrotransfer.

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Transfer to nitrocellulose in low salt (phosphate/citrate) is also improved by adjusting the pH to 3.0 (Smith et al., 1984). However, nylon should be chosen whenever possible. 9.2.2. Northern blotting

Northern analysis (Alwine et al., 1977, 1979) of RNA after electrophoretic fractionation was initially carried out on diazotized (DBM) paper, but its detection limit was only about 500 pg RNA (using a probe of lo8 cpm/p,g). Subsequently, Thomas (1980, 1983) succeeded in detecting less than 1 pg on nitrocellulose. Diazotized (APT) paper was also found to be superior to DBM paper (Seed, 1982), however nitrocellulose and charged nylon (Reed and Mann, 1985) have become the preferred membranes. RNA can be detected at less than 0.01% (10 pg loaded) on nitrocellulose or, particularly on nylon which usually has an increased background, however. Nylon membranes are required if reprobing is desired. Although RNA can bind as well as DNA to nitrocellulose, reproducible results are obtained only if RNA is carefully denatured. Among formaldehyde, glyoxal/DMSO, methylmercuric hydroxide, alkali and heat, the first two are most effective and give best binding, whereas alkali pretreatment can even be counterproductive (Thomas, 1983; Henderson et al., 1991). Recently, some attractive alternatives to these standard methods have become available (see below). The transfer of RNA from gel to membrane is as for DNA, of which the downward alkaline capillary transfer is the most attractive (Table 9.11A). Generally, radioactive probes are used for the detection of RNA, notably for rare transcripts. However, several powerful nonradioactive systems, such as DIG-labeled probes, have been developed to eliminate health hazards, probe instability and cost. Nonradioactive systems are unlikely to work if it takes longer than 36-48 h with a 32P-probeto obtain a signal. The modification of the standard Northern method by Kroczek and Siebert (1990) (Table 9.11B) involves staining of the RNA sample by heating in the presence of ethidium bromide, a low formaldehyde concentration in the gel and vacuum blotting. This

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made it possible to monitor the integrity and migration of RNA during electrophoresis (detection limit 30 ng/band) instead of after hybridization. Moreover, the efficiency of the transfer can be checked. The use of low concentration of formaldehyde, possible since the high voltage electrophoresis run is short, may be resposible for the efficient transfer of RNA by vacuum. Low quality RNA is recognized by smearing and disappearance of the rRNA bands. Rosen et al. (1990) observed that the quality of the gel determines in large measure the end result, e.g., the gel thickness should not exceed 4 mm and the gel should be evenly poured. The negative effect of ethidium bromide staining on hybridization was minor in contrast to earlier reports (Thomas, 1983) (about 15%). A ruler placed next to the membrane or marking on the membrane allows the relative positions of the rRNA markers to be documented. Although RNA ladders (BRL) can be used, Chaudhari (1991) showed that denatured plasmid restriction fragments (stored at - 20°C) are also accurate markers for Northern blots (1-20 pg DNA per band). These do not require a separate probe if the specific probe contains plasmid sequences. The Northern blotting variant described by Wilkinson et al. (1991) (Table 9.11C) also allows an assessment of the integrity and amount of RNA after electrophoresis and a determination of the efficiency of transfer. Acridine orange ( A 0 stain) is used to stain the RNA after electrophoresis in a formaldehyde-agarose gel whereas staining of the membrane with methylene blue (MB) stain allows the detection of as little as 15 ng (visible band; size markers can be indicated on the blot). MB stain is then removed if the 30% decrease it causes in hybridization signal is important. A 0 stain does not affect the hybridization signal and distinguishes ds from ss nucleic acid (McMaster and Carmichael, 1977). Hybridization of Northern blots calls for complicated buffers (Chapter 8). However, as for DNA (Church and Gilbert, 19841, it is possible to use single buffers with high SDS concentrations for (pre)hybridization (Virca et al., 1990). (Prelhybridization is carried out in a solution containing 50 mM PIPES, 1 mM EDTA and 5% SDS for 15 min and hybridization in the same (fresh) buffer containing lo6 cpm/ml of probe (or equivalent nonradioactive probe)

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TABLE 9.11 Northern blotting A. Downward alkaline capillary transfer (1.5 h) The alkaline conditions used in this method do not affect the hybridization results appreciably (signal about 80% stronger with this method than with the standard methods). Glyoxylated RNA gels can be used directly since glyoxal dissociates from RNA in alkaline conditions. In the case of formaldehyde gels, a concentration of 0.41 M is usually adequate. 1. Proceed, after electrophoresis, as in Table 9.8B, steps 3-5. Gel washing before transfer is not required. 2. Drying should be kept at 15 min as prolonged fixation decreases signals (see Table 9.8 B).

B. Optimized method after staining of RNA before electrophoresis Direct staining of RNA before electrophoresis makes it possible to establish the quantity and quality of RNA and to evaluate the efficiency of each step. Diamond (1992) proposed a borate buffer as an alternative to MOPS since the latter is light-sensitive and has limited stability (20 X RNA borate buffer: 24.73 g tetraborate, 38.14 g borate and 8 ml 0.5 M EDTA per liter). 1. Dissolve vacuum-dried RNA in sample buffer (10 p,g/50 pl; 1X MOPS buffer, 5 mM NaOAc and 1 mM EDTA, pH 6-7, 6.5% formaldehyde, 50% deionized formamide) and add then 10 p,l of loading buffer (1 mM EDTA, pH 8.0, 0.25% bromophenol blue, 0.25% xylene cyanol, 50% glycerol) and 2 p1 of ethidium bromide (0.5 p,g/pl). Heat the sample for 15 min at 55T, quench on ice and load on formamide gel (Table 9.7; 1.2% agarose, 1.1% formaldehyde). Run electrophoresis at 5 V/cm with recirculation of 1 XMOPS buffer. In this procedure, only sample and loading buffer and MOPS electrophoresis buffer are DEPC-treated. Separation of RNA can be followed by the separation of the 18s and 28s rRNA bands (MOPS buffer as in Table 9.7 B). 2. After electrophoresis, soak the gel for 20 min in transfer buffer and vacuum-blot to a nylon membrane (4 h) or as above (A). 3. Fix RNA by UV irradiation and proceed with hybridization as described in Section 8.2. C. Acridine orange and methylene blue staining prior to and during Northern transfer 1. Stain formaldehyde-agarose gel after electrophoresis for 3 min with A 0 stain (15 pg/ml acridine orange in 10 mM sodium phosphate buffer, pH 6.5 and 3% formaldehyde). The same solution can be used repeatedly but it may be necessary to prolong staining. 2. Destain gel for 20 min in the phosphate buffer containing 3% formaldehyde. 3. Transfer RNA to a nylon membrane, UV irradiate and immerse in MB stain (0.03% methylene blue in 0.3 M sodium acetate, pH 5.2) for 1 min and rinse with water for 2 min. Identify bands (e.g., photography or marking on blot). 4. Remove MB stain by washing for 10 min with 1% SDS/l XSSPE. Proceed with (pre)hybridization.

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overnight with both incubations at the same temperature. Posthybridization washes are in 5% SDS and 1 X SSC (room temperature for the ‘hot rinse’ and hybridization temperature, respectively). For oligomeric probe incubations, temperatures should be strictly 543°C below the T,. Nonspecific signals increase rapidly below this optimum whereas at higher temperatures, the signals become weaker (Henderson et al., 1991). Formamide should not be used with oligomer probes.

9.3. Hybridization in the gel or before electrophoresis Most of the target nucleic acid is lost during the transfer or during hybridization (Gamper et al., 1986). Strategies have been developed to eliminate the transfer steps and to decrease losses during hybridization: (i) hybridization prior to electrophoresis, drying of the gel and detection in the gel; (ii) hybridization in the gel after electrophoresis. Solution hybridization of the probe with the target and electrophoretic fractionation has led to the S1 and RNase blocking techniques described in Chapter 12 and reverse Southern hybridization described below. Hybridization in the gel Cunblot’) may be a good alternative, particularly with oligomer probes. Advantages of these methods are their speed and efficiency. Drawbacks are the loss of small fragments ( < 1 kbp) during hybridization in gel or the necessity of specially modified oligomers in the reverse Southern method, and that their simplicity does not match that of downward capillary transfer which is equally fast and efficient.

9.3.1. Reverse Southern analysis In reverse Southern hybridization (Gamper et al., 1986), a crosslinkable DNA oligomer probe is hybridized in solution to its target prior to denaturing electrophoretic separation. The probe is kinased with 32P (Section 7.7.1) and HMT (4‘-hydroxymethyl-4,5’,8-trimethylpsora1en)furan thymidine adducts are formed by photofixation simultaneously with hybridization (‘photochemical pumping’). Excess of probes is removed after hybridization in solution by Centricon cen-

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trifugation (Section 3.1.4.2) and the crosslinked hybrid will withstand strand displacement and is stable during denaturing gel electrophoresis (in 30 mM NaOH, 1 mM EDTA).

9.3.2. Hybridization in gel after electrophoresis of DNA Although hybridization of DNA in agarose gels in situ has been described simultaneously with the Southern method (Shinnick et al., 1975; Southern, 1975), it is becoming popular only in recent years after its rediscovery in 1983 (Purrello and Balazs, 1983; Tsao et al., 1983). Following electrophoresis and photography of the gel, DNA is denatured by adding 5 vols. of 0.5 M NaOH/O.15 M NaCl to the gel and incubating for 20-30 min and subsequent neutralization in 0.5 M Tris-HC1 (pH 8)/0.15 M NaCl for 20-30 min with occasional shaking (no depurination). The gel is then placed on a dialysis membrane or two sheets of 3MM paper and dried. Typically, the gel is trimmed (to make the gel as small as possible for hybridization) and covered with a plastic wrap. The sandwich (3MM paper, gel, plastic) is then placed in a gel dryer, covered with the neoprene sheet and dried with only vacuum for 30 min and then with heat to 60°C for another hour. The gel becomes brittle if too dry whereas at 80"C, it will melt. However, background increases if the gel is not dry enough. The gel should resemble a dialysis membrane in consistency and thickness and can be stored at room temperature until hybridization. Prior to hybridization, the gel is wetted until it can be removed (but leave on backing for easier handling) from its backing (the gel does not rehydrate significantly but becomes very flexible). The gel is then hybridized in situ with probe (e.g., lo6 cpm/ml in 5 X SSPE, 0.1% SDS and 10 pg/ml carrier DNA; 50 pl/cm2 gel at 58°C for a 22-mer with 50% GC, but five times less salt for a 30-mer; prehybridize, if necessary, by using prehybridization solution during wetting of dried gel), washed at room temperature to remove the bulk of unhybridized probe, followed by stringent washes (a few minutes, but longer if probes are tailed). Do not increase the temperature to above 60°C for increased stringency, but use lower salt concentration instead (e.g., for longer oligomers). Reprobing is possible after denaturation/neutralization as described above. The gel is then

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wrapped in a plastic sheet and exposed to X-ray film with an intensifying screen. Signals can be increased by drying the gel before exposure (complete drying makes additional washing impossible in the case of high background). In-gel hybridization is not restricted to oligomer probes. In fact, the first reports were based on experiments with long probes. Ehtesham and Hasnain (1991) used nick-translated probes of up to 2 kbp and up to 1.2% agarose gels. However, this method is most suitable for short probes (nick-translated probes can be made shorter by increasing the DNase activity, Section 7.6.1) and long target molecules ( < 0.5 kb fragments hardly detected).

9.3.3. Hybridization in gel after electrophoresis of RNA RNA, if not too small, can also be detected by in-gel hybridization. Instead of denaturation/neutralization, the formaldehyde-containing gel is washed for 30 min in 0.1 M Tris-HC1 (pH 7.5) (Wallace and Miyada, 1987). Ahmad et al. (1990) fractionated RNA in a formaldehyde-containing agarose gel in MOPS buffer (Section 9.1.31, washed the gel in DEPC-treated water (twice) and placed it between two sheets of gel blot paper (S & S GB002) before drying in a gel dryer as described in the previous section. They hybridized with cDNA at 42°C in 50% formamide, 5 X Denhardt’s solution and 5 X SSPE as described in Section 9.3.2.

9.4. Hybridization after transfer to membranes Target nucleic acid is detected as in slot/dot blot hybridization (Section 8.2). It is important that all agarose fragments are removed from the membrane, e.g., by gently rubbing, to avoid nonspecific signals. If bands from large DNA fragments appear fuzzy, depurination has most probably proceeded too far. High molecular weight DNA also tends to adsorb kinased oligomer probes nonspecifically and restriction often remedies this problem. Probes to ubiquitous, short interspersed repetitive sequences (SINES or ‘simple D N A ) are useful to detect many independent loci and to obtain DNA finger-

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prints (Jeffreys et al., 1985; Ali et al., 1986; Schafer et al., 1988; Carter et al., 1989). As for dot blot hybridization, RNA probes can be used to detect RNA targets but these probes could not be stripped from the membrane due to the higher stability of RNA:RNA hybrids (Srivastava and Schonfeld, 1991). Moreover, RNA probes make very stringent washes mandatory (e.g., (prelhybridization at 60°C and in 5 X SSPE, 50% formamide, 0.2% SDS, 200 Fg/ml denatured carrier DNA and 200 Fg/ml yeast tRNA and washes, after a 'hot rinse', in 0.1 X SSPE, 0.1% SDS at 60°C) and may still lead to spurious binding of probes to rRNA. Normalization of blots by rehybridizing with a probe to a cellular function suffers from the variation in the level of expression of some genes. Instead, p-actin or 28s rRNA probes (Barbu and Dautry, 1989) are suggested. Note added in proof A.D. Stiles and B. Moats-Staats noted in the Winter 1993 Red Book Bulletin that riboprobed Northerns can be routinely stripped and rehybridized (on Genescreen nylon). The stripping solution contains 10 mM sodium phosphate (pH 6.8) in 50% formamide. The membrane is incubated in 100 ml for 1 h at 70"C, rinsed with 25 mM sodium phosphate, and submitted to autoradiography to check whether any signal remains.

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CHAPTER 10

Colony and plaque lift hybridization A single colony or plaque contains enough DNA for detectable hybridization to a labeled probe. Colony or plaque hybridization thus allows the detection of particular plasmid or bacteriophage clones among a large number in a genomic or in a cDNA library to facilitate their selection. This technique is complementary to phenotype selection, immunoscreening or hybrid selection of mRNA and translation. If on the other hand a partial sequence is known (e.g., deduced from N-terminal sequence from a protein), then it is straightforward to synthesize a biotin-labeled oligomer and to select the appropriate mRNA from the cells (Table 3.16) to generate cDNA clones from the selected mRNA or even to amplify directly through a PCR method (Chapter 5). Many libraries are available commercially or from colleagues. Many journals require that libraries or clones discussed in their publications become freely available to the scientific community. When generating libraries (Chapter 31, it is essential that both vector and target DNA are free of contaminants detectable by the probes since specific sequences may represent only lo-’ of the total target. In addition to screening libraries of bacteria containing recombinant DNA, this method is also useful for genetic characterization of a variety of organisms. An example is the detection of enterotoxigenic E. coli (Moseley et al., 1980; Maas, 1983). Colony and plaque hybridization follow the same essential steps: (i) distribution and growing of bacteria, phage, etc., on plates (‘master plates’) and replica plating on membranes; (ii) processing of the replica membranes by lysis of the bacteria or other cells or phages in situ and hydrolysis by RNase; (iii) fixation of DNA and hybridization; (iv) location of a positive signal and picking a corresponding clone on the master plate. For the uninitiated, it is useful to mix specific

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recombinant clones (e.g., 100 colony-forming units) with, e.g., 1000 colony-forming units of nonrecombinant bacteria (or wildtype phage with recombinant phage, 50 plaque-forming units each, etc.) and gain experience before using precious material.

10.1. Colony and plaque hybridization: different approaches The probability that a sequence of interest is present in a library can be calculated as shown in Table 10.1. The optimal digestion of the target (e.g., partial digest with Suu3A) to obtain maximum randomness of cloning is achieved when the most abundant insert size equals the vector capacity (Seed et al., 1982). A problem which may frequently occur is the change of representation of clones in the libraries during amplification. Some clones may be lost or become under-represented just because they reproduce slightly less rapidly. Independent libraries may then have to be screened. False positives also occur often, even if utmost care is taken to prevent contamination. In the case of cDNA libraries, messages can range from lo-' to lop5 of total mRNA. The size of the cDNA library required is directly related to the abundance of the message. Abundant messages can be cloned into plasmids but for relatively rare transcripts it is advantageous to use lambda vectors since they have a high cloning efficiency (e.g., lambda gtlO). Alternatively, vectors such as lambdaZAP I1 can be used which allow the excision of plasmids or ss DNA. Libraries should always be obtained from cells which express the mRNA most abundantly. Since bacterial colonies or phage plaques contain large amounts of a clone, the inserts can be screened directly by hybridization (Grunstein and Hogness, 1975; Benton and Davis, 1977). Large numbers of clones can be grown on agar plates and replica copies on nitrocellulose membranes are prepared for rapid screening. It is essential to avoid handling membranes with bare hands or contaminated gloves and to mark membranes for future orientation. One of the major problems is the occurrence of false positives.

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TABLE 10.1 Probability of presence of sequence of interest in random rDNA library The likelihood P that a sequence of interest is present among the clones obtained can be calculated easily if clones represent completely random fragments of the nucleic acid cloned. If f is the fraction that the average clone represents of the total target genome (i.e., bp of average clone/bp of target genome) and T the total number of screened clones, then P is the probability that the sequence of interest is present among T clones. Two different approaches to calculate P (or T required to realize a certain P ) are: 1. The method of Clarke and Carbon (1976): the probability that the sequence of interest is not present in one clone equals 1-f. The probability that it is not present in two clones equals (1 - f)', etc., or in general (1 - f ) T . Consequently, the probability P that the sequence of interest is present is P = l - ( l - f ) T or (1 - P) = (1 - f ) T . Therefore, T = I d 1 - P)/ln(l- f). 2. The probability can also be calculated from the Poisson distribution (Seed et al., 1982) since given sequences are rare. The probability that the sequence of interest is absent is P(0). According to the general Poisson distribution, P(0) = e-"' X m'/O!=e-"' where m is the average presence of the sequence of interest (i.e., average of f times the number of clones or fr). Therefore, P(0) = e-fr or InP(0) = - fr or fr = In 1/P(O). Thus, T = (l/f)ln(l/(l- PI). Note: A. (l/f)ln(l/(l- P))= In(1- P)/ln(l - f)only when f is small (a prerequisite of the Poisson distribution). B. The number of clones to be screened can be calculated rapidly since T =(I/?) x4.6 if P = 0.99 and T = ( l / f ) x 2.3 if P = 0.90. Thus, to have a 99% probability of isolating the desired sequence, one should screen a number of clones of which the combined size is 4.6 times larger than the target genome. For a 90% and 50% probability, this drops to factors 2.3X and 0.69X the size of the target genome, respectively. Enrichment of mRNAs is recommended when f <

Membranes with negative and positive controls should, if possible, be included (e.g., clones of vector without an insert and clones with the probe insert). A second aid to reduce the number of false positives is to screen membranes at least in duplicate and to retain only colonies with signals in identical locations. Nitrocellulose (reinforced) membranes with 0.45 pm pores are usually chosen over nylon since false positives are the main problem. However, nylon is more flexible, more durable, can be used with TMAC and allows more reprobing with different probes to reduce

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the number of false positives. Whatman 541 filter paper is a cheap alternative, has a high wet strength and is useful for arrayed colonies which may yield strong signals (Gergen et al., 19791, but has also been adapted for the detection of single genes (Maas, 1983). For randomly dispersed colonies or plaques, nitrocellulose and nylon are preferred. Most nitrocellulose membranes contain a small amount of detergent or wetting agent (e.g., Triton X-100); these (e.g., Millipore HAWP) are not recommended with a small bacterial load and, e.g., Triton-free Millipore HATF should be used instead. With arrayed colonies (Table 10.2A), detergents are less of a problem and HAWP-type membranes can be used.

10.2. Colony hybridization 10.2.1. Identification of bacterial colonies harboring specific plasrnids or cosmids Specific clones are identified on membranes (replicates) and then recovered from the agar (master) plates. Two approaches are possible to obtain an array of colonies on membranes: (i) growing bacteria on a membrane placed on agar in a Petri dish; (ii) growing bacteria on agar and contact-transfer of a fraction of each colony to a membrane. Different approaches can also be distinguished according to the size of the library. Small libraries (a few hundred clones) are transferred to a master plate and in tandem to agar plates overlaid with a membrane in the same grid pattern (up to about 100 colonies per plate). After the colonies have developed on both the master plate and on the membrane on the agar, specific clones are identified by hybridization and the specific clones recovered from the master plate (Table 10.2A). Large libraries are analyzed in a different way. In this case, transfected bacteria are plated on agar plates (Table 10.2B)or on membranes on agar (Table 10.2C). When colonies have developed, replica copies are prepared and positive clones identified. The positive clones are recovered from the master plates and replated until pure colonies are obtained.

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TAEILE 10.2 Colony hybridization A. Small number of colonies ( < 250) 1. Place 0.45 Frn of nitrocellulose membrane on an agar plate (not necessarily sterile, if antibiotics are used, or detergent-free). Stick paper with the same grid pattern (e.g., photocopy) underneath a parallel (master) plate. 2. Streak individual colonies using sterile toothpicks (without special flavors) onto the membrane and the corresponding location on the master plate. Also streak a negative control clone (containing the same vector without insert). 3. Incubate plates inverted at 37°C until the colony streaks are up to 1 mm wide. 4. Mark the membrane by punching at three asymmetrical sites. Seal the master plate and store at 4°C. 5. Proceed with bacterial lysis and hybridization (Section 8.2). Nore: nitrocellulose membranes will shrink upon heating. They can be preshrunk by autoclaving for 5 min. B. Large number of colonies grown on membrane 1. Mark detergent-free nitrocellulose membranes with a soft-lead pencil, place between Whatman 3MM papers, wet with water and wrap stack in aluminum foil. Sterilize at 121°C (liquid cycle, 15 mid. Prepare at least three for each plate (one master and two replicas). 2. Place the sterile membrane on agar (best when a few days old) with blunt tweezers and turn over when thoroughly wet so that the marked side is up. 3. Spread titered bacteria on the membrane using a Pasteur pipette bent above a flame into the form of a hockey stick. About 0.1 ml with 103-4 bacteria are suitable for a 82 mm diameter plate (depending on experience; avoid spreading on the 3-5 mm edge of the membrane). Nore: instead of spreading bacteria on the membrane on agar, a more even distribution can be obtained by filtration of a suspension of bacteria (in 5 ml for a 82 mm membrane) through the membrane on a sterile sintered glass filter with a vacuum. 4. Dry the membranes for a few minutes and incubate on agar at 37°C until small colonies appear (about 0.2-0.5 mm diameter). Bacteria can be stored on the membrane at -20°C until further processing by placing the membrane first for 2 h at 37°C on an LB agar plate with 25% glycerol and sealing the dish with Parafilm. 5. Peel the nitrocellulose membrane from the master plate and place, colonies up, on sterile 3MM paper. (If frozen, plates are first thawed in inverted position). Place a damp, sterile membrane on top of the master membrane by bending the replica membrane so that contact is made from the center outwards to avoid trapping air bubbles. Press the membranes together with foam with a glass plate on top and punch asymmetric holes with an 18-G needle (can later be viewed with backlighting). Peel membranes carefully apart (no sliding), place the replica on an LB agar plate with the appropriate antibiotic) and incubate until colonies appear ( -- 5 h). Several replicas can be made using the same needle holes as reference. 6. Incubate the master membrane for 1 h at 37°C on an LB agar plate with appropriate antibiotic and 25% glycerol to regenerate the colonies. 7. Proceed with bacterial lysis (Table 10.2D) and hybridization as in Section 8.2.

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TABLE10.2 (continued) C. Large number of colonies grown on agar 1. Spread bacterial suspension, which has been titered, directly onto appropriate agar as in step B3 and incubate overnight at 37°C. For best results, the agar should be a few days old. 2. Chill plates for 1 h at 4°C and number dry, Triton-free nitrocellulose membranes and sterilize membranes as in step B1. 3. Place the membrane, numbered side down, on agar as in step B5 and leave for 10 min. Punch the membrane and agar as in step B5 and peel off the membrane with blunt tweezers. This membrane either serves directly for hybridization or is placed on an agar plate to obtain larger colonies or on a plate containing chloramphenicol (200 kg/ml) to obtain an amplification of vector or to obtain more replicas. 4. Regenerate colonies on the master plate by incubation for = 5 h at 3TC, seal the disk with Parafilm and store at 4°C. 5. Proceed with bacterial lysis (Table 10.2D) and hybridization on replicas as described in Section 8.2.

D. Bacterial lysis and binding of DNA to nitrocellulose 1. Saturate one piece of 3MM paper with 10% SDS, one with denaturation solution (0.5 N NaOH+ 1.5 M NaCI), one with neutralization solution (1.5 M

+

NaCl 0.5 M Tris-HCI, pH 7.4) and one with 2 X SSC in separate trays. Excess liquid should be avoided. 2. Incubate the nitrocellulose membrane, colony side up, on these papers (3, 5, 5, and 5 min, respectively) in the same order. Scrape the membrane over the edge of the tray when transferring to the next tray to remove as much liquid as possible from the underside of the membrane (otherwise place briefly on dry paper towel). Air-dry the membrane on the bench for 1 h. 3. Fix by baking and detect DNA (Section 8.2). Scrape bacterial debris from the membrane in prehybridization solution (with a glove). Note: avoid air bubbles between the 3MM paper and the membrane and avoid liquid on top of the membrane (causes smearing).

Contact printing of membranes, relying on fitting the membrane to the colonies-bearing membrane (applying an even pressure and separating the membranes carefully) very often results in smeared colonies or the formation of satellites. Replica plating to membranes can be a useful alternative. Replica plating was introduced by Lederberg and Lederberg (1952) and their use of (sterile) velvet stretched over a cylindrical support has been followed widely. FMC BioProd-

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ucts introduced the Repliplate, a polyurethane foam transfer pad, which has a porous and irregular surface which tends to be less reliable for small colonies although not as spotty as the velvet pad. Schleicher and Schuell offers a nylon velvet replica plater (AccutranTM). Plates are lowered onto the Accutran surface to obtain full contact between the colonies and the transfer surface (can be seen through the agar). Excellent results are obtained with a variety of microorganisms and resolution of closely spaced colonies is very good. Problems in replica plating may include the extensive formation of satellite colonies (aerosol-generated, particularly with velvet), spreading of colonies due to build-up of moisture and streaking due to sliding of the surfaces. After the master plate containing the colonies on agar and the replica plates (membranes on agar) have reached room temperature and are properly identified and marked, the colonies on the master plate are transferred to the Accutran replica plater and each receiving plate with membrane is inoculated with the replica plater in sequential order. The receiving plates are then incubated at 37°C to regenerate the colonies and processed for hybridization. Microtitre plate methods can be useful for relatively small numbers of bacterial colonies (up to about lOOO), e.g., for cDNA from mRNA with an abundance > 0.4%. Colonies are picked and grown to saturation in individual wells of microtitre plates with 96 wells and covered with wide adhesive film and can be stored at -70°C after adding 0.5 vol. of 80% sterile glycerol. Two wells per tray are left uninoculated as a sterility check. A replica device (multipronged, stainless steel, uniformly ground pins fitting a microtitre plate) can be constructed for the transfer of bacteria to nitrocellulose. The distance between the centers of the colonies will be 9 mm. It is also possible and less wasteful of nitrocellulose to use a 12- or 8-tip propipette to transfer a small amount of the suspension to the premarked membrane. After the transfer of the bacteria, the tips are rinsed once in water and placed back in a tip rack (can be reused after autoclaving). This method also allows perfect replicas to be made for multiple screening. The bacterial load on the membranes can be increased by incubation on agar overnight at 37°C.

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10.2.2. Colony hybridization of lower eukaryotes Plasmids are also encountered in eukaryotes, although less commonly than in bacteria. Examples are the ‘2-ILcircle’ in yeast, the senescence (aging) plasmids in the fungus Neurospora and the plasmids associated with male sterility in corn. In addition, there are many other extrachromosomal genomes such as those from mitochondria, chloroplasts and viruses. In addition, colony hybridization type experiments can be carried out on single genes or on transcripts and may resemble in situ hybridization, except for the transfer to membranes. The usefulness of colony hybridization for the detection of genes in fungi has been demonstrated by Stohl and Lambowitz (1983), e.g., for the detection of shuttle vectors. In their example, Neurospora colonies are formed on 1.5% agar containing Westergaard’s minimal medium plus 1% sorbose and 0.025-0.05% glucose. Nitrocellulose membranes are washed twice in Westergaard’s sorbose medium and then carefully placed on top of the agar plates with the same medium. A loopful of a suspension of conidia is spread on the membrane and the dishes are incubated for 1-3 days at 30°C until the colonies have formed. Membranes are then transferred to a stack of three Whatman 3MM papers saturated with 50 mM EDTA (pH 8.0), 2.5% 2-mercaptoethanol (or 20 mM D l T ) for 15-30 min and then to a stack of 3MM papers saturated with zymolase (2 mg/ml in 20 mM phosphate and 1 M sorbitol, pH 7.0) and incubated for 3 h at 37°C (dense colonies may require an extra drop of the zymolase solution). The membranes are then placed on a solution containing 1.5 M NaCl and 0.1 M NaOH for 2 min after which excess liquid is removed by filtration through a Biichner funnel for 2 min. The membranes are washed three times with 50 ml of 2 X SSC, 0.2 M Tris-HC1 (pH 7.5) and dipped for 1 min in chloroform, respectively, before drying and baking in vacuo for 1 h. This is followed by standard hybridization procedures. Maniak et al. (1989) applied the colony-blot technique for the detection of transcripts in Dictyostelium and Saccharomyces. Since this technique proved applicable to the thick-walled yeast cells, it is thought to be generally useful for eukaryotes. Cells are grown on

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agar plates on nitrocellulose membranes which are then placed, colony side up, in a drop of GuSCN solution (4 M GuSCN, 25 mM sodium citrate, pH 7.0, 0.5% sarkosyl and 0.1 M 2-mercaptoethanol; 15 pl/cm2) on a piece of Saran wrap and incubated for 5 min. The membrane is then transferred for 5 min onto a drop of 20 X SSC, followed by baking for 2 h at 80°C. (Prelhybridization is then performed as for Northern or RNA blots. 10.2.3. Adaptation of colony hybridization for higher eukaryotic cells

Single cell-derived colonies or foci are readily analyzed by cell colony hybridization (Avraham et al., 1989). Although cell colony hybridization on animal cells was described early (Villarreal and Berg, 1977), the subsequently developed dot/slot blot hybridization is normally preferred. Colony hybridization can have some particular advantages, however, such as the quantification of transfected cells or foci, the isolation of certain foci or colonies and may be useful for screening of stable somatic cell hybrids. The bacterial colony hybridization technique can be adapted easily to eukaryotic cells (Avraham et al., 1989). Nitrocellulose membranes, autoclaved for 30 min, are placed in a sterile plate containing sterile PBS and the monolayer cultures are washed with sterile PBS twice (PBS is Ca2+ and Mg2+ free). Three drops of PBS are added to each plate and the membrane is placed on top of the cells and left for 2 min (no air bubbles; both membrane and plate marked). The membranes are then gently lifted and fresh medium is added to the cells to regenerate in a 37°C incubator. Fixing cellular DNA to nitrocellulose is done exactly as fixing bacterial colony DNA to nitrocellulose (lysis, neutralization, baking). Hybridization is according to standard procedures. A useful technique is tissue printing on membranes. This allows a variety of proteins and nucleic acids to be detected and localized. Tissue print hybridization is a useful addition to in situ hybridization for detecting organ- and tissue-specific gene expression (McClure and Guilfoyle, 1989a). As an example of the study of rapid redistribution of auxin-regulated RNAs during gravitropism, McClure and Guilfoyle (1989b) gently but firmly pressed a free-hand section

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( = 300 km thick) onto a nylon membrane for a few seconds before

drying and hybridization. Clear Northern prints were obtained and could be correlated to different tissues. This method is promising for a number of applications. 10.2.4. Useful modifications in colony hybridization

Chloramphenicol-amplifiable plasmids can be treated with this antibiotic to inhibit protein synthesis while continuing the replication of plasmids. The replica membrane, colony side up, is placed onto agar plates with chloramphenicol (150 p,g/ml) and incubated overnight before proceeding with lysing of the bacteria and fixation of DNA. Gergen et al. (1979) used the cheaper 541 Whatman filter paper, a high wet strength paper, to simplify the method. DNA is rapidly immobilized on this paper and does not require baking. The method of Maas (1983) has a higher detectability of single genes since it improves lysis of the cells by a steaming step. Whatman 541 paper is placed over the bacterial colonies while taking care not to trap air bubbles (otherwise removed with a glass spreader) and left for 1-2 h on the agar. The paper is then removed with sterile tweezers and placed, colony side up, on Whatman 3MM paper saturated with lysing solution (0.5 M NaOH and 1.5 M NaCI). When the 541 paper becomes thoroughly saturated, the open Pyrex dish with these filters is then placed above boiling water and steamed for about 3 min to enhance lysis and DNA fixation to the filter. The 541 filter is then immersed in a fresh lysis solution at room temperature for 1 min and then in a neutralization solution (1 M Tris-HCI, pH 7.0 and 2 M NaCl) for 4 min. The 541 papers are then blotted and left to dry at 37°C. They are now ready for hybridization but can also be stored in a Petri dish or wrapped in a tinfoil. Maas (1983) used a 10-fold probe excess and observed that a reduction to a 5-fold excess produced a weaker signal which could not be compensated for by doubling the hybridization time. This observation can be useful to dilute out probe contaminants. Buluwela et al. (1989) established a rapid procedure in which lysis, DNA denaturation and fixation is achieved in a single step using positively charged nylon membranes for lifts and a microwave oven

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treatment (caution: nitrocellulose may ignite or explode during microwave treatments). Bacterial colonies are lifted from the agar by placing a dry nylon membrane onto the plates and applying a gentle pressure to obtain an even contact. The membrane is peeled off and the agar plate (master plate) is regenerated for a few hours at 37°C or overnight at room temperature. The membranes are placed on Whatman paper soaked in 2 X SSC/5% SDS and left for 2 min. The dish with membranes is then transferred to a microwave oven with a rotating turntable and treated for 150 s at full settings. The membrane is then wetted with 5 x SSC/O.l% SDS and placed between two Whatman filter papers soaked in 5 X SSC in hybridization bags before proceeding with hybridization. Prehybridization for 30 min, hybridization for 4 h and autoradiography for 3 min thus allow same day picking of colonies if the colonies are well separated and relatively large. The same technique can be used for plaques but autoradiography needs to be longer, as for high density plasmid and cosmid screening.

10.3. Plaque hybridization Phage libraries are attractive because inserts can be large, the packaging and infection is efficient (10-50 more efficient than plasmids and thus more representative of the target sequences), phage libraries can be stored for long periods in bacteria-free, chloroformsaturated buffers in glass and the titers can be well established. Benton and Davis (1977) developed the plaque assay for the screening of lambda gt recombinant clones by hybridization to single plaques in situ. They plated a lambda library, applied a nitrocellulose to the surface of the top agar to allow transfer of phage to the membrane, fixed the DNA to the membrane followed by hybridization. 10.3.1. Standard plaque hybridization methods

A library can be either amplified or screened directly. Amplification will yield a library that can be a source for many experiments. However, amplification may also change the composition of the

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library due to a different reproduction rate of the individual phages. Primary libraries are therefore screened directly if enough material is available or when a limited screening will be required. Otherwise, only a fraction of the primary library should be amplified. It is useful to keep in mind that to maintain the integrity of lambda phage particles Mg2+ is necessary and that maltose induces the expression of the lambda phage receptor. The inclusion of MgSO, and 0.2% maltose in the media for the bacteria and MgSO, in the SM buffer, used for the dilution of phage, is recommended but not obligatory once phage has injected the DNA into the cells (during the 15 min incubation at 37°C). Moreover, the presence of nonviable cells should be minimized since they are ‘dead ends’ for phage and decrease the efficiency of infection. The number of plaques that is required to obtain the desired clone with a given probability, can be calculated as described in Table 10.1. For cDNA libraries this is usually between 100 and 200000 plaques whereas for genomic libraries this can be 100 times larger. Titration allows the desired number of plaques per plate to be obtained. The standard method is described in Table 10.3. When plates are too wet, plaques tend to smear over the plate. If the plaques remain small, the plates may have been too dry or the cell density too high (a multiplicity of infection of 0.01-0.05 phage particles per cell is optimal). The low background of nitrocellulose membranes is sometimes sacrificed for more durable nylon membranes. This choice is also determined by the abundance of the target. At low abundance, it becomes more important to have lower background levels and nitrocellulose is recommended. Nylon membranes do not offer a higher detectability of the target than nitrocellulose due to the high target concentration. In a survey cited in Table 8.2, about 2/3 of researchers use nitrocellulose (particularly from Schleicher and Schuell) whereas the rest use nylon membranes, primarily from DuPont NEN and Amersham. 10.3.2. Modified plaque hybridization procedures

Several replicas can be made from a master plate but it is often useful to amplify plaques in situ to increase the signal/noise ratio,

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TABLE 10.3 Plaque hybridization

1. Plate phage, as described in Table 4.7, so that about 10000 plaques are obtained per 135 mm plate (or about 5000 per 82 mm plate; note that for best results dishes are prepared a few days before use). 2. When plaques have developed, but are not yet confluent, cool plates for 1-2 h at 4°C (not overnight, then place at 4°C and let plaques develop the next day). If plaques develop poorly (poor bacterial growth), then top agar was probably too warm during plating. 3. Remove plate covers and let agar air-dry for about 30 min at room temperature. Label each plate and corresponding membrane with a pencil and carefully apply, without trapping air bubbles, to the surface of the top agar (ink-side up). The membrane will wet in about 1 min. Make matching marks by punching the edge of the membrane and the agar with a clean 18-G needle (patterns should be different for the various membranes). Let membrane sit on the agar for about 2-10 min. Peel the membrane off carefully (leave top agar intact). If problems are encountered, longer cooling or higher concentration in top agar (agarose) may be required. Up to five replicas can be made from a plate without regenerating the plaques. 4. Alternative: in situ amplification of plaques. Resuspend bacteria in LB+ 10 mM MgSO,. Label the membrane, dip in bacterial suspension and air-dry briefly. Lay the membrane on a plate with plaques (phage will come into contact with bacteria and infect them). Place the membrane, plaque side up, on a fresh LB+Mg2* plate and incubate inverted overnight (considerable amplification of phage will occur). Do not forget to apply matching marks to the membrane and master plate. 5. Dry membranes for about 30 min on the bench top and place them on 3MM paper saturated with 0.2 N NaOH+ 1.5 M NaCl (plaque side up) for 2 min. Transfer then to 3MM paper saturated with neutralization solution (1.5 M NaC1+ 0.5 M Tris-HCI, pH 7.4) and with ~ x S S C ,respectively, 2 min each. Dry membranes and continue with hybridization (Section 8.2). Replica membranes can be used to increase contact with each solution to 1 min; however, the NaOH treatment should be kept to less than 5 min for nitrocellulose. 6. Pick colonies, corresponding to the positive signal on the film, with a Pasteur pipette and resuspend in 500 p,I of SM buffer (Table 4.7A, step 4), add a few drops of chloroform and vortex for a few seconds. Spin at 3000 X g for 3 min and collect the supernatants (add a few drops of chloroform). 7. Dilute phage stock to = 200 plaques/O.l ml and replate (Table 4.7). Repeat plaque hybridization and pick well-isolated plaques. Note: Nylon membranes do not offer a higher detectability than nitrocellulose due to the high concentration of the target in the plaques.

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particularly if oligomers are used as probes. In the method of Woo (1979) and Vogeli and Kaytes (19871, a membrane is dipped in a bacterial suspension before the plaque lift and then incubated on fresh agar plates. The transferred phages infect the growing bacteria and the signal may be increased five-fold. In detail, plaques are allowed in the master plate until = 0.5 mm (if not too dense) and then incubated at 4°C for 1 h to harden the agarose. Although both Woo and Vogeli and Kaytes dipped the numbered membrane into a suspension of bacteria in fresh LB medium with 10 mM MgSO,, this is not essential since the lawn provides enough cells during transfer. The membranes are placed on the lawn with plaques and are marked by stabbing with waterproof black drawing ink and incubated for a few min at 4°C. Several replicas are then placed, phage side up, on fresh LB Mg2+ plates and incubated until visible plaques appear (6- 10 h; bacterial growth on nylon is slower). Membranes are then processed as described in Section 10.3.1. The lambda phage method is readily adapted to the screening of M13 plaques (Mason and Williams, 1985). However, best results are obtained when the lifts are directly baked, without lysis and denaturation steps (M13 is ss). It is also advantageous to use ds probes or to prepare probes complementary to the strand packaged in M13.

+

10.4. Hybridization to colony or plaque nucleic acids Detection of pertinent clones requires three steps: (i) prehybridization to block nonspecific sites on the membranes; (ii) hybridization; (iii) washing and detection of positive signals. The large majority of plaque or colony hybridizations have been achieved with radioactive probes. Biotinylated probes often give background signals with bacterial material and require special steps to reduce these. However, DIG probes have become an attractive alternative with very little background, lack of nonspecific hot spots on the film and very good resolution. Moreover, it is fast and safe. As with dot blot hybridization, it is possible to choose between formamide-containing and aqueous hybridization solutions. For-

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mamide, requiring lower hybridization temperatures, prolongs the useful life of the nitrocellulose membranes. However, aqueous conditions tend to give superior results (typically 30 min prehybridization and 6 h hybridization at 65°C). Bacterial debris can be removed before hybridization, but after DNA fixation, by washing for 60 min in 5 X SSC and 0.5% SDS at 65°C and scraping the debris off gently with gloved hands or with Kimwipes soaked in the same buffer. 10.4.1. Radioactive probes

Similar to dot blot hybridization (Section 8.21, a large number of different probes can be used ranging from polynucleotides to nicktranslated fragments to oligomers (guessmers, (degenerate) oligomers). Since related sequences may show up and false positives may account for a large proportion of the signals, it is useful to avoid drying of the membrane so that, after exposure, increasingly stringent washes can be made. This is best achieved empirically although Southern or Northern blots often may be used to optimize conditions. Well-separated colonies contain large amounts of target DNA and can be detected with probe at about (1-5) X lo5 cpm/ml whereas dense screens require about 5 x lo6 cpm/ml. The probe is alkali-, microwave- or heat-denatured and added to fresh hybridization solutions as described in Section 8.2.2.2. After washing, wet or damp membranes are placed on used X-ray film covered with Saran wrap (used as support; marked with phosphorescent pencil for orientation of membranes) and then covered with Saran wrap before placing on unexposed X-ray film. The length of autoradiography depends on the amount of label but can be as short as a few minutes (Buluwela et al. (1989) but is usually 1-24 h. Plaque hybridization may require up to ten times longer autoradiography. Probes can also be stripped (Section 8.2.5) in order to rehybridize with a different probe although the same information can be obtained from replicas. Since only a very small amount of the probe will hybridize, it is possible to recover the probe, denature (important!) and reuse again. Denaturation in aqueous solutions should be at

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

= 100°C for 15 min, whereas for solutions containing formamide, 15 min at 70°C should suffice. 10.4.2. Nonradioactive probes Despite the wide use of radioprobes in colony or plaque hybridization assays, nonradioactive probes can be advantageous. The use of biotinylated probes, initially the most common among nonradioactive detection systems, is limited since biotin-streptavidin systems tend to give high background levels with bacterial material unless specific measures are taken. The more recently developed DIG (Table 7.21, but also other hapten-antibody systems such as sulfonated probes, are very attractive alternatives. The main restriction is that monoclonal antibodies (commercially available) should be used since polyclonal antisera often contain antibodies against bacteria. The main drawback of nonradioactive probes is the ability to reprobe the same membrane. It is possible, however, to strip a membrane of its probe after a colorimetric detection and to perform a chemiluminescent detection or vice versa. Biotinylated probes have been applied in colony hybridization (Haas and Fleming, 1986, 1988) and plaque hybridization (Kincaid and Nightingale, 1988). They require proteinase K and chloroform treatments to reduce background. After lysis and neutralization, as described in Section 10.2.1, the membranes are incubated in 30 ml of 1 X SSC, containing 200 pg/ml proteinase K, for 1 h at 37°C. After rinsing twice for 2 min in 30 ml 90% ethanol (wt/wt, not v/v otherwise it exceeds the tolerance level of some batches of nitrocellulose), the membranes are dried and 100 ml of chloroform are passed through each membrane using the filtration device described by Grunstein and Hogness (1975) (commercially available from Schleicher and Schuell), air-dried and incubated for 5 min in 0.3 M NaCl (30 ml/membrane; optional) and baked in vacuo at 80°C. (Pre)hybridization is in the presence of 45% formamide at 42°C and 100-200 ng/ml biotinylated probe is used (Section 8.2). Hybridization solutions can be reused at least 10 times over a time span of at least 5 months if stored at -20°C. Background levels increase with the density of the bacterial colonies but positive colonies can still be

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distinguished at very high densities. Note that the chloroform treatment tends to shrink the membranes and makes them brittle. Colony and plaque hybridization with the DIG system is very useful (Martin et al., 1990). Following standard transfers to nitrocellulose membranes (Sections 10.2.1 and 10.3.1) and baking, membranes are prehybridized in 5 X SSC (containing 0.5% BM blocking reagent (Section 8.21, 0.1% sarkosyl and 0.02% SDS) for 1-8 h at 68°C. Fresh buffer with 5-200 ng/ml DIG-probe is used for the overnight hybridization at 68°C. Posthybridization washes include two rinses for 5 min at room temperature in 2 X SSC and 0.1% SDS and stringency washes at 65-68°C with 0.1% SDS in 0.1-0.5 x SDS (depending on the degree of stringency required). For immunological detection, membranes are washed for 1 min in wash buffer (0.1 M Tris-HC1, pH 7.5, containing 0.15 M NaCI) and then blocked with BM blocking buffer (for colorimetric detection 0.5% blocking reagent and for chemiluminescent detection 2% blocking reagent in wash buffer). After blocking, anti-DIG Fab fragments conjugated with alkaline phosphatase (Boehringer Mannheim), diluted 1/5000 in blocking buffer, are allowed to react for 30 min. Enzyme is detected as described in Sections 7.3.3.4.1 and 7.3.3.5.1. Colonies can be detected in 10-60 min and background staining is minimal although it increases with time. In the case of nylon membranes, 7% SDS in 50 mM phosphate buffer is used during (prelhybridization and for the blocking buffer increasing the concentration of blocking reagent to 1% and adding 50 kg/ml carrier DNA. It is then very important to rinse the membranes well with the Tris buffer between hybridization and immunological detection since phosphate is a potent competitive inhibitor of alkaline phosphatase. 10.4.3. Reduction of background Background signals should be identified as much as possible by: (i) use of replica membranes and increasing stringency; (ii) use of negative control and, if available, positive control membranes; (iii) careful preparation of probes; (iv) optimal hybridization conditions. Particularly with radioactive probes, the spontaneous signals (from nonspecific radioactive spots to static electricity-generated dots) can

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be easily recognized from duplicate membranes. Only those present on both membranes are considered. ‘Static dots’ can be reduced by using a sheet of paper, as found in the X-ray film box, instead of or in addition to the Saran wrap. Negative controls (clones with irrelevant insert) and positive controls (clones with probe insert) are excellent indicators of the quality of the procedure. Whenever possible, an irrelevant insert should be chosen which has a higher GC content and is longer than the relevant insert. Probes should be carefully prepared. Particularly those with GC tails or GC-rich regions are prone to give high background levels. Radioactive probes should always be filtered before use (Section 8.2.2.2). Since colony or plaque membranes may contain a considerable amount of bacterial debris, prehybridization solutions should never be used, contrary to slot blot hybridization, as hybridization solutions. Probe should be added to fresh solutions. Oligomer probes tend to adhere nonspecifically to colony proteins on the membranes. Special grade colony/plaque membranes and the use of SDS in the hybridization solution reduces this problem somewhat.

10.5. Selection, picking and purification of clones The size of the signals depends on the size of the area occupied by the recombinant clones but can be as small as pinpoints for high density screens. The positive spots are circled with a red pencil and the autoradiogram, or its mirror image (depending on orientation marks) by reversing the film, is oriented with respect to the master plate on a light box. Positive colonies are picked with a tooth pick if very well separated or all bacteria within the positive area with a sterile wire loop. Several dilutions are made and those with about 25-1000 colonies per 150 mm plate are screened again by colony hybridization until a well-isolated colony is obtained. Positive plaques are picked with the wide (‘wrong’) or narrow end (depending on the resolution of plaques) of a Pasteur pipette and the agarose plugs are added to SM buffer (Table 10.31, 100 p1 of

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chloroform are added, vortexed and incubated for 30 min. After centrifugation for 3 min at 3000 Xg, phage is plated at different dilutions as described in Table 10.3 and the plaque hybridization is repeated for plates containing 25-1000 plaques/l50 mm plate. Well-isolated plaques should then be obtained.

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CHAPTER 11

In situ hybridization In situ hybridization (ISH) has been used to study nucleic acids in cells for over 20 years (early reviews: Gall and Pardue, 1971; Tijssen and Kurstak, 1977). Important advances in molecular biology and the overcoming of several technical problems led to the transition to application of ISH in the study of both mRNA and genomic DNA. ISH was originally realized with radioprobes (particularly 3H and I2’I labels), but the long exposition times required and the relatively poor resolution limited its application to abundant DNA, e.g., in amplified polytene chromosomes or in highly reiterated sequences of metaphase chromosomes. Statistical analysis of grains in autoradiograms from many metaphase chromosomes was required to map genes. Simple and rapid nonradioisotopic detection methods notably extended ISH methods and made it easier to combine ISH with immunohistological methods (e.g., to answer the question whether the RNA detected is also expressed). Nevertheless, nonradioactive techniques have been used primarily for relatively abundant nucleic acids or for large targets whereas autoradiography remained in use for small targets requiring a high detectability. This is most probably due to a lack of optimization of the nonradioactive methods (McNeil et al., 1991). ISH allows the detection of genes or their expression in cells and complements studies on extracted nucleic acid by filling the gap between cell and molecular biological methods in the study of gene localization, translocation, amplification, transcription and transcript processing. This histological technology has several unique features which enables it to answer important biological questions such as: (i) the localization of particular nucleic acids in single cells or the mapping of particular sequences on condensed chromosomes (possibly due to an increased topographical resolution by nonradioactive

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detection methods and an increased knowledge on the mechanisms and meaning of banding of mitotic (Holmquist et al., 1982) or meiotic (Ambros et al., 1987) chromosomes) or even on interphase chromatin; (ii) simultaneous detection of two or more target nucleic acids in a single cell (by variant detection methods); (iii) distinguish subsets of cells or follow cell development with respect to the target nucleic acid (e.g., to determine whether only subsets of cells express a given gene or whether many cells have a low expression or a few cells a high expression); (iv) detect both particular nucleic acids and proteins encoded by them in the same cell by complementary techniques (ISH and immunohistochemistry) (Tijssen, 1985, Chapter 17). Although ISH was primarily used by cytogeneticists, this technique is rapidly becoming an essential tool for molecular biologists, particularly for the study of the mouse, as different mutant strains exist and transgenic animals can be generated (e.g., by microinjection, retroviral vectors or manipulation of pluripotent embryonic stem (ES) cells) (Lee et al., 1990). In addition, homologous recombination in ES cells can be used to generate null or modified alleles of cloned genes (De Chiara et al., 1990). ISH permits the assessment of the karyotype of the cell and the localization of endogenous sequences. ISH has a very high detectability, down to 1-20 copies/cell. mRNA can often be detected where the expressed protein cannot (Bloch et al., 1986) or when it is not expressed, if care is taken to prevent RNA degradation.

11.1. Theoretical considerations The technical details for hybridization to nuclear DNA differ considerably from those to cytoplasmic RNA. The requirements for the preservation of morphology are less stringent for hybridization to cellular DNA; generally only the chromosomes and the nuclear morphology need to be maintained. The morphology of the entire cell needs to be preserved for most studies on the localization of RNA in cells. In cases where DNA is the target nucleic acid, RNA hybridization often complicates the results unless RNase treatments have been included. If the target nucleic acid is RNA, background

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hybridization to DNA is generally not a problem since: (i) the number of RNA transcripts far exceeds the number of corresponding DNA sequences; (ii) the target RNA is single-stranded so that no denaturation step is required as for the double-stranded DNA, which in turn will not react with the probe; (iii) RNA is usually located in the cytoplasm; (iv) some fixatives, such as formaldehyde, used for TABLE11.1 Steps in in situ hybridization A. Cytological procedures Cultured cells

Fixation

I

Dehydration

I I

Embedding Sectioning

I

Freezing

I

Cryosectioning

Fixation

I

Fixation

I

Dehydration

I

Storage

I

Drying

Storage

I

St rage

B. In situ hybridization procedures Rehydration of cells

I

(Pretreatments)

I

(Prehybridization)

I

Hybridization

I

'

Washing (stringency)

I

Detection

Autoradiography

I

Contrast staining

Nonradioisotopic detection

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RNA ISH yield poor results for DNA (Pardue, 1985). Electron microscopic applications of ISH are still in their infancy. ISH is not standardized, since standardized tissue does not exist, and can be accomplished in any of several approaches, the steps of which are not always parallel (Table 11.1; e.g., sectioning after/before embedding). Optimization of ISH for a given tissue requires finding the optimal combination of the different steps, controls, buffers and probes (Table 11.2). Most often, fixation is the crucial step.

11.1.1. Nature of probes Small probes often reach the target more efficiently than large probes due to its cellular localization, yet very small probes do not form stable hybrids. Short probes also require a higher specific activity (cpm/p,g) to obtain the same amount of cpm/mol and will have a suitable shelf-life which is exponentially shorter (not a problem for 3H). On the other hand, unhybridized large probes diffuse with more difficulty from the cells during posthybridization washes resulting in higher background levels, particularly in the case of nonradioactive probes. The ideal length is about 100-500 bases. The abundance and stability of the target will dictate the specific activity required. RNA probes, oligomers or cDNA probes are the most frequent choice. When nick-translated probes are prepared, the DNase concentration can be increased to obtain smaller probes (Section 7.6.1). PCR probes and in vitro transcription allow better control of the probe size and of the particular sequences which serve as a probe. The use of oligomer probes to different regions of the target provides an excellent control for the specificity of the hybridization, especially if the target is a member of a gene family. The increased specificity of oligomers is often at the cost of detectability. Nuclease treatments can indicate whether an ISH signal was not due to a nonspecific adsorption to cellular components. RNA probes have several advantages since they (i) are singlestranded, (ii) have high thermal stability, (iii) allow a background reduction simply by posthybridization RNase treatment, (iv) are easily produced in defined form and in large quantities and (v)

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TABLE 11.2 Solutions and materials for cytological procedures A. Solutions

1. 1 x PBS = 10 mM sodium phosphate + 130 mM NaCl (pH 7.0) 2. Depolymerized formaldehyde, 4%. Paraformaldehyde (Baker) is dissolved at 7 g/100 ml in warm water (60"C, not higher) by continuous stirring on a heating plate in a fume hood. After 1 h, 2-6 drops of 100 mM NaOH are added until the solution clears. The solution is removed from the heating plate and mixed with 0.6 vol. of 3 XPBS (see above). Diluted HCI is added to bring the pH to 7.2 and water to bring the volume to 1.75 of the original. The 4% formaldehyde fixative is then stored at 4°C but tends to polymerize over time with increasingly poor results. 3. Subbing solution (5 X ). Gelatin is dissolved in water at 65°C to make a 0.5% solution. This solution is cooled to room temperature and made 0.05% with respect to chrom alum ( = chromium potassium sulphate; CrK(S04),. 12H20). Sterile water is used for further dilutions of the subbing solution.

B. Materials 1. Gelatin-subbed glass slides. Gelatin-subbed glass slides are prepared, first by washing the slides with acid or detergent, thoroughly rinsing for 30 min and then immersing for 2 min in a 1 X gelatin-subbing solution. Slides are set on their side to allow excess solution to drip off and allowed to dry for several hours. Slides can be stored for prolonged periods in dry, dust-free containers. Alternatively, glass slides, after acid cleaning, are treated with 1-5% Elmer's white glue or Kodak Photoflo. 2. Poly-L-lysine coated glass slides or coverslips. Clean slides or coverslips are dipped into a poly-L-lysine hydrobromide solution (0.5 mg/ml; Sigma cat. no. P 1399), air-dried and stored in a dust-free environment at 4°C (at most for a week). 3. Siliconized coverslips to prevent adherence of specimens. 4. Plastic chambers for incubations.

permit a larger probe size range to be used than with DNA probes. The stability may be lower than that of DNA probes as well as the tendency of increased secondary structures may make DNA probes a viable alternative (Godard and Jones, 1979). Among the radioisotopes, 35Sis often superior for ISH due to the relatively high specific activity, the high autoradiographic efficiency (10-25 times higher than 3H), low nonspecific adherence, reasonable half-life both for the radioisotope and the probe and is safer than

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HYBRIDIZATION WITH NUCLElC ACID PROBES

1251 or 32P. However, the phage RNA polymerases do not use [35S]UTPas efficiently as UTP (Table 7.18); oxidation of S may lead to high background (include DTT or 2-mercaptoethanol!) and resolution with this isotope is much less defined. Nonradioactive probes have become popular since they allow superior definition, are rapid, safe and cheaper. Nonradioactive probes also permit different targets to be detected simultaneously in the same cell (e.g., probes with different enzymes, enzyme/FITC, rhodamine (red) and coumarin (blue) fluorescence, etc.) (Mullink et al., 1989, Wiegant et al., 1991). Wiegant et al. even used triple hybridization (fluoresceinated, biotinylated and DIG-probes) but observed (as McNeil et al., 1991) that direct detection, instead of immunocytochemical amplification, yields a better resolution with less background. Fluoresceinated probes have a detectability of 50100 kb in the visual mode, but digitalization of hybridization images with a CCD camera and image recording and analysis increases the detectability at least 30 times. This method (FISH, fluorescent ISH) can be very useful for the detection of trisomies or monosomies and for the study of translocations, rapid assessment of numerical chromosome aberrations in interphase cells, e.g., in tumor cytogenetics (Amoldus et al., 1991) and gene mapping. Biotinylated probes can be detected with avidin, streptavidin or modified avidin (Yehle et al., 1987). Replacing phosphate-buffered saline by SSC during hybridization reduces the background of unmodified avidin (fluoresceinated) making it superior to modified avidin for single-copy detection (McNeil et al., 1991). 11.1.2. Requirements of tissue preparation The material to be investigated may be: (i) whole, small animals or organs; (ii) large pieces of tissues, directly from the organism or maintained as explants; (iii) cell suspensions or cells grown in vitro, which do not require sectioning, if rendered permeable to facilitate the access of the probes to the target nucleic acid. Whole animals can be studied if precautions are taken to prevent shattering of the sections during cutting.

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Cells from suspensions or in vitro cultures and even small pieces of tissue teased from freshly dissected tissue can be permeabilized simultaneously with the fixation. This treatment should not (i) modify the target nucleic acid, (ii) (locally) elute the target nucleic acid, (iii) allow the redistribution of the target nucleic acid and (iv) change the morphological features of the tissue preparation. When a cellular monolayer is used, best results are obtained when the layer is only about 70% confluent. Morphologic analysis is optimum when the cells are flat and adhere tightly to the support. Moreover, a low cell density improves the signal/noise ratio and avoids cell overlapping. The pH of the formamide solutions used in hybridization is very important for the preservation of cellular morphology and optimal hybridization (Lomholt et al., 1989). Concentrated formamide has a pH of about 4.5 (note that pH measurement of undiluted formamide is incorrect), but after dilution with SSC (pH 7) and EDTA to 70% formamide, 2 X SSC and 1 mM EDTA it has a pH of 9.5. Chromosome preparations denatured with this solution have a poorly conserved morphology whereas titration of the solution to pH 7 with HCI produced both good morphology and increased specific hybridization. Moreover, if DeSO, is used, autoclaving this accelerator reduces background. 11.1.3. Positive and negative hybridization controls Controls should include determinations of the detectability, specificity and reproducibility of the probe. The detectability and specificity can be established on slot/dot or Northern blots. At least 1 pg of radiolabeled probe should be readily detectable. Moreover, if positive cells are available, reproducibility of the technique should be tested. Negative controls for both background and specificity are required. Background levels can be established using blank slides, omitting the probe, or using negative cells, or only the detection system. These should have extremely low grain density or nonradioactive signals. The specificity can be investigated by hybridization with sense probes (for the detection of mRNA), different probes to different sequences on the same target and heterologous probes

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(e.g., plasmid). Single-labeled probes, each with a different color or fluorescence, to the same target makes multicolor labeling of that target possible (Nederlof et al., 1990). Sometimes, competitive hybridization with unlabeled antisense RNA probe is used in mRNA detection; however, the results are usually ambivalent. Pretreatment with RNase is often tricky since it not only removes all RNA from the cells, but residual nuclease may degrade the probe in subsequent steps. Ca*+-dependentmicrococcal nuclease (Williamson, 19881, used at pH 8.8 with 1 mM CaCI, and subsequently inhibited with 2 mM EGTA, avoids this problem. Chemicals may sometimes induce the formation of grains Vpositive chemography’) and some control sections should be treated with buffers only. Similarly, for nonradioisotopic detection it is necessary to establish that the tissue studied does not yield nonspecific signals (e.g., cellular peroxidase). Negative chemography (loss of grains due trJ chemicals in tissue) or inhibition of nonradioactive detection systems may also occur. Protein-probe interactions, e.g., by DNA-binding proteins, or charge interactions between positively charged proteins (evident from binding of labeled nonspecific probes) and nucleic acid can be masked by acetylation or glycine treatments (see below).

11.2. Cytological procedures Different cytological procedures, discussed separately here, are used in the different hybridization methods according to the sequence given in Table 11.1. From large pieces of tissue, paraffin sections of fixed tissue or cryostat sections of fresh or fixed tissues are prepared (Table 11.3). Many laboratories prefer either one or the other. Cryosectioning is rapid and relatively easy but morphology may be better preserved with paraffin sectioning. Methacrylate sections are used rarely for ISH. The sensitivity of ISH on paraffin-embedded tissue sections or on cryosections has rarely been compared (Lum, 1986; Tournier et al., 1987) but does not seem to differ greatly. This may depend on the type of tissue and the nature of the target nucleic acid.

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Whole animal or organ section is a useful tool for the study of the distribution of target molecules. Animals are anesthetized for sacrifice through deep axillary incision and are then shaven and frozen into blocks of 3.5% carboxymethyl cellulose mounting medium in a bath of dry ice/ethanol. Cryomicrotome sections of 20-40 p,m are collected by firmly sticking a 3M 688 tape to the frozen surface of the section block and cutting with the cryomicrotome to release the tape with the section. For protein blotting, the section is immediately transferred to a nylon membrane, whereas for ISH the section is allowed to thaw and dry for 10-30 min at room temperature before fixation in 4% paraformaldehyde and hybridization (Blount et al., 1986). 11.2.1. Permeabilization and fucation of tissues and cells Optimal fixation is the key element of successful ISH. Advances in tissue preparation and fixation now allow the study of a wide range of material. Two types of fixatives are used: (i) ‘precipitant fixatives’, which remove lipids from the tissues and precipitate proteins; (ii) ‘cross-linking’ fixatives which cross-link macromolecules. Initially, precipitating fixatives (e.g., Carnoy’s fixative, acetic acid/ethanol) were used since cross-linking fixatives were expected to make cells impermeable for the probes. Several research groups have reported that cross-linking fixatives (e.g., formaldehyde, glutaraldehyde) yield superior results, particularly with mRNA (Godard and Jones, 1979; Angerer and Angerer, 1981; Lawrence and Singer, 1985; Moench et al., 1985) due to a better retention of the target nucleic acid and a better preservation of the morphology of the cell. This conviction is strongly challenged by other groups (e.g., Bresser and EvingerHodges, 1987) implying the absence of a universal fixative. The effects of the fixatives on preservation of cellular morphology, preservation and accessibility of target and relative hybridization efficiency depend on the cell type. Optimal fixatives have to be determined empirically for each tissue and optimal fixation is the most important step in ISH. Good starting points for mRNA detection are 50% methanol and 50% acetone for 20 min at -20°C (precipitating fixative) or, most often, 4% paraformaldehyde in PBS

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TABLE11.3 Cytological procedures A. Preparation of cultured cells Poly-L-lysine-coated glass slides or coverslips (Table 11.2) are particularly useful for hybridization at high temperatures. However, they require an acetylation step to decrease nonspecific retention of probes. At lower temperatures, < 45-5OoC, gel-coated slides (Table 11.2) give cleaner results. The retention on gel-coated slides can be improved by incubation in PBS containing 2.5% glutaraldehyde for 15 min before adding specimens. After fixation, aldehyde groups can be blocked by incubation for 15 min in 1% hydroxylammoniumchloride in PBS, washing in PBS and dehydration. 1. Place 10 P I ( = lo7 cells/ml, in serum-free medium) onto a slide (or coverslip) and allow the cells to settle (20 mink alternatively, these cells can be grown on these slides or slips, to about 70% confluency and directly fixed after rinsing in serum-free medium. Coverslips for the culture of cells should have been boiled in 0.1 N HCI, rinsed and autoclaved and subbed with 0.5% gelatine (type Bloom 275). 2. Remove excess liquid and dip the slides into the fivative at the appropriate temperature and optimum time (Section 11.2.1). 3. Rinse fixed specimens for 2 min in 3XPBS and 2 min in lXPBS and pass through graded alcohol (50, 70, 95 and lOO%), dry and store desiccated at - 70°C. B. Preparation of metaphase chromosomes For mapping, BrdU is added to enhance chromosome elongation and banding for metaphase chromosomes and to help distinguish G1 from S and G2 phase nuclei in the case of interphase chromosomes. Treatment of the cells for a few minutes with hypotonic solutions to make them swell and exposure to low temperatures, thus interfering with the stability of spindle fibres, can improve the preparations. 1. Add BrdU to 3 mM, in fresh medium, to the culture, 7 h before harvesting. Alternatively, cells should be fed with fresh, complete medium 3-4 h before adding colcemid to ensure optimal growth conditions. 2. Add colcemid to 0.01 pg/ml to the culture, 1 h before fixation, to arrest the cells at metaphase and leave at 37°C. 3. Wash cells once with 1X PBS, trypsinize if necessary, resuspend in complete medium in a 15 ml centrifuge tube and collect by centrifugation at 5OOX g . 4. Remove the supernatant, loosen the cell pellet and slowly add 5 ml of prewarmed (37"C), hypotonic KCI (0.56% salt) while shaking the tube gently to maintain a single-cell suspension. 5. Centrifuge for 15 min, remove the supernatant and loosen the cell pellet. 6. Fix the cells by very slowly adding 5-6 ml of 3 : 1 methanol/glacial acetic acid (mixed just before use), while continuously shaking the tube to prevent clumping of the cells.

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TABLE 11.3 (continued ) 7. Repeat steps 5 and 6 twice (cells can be stored in fixative at -20°C for a few weeks but quality will decrease). 8. After a final centrifugation, resuspend cells in fresh fixative to an optical density of about 0.3 at 600 nm (light scattering depends on the configuration of the spectrophotometer and higher or lower values may be needed) and drop about 200 p1 from about 5 cm on slightly slanted slides. 9. Air-dry the slides and store at -70°C. If preparations dry too fast, chromosomes will not spread well (usually when the relative humidity is too low; then use a humid chamber or ice-cold wet slides). 10. Bake for 2 h at 65°C immediately before use.

C. Perfusion with formaldehyde (example with mice) Animals are anesthetized by intraperitoneal injection of 60 mg/ml pentobarbital or as in step 2. Extensive perfusion can lead to overfixation. 1. Fill one syringe (equipped with a 2 3 4 needle) with 25 ml of l x P B S and another with 25 ml of 4% formaldehyde and leave at 4°C. 2. Kill the mouse in a CO, gas bag; following respiratory arrest, the mouse is quickly laid on its back and the thorax carefully, but quickly, opened and the diaphragm removed to gain access to the heart. About 0.5 ml of heparin can be injected transcardially (optional but can be beneficial). 3. Carefully insert the needle of the syringe with PBS into the left ventricle (lower right chamber of heart) and cut the right ventricle for drainage. The PBS is perfused at a constant rate followed by 25 ml of the formaldehyde solution (needle inserted into the same puncture). Blood-rich organs (liver, kidney) should turn greyish. Including sucrose to 0.5 M in PBS after fivation may improve the morphology. 4. Dissect tissues or organs and transfer into snap-cap vials with 4% formaldehyde in PBS. D. Paraformaldehyde fixation and paraftin embedding of tissues 1. Fix dissected tissues, organs or embryos in glass vials with 4% paraformaldehyde in PBS at 4°C. Small tissues (e.g., young embryos) are fixed rapidly (20 min) whereas larger samples (e.g., old embryos) may require several hours (for large organs, perfusion is recommended). Over- or underfixation may lead to failure in sectioning. 2. Transfer tissue to graded alcohol (50, 50, 50, 70, 95 100 and 100%; 5-20 min, depending on size of tissue). 3. Transfer to xylene, three changes, each for 10 min. 4. Add 5 ml of xylene and 5 ml of molten paraffin wax (60°C; with a hot glass pipette) to each specimen, mix and leave overnight at room temperature. 5. Remelt the wax/xylene mixture by transferring to 60°C, replace by molten paraffin wax without losing specimens (do not allow paraffin to harden) and leave for 1 h at 60°C.

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TABLE 11.3 (continued) 6. Repeat step 5 twice. 7. Fill embedding molds according to the manufacturer’s recommendations, transfer the specimen, leave at room temperature to harden and remove molds. 8. Trim the block into a trapezoid and prepare 4-8 p,m sections with a microtome according to standard procedures. 9. Transfer ribbons of sections into 0.2xsubbing solution on subbed slides. Place on a slide warmer at =4YC to stretch the sections, remove excess subbing solution and dry at 42°C for 24 h.

E. Cryosectioning If no other histochemical techniques are needed, fresh tissue can be used. The block must be frozen quickly without cracking. Tissue is cut in small blocks with a new razor blade (washed in soap to remove oil). An alternative to the method below is freezing tissue on aluminum foil on top of dry ice and surrounding with crushed dry ice. However, the perimeter of the tissue is usually poorly preserved with this method. For RNA detection, DEPC is included at 0.02% in the subbing and fixation solutions. Poly-L-lysine coating may be superior at temperatures > 50°C since gel from gel-coated slides may detach at higher hybridization temperatures. It is convenient to use only some of the slides for immediate hybridization and store the rest for future reference. 1. Place a beaker in liquid N, and fill with Cryokwik, isopentane or Freon. These coolants freeze tissues much faster than N, and prevent the formation of large ice crystals that would damage the cellular structures (Terracio and Schwabe, 1981). Stirring of the coolant can be beneficial as it increases the cooling rate. 2. Place the specimen on a filter paper strip or in an aluminum foil pocket (pierced at the bottom) marked at the other end and immerse immediately, but slowly so that tissue freezes just ahead of immersion in the coolant (frozen tissues can be stored at - 70°C). 3. Mount specimens on a cutting chuck with OCT mounting medium (Tissue Tek 11, Miles) in a cryostat chamber and cool until the OCT solidifies. 4. After 15 min, trim the block, section the block and collect sections onto warm subbed slides or coverslips coated with Histostik (Accurate Chemical and Scientific, Co.) with standard methods. 5. Fix the cells by incubation in 30 ml/slide at an appropriate temperature (Section 11.2.1). Remove fixative, incubate three times in PBS, once in 5 mM DTT and dehydrate by passing through graded ethanol (specimens can now be stored dry at -70°C or in a vacuum desiccator for use the next day). Alternative fixation in methanol and acetone (1 : 1) for 20 min at -20°C and rinsing in 2 X SSC plus 0.02% DEPC.

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for 20 min at 4°C (cross-linking fixative) for small tissues and longer for larger tissues. For the detection of cellular DNA, precipitant fixatives (e.g., 3 : 1 methanol/acetic acid) are generally used. Formaldehyde adversely affects the denaturation of DNA targets and should be avoided whenever possible. The most frequently used precipitant fixatives are alcohol- or acetone-based. Slides or coverslips carrying the tissue or cell preparations are submerged in either of these solvents for about 15-20 min and washed with physiological saline or buffer until the pH is around 7. Although the tissue-specific effect of these fixatives was investigated by Bresser and Evinger-Hodges (1987) for the detection of mRNA, their results indicate that optimal fixatives have to be determined for each tissue type to be assayed. For the detection of RNA, freshly prepared formaldehyde is a versatile fixative since it cross-links proteins less than glutaraldehyde, permeabilizes cell membranes and provides an improved penetration of the probes due to the limited cross-linking. Moreover, formaldehyde has the added advantage of its compatibility with other fixatives. Fixation with formaldehyde is highly pH dependent (Berod et al., 1981) and is extremely slow at pH 6.5. This phenomenon can be used to advantage, particularly for large tissue fragments, by first distributing the fixative uniformly throughout the tissue at low pH and then cross-linking the preparation evenly by raising the pH (with sodium borate). Depolymerized paraformaldehyde should be used since commercially available formaldehyde solutions (37%) contain stabilizers and methanol, which may be deleterious (Farr and Nakane, 1981). The fresh fixative is prepared as presented in Table 11.2. Its use is the least technically demanding and therefore allows for slight variations without apparent effects. The superiority of paraformaldehyde for ISH of mRNA was confirmed in a study by Singer et al. (1986a,b) in which cellular RNA was labeled with 'H and probes with 32P and (i) the retention of RNA, (ii) the amount of probe hybridized and (iii) the preservation of cellular morphology were evaluated. Acridine orange staining after each step before hybridization also showed a superior RNA retention after paraformaldehyde fixation (Hafen et al., 1983). The best fixative for the preservation of cellular morphology is

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glutaraldehyde (Sabatini et al., 1964; Hayat, 1970). Unfortunately, glutaraldehyde has several drawbacks for ISH: extensive cross-linking reduces permeability, it is reactive with nucleic acids and introduces reactive groups which may enhance background staining. Reactive groups may be blocked, however, by treatment with 50 mM NH,C1 before hybridization. Due to the potency of glutaraldehyde, subsequent treatment of the sample with a protease is generally beneficial for the accessibility of target nucleic acid, as is the use of short probes. This method requires thus a delicate compromise and is only chosen when others fail. Fixatives at concentrations which are separately suboptimal, can be adequate in combination (Cox et al., 1984; Lum, 1986; Dutilh et al., 1988). Fixed cells can be dehydrated in graded alcohol baths and stored under RNase-free conditions for at least 6 months at room temperature.

11.2.2. Sectioning and mounting of cells and tissues Sections cut from frozen or embedded tissues are most frequently used for ISH, although single cell suspensions, such as hematopoietic cells, can also be mounted (Giovanni et al., 1986; Harper et al., 1986; Bresser and Evinger-Hodges, 1987) using nuclease-free physiological buffers. Different supports have been used such as 3M ScotchTMtape (excellent retention of cells and high signal/noise ratio for radioactive probes), GeneScreen Plus (NEN) or nitrocellulose (Southern et al., 1984; Davis, 19841, but treated microscope slides are convenient and satisfactory. To promote the adherence of cells and to prevent cells or sections from lifting off during the various hybridization steps, slides can be ‘subbed’ or coated with poly-L-lysine,whereas the coverslips can be treated with silicone to prevent sticking of the cytological preparation to the coverslips (Table 11.2). Tourtellotte et al. (1987) described a method for covalent binding of paraformaldehyde-fixed paraffin embedded sections to silanized slides (Maples, 1985). They observed that tissue adhesion was very high and that nonspecific probe binding was absent. This technique is also applicable to cryosections and cultured cells. Brigati et al. (1983) coated

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coverslips with egg white, irradiated them for 30 min under shortwave UV and used them to culture cells. For this purpose, acidcleaned coverslips are incubated for 2 h at 65°C in a solution of denatured egg white (one large egg white in 500 ml of distilled water, to which 1 ml of 10 N NaOH and 2 ml of 50% PVP are then added, stirred for 2 h and filtered through Whatman no. 1 paper), dipped in distilled water, fixed in ethanol/acetic acid (3 : 1) for 20 min at room temperature, air-dried, acetylated (Section 11.3, Table 11.4) and dehydrated in graded ethanol. In addition, humid chambers are needed to prevent evaporation during the hybridization steps. Any tight-fitting box with a plastic lid (to prevent the formation of large condensation drops; no glass!) can be used with a paper towel with a small amount of buffer (same ionic strength as on preparation to prevent distillation) on the bottom. ISH is often possible on routinely processed formalin-fixed paraffin-embedded tissues. Retrospective studies of routinely processed human material are therefore feasible. Protease treatments prior to the hybridization may be necessary since the duration of fixation in autopsy material is often unknown. Sections of the same block have then to be examined to establish the optimum degree of protease digestion. Plastic embedding media have been used for sections less than 1 p m thick, e.g., for electron microscopy (Falser et al., 1986; Binder et al., 1986) for cases where superior morphological detail and resolution are required.

11.3. Hybridization procedures General ISH procedures are given in Table 11.4. Sectioned tissues or cultured cells are used and probes are detected, after hybridization and washes, by autoradiography or nonradioactive methods. Usually, very low probe concentrations, e.g., 50 ng/ml, are used to prevent background accumulation (Gerhard et al., 1981; Harper et al., 1981). However, when hybridization is optimized background levels increase only marginally with much higher probe concentrations. For instance, McNeil et al. (1991) observed that concentrations

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TABLE 11.4 Prehybridization treatment of cells When peroxidase is used as the label, endogenous peroxidase should be inactivated (e.g.. immersing slides, after fixation, in 1% H 2 0 , in methanol for 30 min, washing in methanol and air-drying. A. RNA in cryosections McCabe and Pfaff (1989) found tissue pretreatment to be beneficial for RNA probes but not for DNA probes. Both glycine and acetylation treatments decrease background levels. 1. Rinse slides with cryosections warmed to room temperature, with PBS plus 0.02% DEPC, two rinses with PBS plus 0.1 M glycine and twice with PBS for 3 min each, then for 15 rnin in 0.3% Triton X-100 in PBS and another two rinses in PBS, 3 rnin each, all at room temperature. 2. Incubate slides for 30 rnin at 37°C in predigested proteinase K (0.5 pg/ml in 0.1 M Tris-HCI, pH 8.0 and 50 mM EDTA) at 37°C (30 rnin predigested). Alternatively pronase, in 20 mM Tris-HCI (pH 7.5) and 5 mM EDTA, can be used and the reaction stopped by rinsing for 30 s in PBS containing 0.2% glycine. 3. Postfix with 4% paraformaldehyde in PBS (5 min) and rinse twice with PBS containing 0.2% glycine, for 3 min at room temperature; 30 ml/slide. 4. Acetylate specimen by passing through 0.1 M triethanolamine (solubilized by adding NaOH to pH 8.0), first without and then with acetic anhydride (very short half-life) added directly to 0.25% (10 min) and passing twice through 2XSSC and graded alcohol (Hayashi et al., 1978). Dry slides and use immediately for hybridization. This step is important with poly-L-lysine-coated slides and after formaldehyde fixation. Prehybridization with hybridization solution without probe or DeSO, or PEG 6000, is for 2 h at the hybridization temperature. B. RNA in paraffin sections and cultured cells 1. Dewax and rehydrate sections: warm slides to room temperature and pass through three changes of xylene, and then 100, 95, 70 and 50% ethanol (2 min each). Some authors suggest denaturing proteins and nicking DNA by passing the slide through 0.02-0.1 N HCI for 20 min (see C below). 2. Denature RNA for 15 rnin at 70°C in 2 X SSC or 50°C in 0.1 X SSC and rinse in PBS for 2 min. 3. Proceed with steps 2-4 from section A above. C. DNA target detection 1. Hydrate cells briefly in PBS and place for 10 min in 0.02 N HCI. 2. Wash three times in PBS and once in PBS with 0.02% Triton X-100 (2 min each). 3. Optionally (omit if not absolutely required): treat with pronase as in A2 and treat cells with RNase A (100 pg/ml) and RNase T1 (5 pg/ml) in PBS with 5% glycerol (not with micrococcal nuclease!) at 37°C and proceed as in A3,4.

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up to 10000 ng/ml could be used (20 pl applied to the slide). Instead of days, a 1 h hybridization is then sufficient (after 10 min, about 1/3 of maximal signal). High concentrations of probe also yield much higher signal/noise ratios and allow nonstatistical detection of single sequences in metaphase or interphase cells. Suppression hybridization (Section 12.1.2) may be required if the probe contains repetitive sequences (e.g., lambda, cosmid or YAC probes) which would hybridize much faster and give a stronger signal than the specific sequence. It is also recommended to use low salt concentrations (e.g., 1 X SSC). Double labeling can then avoid statistical analysis of many metaphase chromosomes (Lawrence et al., 1990).

1 1.3.1. Prehybridiza tion trea trnents Accessibility of the target can often be improved by different techniques. They all intend to improve the diffusion of the probe to the target and to prevent nonspecific trapping of probe in tissues. Mild detergent treatments, proteinase K or pronase digestion, heat and mild HCl treatments have been suggested (Brahic and Haase, 1978; Willingham et al., 1978). These treatments can also include nonspecific blocking with the usual prehybridization methods. Permeabilization can sometimes be improved by rinsing with 0.1% Triton X-100 in PBS for 5 min (Smith, 1987). Often, however, pretreatments are unnecessary or even detrimental after paraformaldehyde fixation but remain beneficial after glutaraldehyde fixation (Singer et al., 1986). Although pretreatment can be critical to the accessibility of the probe, it can be disruptive of cellular morphology, it can lead to RNA degradation (autodigestion of proteases before use recommended) and it may cause cells to lift from the support. Pretreatments should therefore be optimized by serial two-fold dilutions/incubation times. 11.3.2. Hybridization to DNA

DNA detection usually does not require the same degree of cellular integrity as RNA detection. Optimization involves (i) fixation condi-

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tions and time, (ii) denaturing method, time, temperature and solution, (iii) (pre)hybridization conditions and probe concentration and (iv) posthybridization conditions. TABLE 11.5 Hybridization and posthybridization A. RNA probe: RNA target hybridization 1. Remove prehybridization solution without permitting the specimens to dry and add probe in hybridization solution (radiolabeled probes 10-600 ng/ml; nonradioactive probes up to 10 times more). Hybridization solution contains: 50% formamide, 0.5 M NaCI, 10 mM Tris-HCI (pH 7.61, 2 mM EDTA, l x Denhardt's solution, 20 mM DTT, 300 wg/ml tRNA, 300 Fg/ml poly(A) or/and poly(C) (optional), 10% PEG 6000; RNase-free. 35S label may form nonspecific disulfide bonds in the cell if reductants, such as DTT or 2mercaptoethanol, are omitted. , 2. Cover specimen with small amount of hybridization solution, e.g., 10 ~ 1 and cover with coverslip without air bubbles as described in Section 11.3.2. 3. Incubate 1 h to overnight in a moist chamber at 25-55"C, depending on target abundance and probe concentration.

B. RNA probe: DNA target hybridization 1. Denature DNA with any of the following procedures (establish which, in addition to adequate denaturation, preserves morphology satisfactorily; it is important to neutralize the formamide solutions): a. 2 min in 70% formamide and 0.6 X SSC (or 2X SSC) at 70°C; b. 15 min in 95% formamide and 0.1 X SSC at 65°C; c. 10 min in 50% formamide and 2 X SSC at 80°C; d. 10 min steaming above hot water (90°C); e. 0.15 N NaOH in 70% ethanol for 5-10 min (only for fresh preparations); f. microwave treatment (Section 11.3.2). 2. Dehydrate in graded ethanol, air-dry and proceed as in A. C. RNA probe posthybridization 1. Remove unhybridized probe in a small volume of wash buffer (3 X SSC, 5 mM DTT or 20 mM 2-mercaptoethanol, with or without formamide, depending on stringency) and wash in the same buffer three times for 20 min at 20-50°C (last two times with 0.5% Triton X-100). 2. Wash with wash buffer without formamide or Triton. Incubate with RNase solution (40 kg/ml RNase A and 2 kg/ml RNase T1 in 10 mM Tris-HCI, pH 7.5, 1 mM EDTA and 0.5 M NaCI) for 30 min at 37°C. 3. Wash for 30 min in 2XSSC plus 2-mercaptoethanol at 37°C and twice with 2xSSC for 10 min at room temperature and overnight at 0.lxSSC plus mercaptoethanol at room temperature or higher (stringency).

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TABLE 11.5 (continued) 4. Proceed as:

a. For autoradiography, dehydrate through (1 min each) 0.3 M NH,OAC/ ethanol (1: 1, 3 :7, 1 :9) and ethanol and dry slides. b. For fluorescein-avidin detection, stain with fluorescein conjugate (10 p,g/kI in 2X SSC and 1% BSA) for 1 h at 37°C; rinse three times with 2X SSC, once with 2X SSC/O.O5% Triton X-100 and once with 2 x SSC (10 min each). c. For anti-hapten-antibody conjugates, proceed as in 4b with the proper dilution of the conjugate (e.g., 1: 1000 anti-BrdU-fluorescein conjugate or 1 :200 anti-DIG monoclonal antibody conjugate).

D. DNA probe hybridization 1. Denature probes and targets by one of the following methods: a. Separately (RNA but also DNA target) for 10 min in hybridization solution (step lb) at 95°C. Denature target DNA simultaneously as described in step B1. Alternatively, nick-translated ds DNA probe is lyophilized and resuspended in 10 pl of 80% neutralized formamide and denatured for 15 min at 85°C (avoid evaporation) before adding a solution containing 20% DeSO,, 4 X SSC, 2 mg/ml BSA and 20% formamide (neutralized to pH 7). b. Simultaneously with target DNA by adding probe in hybridization solution (50% formamide, 2 x SSC, 200 pg/ml carrier DNA, 4 X Denhardt's solution (optional) and (optional, depending on size and concentration of probe) DeSO, or PEG), covering with a coverslip and sealing with rubber cement, followed by incubation at 80°C for 5 min for paraffin sections or 2 min for cryosections. 3. Incubate for 1 h to overnight at 20-45"C, depending on target abundance and probe concentration, in a moist chamber or under a coverslip or autoclavable plastic. E. DNA probe posthybridization 1. Rinse in small volume of 2 x SSC plus 50% formamide to remove the bulk of the probe and then twice for 10 min in large volume of the same buffer (30 ml/slide) 2. Wash overnight in O.l-OSXSSC, containing 5 mM DTT or 20 mM 2mercaptoethanol for 35S-probes, or 0.05% Triton X-100 if nonradioactive probe is used. 3. Proceed as in step C4.

For DNA, fixation with a precipitant fixative is far superior to formaldehyde. RNA may yield false signals if DNA has to be detected since (i) the number of RNA transcripts exceeds the number of corresponding DNA sequences, (ii) the target RNA is ss and

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(iii) RNA is mostly located in the cytoplasm which the probe has to pass. Therefore, cells are treated with DNase-free RNase, followed by inactivation of RNase. Alternatively, ss probes with the same polarity as mRNA can be used. When ds probe is used, then probe and target can be denatured simultaneously. A large number of variants have been described (50-95% formamide, 0.1-2 X SSC, 60-100°C; Table 11.5): (i) for simultaneous denaturation usually 50% formamide in hybridization solution with probe for 5 min at 70-80°C and then overnight at 37°C; (ii) for denaturation of only target higher formamide and lower SSC concentrations can be used (often without any significant difference in denaturation efficiency but with considerable differences in the preservation of morphology) (Raap et al., 1988). DNA can also be denatured in steam over a 90°C water bath for 10 min (e.g., Chantratita et al., 1989). Microwave irradiation to denature DNA has been shown to be particularly useful in order to increase the hybridization signal (Coates et al., 1987). For this purpose, several slides can be placed simultaneously in a slide tray with towel and 25 ml of 2 X SSC, irradiated for 1 min at moderate setting and 7 min at low setting (excellent also for simultaneous denaturation of probe and target). Specimens can be covered with hybridization solution and then with a coverslip and sealed with rubber cement or they can be surrounded with clear nail polish (hydrophobic) and incubated in humid chambers. Optimized conditions, especially probe concentrations and posthybridization washes, can be narrowed down using 32P-labeledprobes in ISH. 11.3.3. Hybridization to RNA

Hybridization to mRNA presents several difficulties such as (i) lability of RNA, (ii) solubility and (iii) dispersal of RNA throughout the cytoplasm. DEPC should be added to the different buffers, even to the subbing solution, to 0.02% throughout the whole procedure. The probe concentration varies widely in different reports (505000 ng/ml). Although high concentrations increase the probe diffusion rate and reduce the hybridization time, background also increases. For radiolabeled probes, it is best to maintain relatively low

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concentrations whereas for nonradioactive probes higher concentrations can be used. Bresser and Evinger-Hodges (1987) used very high concentrations (5 kg/ml) for 45 min hybridizations. The hybridization buffer usually contains 5 x SSC, 50% formamide, 100 pg/ml carrier DNA/tRNA, 0.1% Triton X-100 and 20 mM vanadyl ribonucleoside in addition to denatured probe. In the case of optimal probes of 150 bases (40-60% GC), the optimal hybridization temperature ( T , - 25°C) in this buffer would be about 56-70°C for RNA probes and 37-61°C for DNA probes (eqs. (11) and (121, Chapter 2). However, very high temperatures, e.g., > 50"C, affect tissue quality. Usually, incubation is at 20-50°C in a humidified environment. 11.3.4. Posthybridization and detection of probes Extensive posthybridization washes are usually not necessary or do not make a difference. Standard methods (Table 11.5) or a few rinses in 2 X SSC suffice. Radiolabeled probes are detected in an autoradiographic emulsion or stripping film (Section 7.2.5.21, by film (Section 7.2.5.1) or using a scintillation counter. Nuclear track emulsions require an exposure time of about 2-15 days for 35Sand 15-100 days for 3H. For 35S, it is possible to detect signals first with a film. At a specific activity of 2 X lo7 cpm/pg for 3H, 1000 copies/cell of mRNA can be detected in 2 months with a signal/noise ratio of 50-100. The detectability is thus about 20-40 copies/cell if a signal/noise ratio of 2 is chosen as the minimal specific signal. However, it can be calculated that a unique gene of 1000 bp would require a tritiated probe of lo8 dpm/p,g in order to obtain 1 silver grain after 100 days of exposure (assuming an efficiency of 15 disintegrations per grain; this ratio may be five times higher). In contrast, 35Sis about 10 times faster than 3H but with a decreased resolution. 35S has long track lengths and grains may form in emulsion around the labeled cells. Counterstaining is usually needed to reveal the cellular outlines (Table 11.6). Haptens in the probe are detected using streptavidin, for biotin, or antibody conjugates. The size of a biotinylated probe is critical to keep noise at a low level and the signal/noise ratio is often between

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TABLE11.6

Counterstaining after autoradiography A. Toluidine blue (TB), primarily for embryo tissues 1. Dilute TB stain (0.5 g of TB, 1 g of sodium borate in 100 ml of water) so that optimal counterstain will be obtained (50-500 times). 2. Dip slides briefly in diluted stain, rinse several times with water and mount. B. Hematoxylin/eosin, primarily for adult tissues 1. Pass slides in a rack through: a. 30 s in hematoxylin stain (Harris-modified, supplied by Fisher; do not overstain). b. Twice 2 min in water. c. Quickly in 0.1% NH,OH, too long, then emulsion may detach. d. Twice 2 min in water. e. 30 s in eosin (12 g of eosin Y and 3 g of phloxine B (both Fisher) in 500 ml of 70% ethanol. f. Dip 5-10 times in 95% ethanol and 5-10 times in 100% ethanol. 2. Dehydrate for 5 min in 100% ethanol and mount. C. Hoechst fluorescence staining of nuclei 1. Cover sections with diluted Hoechst stain (1:500of 1 mg/ml Hoechst 33258 dye in DMSO) and incubate for 2 min. 2. Wash slides in water and air-dry. 3. Incubate dry slides for 1 h at SOT, bring back to room temperature and mount in Canada balsam.

D. Fluorescent counterstain 1. Prepare antibleach medium with fluorescent counterstain: 90% glycerol and 2.5% 1,4-diazabicyclo[2.2.2]octane (DABCO antifade, Sigma) in 1X PBS to which either 1 mg/ml 4,6-diamidino-2-phenylimdoledihydrochloride (DAPI, Sigma) or 5 mg/ml propidium iodide is added. 2. Mount slides.

10 and 100. Acetic anhydride treatment before hybridization may reduce nonspecific adherence of these probes. Bresser and EvingerHodges (1987) observed that streptavidin conjugates can be added directly to the hybridization mix (containing formamide), 1: 1, incubated for 20 min at room temperature and then 10 min at 37"C, just before posthybridization washes (2 X SSC and 0.1% Triton X-100 containing RNase A and T1 to reduce background with RNA probes or with S1 nuclease and S1 buffer (Section 12.3) to reduce back-

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ground with DNA probes) (Cox et al., 1984) three times for 5 min in large volumes. Higher stringency washes can be added. Simultaneous detection of different probes is possible with different fluorescent tags, enzymes, radioisotopes or combinations thereof. Double-label ISH with radioisotopes can be achieved by exploiting the difference in energy of the nuclides. For example, a thin layer of clear plastic over the specimen prevents 3H from penetrating the emulsion while 35Scan be recorded in the second layer. Haase et al. (1985) fine-tuned this technique by color microautoradiography in which grains in the first layer are converted to a magenta color and those in the second layer to a cyan color.

11.3.5. Turbohybridization in situ A high speed hybridization procedure (total, including fixation and detection, 1 h) has been described by Bourinbaiar et al. (1991). They suspended washed lymphocytes, for the detection of HIV, in a mildly hypotonic medium (1 part medium and 2 parts distilled water) and spread 25 p1 (= lo5 cells) in 8 mm wells of Teflon-coated multiwell glass slides and fixed the cells in Carnoy’s I1 solution (60% methanol, 30% chloroform, 10% glacial acetic acid) for 10 min at room temperature and then for 10 min in absolute acetone. Five microliters of biotinylated probes (100 ng/ml) is added to the dried cells and the slides are covered with autoclavable plastic (cut from biohazard bag) and placed in a microwave oven. The position of the slide in the oven and the incubation time are crucial for this hybridization step. In microwave ovens with a carousel, the slide is placed about 6 cm from the center and a 150 ml beaker filled with tap water is placed in the center. The slide is then irradiated for 30 s on ‘Defrost’. The probes can be detected after a rinse with 2 X SSC.

11.4. Emerging in situ hybridization techniques 11.4.1. Quantitative analysis of mRNA Quantitation of mRNA copies is convenient with ISH since it is rapid and can be done with a small amount of cells. It is less precise

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than solution hybridization (sandwich or capture assays) since target may have been lost or become inaccessible during the various steps. Singer et al. (1986), in a model system, observed that with 32P probe at 2 p,g/ml, about 0.2 ng of probe was retained at the saturation point by 2 x 10’ cells on a coverslip (by cutting the coverslip in half with a diamond pencil and counting Cerenkov radiation in a scintillation counter). This corresponded to about 1000 mRNA copies/cell, which is probably about 50% of the real number. In another approach, Cash and Brahic (1986) quantitated ISH using initial velocity measurements and observed a linear relationship between the number of autoradiographic grains and the number of viral genomes per cell (between 600 and 60000). This method avoids the requirement of achieving saturation. 11.4.2. Combination of in situ hybridization with immunohistochemistry Double-label techniques of ISH and immunocytochemistry (e.g., Brahic and Haase, 1989) offer a powerful means of studying both the fate of nucleic acid and its expression in the same cell. The main problem is again fixation where both antigenic reactivity and nucleic acid must be preserved in a way compatible with both techniques. Fixation conditions are thus best determined empirically and the guidelines given here are intended only as good starting points. Three fixatives have been used successfully: (i) 2-4% paraformaldehyde in PBS (pH 7.4); (ii) 0.5% formaldehyde, 0.5% glutaraldehyde, 0.1 M phosphate buffer (pH 6.0), 1.6% glucose, 0.02% CaCl,, 1% DMSO (Morris and Barber, 1983); (iii) 2% formaldehyde, 0.075 M lysine, 0.01 M NaIO,, 0.037 M phosphate buffer (pH 7.5) (McLean and Nakane, 1974). Mice are perfused with 20 ml PBS and 20 ml fixative (Table 1 1 . 3 0 and tissues immersed in fixative at 4°C for 30 min. Fixation with formaldehyde is quenched in cold 0.15 M triethanolamine (pH 7.5) for 30 min and two 5 min washes in cold PBS. Tissues are dehydrated in graded ethanol and embedded in paraffin. After sectioning and adhering to a coated slide, paraffin is removed by dipping three times 5 min in xylene and twice 5 min in ethanol.

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Antigens are now detected by standard immunocytochemical techniques (Tijssen, 1985). It should be noted that some sera contain high concentrations of RNase (add 2% calf serum and 0.5% heparin or 0.4% DEPC to antiserum). Some authors suggest including a pretreatment at this point of 0.2 N HCI for 20 min, 2 x SSC at 70°C for 30 rnin and proteinase K at 1 pg/ml for 15 rnin at 37°C. Some products generated during immunodetection, such as diaminobenzidine polymers, may nonspecifically bind probes. This problem may be alleviated by acetylation by immersion in a solution of 0.25% acetic anhydride in 0.1 M triethanolamine, vortexed vigorously for 10 min at room temperature, followed by two washings in water and dehydration in 70% and 90% ethanol. The subsequent hybridization and probe detection conditions are standard. 11.4.3. In situ hybridization at electron microscopic level Ultrastructural ISH has been applied to study different aspects of mRNA transport and localization and, to a lesser degree, of DNA, using primarily biotinylated or haptenated probes. Colloid-gold conjugated antibody or streptavidin is convenient as the detector system (Binder et al., 1986). In a typical application for whole-mount electron microscopy (Singer et al., 19891, cells cultured on a coverslip-grid-formvar assembly (sterilized by gamma irradiation) are washed with an isotonic buffer (0.3 M sucrose, 0.1 M NaC1, 10 mM PIPES, 3 mM MgCI,, 10 FM Leupeptin (Sigma) and a 1:40 dilution (or 12 mM) of vanadyl ribonucleoside complex solution), extracted for 90 s with the same buffer containing 0.5% Triton X-100 (to facilitate probe penetration), followed by another wash in the isotonic buffer. After fixation with 4% glutaraldehyde in PBS and 5 mM MgCI,, the specimens are passed through an ethanol series (30,50 and 70%) and stored in 70% ethanol at 4°C. For hybridization, cells on the grids are rehydrated in PBS with 5 mM MgCl, for at least 10 min and 0.1 M glycine and 0.2 M Tris-HC1 (pH 7.4), also for 10 min, and then kept in 50% formamide and 2 x SSC for 15 min prior to hybridization and label detection. With probes of 150 bases, hybridization is allowed to proceed for 3 h in a humidified chamber at 37°C. The cells are left in

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1 x SSC before label detection with antibody conjugates. After the detection reactions and dehydration in graded ethanol, grid assemblies are critical-point dried before viewing with the electron microscope. Instead of whole-mount cells, it is also possible to prepare thin sections. Cells are grown on plastic coverslips, fixed (glutaraldehyde concentration lowered to 2%), hybridized and the label detected as described above. The cells are then postfixed with 2% OsO, in PBS for 30 min and dehydrated in graded alcohol. They are then passed through 100% alcohol/Epon (1 : 1) for 1 h at room temperature and 1 h in 100% Epon. Beem capsules filled with Epon are quickly inverted onto the surface of the coverslip and oven-baked at 60°C for 48 h. The Beem capsule and coverslip are removed followed by routine ultrathin sectioning and processing. 11.4.4. Gene localization

Since this methodology was initially described (Gall and Pardue, 1969), ISH has been applied widely to detect DNA sequences in polytene chromosomes or highly reiterated sequences. Recent developments have made it an increasingly important molecular tool for the localization of single-copy genes on chromosomes using *''I- or 3H-labeled probes (Gerhard et al., 1981; Harper et al., 1981). However, the scatter of radioactive disintegrations limited this technology to the analysis of large chromosome segments only and required statistical analysis of autoradiographic grain distributions in up to 110 metaphase figures. More recently, Lawrence et al. (1988) developed a high resolution mapping method of single-copy sequences down to 5 kb in both interphase nuclei and metaphase chromosomes by ISH. The high resolution obtained of only 130 kb was not the result of a single step but depended on the optimization of all experimental parameters for hybridization in interphase nuclei. The use of nonradioisotopic detection techniques was very important since grain scatter represented a lower limit of resolution. For example, the scatter of tritium is 1-2 Fm in both directions (Rogers, 1979), whereas Lawrence et al. (1988) obtained a 0.1 pm resolution using fluorescein-labeled avidin, thus a 10-20-fold greater precision,

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while maintaining a very low background (allowing high probe concentrations). Moreover the absence of a photographic emulsion allows other cytogenetic banding techniques. Interphase chromatin mapping may contribute significantly to the understanding of gene and chromatin organization and function. The 5-11 times less condensed state of chromatin makes it possible to obtain correspondingly higher resolution. Combined with the higher resolution obtained with fluorescence, this results in an approximately 100-fold improvement of resolution over autoradiographic methods on chromosomes. Metaphase chromosomes are condensed and allow cytogenetic banding. The extent of condensation will influence the relation between metaphase distance and linear DNA. The genetic map is expressed in cytogenetic bands which, particularly in the combination of Q-banding (Table 11.7A) and fluoresceinated probes (Lawrence, 1991), provides a more accurate localization and is independent of condensation. Signals along the chromosome, but not across its width, are useful for ordering. Due to the condensation, the lower limit of resolution is about 5 megabases. The relationship between linear DNA and interphase chromosome distance is not as well validated (Trask et al., 1989) since the DNA molecule may be lo5 times longer than the diameter of the nucleus. The interphase distance/linear DNA distance ratio varies with the particular region of the genome. The interpretation becomes increasingly more difficult when a signal is farther from the reference signal. Dual-band filters allow two fluorochromes to be viewed simultaneously. This avoids the requirement of references for both fluorochromes when the image is shifted which would make it difficult to resolve closely located probes. Moreover, the use of CCD cameras (Section 11.1.1) and image analysis makes very high resolution possible (Wiegant et al., 1991). Metaphase chromosomes are often G-banded, karyotyped and destained before ISH, although there is some loss of hybridization signal (Table 11.7). The tissues or cells should be actively dividing (hematopoietic tissue, primary cell culture). Mitogen stimulation often has a different effectiveness for different tissues or for similar tissues from different animal species (Davisson and Akeson, 1987).

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HYBRIDIZATION WITH NUCLEIC ACID PROBES

Concanavalin A can be added to the medium to increase the mitotic activity (Davisson and Meson, 1987), methotrexate, an inhibitor of thymidine synthesis, can be used to promote synchronization of the culture (Lee et al., 1990) and colcemid to increase the number of metaphase chromosomes (Table 11.3B).

TABLE 11.7 Banding of metaphase chromosomes The identification of the chromosomes requires either banding or reference probes (commercially available). Chromosomes can be Q- or G-banded, photographed and then hybridized and compared to the original photograph. G-banding depends on the removal of chromosomal proteins by trypsin and subsequent staining of remaining proteins by Giemsa. The extent of trypsin treatment required depends on the preparation (nature of preparation, storage) and on its age (Sun et al., 1974; Lee et al., 1990). A. Quinacrine staining of metaphase chromosomes 1. Pass chromosomes, rinsed in 2 X SSC, quickly through two rinses of McIlvaine’s buffer (pH 7, 82.4 ml of 0.2 M Na2HP0, and 17.6 ml of 0.1 M citric acid). 2. Stain slides for 30 min in quinacrine mustard solution (12 mg of quinacrine mustard dissolved in 50 ml of H 2 0 and diluted 1 : 1 with McIlvaine’s buffer). 3. Rinse twice with McIlvaine’s buffer and once in water, air-dry and mount. B. G-banding with Giemsa stain 1. Dip slide in trypsin solution (0.005-0.05% trypsin in 25 mM potassium phosphate and 0.5 mM EDTA, pH 6.8) for 10-60 s. 2. Dip immediately three times in 70% ethanol to stop the reaction and dip slide several times in water. 3. Dip slide for about 3 min in Giemsa stain solution (prepared by mixing 72.5 ml of 25 mM potassium phosphate buffer (pH 6.81, 10 ml of methanol and 1 ml of Giemsa stock solution; the latter is prepared by dissolving 1 g in 66 ml of glycerol at 56°C for 2 h and adding, after cooling to room temperature, 66 ml of methanol and aged for at least 2 weeks at 4°C). To prevent the metallic film that forms on the solution adhering to the slide when it is pulled out, water is added to overflow before removal of the slide. Dip several times in the phosphate buffer and water. 4. Air-dry, examine and photograph the slide by microscope (oil immersion). Remove the oil by a quick dip in xylene before destaining and ISH. Destain by dipping slides three times for 5 min in 70% ethanol and once in 95% and 100% ethanol, 5 min each, and air-dry.

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TABLE 11.7 (continued) C. Potential problems in banding 1. No banding, i.e., no staining: a. Incomplete fixation b. Preparations were insufficiently ‘hardened’ or overtrypsinized c. Preparations were not equilibrated with room temperature d. Stain was too old or slides were insufficiently stained 2. Heavy staining: a. Not properly stored b. Undertrypsinized c. Overstained; a rinse in methanol may help somewhat 3. Short chromosomes: a. Colcemid incubation excessively long 4. Clustered chromosomes: a. Insufficient treatment with hypotonic buffer b. Incomplete fixation; otherwise increase acetic acid from 25 to 33% c. Insufficient spreading since slide was not slanted enough during dropping of the cell suspension or height of drop was insufficient 5. Loss of chromosomes: a. Cell lysis during fixation (no vortexing) b. Spreading too extensive (slanting of slide, height of drop)

11.4.5. Troubleshooting Any step in ISH may lead to specific problems but a systematic analysis may help to pinpoint the cause (McCabe et al., 1986; McCabe and Pfaff, 1989). The main problems are no signal, low signals, high background or poor tissue morphology. Problems with metaphase chromosomes are summarized in Table 11.7C. The lack of autoradiographic grains can be due to a problem with the probe or with the hybridization procedure. Hybridization with a nonradioactive probe may help to distinguish between these possibilities. Possible problems with radiolabeled probes include latent image fading due to moisture (improve storage), negative chemography (determine which step and chemical prevents latent image formation) and photodevelopment (emulsion or photochemicals are expired). Possible problems with hybridization include loss of target (nuclease contamination, prolonged pretreatment, diffusion of tar-

524

HYBRIDIZATION WITH NUCLElC ACID PROBES

get), lack of hybridization (probe incorrect, poor labeling, hybridization conditions incorrect; check with Northern or Southern blots) and inaccessibility of target (overfixation, not enough pretreatment). Low signals indicate a low degree of hybridization to target, if more target should be present. If fixation is adequate, the nature of probe and the hybridization procedure should be investigated. Probes of about 150 bases (or 50 bases for oligomers) are usually optimal. They should be ss and have a high specific activity. More energetic probes (32P or 35S)can be used to optimize the technique. The hybridization should be optimized with respect to probe concentration, hybridization temperature and duration. Including dextran sulfate during hybridization and increasing the duration of autoradiography may also help. Some lots of coverslips leach significant amounts of alkali and interfere with ISH. High background in autoradiography may be due to positive chemography, to too long exposure times at too high temperatures. If the emulsion on the glass slides is dried too rapidly, nonspecific signals may increase substantially. High background may also be due to insufficient stringency or duration of posthybridization washes. Nonspecific binding to cellular components can be alleviated with acetic anhydride or glycine treatments, with prehybridization and prevention of oxidation of 35S-labeled probes. Crosshybridization (GC or poly(A) stretches) should also be avoided or competing polynucleotides should be included in the hybridization mix (Section 8.2). Poor tissue morphology can, among other things, be due to excessive deproteination during the pretreatments or the formation of ice crystals during freezing. Clearly, the use of nonradioactive systems is particularly helpful for a rapid optimization of ISH.

CHAPTER 12

Selected applications of hybridization Hybridization is the cornerstone of many techniques in diagnosis and research not yet discussed. Although hybridization is essential in many methods in genetic engineering and molecular biology, only those in which the nature and method of hybridization are critical and dominate the experimental setup, are reviewed here.

12.1. Aims of subtractive or suppression hybridization In a mammalian cell, a large number (about 10000) of mRNAs have a low abundance, i.e., a few copies per cell. Many strategies have been designed to enrich for specific mRNAs or to recognize certain clones (Section 2.5.2.2); otherwise many clones would have to be analyzed (Table 10.1). Subtractive hybridization depletes the sample of nonspecific mRNA in order to enrich for the mRNA sought. It is particularly suitable for isolating tissue-specific or developmental regulated sequences or clones derived from mRNA induced by external factors. Suppression hybridization is used when repeated DNA is present in both target and probe sequences. Since these repeat sequences are present in a relatively much higher concentration and since they have a lower complexity, they would give a much stronger signal than less abundant sequences. These nonspecific hybridization signals can be suppressed by hybridizing first with unlabeled repetitive DNA probes. Such probes are now commercially available for different species (e.g., from BRL).

12.1.1. Subtractive hybridization Two situations can be distinguished: (i) when there is a significant difference between two cell cultures or tissues in the level of expres-

526

HYBRIDIZATION WITH NUCLEIC ACID PROBES

sion of the mRNA studied (high ratio subtractive hybridization); (ii) systems with only slight differences in level of expression (low ratio subtractive hybridization). If we assume that the cDNA from the ( + ) library contains the target and the cDNA from the ( - library contains less or no target (differential expression), then the ( - ) sequences can be used to deplete the (+) library from nonspecific targets. Most strategies are based on hybridization of an excess of the (-) library to the (+) library and selective cloning of the unhybridized targets in the (+ ) library. Although solution hybridization is most common, immobilized driver DNA can also be used to enrich for clones unique to a library (Love and Deininger, 1991). In low subtractive hybridization, the amount of (-) added to the (+) library is more critical. Only a few among the most convenient methods are discussed here. Several ( + 1 and ( - ) lambda or plasmid libraries are commercially available (e.g., Clontech).

12.1.1.1. High ratio subtractive hybridization by cloning inhibition In the method described by Klickstein (19871, ( + ) cDNA with certain restriction sites (e.g., EcoRI) and a fragmented ( - cDNA library lacking these restriction sites are mixed in a proportion of 1 :50, denatured and hybridized so that only ( + DNA unhybridized with (- ) DNA can be cloned in EcoRI-digested vectors (Fig. 12.1(1)). In this method, lambda libraries are made from both the ( + and the ( - ) targets. A large prep (2 1) is made of both libraries. The (+ ) and (-) library DNA is digested with EcoRI and purified on a 10-40% sucrose gradient, containing 20 mM Tris-HC1 (pH 7.51, 1 M NaCl and 5 mM EDTA, in an SW28 rotor (26000 rev./min overnight). The inserts will remain near the top whereas lambda DNA will move about midway into the gradient. The insert DNA is collected from the top (avoid contamination with lambda and verify by agarose gel electrophoresis) and is precipitated with ethanol (Table 3.1). The ( + ) DNA is resuspended in TE to a final concentration of 0.2 pg/ml (about 50-75 pg can be expected from 1 mg of lambda) and all of the ( -1 DNA is resuspended in 100 p1 of TE. To remove EcoRI sites, 90 p1 of the (-1 DNA is added to 10 j ~ l of 10 X S1 buffer (0.5 M NaOAc, pH 4.5, 10 mM ZnOAc, 2.5 M NaCl and 0.5 mg/ml BSA) and 2 units of S1 nuclease and incubated

Ch. 12

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SELECTED APPLICATION OF HYBRIDIZATION

[I (+)mRNA

+ )mRNA

( - )mRNA

cDNA

I

---A 4

(-)mRNA

4

4

7 cDNA

_J

Cloning into A vectors

4 4 Restriction with EcoRl 4 4 inserts Purification

"[

photobiotud

+ 4.

4

+

Removal of biotin-containing

+

duplexes and cloning

111

i.f. Further fragmenting by blunt-end endonucleases

BlOj-cDNA

Hybridization

of

Removal of overhangs created by EcoRl

4

cDNA

(+)rnRNA

Denaturation and

( - ) m r

hybridization ( 5 0 x excess of (-)DNA)

4 4

Ligate remaining homoduplex (+)DNA La AgtlO Plate and evaluate plaques by +/- scrcening

Hybridrzation

4 4

Removal by poly(A)

+

tails

Cloning

Fig. 12.1. Examples of subtractive hybridization systems. Example I shows the 'EcoRI method' in which the proportion of over-represented mRNA (in the '+' library) is increased in the final library. Simple alternatives are based on the selective removal of common sequences in ' - ' and ' + ' preparations, either by biotinylated ' - ' cDNA (11) or poly(A) tails from ' - ' RNA hybridized to ' + ' cDNA (111).

for 30 min at 37°C. The reaction is stopped by adding 5 pl of 0.5 M EDTA, 200 pI of TE and extraction with 300 pl of phenol/chloroform. After ethanol precipitation, ( -) DNA is resuspended in 100 p1 of TE buffer and further fragmented by digestion with the frequent cutters A h 1 and &aI, then extracted with phenol/chloroform and precipitated with ethanol. The (+) DNA (0.2 pg) is hybridized with 10 pg of (-1 DNA in 50 pl containing 50% formamide, 5 x SSC, 10 mM sodium phosphate (pH 7.0), 1 mM EDTA, 0.1% SDS and 200 Fg/ml tRNA for = 24 h at 37°C. Evaporation in the tube should be minimized (no cold top, e.g., plunger above liquid). To extract the sample, 450 ~1 of TE and 500 pl of phenol/chloroform is added. The DNA is precipi-

528

HYBRIDIZATION WITH NUCLEIC ACID PROBES

tated and resuspended in 12 p1 of TE. For ligation to lambda gtlO, 10 pg of phosphatased phage arms in 10 p1, 2.5 pl of 10 X ligase buffer (Section 4.5) and 0.5 p1 of T4 DNA ligase (200 units) are added and incubated overnight at 15°C. The next morning, the DNA is packaged (according to the manufacturer's recommendations) and the library plated with E. coli C600hfo1. The plaques are evaluated by hybridization: one lift with ( + >DNA probes and a parallel lift TABLE 12.1 Subtractive hybridization A. Subtractive hybridization with biotin-driver DNA and partitioning hybrids into phenol/chloroform phase (Herfort and Garber, 1991) 1. Prepare independent, directionally cloned cDNA libraries in phagemid vectors (e.g., pBluescript) from ( + ) and (-1 poIfiA)' RNAs as described in Section 4.5. 2. Amplify libraries by growing clones to saturation in 100 ml and save as glycerol stocks (25% glycerol added and stored at - 70°C). 3. Prepare ss DNA from ( - ) library by adding 4 X lo9 plaque-forming units of M13K07 (e.g., Stratagene) when bacteria in 200 ml attain a density of 0.4 OD,,. After another hour of shaking at 3TC, add kanamycin sulfate to 70 pg/ml and continue culture overnight (yield will be about 1 pg/ml of ss DNA). 4. Purify ss DNA from supernatant as described for M13 in Section 4.3.2.4. Photobiotinylate the ss DNA as described in Section 7.8.2 (25 pg in 50 pl). 5 . Digest an aliquot of the (+) DNA to completion, using the enzymes used for directional cloning and purify. Mix biotinylated ss DNA (8 Fg) to 400 ng (+) DNA (molar ratio of 40) in 8 pI of H,O in a siliconized microcentrifuge tube. Heat to 65°C and then add 8 p1 of deionized formamide, 2 pI of 5 M NaCI, 1 ~1 of 1 M HEPES (pH 7.6) and 1 pI of 4% SDS. Mix well, boil for 5 min and hybridize at 42°C for 36 h. 6. Add 350 FI of 0.5 M NaCl in TE, mix and add 6 pg of streptavidin and incubate for 1 min at room temperature. Extract with phenol/chloroform and collect the aqueous phase. Extract phenol/chloroform with 50 p1 of TE with 0.5 M NaC1. Repeat streptavidin addition and extraction of pooled aqueous phases. 7. Place in a SpeedVac to remove residual chloroform, dilute to 2 ml with TE! and pass twice through a Centricon-100 (Section 3.1.4.2). Lyophilize retained sample ( = 50 ~ 1 ) . 8. Repeat subtraction (if desired) or continue with ligation to phagemid and transformation.

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TABLE12.1 (conrinued) B. Low ratio subtractive hybridization (Fargnoli et al., 1990) 1. Prepare cDNA from poly(A)+RNA with MMLV reverse transcriptase (100 pg of RNA should yield about 50 pg of cDNA). Wash the cDNA extensively with TRES (10 mM Tricine, pH 6.8, 0.4 mM EDTA, 0.1% SDS; RNase-free) in a Centricon-30 microconcentrator (Section 3.1.4.2). Recover RNA/DNA (about 50 p 3 , save 2% of the sample and concentrate the rest in a SpeedVac to 15-20 2. Add sodium phosphate and water to obtain a final volume of 25 FI and 0.4 M salt at pH 6.8. This sequence of addition is important to prevent aggregation of RNA. After heating briefly at 95”C, hybridize to Rot = lo3 (assuming 10.3 R,t/h per pg/kI at 65°C; usually about 25-36 h). 3. Separate ss cDNA from duplex DNA: RNA and RNA by chromatography on hydroxyapatite columns (Section 8.1.1.5) at 62°C in HAP buffer (0.12 M sodium phosphate, pH 7.5, 1 mM EDTA and 0.2% SDS; ss DNA will elute with several washes of HAP buffer). Wash the ss DNA again in the Centricon-30 with TRES. Remove the duplex from the column with > 0.4 M sodium phosphate. 4. Add (+) poly(A)+ RNA (the same as used for cDNA synthesis) and continue as in step 2 but to 0.3 M sodium phosphate and incubate for 24 h at 65°C to remove A/T rich cDNA. 5. Pass again on hydroxyapatite but collect cDNA:RNA duplex. Add NaOH to 0.1 N, heat to 65°C and neutralize with 2 M Tris-HC1 (pH 7.5) and wash with TRES; save 10% of cDNA for screening. 6. Make ds cDNA using random primers (Section 4.4) and clone according to standard methods (Section 4.5). For colony hybridization, use enriched (after step 5) and unenriched probes (after step 1) on parallel blots.

with ( - DNA probes (Section 10.3). The number of positives with the (-1 probe should be small. 12.1.1.2. High ratio subtractive hybridization by biotin- or poly(A) capture Elegant and efficient subtractive hybridization procedures have been developed from the original procedure described by Sive and St John (1988) and Duguid et al. (1988) in which driver cDNA is photobiotinylated, allowed to react with the ( ) DNA and the hybrids then eliminated with streptavidin agarose. Schweinfest et al. (1990) used the lambda ZAP I1 phage vector system to produce by in vivo excision a large amount of ss DNA for biotinylation. Recent methods take advantage of the property of biotinylated nucleic acids + -

+

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HYBRlDlZATlON WITH NUCLEIC ACID PROBES

confirm higher expression of RNA in experimental tissue

Fig. 12.2. Low ratio subtractive hybridization flow chart.

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531

complexed with streptavidin to be partitioned into organic solvents (Herfort and Garber, 1991; Lebeau et al., 1991; Table 12.1; Fig. 12.1(11)). They succeeded in an enrichment of at least lo4 allowing the detection of cDNA clones present at a frequency <0.001%. Lebeau et al. also used PCR, using plasmid sequences (T7 and SP6 promoters) for primer target, to produce enough driver DNA. Batra et al. (1991) subtracted ( + ) cDNA against poly(A)+RNA, removed the heteroduplexes by the poly(A)+ tails and used the subtracted ss DNA as template for cloning. They observed an enrichment of > 300-fold in their system. 12.1.1.3. Low ratio subtractive hybridization The method described by Fargnoli et al. (1990) (Table 12.1B) allows a substantial enrichment of low abundance transcripts (Fig. 12.2). This method may also be used for ‘housekeeping’ genes whose levels are fairly constant but change with age. On the other hand, the use of whole tissue may yield RNA of high complexity and variation in expression. cDNA quality has a considerably higher impact on low ratio subtractive hybridization than at high ratios. It is important to note that the relative molar amounts of cDNA and driver RNA are crucial in this method. MMLV reverse transcriptase tends to produce larger cDNA and thus be better suited. The cloned MMLV, lacking RNase H, may even be superior. Nevertheless a substantial fraction of ss cDNA, produced with noncloned MMLV enzyme, cannot form stable duplexes. Therefore, Fargnoli et al. also hybridized the subtracted cDNA with ( ) RNA to recover hybrids and eliminate nonhybridizing cDNA. The use of Centricon-30 microconcentrators (Section 3.1.4.2) in this procedure instead of ethanol precipitation is important to prevent aggregates of RNA. Moreover, phosphate precipitates in ethanol. A 30-fold enrichment is usually obtained allowing < 6 transcripts per cell to be identified. It is essential to complete the hybridization reaction as much as possible without having a large excess of ‘driver’. Clones tend to be small with this method. A major drawback is that variation between ( ) and ( - ) expression of different genes may reduce the efficiency of low ratio hybridization subtraction.

+

+

532

HYBRIDIZATION WITH NUCLElC ACID PROBES

12.1.2. Suppression hybridization Mammalian genomes may contain a considerable amount of repetitive DNA, e.g., 900 000 A h sequences are interspersed in the human genome. Genomic clones, particularly large ones such as cosmids or YAC (yeast artificial chromosome), have a high probability of containing these repetitive elements. When such probes are used on Southern blots or in ISH a very high, usually dominating, background signal is obtained, This problem is usually solved by the time-consuming characterization of the clones and subcloning or by hybridization with total genomic DNA (Sealey et al., 1985; Landegent et al., 1987). Nisson et al. (1991) described an elegant method of suppression hybridization in which repetitive DNA is selectively purified and then used to block repetitive sequences in the target prior to specific hybridization. DNA from the same species as the target is extracted and sheared, denatured and renatured to a Cot = 1. The remaining DNA (including single-copy sequences) is then digested with S1 nuclease (Section 12.31, whereas the ds DNA is extracted with phenol/chloroform, precipitated with ethanol and resuspended in TE. BRL offers this so-called Cot-1 DNA from many species including human. The background reduction with Cot-1 DNA in Southern blots or ISH is far superior than that obtained with total genomic DNA (Lichter et al., 1990; Johnson et al., 1991; Nisson et al., 1991). When cosmid clones are used as hybridization probes, repetitive sequences usually interfere. In addition to saturating the target sequence with total DNA (100 pg/ml, 15 h), Djabali et al. (1990) used the PERT method (Section 2.3.1.1) to prehybridize the random-primed cosmid clones to eliminate repetitive DNA in the probe. Cosmid clones (50 ng) are labeled by random priming (Section 7.6.2) and then mixed with 100 pg of sonicated total DNA (average size 500 bases) in 1 ml of solution containing 0.1 M sodium phosphate, followed by the addition of 500 pl of water-saturated phenol (shaken for 15 h at room temperature). After centrifugation, the probe in the aqueous phase can be used for hybridization. Cot-1 DNA instead of total DNA should also be useful in this procedure. Finally, it is usually beneficial to avoid high salt concentrations during hybridization (e.g., 1 x instead of 5 x SSC).

Ch. 12

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12.2. Hybrid selection of mRNA RNA to be studied can be selectively purified by hybridization on solid supports or in agarose chromatography or centrifugation. Conventional solid phases have included nitrocellulose or activated cellulose and affinity columns. Recently, nylon or, particularly, paramagnetic beads have become very useful since hybridization is much faster, reaches completion rapidly, leaches less capture nucleic acid and they allow convenient washes. These techniques require the availability of DNA complementary to the mRNA to be isolated. This can range from genomic clones to oligomers, either perfectly complementary or degenerated oligomers or guessmers (Section 6.2). These DNA molecules are immobilized on a solid phase and isolated RNA is allowed to hybridize with this target. After washings, the hybrid-selected RNA is eluted. The RNA selected can be specific for a certain family or mRNAs with common exons, for a unique gene or can be used to isolate poly(A)+RNAs (using poly(dT) capture molecules).

12.2.1. Procedures of hybrid selection 12.2.1.1. Membranes and hybrid selection Nylon, particularly the positively charged membranes, has largely replaced nitrocellulose or diazotized paper for hybrid selection. DNA, e.g., from linearized plasmid, can be applied in 0.4 N NaOH to denature the DNA and to promote a strong fixation. The nylon membrane (2-cm squares) is soaked for 5 min in water and then 30 min in 0.4 N NaOH. Concurrently, DNA is denatured for 10 min in 0.4 N NaOH. DNA is then applied repeatedly with drying between applications. The membrane is then washed twice with 1 M NH,OAc and twice with 1 x SSC. The membrane is blotted and dried (may contain > 100 p,g/cm2). An alternative method is presented in Section 8.3.1. Disks of 0.5 cm are then punched out with a sterile one-hole paper punch. It is also possible to use Southern blots, or fragments thereof, for hybrid selection.

534

HYBRIDIZATION WITH NUCLEIC ACID PROBES

The major problem in hybrid selection is degradation of mRNA by contaminants of formamide. Formamide is required to lower the T,, to prevent RNA degradation due to high temperatures and should be deionized (stir 10 ml of formamide with 1 g of AG-501SA from Bio-Rad for 1 h at room temperature, filter and store at - 70°C). Prehybridization is carried out in 0.3 ml of 50% formamide, 0.75 M NaC1,O.l M PIPES (pH 6.4), 8 mM EDTA, 0.5% BLOTTO, 100 p.g/ml tRNA (extracted several times with phenol) and 0.5% SDS in sterile round-bottom plastic tubes (e.g., Nunc, 1.8 ml; up to 10 disks per tube) at 37°C overnight (Jagus, 1987). The membrane is then washed 10 times with 1 X SSC, 2 mM EDTA and 0.5% SDS at 60°C and rinsed with preheated hybridization buffer (0.4 M NaCl, 10 mM PIPES, pH 6.4, 8 mM EDTA, 0.5% SDS and formamide). Hybrid selection is optimal with poly(A)+RNA (> 100 p,g/ml) and 65% formamide for abundant RNA and 50% formamide for low abundant RNA in preheated hybridization buffer. If total RNA is used, the concentration should be 10 mg/ml. For high abundant RNA, 4 h at 50°C are sufficient otherwise 37°C overnight is used. After hybridization, wash the membranes ten times with 1 x SSC, containing 0.5% SDS, at 50°C and five times with 1 X SSC. Wash the membrane once with 5 mM Tris-HC1 (pH 7.5) and 1 mM EDTA (30 s) and elute by boiling for 5 min with 5 mM KCl, 2 mM EDTA and 10 pg/ml tRNA and collect the supernatant. Repeat and pool the eluates. 12.2.1.2. Ultracentrifugation

Ultracentrifugation in CsCl gradients containing guanidine HCl (Enea and Zinder, 1975) can be used to enrich or deplete for a specific mRNA species (Lubbert et al., 1987). This method is based on the observation that DNA duplices and RNA duplices have very different buoyant densities in such gradients and that DNA :RNA hybrids have an intermediate density. The complementary DNA should be ss (in ss vector: M13 or phagemids such as pBluescript or 1ambdaZAP). The mRNA-enriched fraction then corresponds to, the RNA :DNA band whereas the depleted fraction corresponds to the RNA band (lowest in the gradient).

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12.2.1.3. Hybrid selection through biotinylated DNA Soh and Pestka (1990) described a convenient method to purify specific mRNAs using probes generated by asymmetric PCR and the primer of which was biotinylated (biotin can be introduced directly during oligomer synthesis). The biotin primer is in a 100-fold excess during PCR (50 pmol versus 0.5 pmol) and about 0.1 pg of the product is used in the hybridization mix (RNA in 50 pl of 1 M NaCl, 50 mM PIPES, pH 7.0 and 2 mM EDTA, 2 min at 85°C and then at 55°C for a period depending on the RNA abundancy). Hybrids can be retained on streptavidin-agarose, streptavidin paramagnetic beads or by streptavidin-coated microtiter plates (Section 8.1.1.4). Streptavidin agarose (60 pl, from Sigma) is first washed three times with 0.5 ml of 0.25 M NaCl and 10 mM Tris-HC1 (pH 8.0) and then incubated with the same buffer containing 10 bg/ml tRNA (15 min shaking). The hybridization mix and 50 pl of water are added and incubated for another 90 min. The agarose is washed twice with 150 p1 of water, recovering the agarose by centrifugation, and the mRNA is eluted with 200 pl of 10 mM Tris-HC1 (pH 7 8 , containing 30% formamide at 60"C, snap-cooled and the mRNA recovered from the supernatant by ethanol precipitation (Table 3.1). 12.2.1.4. Hybrid selection through capture assays Several of the previously described methods are readily adapted for the purification of specific mRNAs. Most convenient is the system based on the use of paramagnetic beads (Table 3.16) in which the oligo(dT) should be replaced by specific oligomers. Alternatively, the (reverse) capture or sandwich assays described in Section 8.3 are well suited for hybrid selection.

12.3. S1 analysis of mRNA with DNA probes S1 analysis is widely used to determine the ends of an mRNA molecule, the sites of introns or to quantify mRNA. Recently, RNase protection assays have become increasingly popular since they are more sensitive and can be used to obtain the same information. However, background is much less of a problem in S1 analysis. The

536

HYBRIDIZATION WITH NUCLEIC ACID PROBES

1

5’-ends

-1 -

mRNA

+

5’

1

5’

DNA probe (kinased) heteroduplex

S1 nuclease

5’

~

mRNA DNA probe heteroduplex

I11

~ntrons

mRNA

5’

+ genomic DNA probes

01

heteroduplex

S 1 nuclease

1 electrophoretlc sizing of DNA fragments

Ch. 12

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537

end-labeled probes are also more stable than the internally labeled probes used in RNase protection assays (5-8 times).

12.3.1. Principles and goals of S l analysis The principles of S1 analysis are shown in Fig. 12.3. This method requires intact ss DNA that is usually obtained by end-labeling although internally labeled probes can also be employed (Ley et al., 1982). In the original method, Berk and Sharp (1977) exploited the observation of Casey and Davidson (1977) that DNA: RNA hybrids are more stable than DNA:DNA hybrids and hybridized DNA specifically to RNA by choosing conditions between the two T, values. Now, it is possible to obtain ss DNA probes from any position to a predetermined position and in the desired orientation. This simplified the S1 analysis considerably. At a 5-10-fold excess of probe, a pseudo-first-order reaction describes the kinetics of hybridization (Section 2.3.3):

T / T o = epkP' which demonstrates that the fraction of target ( T ) not hybridized at time f is independent of its prevalence (as long as probe P is in excess). Variations due to small experimental differences in the fraction hybridized will become insignificant at large t when T / T o < 0.05. Quantification of target is then independent of hybridization kinetics. The choice of the probe concentration is thus important. Fig. 12.3. S1 nuclease method for the analysis of RNA. Mapping of S'-ends requires 5'-end labeled probes with a 3'-overhang in the heteroduplex (I). Digestion with S1 nuclease is followed by gel electrophoresis (denaturing polyacrylamide/urea gel). Similarly, the 3'-end can be mapped with 3'4abeled probes and the sequence should exceed the template-specific sequence of the RNA (11). The poly(A) tail of the mRNA, however, can be longer than the probe. Introns can be mapped by probes connecting two consecutive exons. S1 nuclease digestion at 20°C occurs mainly in the DNA loops whereas at 40-45"C, RNA is also digested at the segments facing the intron loops. Migration of labeled probe fragments on alkaline gels will be similar for the two situations whereas the 45°C digest yields multiple bands and the 20°C digest primarily a single band on neutral gels.

538

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Too high a concentration yields a high background with respect to the specific signal. Too low a concentration, on the other hand, prevents the reaction from reaching completion. The lowest amount of probe that can be used is about 1 ng/ml (completion of reaction in about 60 h). To determine the 5’-ends of an mRNA molecule, a 5’4abeled ss DNA probe is hybridized to the RNA in which a ss overhang of the 3’-end of the DNA and a hybridized 5’-end of the DNA is obtained. Digestion with S1 nuclease (ss specific for both RNA and DNA) removes the 3’-overhang. The S1 enzyme is eliminated and the exact size of the S1 probe, protected by the mRNA, is determined on a sequencing gel using a sequence ladder as a marker. Similarly, the 3’-ends of an mRNA molecule can be established using a 3’4abeled ss DNA probe. Several S1 analysis methods have been used to establish the presence and location of introns. DNA probe, derived from a cloned gene, is hybridized with mature, spliced mRNA whereby the label should be protected. Intron sequences in the probe will not hybridize and loop out whereas the exons will be protected by the mRNA. After S1 digestion of the intron sequences, the remaining exon(s) can be analyzed on a denaturing gel. If only the 3’ or 5’-end is labeled, they will be the only ones observed. Internal labeling reveals all exons. It is also possible to use unlabeled DNA probes for S1 analysis (Favaloro et al., 1980). The hybrid formed between the probe (cloned genomic DNA) and the spliced mRNA is submitted to S1 digestion, either at 45°C or 20°C. At 45”C, introns are removed and the RNA digested at the exon/intron boundaries. The number of bands on neutral or alkaline gels both correspond to the number of exons. At 20”C, however, introns are efficiently removed while RNA is less efficiently digested so that on neutral gels a single band (size of mRNA) and on alkaline gels a number of bands corresponding to the number of exons is obtained. Recent developments in PCR make it the preferred method to determine exon/intron boundaries (Bergeron et al., 1993). Finally, mRNA is readily quantified by S1 analysis. Preferably, long oligomers are added in excess to the mRNA preparation, digested and the amount of protected DNA is determined.

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Instead of S1 nuclease, mung bean nuclease (cleaner results with the same enzyme activities but much more expensive) or exonuclease VII can be used. Exonuclease VII is interesting since it requires a free 3'- or 5'-end for digestion and introns will be resistant. However, exonuclease VII is useful in combination with S1 nuclease, particularly for short introns. 12.3.2. SI analysis methods 12.3.2.1. Preparation of DNA probes for S l analysis Methods to obtain ss DNA labeled at either the 5'- or the 3'-end have been described in Section 7.7. Particularly the methods based on PCR are useful. Submitting one of the primers used for amplification to kinasing with [ y-32P]dNTPrenders the strand containing this 5 '-phosphate group resistant to lambda exonuclease and only these specific strands will survive digestion. The 3 '-ends are labeled, after specific restriction enzymes have produced a protruding 5'-end, by a fill-in reaction that allows only one label to be introduced (e.g., labeling with dATP with an overhang with 2 Ts should be avoided). Reverse transcriptase or Sequenase are the preferred enzymes for this fill-in although the Klenow fragment has been widely used. The 3' + 5' exonuclease activity of Klenow, however, may remove the protruding template end. A widely used method for preparing ss DNA is by extension of kinased primer on a M13 clone. Details of kinasing of oligomers are given in Section 7.7.1. Kinase enzyme is then heat-inactivated for 5 min at 65°C and 20 pg of the M13 clone and 100 ng of the oligomer are hybridized for 15 min in 50 pl of 15 mM Tris-HC1 (pH 8.0) and 15 mM MgCl, after which 25 pl, containing 1.2 mM of each dNTP and 10 units of Klenow fragment, is added and the mixture incubated for 30 min at 37°C. Klenow is then heat-inactivated (5 min 65°C). The mixture is placed on ice before restriction to obtain uniform 3'-ends. The buffer in the sample is adjusted for the restriction enzyme to be used, 25 units of the appropriate enzyme is added and the sample incubated for 1 h at the temperature dictated by the enzyme. The DNA is ethanol-precipitated (Table 3.1) and then purified by gel electrophoresis, either on denaturing polyacrylamide

540

HYBRIDIZATION WITH NUCLEIC ACID PROBES

gels (5%) or alkaline low melting agarose gels (1.2%). Agarose gels are run at low voltage (1.5 V/cm), otherwise they will melt, for 5 h and the position of the ss probe is located as described in detail in Section 12.4.2.4. The probe is excised from the gel and the slice is heated to 65°C and an equal volume of TE buffer added. After an incubation for 10 min at 65"C, DNA is phenol-extracted (do not use chloroform!), 10 pg of tRNA are added and the nucleic acid is ethanol-precipitated. The probe is resuspended in 100 pl of 0.3 M NaOAc. 12.3.2.2. S l mapping of mRNA Shorter DNA probes will tolerate more degradation of target RNA, while detectability does not change due to the end-labeling, but the potential derived information is less. The amount of total target RNA required depends on the abundance of the specific RNA. A good starting point is 10 Fg, i.e., RNA from about 5 X lo5 cells, but pilot experiments, as shown for RNase protection assays (Section 12.4.2.2), are often necessary. Do not use more than 20 pg of RNA (then use poly(A)+RNA instead) since this may require large amounts of enzyme. RNA is ethanol-precipitated, the supernatant carefully removed with a drawn-out Pasteur pipette (rinsed in 1% SDS) and the pellet spun again for a few seconds to eliminate the last drop of liquid. The pellet should not be dried before adding 30 p1 containing 80% formamide and 20% of 2 M NaCl and 5 mM EDTA and 2 mM PIPES (pH 6.4) (prepared from disodium salt and pH adjusted with 1 N HCI). Radiolabeled probe is added to 0.01 pmol (= 1 ng of a 250-mer) of which a high proportion should have a label (kinased with [y-32P]ATPat 3000-7000 Ci/mmol). The sample is heat denatured for 5 min at 80°C and incubated overnight at 30°C. Note that the formamide concentration can often be lowered to 50% thus increasing the hybridization rate about five-fold. Formamide allows the T, to be lowered to prevent degradation of RNA and lower formamide concentrations may require higher incubation temperatures. For digestion, use S1 buffer (300 p1 containing 0.28 M NaCI, 50 mM NaOAc, pH 4.5, 4.5 mM ZnSO,, 20 pg/ml denatured carrier

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54 1

DNA) and 100 units of S1 nuclease and incubate for 60 min at 20 or 45°C. This nuclease requires Zn2+ ions and the reaction is stopped by adding 100 pl of 50 mM EDTA, 4 M NH,OH and 5 pg of tRNA, the nucleic acid is ethanol-precipitated. For mapping, the pellet is dissolved in 4 p1 of TE (pH 7.5) and then 6 ~1 of formamide loading buffer (80% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue and 10 mM EDTA) is added. The nucleic acid is heated for 5 min at 95°C and loaded on a denaturing polyacrylamide/urea gel (1 X TBE, 8 M urea and 5% polyacrylamide (depending on the size of the fragment, Table 9.2) while a sequence ladder is loaded as a reference. Keep 1/2 or 2/3 of the sample for additional runs.

12.3.2.3. Quantitative S l analysis of mRNA Long oligomers ( = 60-mers1, labeled by kinasing at the 5'-end are generally used. Again, the highest specific activity ([Y - ~ ~ P I A T P , 3000-7000 Ci/mmol) should be used for labeling of 2 pmol of an oligomer (corresponding to 40 ng of a 60-mer) according to the method in Section 7.7.1. After adding 10 pg of tRNA, labeled oligomer is ethanol-precipitated. Using 2 M NH,OAc as the precipitation salt reduces the co-precipitation of ATP. Probe is added to 3 M NaCl, 0.5 M HEPES (pH 7.5) and 1 mM EDTA to a final concentration of 3 pg or = lo4 cpm per FI ( = hybridization mix; stable for 4-6 weeks, although activity will then decrease four- to eight-fold and background will increase). In quantitative S1 analysis, it is essential that (i) excess probe is added to the target RNA, (ii) hybridization goes to completion, (iii) S1 nuclease is active (may have to be titrated) and (iv) hybrids are stable at the labeled end. In pilot experiments, the level at which probe is sufficiently in excess can be determined (i.e., when adding more probe does not increase the signal (in practice signal increases somewhat despite excess probe since unhybridized probe is often not completely digested) or when increasing target, with constant amounts of probe, the signal increases proportionally). Adjusting the hybridization temperature minimizes the formation of nonspecific duplices and optimizes the hybridization rate. Moreover, the hy-

542

HYBRIDIZATION WITH NUCLEIC ACID PROBES

bridization period should be long enough to reach completion: longer periods do not increase the signal. For hybridization, 20 pl of target RNA (or controls with known amounts of complementary RNA) is mixed with 10 pl of the hybridization mix. The nucleic acid is denatured for 10 min at 80°C and then incubated, e.g., overnight at 60°C, until completion of hybridization. For digestion of unhybridized sequences, the tube(s) is briefly centrifuged and 10 vols. of S1 buffer and S1 nuclease are added (Section 12.3.2.2) and incubated for 1 h at room temperature. Protected nucleic acid is precipitated and run on a denaturing polyacrylamide gel as described in the previous section. The amount of RNA can be determined by densitometry, in comparison to known amounts of RNA in the controls. This method is also suitable for simultaneous quantification of several mRNA species (O'Donovan et al., 1991). Different lengths of oligomers are used to the different mRNAs and an internal reference such as p-actin. It is necessary that these probes have the same specific activity (e.g., by kinasing) and the same hybridization rate and stability. 12.3.3. Critical parameters of S l analysis It is important that the residue containing the label hybridizes well with the target, otherwise it will be removed by S1 nuclease. It is useful to choose a residue in a well-conserved domain in a G/C rich area to decrease breathing of the heteroduplex and prevent digestion from this end. Breathing can also be decreased by using lower temperatures during the S1 digestion or increasing the salt concentration. It is also useful to choose target sequences which lack rU :dA stretches since perfect heteroduplexes of this type are known to be susceptible to S1 nuclease (Miller and Sollner-Webb, 1982). S1 nuclease may have to be titrated for optimal performance since activity of different lots may differ considerably. Controls should include a test for the integrity of target RNA (e.g., by electrophoresis in agarose gels to show the sharpness of the rRNA bands), S1 probe

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543

with control RNA (to test S1 nuclease activity), control RNA spiked with RNA complementary to the probe. S1 probes should also be designed so that at the unlabeled end they are not complementary to the target to allow protected probe to be distinguished from the original probe. For long oligomers, an internal probe should be synthesized (to one exon) with a noncomplementary 3'-end. This noncomplementary end should be short enough to prevent nonspecific annealing. Multigene families may complicate the interpretation since small mismatches are relatively resistant to S1 nuclease (higher concentrations can be used) or a single probe molecule may be protected by several related RNA molecules (Lopata et al., 1987), particularly if another abundant RNA species hybridizes to an adjacent region of each probe molecule. Results obtained with S1 mapping may not be free of artifacts and PCR amplification and sequencing around the suggested map positions is recommended for confirmation. Alternatively, a combination of RNase H and S1 nuclease analysis may circumvent the problem of poor S1 digestion at introns by hybridizing a DNA fragment to the intron, followed by RNase H treatment and then by S1 analysis (Sisodia et al., 1987). Leone et al. (1989) mapped the introns by specific digestion of exons by RNase H, Le., RNA in the DNA : RNA hybrid.

12.4. RNase protection assays RNase protection assays (RPA) are based on the property of RNase to digest ss RNA, but not ds RNA, and its principles and applications resemble those of S1 analysis (Lynn et al., 1983; Zinn et al., 1983) (Fig. 12.3). The sequence of interest is inserted in a plasmid downstream of a bacteriophage promoter (e.g., pUCll8, pT7, etc., Table 4.4). The purified plasmid is then restricted downstream of the inserted DNA and the linearized plasmid is transcribed in the presence of a labeled rNTP precursor. The transcript should be complementary to the RNA to be studied and an excess of probe is hybridized to its target. Any RNA remaining ss is then digested by one or more RNases. The size of the RNase-resistant probe and the

544

HYBRIDIZATION WITH NUCLEIC ACID PROBES

amount of label is indicative of the size and amount of the target. Several kits (e.g., Ambion) are on the market.

12.4.1. Advantages of RNase protection assays over S1 analysis Melton et al. (1984) observed that RPA allows the detection of as little as 0.1 pg of mRNA, which is at least ten times better than S1 analysis. Moreover, RPA are easier and more reliable. Low abundance mRNA are more readily detected than with Northern blotting and quantitation is more accurate. Protected RNA is fractionated by polyacrylamide gel electrophoresis which allows, due to its high resolution, the mapping of the 5’ and 3’-ends of the transcripts or the exon/intron boundaries (Calzone et al., 1987; Kekule et al., 1990) and even small differences between probe and target, e.g., after mutations, by adjusting the RNase concentrations (Genovese et al., 1989; Takahashi et al., 1989). RPA are most reliable for relatively short probes (200-400 bases): the longer the probes, the stronger the need for intact mRNA. In contrast to S1 analysis, gel purification of the probe is not required, although recommended, after runoff synthesis. Another important advantage of RPA is that probes are internally labeled so that the specific activity of RNA probes is determined by specific activity of precursors and not by the activity of kinase (as in common S1 probes) although internally labeled DNA probes have also been used in S1 analysis (Ley et al., 1982). Internally labeled probes have a much higher specific activity than end-labeled probes and improve the detectability considerably. Finally, RNases are not as sensitive to reaction conditions as S1 nuclease and thus yield more reproducible results. RPA consist of four steps: (i) preparation and purification of RNA probes; (ii) hybridization of probe with target RNA; (iii) digestion of ss RNA; (iv) analysis of protected fragments or quantification of label remaining in probe. As for S1 analysis, it is important to maintain a probe excess to obtain pseudo-first-order kinetics while avoiding a large probe excess that would lead to excessive background.

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12.4.2. RNase protection assay methods 12.4.2.1. Preparation of full length RNA probes and purification Radiolabeled RNA probe is synthesized as described in Section 7.6.6 and its quality is critical for RPA. Full length transcripts are important and easier to obtain with smaller templates or higher concentrations of the labeled rNTP in the reaction mixture (Table 7.18). Sometimes, the use of another rNTP as labeled precursor can improve the proportion of full length transcripts. For very long transcripts (> 800 bases), cold rNTP can be added to 25 or even 50 pM while maintaining a good detectability. Moreover, Krieg (1990) observed that transcription at room temperature or even 4°C instead of the commonly used 37°C maximizes the proportion of full length transcripts. At 4"C, however, spermidine in $thereaction mixture may precipitate. It is useful (Section 12.4.3) to produce transcripts that include plasmid sequences so that they can be distinguished from protected probe. The success of RPA is also determined by the size of the target. The shorter the probe, the more degradation of target can be tolerated. Probes shorter than 150-200 bases will have less label incorporated (at 600 Ci/mmol, only 1 in 15 radiolabeled precursor nucleotides is labeled and thus, on average, 1 per 60 bases in the transcripts). Secondly, short probes are not as efficiently precipitated. Purification of probe is recommended for RPA. In contrast to other RNA probe applications, it is beneficial to remove template DNA by DNase I treatment and phenol/chloroform extraction to prevent spurious background bands. The removal of incomplete transcripts is also recommended since these would give a background smear below the full length probe. This is most easily achieved on a 0.8 mm thick denaturing polyacrylamide gel (8 M urea, 1 X TBE, 4% gel; sample in 1 X TBE, 80% formamide and 0.1% bromophenol blue) at 200 V until the bromophenol blue has run at least one-half of the gel (Section 9.1.3). The plates are separated and the gel on one of the plates is covered with Saran wrap and placed in the corner of a box. A n X-ray film (one corner bent) is placed on the gel against the edges of the box for future alignment for 1-5 min and

546

HYBRIDIZATION WITH NUCLEIC ACID PROBES

developed. The film is aligned again with the gel in the box, the film gel plate is carefully flipped over and the plate removed, and the full length RNA band (slowest migration) is cut out using the film as a template. RNA is eluted from the gel in 0.4 ml 0.5 M NH,OAc, 1 mM EDTA and 0.5% SDS at 37°C. After an overnight incubation, 10 pg of tRNA is added to the eluate and the RNA precipitated with 2.5 vols. of ethanol (Table 3.1). Counts in the gel slice, ethanol pellet and supernatant are monitored to ensure successful purification. RNA pellets are easily lost from polypropylene microcentrifuge tubes and supernatants are best removed with drawn-out Pasteur pipettes (rinse with 1% SDS prior to use to denature RNases).

12.4.2.2. Hybridization of RNA probe with target Particularly for quantitative determinations, it is important to have a molar excess of RNA probe. If we assume that total cellular RNA contains 3% mRNA and that the target mRNA is 1 kb long and abundant (e.g., O.l%), then for 10 pg of RNA and a five-fold molar excess of a 250-base probe about 375 pg of probe would be required. At a specific activity of 2 X lo8 cpm/pg, this corresponds to 7.5 X lo4 cpm. Pilot experiments allow the optimization of probe and input total cellular RNA. For this purpose, a constant amount of probe (e.g., 400 pg) is hybridized with variable amounts of target RNA (Table 12.2) as described below. Usually, 10 pg of target RNA is sufficient to detect a few copies per cell but for < 1 copy/cell, it may be necessary to increase total RNA up to a maximum of about 100-150 pg. Controls are important, both for background (e.g., with yeast tRNA) as well as for positive signals (spiking the sample with sense, cold RNA generated from plasmid or using another probe, e.g., to p-actin mRNA which has an abundance of 0.05%). In hybridization, a suitable volume of probe (e.g., 3 X lo5 cpm at a specific activity of lo9 cpm/pg; determined as in Table 7.15) and sample RNA suspensions or controls are mixed in freshly prepared hybridization buffer (80% formamide and 20% of buffer containing 0.2 M PIPES, pH 6.4, 2 M NaCl and 5 mM EDTA, PIPES is prepared by dissolving its disodium salt and adjusting the pH by adding 1 N HCl) in a microcentrifuge tube. Precipitate RNA with 100% ethanol, place tubes at - 20°C for 30 min and collect the pellet

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TABLE12.2 Optimization of RNase protection assays Purpose Adjustment of target RNA

Adjustment RNase conc.

No. 1 2 3 4 5 6 7 8

9 Control (negative) RNA Control (positive) RNA Control (probe) RNA System control * *

10 11 12 13 14 15

Amount of sample (pg) *

RNase mixture

*

Standard Standard Standard Standard 0.5 X standard 2 X standard 4 X standard 10 x standard 1X standard (only T1) 10 (yeast tRNA) 1Xstandard 10 (yeast tRNA) 10 X standard 10 (spiked sample) 1X standard 10 1X standard 10 1 x standard 10 No RNase

1 3.16 10 31.6 10 10 10 10 10

* A constant amount of probe ( = 300 bases) is added, e.g., 3 X lo5 at lo9 cpm/pg; for other specific activities or probe lengths, the amount should be adjusted to maintain the same molarity. Standard RNase is as in Section 12.4.2.3. * * For instance, p-actin probe. by centrifugation for 15 min at 14000 X g at 4°C. Carefully remove the supernatant, spin again for a few seconds and remove the remaining liquid. Add 20 ~1 of hybridization buffer to the pellet, vortex and heat-denature for 5 min at 85"C, rapidly revortex and spin for a few seconds. Incubate the samples overnight at 45°C (sometimes lower or higher temperatures are required: 30-75°C). 12.4.2.3. Digestion of unhybridized target and probe RNA Any RNA remaining ss after hybridization is digested by adding 10 vols. of the RNase digestion mixture (2 pg/ml RNase T1,40 pg/ml RNase A in 10 mM Tris-HC1, pH 7.4, 0.3 M NaCl and 5 mM EDTA) and incubate for 60 min at room temperature. The optimum

548

HYBRIDUATION WITH NUCLEIC ACID PROBES

RNase concentrations depend on the nature of target/probe: (i) how suitable are they as substrate; (ii) do small differences have to be detected or should digestion at mismatch positions be minimized; (iii) what is the specific activity of the RNases; (iv) how strong is the background. It may also be necessary to change the proportion of RNase A and T1. RNase A cleaves 3’ to C and U residues whereas T1 cleaves 3’ to G residues. If the duplex is A/U rich, transient partial strand separation may occur and the duplex would be locally digested by RNase A. This problem can be avoided by lowering the digestion temperature (e.g., 15OC) and using only RNase T1 if A/U regions lack G / C . After RNA digestion, it is necessary to digest the RNases by adding SDS to 0.5% and 100 p,g of proteinase K, incubate for 15 min at 37°C and extract with phenol/chloroform. Alternatively, RNases or proteinase K can be captured by Strataclean (after 2.5-fold dilution in water) or the ds RNA passed through an Immobilon filter (Section 3.1.2.7). The last method has the added benefit that ss probe RNA is also removed. RNA is then precipitated by adding 1 ml of ethanol as described in Table 3.1. 12.4.2.4. Analysis of protected RNA by polyacrylamide gel electrophoresis RNA runs about 5 1 0 % slower than DNA of the same length on a denaturing polyacrylamide/urea gel (also depends on other factors such as voltage). A 5% acrylamide gel, containing 1 X TBE and 8 M urea, is suitable for ss RNA of 50-1000 bases. Both protein and sequencing gel equipment are suitable. Best results are obtained when the gel polymerizes fairly rapidly (15-30 min) and the gel is maintained at room temperature for a few hours before use. The pellets of RNA samples obtained after ethanol precipitation are dissolved in 5-8 p1 of loading buffer (80% formamide, 0.1% xylene cyanol, 0.1% bromophenol blue and 2 mM EDTA). To completely dissolve and denature the RNA, it is necessary to heat the tubes for 5 min at 90°C. After spinning for a few seconds, the samples are loaded on the gel (smaller amounts should be loaded of samples that can be expected to have stronger signals). About 30 V/cm (over the gel length) is usually sufficient and gels are run until

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the bromophenol blue nears the bottom. Gels are exposed as described in Section 7.2.5. 12.4.3. Simultaneous RNA extraction and RNase protection assays

Firestein et al. (1987) described a method for molecular hybridization of RNA probes with target from unfractionated cells solubilized in GuSCN (Thompson and Gillespie, 1987). Cells are harvested and rinsed once with PBS containing 20 pg/ml cycloheximide. The cells are then dissolved in 5 M GuSCN and 5 mM EDTA (pH 7.0), to a final concentration of 10’ ceIls/mI. Aliquots are frozen at - 20°C for future use. Probe (2.5 pl, usually 1 ng) is mixed with 10 pl of solubilized cells, heated for 5 min at 60°C and incubated for 18 h at room temperature. The hybridization mixture is then diluted with 300 p1 of the RNase mix (only RNase A) and incubated for 45 min at room temperature. Proteinase K is added to 200 pg/ml and the samples are incubated for 15 min at 37°C. The digest is extracted with phenol/chloroform and the RNA is precipitated in the presence of glycogen (Table 3.1). Protected probe is then analyzed on a denaturing polyacrylamide gel. 12.4.4. Interpretation, critical parameters and troubleshooting in RNase protection assays The high sensitivity of RPA also makes it susceptible to background problems. A major cause can be transcription: (i) a high proportion of less than full length transcripts (gel purification recommended; Section 12.4.2.1); (ii) degradation of target RNA (smearing of rRNA bands during electrophoresis (Section 9.1.3) indicates degradation) the impact of which can be reduced by using shorter probes; (iii) less than optimal transcription conditions (Section 12.4.2.1); (iv) transcription of sense RNA, particularly by promoter-independent transcription from 3’-overhangs (Section 7.6.6) which can be remedied by using other restriction enzymes or fill-in reactions after restriction; (v) ‘crosstalk‘ with unrelated promoter downstream of insertion (particularly among T3/T7, Section 7.6.6) in undigested template. Another important cause of background is the failure to remove template DNA completely. DNA that is not removed may also

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protect RNA. It is useful to have probes which have also plasmid sequences unrelated to the target RNA (Section 12.4.2.1) so that it is possible to pinpoint the presence of protecting DNA or incomplete RNA digestion. Incomplete DNA digestion will also be suspected when the control target RNA is capable of protecting probe. Probes with considerable secondary structures may also yield protected fragments, which would be usually smaller than the expected fragment and be present in all lanes with probe, including controls. Sometimes, radioactive material remains on top of the gel. This problem is less important with gel-purified probe. The cause of this effect is unknown but a change of brand of microcentrifuge tubes may help. Unexpected results may be due to the target RNA being a member of a multigene family, to allelic differences among targets or the presence of RNA processing intermediates. In the first two cases, a modification of the RNase concentration may be helpful. Since RNA does not migrate exactly as DNA, the use of DNA markers is not advised for mapping of mRNA with respect to exon/intron boundaries and 5’- or 3’-ends if a high degree of resolution is required. It is possible, however, to prepare RNA by in vitro transcription, as above, from inserts of different lengths.

12.5. Triple helices Although triple helices have been known for about 35 years, only recently have they received considerable interest due to improved tools. Natural processes may involve triplexes (Wells et al., 1988; Rao et al., 1991) (Section 2.4), but triplex formation can also be used as experimental tools, e.g., to target specific sequences for transcriptional inactivation (Maher et al., 1990), restriction enzyme or methylase protection (Frangois et al., 1989) and DNA isolation (Ito et al., 1992). There are two basic types of triplex (Section 2.4 and Fig. 12.4 (I)) in which the homopyrimidine or homopurine oligomers bind in the major groove of the duplex DNA. The orientation of the homopurine oligomer is opposite that of the homopyrimidine oligomer and can be

9 I

E’Ig

k’-dn

p’:?

3’-Y

FTTTTFTTTFFTFFT -ACCAAAACGAACGAGGAAG-

11

c.L

R,RRRRRRR,Y,YYYYYYY,--

5’3.-

N

3’ 5’

--TCCTTTTCCTTCCTCCTTC-

3’-R8RRR

F????FF??FF?FF?

n

5’ 7

-AGGAAAACGAAGCACCAACYCCTTTTCCTTCCTCCTTC-

3’ -YiYYY /, CTTTTC

Iv I11

A

B

C

-TACTCTTC

W

I

L noX, noY

G

A

T

C

26.0 17.5 11.0 15.5 21.0 23.5 16.5 20.5 18.0 23.0 22.5 29.0 26.0 @ 25.0 21.0 21.0 23.0 28.5

Fig. 12.4. Triple helix formation. Two basic types and a combination of these can be distinguished (I). Triple helices may occur naturally in a duplex (11) (Wells et at., 1988). One of the applications of triple helices is the protection of a particular site against a restriction enzyme (111). Mismatches are allowed to a certain degree (IV;T , rounded to 0.5”C; data from Mergny et al., 1991). m c m

552

HYBRIDIZATION WITH NUCLEIC ACID PROBES

explained by the hydrogen bonding (Fig. 2.7). It can be seen in this figure that the pyrimidines in the pyrimidine oligomer are the mirror image of those in the duplex and thus have opposite orientation (pyrimidine oligomer parallel with the purine tract in the duplex). The purines in the purine oligomer are the mirror image of the purines in the duplex (purine oligomer antiparallel with the purine tract in duplex) (Pilch et al., 1991). Intrastrand switching (Y RY),(YR * R), (Fig 12.4 (I)) is also possible without any special linkage between the oligopurine and oligopyrimidineblocks (Jayasena and Johnston, 1992) and relaxes the requirement of long oligopurine tracts. Moreover, some mismatch (Mergny et al., 1991) or G - T A base triple (Radhakrishnan et al., 1991) formations are possible (Fig. 12.4). Polyvalent cations, e.g., Mg2+, and some organic solvents may stabilize triplexes (Moser and Dervan, 1987; Jayasena and Johnston, 1992).

12.5.1. Sequence-specific DNA purification by triplex affinity capture Figure 12.5 illustrates schematically the triplex-mediated capture procedure for the purification of specific ds DNA as described by Ito et al. (1992). A biotinylated pyrimidine oligomer is incubated under acidic conditions (pH 4.5-5.01, since the cytosines should be protonated, with a DNA mixture containing the target. The triplex is then captured with paramagnetic beads coated with streptavidin, the beads washed several times and the retained duplex eluted. The stringency of conditions can be easily controlled by a change in pH. At pH 4.5, short T/C stretches may form triplexes but increasing the pH to 5.5-6.0 considerably improves the specificity. A complication in this pH range is the p l of streptavidin 6-61 so that at low pH streptavidin may interact with nonspecific DNA. Ito et al. (1992) observed that adding NaCl to 2 M reduces this background and stabilizes specific triplexes. Alternatively, the capture oligomer may be directly linked to paramagnetic beads as shown in Fig. 8.1 and so avoid both biotinylation and streptavidin-related background. In detail, DNA and the biotinylated oligomer (20 pmol) are incubated in 50 p1 of a buffer containing 2 M NaCl and 0.2 M NaOAc (pH 4.5-5.5, determined at room temperature) at 50°C for 2

Ch. 12

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I

Triplex formation

2biotinylated

~

(>

553

oligo

target sequence

DNA streptavidin-coated Paramagnetic bead

f

Elution

Fig. 12.5. Triplex affinity capture method. The triplex is formed in slightly acidic conditions (I). The complex is captured on paramagnetic beads and washed (11) and the duplex eluted with a mild alkaline buffer.

h. Streptavidin-coated paramagnetic beads (50 11.1) are washed in the same buffer and added to the DNA mixture. After an incubation of 1 h, the beads are separated from the buffer by a magnetic concentrator and washed eight times with 0.5 ml of the same buffer. The DNA is eluted from the beads by an incubation with 1.0 M Tris-HCl (pH 9.0) and 0.5 mM EDTA.

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This method is particularly promising for the isolation of large

DNA fragments where conventional methods lead to an often intolerable degree of DNA strand breakage. Homopurine oligomers seem also applicable in this approach since the stringency of the triplex reaction can also be tuned by changing the incubation temperature. A restriction is the apparent need for a homopurine-homopyrimidine target tract. However, as seen above, alternate strand triplexhelix formation (Fig. 12.4), permissive mismatches (or base analogs) and the use of recombinase proteins (Rao et al., 1991) could extend the applicability of this method. 12.5.2. Inhibition of DNA restriction at specific sites via triplex formation

Franpis et al. (1989) designed a 17-mer homopyrimidine oligomer to recognize, via Hoogsteen pairing, a 17-bp homopurine-homopyrimidine sequence. Inhibition of restriction in this sequence could be obtained in the micromolar range (Fig. 12.4 (111)). In detail, 6 nM DNA was incubated for 20 min at 37°C with the restriction enzyme (0.08 units/pl) and a varying concentration of oligomer in the restriction buffer (but at pH 6.5 to maintain the cytosine protonated). Partial protection was obtained by adding 10 JLMoligomer, while at 100 JLMprotection was virtually complete (50% protection at 20 pM).Protection was poor at pH 7.9. The use of purine oligomers should relax the requirement of acidic conditions.

12.6. Current developments and prospects Hybridization and PCR are among the most important techniques in molecular biology and continue to be fine-tuned for various applications. In this section, some of the spectacular advances in this area are reviewed. 12.6.1. Screening of genetic diseases

In the clinical laboratory, PCR in combination with hybridization proves to have significant utility. Most genetic diseases do not result

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555

from a single invariant mutation but from many different mutations (deletions as in muscular dystrophy, point mutations as in cystic fibrosis, amplifications as in the fragile X syndrome, chromosomal translocations, such as the Philadelphia chromosome). Amplifications yielding triplet repeat mutations in specific regions of genes (e.g., 5’ untranslated region: fragile X mental retardation; 3’ untranslated region: myotonic dystrophy; or in protein coding sequence: spinal and bulbar muscular atrophy) due to unfaithful polymerization in CG-rich areas lead to hereditary unstable DNA (Caskey et al., 1992; Harley et al., 1992; Mahadevan et al., 1992). PCR (e.g., multiplex to amplify multiple exons) combined with reverse dot blot hybridization allows for the simultaneous detection of multiple alleles. A similar approach has been developed to aid cytologic screening of cancer (Sridansky et al., 1991). A portion of the p53 suppressor gene is first amplified through PCR and then cloned into a lambda phage. Hybridization with mutant specific oligomers is performed on recombinant phages immobilized onto nylon membranes. This method allows one mutant cell to be detected among an excess of > 10000 normal cells.

12.6.2. Peptide nucleic acid chimerae Peptide nucleic acid (PNA) chimerae have been constructed in which the deoxyribose-phosphate backbone in the oligomer has been replaced by a peptide backbone. This peptide backbone is composed of (2-aminoethy1)glycine units and is structurally homomorphous with DNA (Nielsen et al., 1991; Egholm et al., 1992). The glycine spacing is optimal for hybridization of a PNA to a target nucleic acid whereas alanine substitutes would decrease the stability. PNA binds also to ds DNA but do not form triple helices since PNA causes a strand displacement (local structure: PNA :complementary strand from duplex and ss noncomplementary strand from duplex) although initially an unstable triplex could have been formed. There is also evidence that two PNA strands can be bound to DNA in the strand displacement complex ((PNA),/DNA triplex by Watson-Crick and Hoogsteen pairing; P.E. Nielsen, personal communication). Binding of PNA to DNA is inefficient at an ionic strength exceeding 0.05 and

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1 mismatch (among 10 units). At lower salt concentrations, PNA :DNA duplices are very stable (much more than DNA :DNA). Short PNAs (e.g., 8 units) may block restriction enzyme recognition sites and block transcription or primer extension. PNA :RNA complex is not a substrate for RNase H. Currently, PNA seems restricted to homopyrimidine PNA and appears not to be taken up by pro- or eukaryotic cells. PNAs are, nevertheless, interesting potential candidates for gene therapeutics and probes.

12.6.3. Matrix array hybridization on silicon wafers (‘genosensors on chips’) Large arrays of probes can be immobolized on small silicon wafers (chips) by means of photolithography. The silicon surface is treated with a silane reagent which bears photolabile protecting groups. Photolysis by successive photolithographic demasking of arrays (selective deprotecting, similar to techniques used in the semiconductor industry) and reaction with deoxynucleotide phosphoramidites, which are also protected with photolabile groups, allows the synthesis of particular probes on specific locations on the chip (Beattie and Fowler, 1991). Using 50 pm X 50 pm arrays and octamer probes, 4’ or 65 536 probes would cover a 1.28 cm X 1.28 cm wafer and require 32 chemical steps in the synthesis. Decreasing the sample locations to 10 pm squares would increase the number of probes 25-fold but the decrease possible is limited by the resolution of the signal. This technology is being developed, among others at laboratories in Oxford (Southern), Palo Alto (Affymetrk Sheldon and Fodor), Oak Ridge National Laboratory (Foote), Moscow (Mirzabekov) .and Houston (Beattie). Different methods for the detection of hybridized probes have been chosen such as confocal fluorescent microscopes, electronic detection of hybridization (dielectric dispersion arising from negatively charged phosphate groups from the hybridized target DNA). Other methods included Betascopcts imaging systems to quantitate 32P-labeled DNA targets or the use of CCD arrays with pixel elements corresponding to different glass-tethered DNA probes.

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Subject index * AAF antibodies, 371 details labeling, 371 extinction coefficient, 371 structure, 288 AAIF structure, 288 Abundance and detectability on Northern blots, 450 mRNA and colony hybridization, 481 mRNA and detection with RPA, 544 Accelerators characteristics, 406 DNA-binding proteins, 407 use in hybridization, 406 Accelerators (see also dextran sulfate and PEG) Accessibility target in ISH, 511 Accuracy definitions, 3, 9 graphic representation, 11 relationship to test parameters, 10 Acid GuSCN/phenol/chloroform (AGPC) see isolation RNA

*

Acridine orange details of RNA staining, 469 effect on hybridization, 468 Acridinium ester half-life, 292 labeling to probe, 293 structure, 290 Adjacent sequences to target PCR, 200 Affinity bonus, 419 vs avidity, 41 9 Affinity columns use in hybridization, 386 Affinity matrix sandwich assay, 418-419 Agarose commercial, 443 quality, 443 Agarose gel see electrophoresis Agarose see electrophoresis Aggregates DNA and restriction, 454 E. coli, 134 RNA, 531

Page numbers in roman type refer to Part I and page numbers in italic refer to Part 11.

576

HYBRIDIZATION WITH NUCLEIC ACID PROBES

AGPC see isolation RNA Alkaline phosphatase see APase Alternating-angle gel electrophoresis see CHEF, TAFE Alu sequences, 532 Amplification use of, 167 Amplification systems comparison, 168 laboratory setup, 169 merits and disadvantages, 170 Amplification systems (see also PCR, IAS, LCR, TAS, Qp-RS) Amplifiers, 318 Annealing calculation of rate, 40 graphic representation steps, 43 rate in reassociation kinetics, 49 Annealing constant criterion RNA-DNA hybrids, 45 effect criterion, 44 effect solvent factors, 44 factors affecting, 42 length and complexicity nucleic acid, 47 mathematical equation, 48 nucleation factors, 42 pseudo-first order, 42 second-order, 42 theory, 243 Annealing constant (see also nucleation rate) Antibiotics inactivation, 131 resistance to, 131 Antibodies background and ionic strength in wash, 410 comparison with Fab fragments, 408 conjugation to APase, 322 conjugation to POase, 323

detection haptens, 328 preparation, 325 purification, 322 to haptens, 325 to nucleic acid, 325 Antisera purification IgG, 322 APase action, 302 activation and inhibition, 303 bioluminescence, 289 chemiluminescence, 290 chromogenic detection (see also BCIP/NBT), 305 chromogenic detection (see also naphthol AS), 307 conjugation, 322 dephosphorylation of nucleic acid, 354 dual enzyme amplification, 309 dual enzyme cascade system, 303 in oligonucleotide ligation assay, 213 inhibition of endogenous, 287 polymerization, 299 properties, 302 purification, 302 Archaeological specimens isolation DNA, 119 Assay parameters, 4 Assay parameters definitions, 3 Autoradiograms, intensification, 283 Autoradiography graphic representation, 282 intensifying screens, 284 latent image, 280-283 nuclear track emulsion, 284-285 preflashing, 281, 283 principles, 280 problems that may occur, 411 resolution, 280 technical details, 284 Avidin coated to paramagnetic beads, 383

SUBJECT INDEX

succinylation to decrease background, 383 Avidin (see also streptavidin) Avidity binder probe, 421 nucleic acids, 1 vs affinity, 419 Azidophenyl structure and reaction, 326

Background acetic anhydride treatment to reduce, 515 alternatives to biotin, 325 and density of bacterial colonies, 490 antibodies and ionic strength in wash, 410 avidin and succinylation, 383 biotinylated probes, 324 clinical samples, 41 1 colony hybridization and biotin probes, 490 colony hybridization, identification, 492 due to avidin, reduction, 500 due to dextran sulfate, 405 due to dextran sulfate or PEG in TRF, 297 due to endogenous APase, 287 due to endogenous POase, 289 due to positive chemography, 524 due to primers, in PCR, 185 due to RNA in ISH, 511 due to silver enhancement, 287, 321 due to tailed homopolymers, 356 from RNA in ISH, 496 in ISH and length of probe, 499 in LCR, 169 in plaque hybridization, 486 in S1 analysis, 538 LCR, 214 noise signal, 5 of conjugates, 321 on nylon membranes, 305, 462

571

reduction in capture assays, 423 reduction in ISH, 499 reduction with nucleases, 516 Background signals, 6 Background vs signal see labels Bacteria antibodies in polyclonal antisera, 490 isolation DNA, 96-98 isolation RNA, 107-108 lysis, 96 lysis and binding DNA to membranes, 480 spreading on membranes, 479 BAHS composition, 399 not recommended for RNA, 400 on nitrocellulose, 400 on nylon, 400 Baking immobilization nucleic acid, 393 optimum conditions, 397 Base stacking nature, 27 sequence dependency, 28 Base-pairing, 2 Base-pairing mismatches, 29 Base-stacking, 2 Basic aqueous hybridization solution see BAHS Basic formamide hybridization solution see BFHS Bayes’ Theorem, 8 BCIP/NBT details of staining, 307 representation of reaction, 306 use for APase, 305 BCR applications, 215 strand displacement assays, 215 variants, 215 Becquerel see radioactivity units

578

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Benzoquinone use in crosslinking, 369 Betascope, 280, 556 BFHS composition, 399 on nitrocellulose, 400 on nylon, 400 Bias see systematic errors Biological probes detectability, 373 use of phenotypic marker, 372 Bioluminescence see APase Biopsies isolation of DNA, 102, 116 Biotin chemical incorporation in oligomers, 23 1 Biotin labeling transaminated DNA, 360-361 Biotinylated probes detection, 411 purification, 335 use in hybrid selection, 535 Biotinylation inactivation APase, 324 of proteins, details, 324 Biotin-probes problems in colony hybridization, 490 Biotin/streptavidin as recognition system, 321 BIO-7-NHS structure and reaction, 326 BIO-nucleotides as substrate for TdTase, 359 incorporation by Klenow, 345 incorporation by T4 polymerase, 345 use in transcription in vitro, 352 Blocking reagents for membranes, 388-391 for membranes, formulations, 390 optimization, 404 recommendations, 399

Blood isolation DNA from blood cells, 102, 116 Blood cells rapid isolation RNA, 118 Blotting amount of nucleic acid, 395 insect squashing, 397 of supercoiled DNA, details, 396 on diazotized transfer media, 398 on nitrocellulose, details, 394 on nylon, details, 394 on PVDF, details, 394 RNA, details, 395 transfer buffers, 396 BLOlTO blocking reagent, 389 formulation and preparation, 399 inhibition streptavidin-biotin reaction, 410 BM blocking reagent use, 389 Bq see Becquerel Branch capture assays (see also BCR) principles, 435 Buffers (see also electrophoresis buffers) PBS, 80 TE, 79 TEN, 79 Cot,532

Cot curves in reassociation kinetics, 50 Cot value and background, 271 Cot-1 DNA reduction background, 74, 532 Capacity of membranes, 379 Capillary transfer bidirectional, 456 characteristics, 453 comparison salts for transfer, 461 conventional, 456

SUBJECT INDEX

details downward (alkaline), 459 details of bidirectional, 459 details of upward transfer, 458 details (alkaline to nylon), 460 details (alkaline, polyacryla, 460 details (neutral to nylon), 460 graphic representation, 457 large fragments, 454 small fragments, 454, 462 unidirectional, 461 Capilllary transfer downward, 456 Capture assays binder probes, 421 capture probes, 420 detectability, 420 direct and indirect probes, 421 microtiter plate (details), 420 optimization, 421 overview, 419 reverse target, 421 reverse target (graphic representation), 422 successive or simultaneous, 421 ternary complexes, 423 use in hybrid selection, 535 Capture hybridization comparison to sandwich assay, 418 with PCR, 177 Carrier DNA formulation and preparation, 390 use in hybridization, 403 Carrier protein choice, 327 to obtain hapten antibodies, 327 Carryover contamination see decontamination Casein for background reduction, 405 Caution ethidium bromide, 81 postamplification, 171 presence infectious material in samples, 103 pre-amplification handling, 170

579

radioactivity, 286 use microwave, 485 CCD camera luminescence quantification, 319 cDNA 1st and 2nd strand synthesis, 158 analysis of quality, 159 cloning, 157 commercial kits, 156 creation blunt ends, 159 first-strand synthesis, 157 full-length RNA for optimal cDNA, 156 liberation RNases during RNA extraction, 156 ligation linkers or adaptors, 161 methylation, 161 procedures, 158 random-primer synthesis, 157 representation of messages in libraries, 476 S1 digestion, 157 strategies for production, 156 subtractive hybridzation, 529 tailing, 160 use adaptors and linkers, 160-161 cDNA probes characteristics, 272 Cellophane membrane use in hybridization, 386 Centricon microconcentrators characteristics, 91 concentrators, 470 elimination of linkers, 165 PCR product purification, 197 postamplification, 171 Centricon-30 microconcentrators, 531 Centrifugal transfer, 463 Cetyl-trimethylammonium bromide see CTAB CG dinucleotide underrepresentation, 25 Chaotropic salts effect on T,,, and annealing, 46 Chargaffs rules, 12

580

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Charged nylon (disladvantages, 380 Charon handling, 151 properties, 146 Charon (see also lambda vectors) CHEF requirements, 445 Chemical labeling overview, 361 radioactive labels, 369 Chemical labeling see labeling Chemicals used in DNA synthesis, 228 Chemiluminescence instead of APase amplification, 310 principles, 289 substrates, 290 systems, 290 Chemiluminescent substrates properties, 316 reaction scheme, 315 use, 314 Chemography see ISH Chi sites see lambda vectors Chloroform see solvents Chloro-naphthol (CN), 310 Circular DNA electrophoretic mobility, 441 Circularization by ligation and length of DNA, 201 Ci see Curie Clones definition, 3 Cloning rDNA, 121 Cloning inhibition, 526 CN

basic protocol, 313 enhancement with heating or W, 313 with phenylenediamine enhancers, 313 Codon bias in different organisms, 226-227 in different tissues, 227 in gene families, 223 Codon degeneracy designing oligomers, 223 number oligomers required, 223 Colony hybridization, 15 Colony hybridization animal cells, 483 bacterial lysis, 480 characteristics, 475 details lower eukaryotyes, 482 distribution of colonies, 478 essential steps, 475 few colonies, 479 higher eukaryotes, 483 hybridization, 488 large libraries, 478 lower eukaryotes, 482 many colonies grown on agarose, 480 many colonies grown on membranes, 479 master plates, 478 membranes, 477 microwave methods, 484 negative controls, 477 nonradioactive probes, 490 oligomer probe problems, 492 on filter paper, 484 positive controls, 477 preparation replicas, 478 radioactive probes, 489 selection and picking cloning, 492 small Libraries, 478 spreading bacteria, 479 tissue printing, 483 use of DIG probes, 491 useful modifications, 484

SUBJECT INDEX

Compatible sites for ligation, 163 Competent E. coli cells preparation, 163 transformation, 164 Complexity and suppression hybridization, 525 RNA, 531 Complexity of DNA, 14 Complexity see probes Concentration error in estimate, 7 precision, 10 Concentration of target definition, 3 Concentrations nonradioactive and radioprobes, 405 Contact printing, 480 Contour-clamped homogenous field see CHEF Cosmid clones, 532 Cosmid probes in ISH, 509 Cosmid vectors cos sites, 151 flow chart of cloning, 145 packaging, 151 stability, 153 structure, 151 Cosmids vectors, 136 Cot-1 DNA, 74, 532 Counterstaining fluorescent (DABCO, DAPI), 516 for cellular outlines in ISH, 515 hematoxylin/eosin (adult), 516 Hoechst fluorescence (nuclei), 516 toluidine blue (embryo tissue), 516 Coupling ratio hapten to carrier protein, 325 CovaLink NH microtiter plates DNA immobilization, 385

581

CPG underrepresentation, 70, 223 Criterion effect on annealing rate, 44 Crosslinking see psoralen Crosslinking graphic representation, 368 Cross-contamination in PCR, 198 Cross-talk between T3 and T7 promoters, 349 CsCl gradients separation DNA and RNA, 119 CsTFA use for DNA purification, 84 use in isopycnic ultracentrifugation, 82 (=TAB DNA isolation from fungal spores, 101 DNA purification methods, 82 DNA purification, flow diagram, 83 for chemiluminescence enhancement, 318 isolation DNA from bacteria, 95 plasmid purification, 139, 141 properties, 82 stock solution, 82 Cultured cells preparation for ISH, 503 Curie see radioactivity units Cytological procedures see ISH

DAB enhancement by imidazole, 312 nickel enhancement, 312 substrate for POase, 312 DAB see also diaminobenzidine Data management, 11

582

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Deamination during transamination, 362 Decomplexing mercury ions, 370 Decomposition dioxetanes, 315 Decontamination amplicons and template, 172 Denaturation, 2, 3, 26 Denaturation DNA, 396 probes, in microwave, 404 Denaturation rate, 26 Denhardt’s solution formulation, 390 glycine instead of BSA, 403 use, 389 Densitometers, 387 DEPC action, 93, 452 effects on mRNA, 94 Dephosphorylation DNA, procedures, 162 lambda DNA, 149 Deprotection oligomers, 230 Depurination for fragmentation of DNA, 454 Detectability, 2-3, 6, 15 Detectability 3’ end labeling compared to 5’ end, 329 AAF-labeled probes, 371 capture assays, 420 definition, 4 fluorochrome labels, 293 graphic representation, 7 in microtiter plates, 385 labels, 271 of conjugates, 321 of protein A, 321 on nylon membranes, 462 requirements and labeling, 276 sandwich assays, 418 Southern vs Northern blotting, 450 vs sensitivity, 295, 432

Detectability index of negatives DN-), 5 Detectability index of positives DI( + ), 5 Detectability indices calculation, 10 Detectability of negatives calculation, 9 Detectability of positives calculation, 9 Detection enzymes, 305 Detection limit see detectability Detection of radioactive probes, 280 Dextran sulfate and background, 405 comparison to PEG 6000, 407 formulation and preparation, 390 properties as accelerator, 406 use in hybridization, 405 Diaminobenzidine (DAB), 310 Diazotized paper blotting, 398 Dichlorotriazinylaminofluorescein see DTAF Diethylpyrocarbonate see DEPC Diffusion constant substrate, 301 Diffusion rate effect on reassociation kinetics, 59 DIG probes detection, 408 plaque hybridization, 488 DIG-probes end-labeled, 359 nick translation, 335 DIG-UTP use in transcription in vitro, 352 Dimerization pyrimidines during Hg decomplexing, 371 Dimethylsulfoxide see DMSO

SUBJECT INDEX

Dioxetanes aggregation, 318 AMPGD, 318 amplifiers, 316 AMPPD, 316 decomposition, 315 half-life, 314 properties, 316 substrates, 314 Discriminability comparison with specificity, 5 definition, 4 graphic representation, 7 Discriminatory power probes membrane hybridization kinetics, 62 DNA amount required signal in blots, 454 clean-up with DNA capture reagent (DCR), 85 clean-up with Immobilon-P, 85 clean-up with Strataclean, 85 denaturation, 396 ethanol-precipitation, 81, 88 ethanol-precipitation (procedure), 90 extinction coefficient, 396 extraction from cells, 393 isolation from mitochondria, 105 PEG-precipitation, 89 purification see isolation DNA spermine-precipitation, 89 supercoiled, blotting, 396 treatment with acid, 221 DNA (see also nucleic acid) DNA concentration impact on hybridization, 5 DNase I stock solutions, 330 DNase-free RNase, preparation, 87 Driver DNA biotinylated, 528 immobilized, 526 PCR synthesis, 531 DTAF labeling details, 296

583

DTAF, alternative to FITC, 296 Duplex antiparallel, 21 axis of the helix, 27 B DNA, 27 base-content-dependent stability, 26 concentration-dependent renaturation, 26 concentration-independent renaturation, 26 double-helix stability, 27 duplex-retention temperature, 26 mismatches and stability, 28 parallel, 21 physical nature stability, 27 retention temperature, 38, 40 retention temperature, example, 41 sequence-dependent stability, 26 solvent factors and stability, 28 stability, 26, 29 stability and mismatches, 30 stability from Arrhenius plots, 29 stability imperfect duplex, 26 structure, 27 thermal stability see stability Duplex DNA major groove, 552

EcoK hsdR subunit, 126 hsdR testing, 126 hsdS subunit, 126 hsdS testing, 126 EcoKm hsdM gene, 126 Efficiency of labeling, 275 Efficiency of transfer DNA fragmentation, 456 Electroblotting characteristics, 465

584

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Electrophoresis agarose gel, 439 buffers, 441, 444 DNA in agarose, 444 DNA mobility, 441 DNA size markers, 441 in agarose, 440 in CGA agarose, 446 in Fast Lane agarose, 446 in formaldehyde agarose gels, 451 in glyoxal/DMSO agarose gels, 452 in NuSieve, 198, 440 in polyacrylamide, 440 in SeaKem, 440 in Seaplaque, 198 large amounts of DNA, 455 large DNA, 443 limiting mobility, 439 principles, 439 pulsed-field in agarose gel, 445 range of separation, 440 RNA, 449 RNA size markers, 450 RNA: details of method, 451 S1 mapping, 541 sieving, 439-440 Electrophoresis tanks RNase decontamination, 451 Embedded targets PCR, 201 Embedded tissue isolation DNA, 119 EMBL handling, 151 properties, 146 EMBL (see also lambda vectors) Endonuclease EcoP1, 123 End-labeling 3’-end tailing, 354 cordycepin, 355 deoxynucleotidyl transferase, 354 exchange reaction, 354, 357 fill-in reaction, 356 graphic representation, 355

kinasing, 354, 357 nonradioactive labeling, 359 of complementary oligomers, 358 radiolabeling, 358 Sequenase for fill-in reaction, 356 specific activity, 358 spin chromatography, 356 T4 polynucleotide kinase, 353 tailing, 356 Energy transfer assays example, 435 Enzyme channeling systems examples, 435 Enzyme immunoassays, 2, 11 Enzymes advantages, 298 choice, 298 chromogenic detection, 305 critical parameters, 298 DNase-free RNase, preparation, 87 labeling density on probes, 299 lysozyme, 86 Michaelis-Menten kinetics, 301 -302 proteinase E, 86 proteinase K, 86 proteolytic enzymes, 86 RNase-free DNase preparation, 87 size, 298 thermophilic protease, 86 used as primary labels, 298 Enzyme-labeled basic polymers labeling to DNA, 366 Equipment basic and additional, 387 positive displacement micropipettes, 172 thermocyclers, 171 Errors random, 10 systematic, 10 Ethanol precipitation co-precipitation ATP, 541 Ethanol see solvents Ethidium bromide, 441

SUBJECT INDEX

Ethidium bromide alternatives, 82 decontamination, 81 mutagenicity and toxicity, 81 stock solution, 81 Ethidium bromide of RNA staining effect on hybridization, 468 Eu-chelates labeling, 295 structure, 294 Eu-labeling transaminated DNA, 360 Exchange reaction end-labeling, 354, 357 Exon trapping use, 74 Exons see mRNA Exonuclease VII use in S1 analysis, 539 Exonuclease (exo) 111 use in replacement synthesis, 345 Exonucleases to produce ss probes from duplex, 206-207 Experimental errors, 12 Extrachromosomal genomes, 482 Extraction streptavidin, 528, 531 Extraction of DNA and RNA simultaneous, 437 E. coli hemimethylation, 123 K-12 strains, 122 lambda lysogens, 123 preparation competent cells, 163 restriction modification systems, 123 transformation competent cells, 164 E. coli handling agar plates, 130 common media, 130 growth phases, 134 liquid media, 130 minimal growth media, 133

overnight cultures, 134 plating, 134 storage, 134 storage of clones, 131 use antibiotics, 131 ‘absorbance’ bacteria, 134 E. coli polymerase I use in nick translation, 330 E. coli strains BB4, 124 BNN93, 124 C600, 124, 146 DH1, 124 genotypes, 124, 125 HBlO1, 124, 127, 139 JC 8111, 135 JC8111, 132 JM101, 124, 126 JM103, 123 JM105, 124, 126 JM107, 124, 126 JM109, 124, 126 JM110, 124, 127 LE392, 124, 146 NM538, 124 NM539, 124, 146 P2392, 124, 146 RR1, 127 Sure 124, 132 XL1-blue, 125, 146 Y1088, 125, 146 Y1090, 125 Y1090hsdR, 146

Fab fragments comparison to antibodies, 408 False negatives, 4 False negatives and multiplex PCR, 196 False positives, 4

585

586

HYBRIDIZATION WITH NUCLEIC ACID PROBES

False positives in amplification systems, 169 in colony hybridization, 476 ‘fast’ blots hybridization, 413 Ferguson plots, 442 Field-inversion gel electrophoresis see FIGE FIGE principles, 445 problems and remedies, 448 pulse controller, 445 size fractionation, 445 technical details, 448 Fill-in reaction end-labeling, 356 for S1 probes, 539 Fill-in reactions characteristics, 272 oligomers, 234 Film for chemiluminescence, 317 Films for chemiluminescence, 289 grain size, 280 protective layer, 281 sensitivity, 280 single/double coated, 281 X-ray cassette, 284 Films resolution, 280 Filter paper blotting, 397 Filters see membranes Filtration manifolds, 387 FITC comparison to other fluorochromes, 293 labeling details, 296 structure, 294 Fixation of tissues for ISH, 506

Fixatives optimal for DNA, 506 optimal for RNA, 506 subsequent PCR, 202 Flucrene labeling characteristics, 273 Fluorene see AAF or AAIF Fluorescein structure, 288 Fluorescein (see also FITC) Fluorescent (DABCO, DAPI) counterstaining, 516 Fluorochrome labels see individual labels Fluorochrome/protein see F/P ratios Fluorography intensifying screens, 280 preflashed films, 280 principles, 280 Formaldehyde comparison to glyoxal/DMSO, 449 depolymerization, 498 use in ISH, 507 Formamide formulation and preparation, 390 use in hybridization, 402 Formamide solutions importance of pH, 501 Fragmentation DNA depurination, 454, 456 Freund’s adjuvant, 327 Fungal spores DNA isolation, 101 F(c) susceptibility to GC content, 24-25 F(c) see nucleic acid F-episome see F-factor F-factor

SUBJECT INDEX

ensuring presence, 123 properties, 123 role, 122 F/ P ratio optimal, 296 GC-rich segments PCR, 193 Gel electrophoresis advantages, 437 Gel retardation assays transcription in vitro, 352 Gene localization interphase chromosomes, 520 resolution, 520 Gene splicing by PCR, 203 Geneclean see glassmilk, 441 Genetic code, 29 Genetic code degeneracy, 222 use to design oligomers, 222 Genetic diseases, 554 Genomic blots improvement by ligation, 455 Genomic DNA electrophoresis, 443 suppression hybridization, 532 Genomic libraries cloning, 135 Genosensors, 555 Genotypes E. coli strains, 125 Glass supports synthesis oligomers on, 385 Glassmilk buffer requirements, 441 purificatioon PCR products, 197 shearing DNA, 443 use, 92 Glutaraldehyde use in ISH, 507 Glycogen use in DNA precipitation, 81

587

Glyoxal formulation and preparation, 390 use in RNA blotting, 397 Glyoxal/DMSO comparison to formaldehyde, 449 Glyoxal/DMSO electrophoresis details, 450 Grains autoradiographic distribution, 520 efficiency of disintegration, 515 lack of, 523 nonspecific, 284 size in films, 280 upper limit, 284 Grains autoradiogram see Poisson distribution Gray see radioactivity units Guanidine (isohhiocyanate see GuSCN Guessmers designing, 223 use in PCR, 185 GuSCN compared to urea, 432 in reverse capture assays, 423 in solution hybridization, 430 simultaneous extraction RNA and DNA, inhibition RNase, 393 GuSCN see also isolation RNA Haptenated basic polymers crosslinking to DNA, 366 Haptens coupling to carrier proteins, 325 detection on membranes, 412 detection by antibodies, 328 structure and reaction, 326 used in detection systems, 321 Hapten/antibody as recognition system, 321 Heavy-isotope-labeled DNA strands, 12 Helicases accelerator of hybridization, 407

588

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Helix structure of DNA, 12 Hematoxylin/eosin counterstaining, 516 Heparin action, 94 formulation and preparation, 391 Heteroduplex breathing, 542 High ratio subtractive hybridization see subtractive Hoechst fluorescence counterstaining, 516 Hoogsteen bonds in triple helices, 67 Horseradish peroxidase see POase Hot-start in PCR, 189 Housekeeping genes, 531 Human genomic DNA, 319 Hybrid selection, 533 Hybrid selection hybridization, 534 membranes, 533 mRNA degradation, 534 prehybridization, 534 procedures, 533 through biotinylated DNA, 535 through capture assays, 535 Hybridization accelerators, 405 acridinium ester probes, 291 after transfer, 472 areas of application, 13 basis, 12 branch capture reactions, 435 capture, 375 capture assays, 419 colony hybridization, 488 conditions, 401, 404 crosshybridization in ISH, 524 definition, 3 details posthybridization washes, 409 energy, 1

filtration of solutions, 403 formats, 375 fraction of G-C, 12 history and development, 12 in concentrated GuSCN solutions, 431 in hybrid selection, 534 matrix array, 556 modified oligomers, 235-236 nonspecific adsorption of probe, 376 on charged nylon, details, 401 on membranes, details, 400 on membranes, graphic representatation, 392 on membranes, steps, 391 on nylon, details, 401 on paper, details, 401 on paramagnetic beads, 381 optimum incubation period, 404 parameters and equations, 52-53 'plaque hybridization, 488 polyethylene bags, 388 posthybridization washes, 407 prehybridization, 401 prehybridization conditions, 402 prehybridization solutions, 402 probe detection, 410 protection assay acridinium ester, 292 rate of renaturation, 12 reuse of solutions, 490 reverse hybridization, 406 sandwich, 375 sandwich assays, 426 sandwich format, 417 semi-solution formats, 416 simultaneous extraction, blotting, 413-414 solid phase formats, 375 solution, 375 solution assay formats, 6 solution (acridinium probes), 292 solutions and buffers, 388 solvent viscosity, 12 strand displacement, 376 strand displacement assays, 433

SUBJECT INDEX

stripping, 391 test performance, 11 turbohybridization in ISH, 517 use of DNA probes in ISH, 513 use of formamide, 402 use of RNA probes in ISH, 512 volume of solutions, 403 ‘fast’ blots, 413 ‘quick’ blots, 414 Hybridization cassettes, 387 Hybridization formats merits and disadvantages, 376 Hybridization in gel see unblot Hybridization incubators, 388 Hybridization kinetics example calculation, 55-57 excess of one of strands, 54 mismatches, 57 on membranes, 58-59 pseudo-first order, 55 rate constant, 54 second-order, 54 tethered target, length, 58-59 theory, 245-248 with competing reactions, 56 zippering, 57 Hybridization ovens, 387 Hybridization theory and nature of target, 71 choice optimal probe, 69 complex vs simple targets, 71 conditions, 72 formats, 75 nature label, 77 oligomers, 70 solution vs solid phase, 75 ss vs ds probes, 69 strategy and probe origin, 69 use in probe design, 68 Hybridization washes stringency, 26 Hybrids see duplex

589

Hydroxyapatite, 529 Hydroxyapatite acridinium probes, 292 use in hybridization, 386

IAS critical parameters, 210 flow chart, 208 for larger fragments, 211 number of cycles, 209 optimized procedures, 210 preferred transcription initiation, 21 1 principles, 168, 207 procedures, 209 selective amplification in presence of ds DNA, 209 yield, 207 Image analysis using CCD cameras, 521 Imidoesters structure and reaction, 326 Immobilization amount of nucleic acid, 395 baking, 393 effect on enzyme catalysis, 300 of supercoiled DNA, details, 396 on nitrocellulose, details, 394 on nylon, details, 394 on PVDF, details, 394 palindromic DNA, 462 RNA, details, 395 salt concentration, 393 simultaneous with extraction, 413-414 targets, 375 transfer buffers, 396 UV-crosslinking, 393 Immobilization DNA in microtiter plates, 420 Immobilization DNA in microtiter plates, 385 Immunization carrier proteins, 325 protocol, 327

590

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Immunodetection on membranes, 412 problems in colony hybridization, 490 In situ hybridization, 16 In situ hybridization see ISH In vitro transcription characteristics, 272 In vitro transcription see transcription in vitro Inactivation antibiotics, 131 APase by biotinylation, 324 endogenous APase, 287 endogenous POase, 289, 510 Klenow fragment polymerase, 539 methylmercuric hydroxide, 449 POase by contaminants, 303 polynucleotide kinase enzyme, 539 protease, 447 RNase by DEPC, 452 RNases, 92-94 p-galactosidase by mercaptoethanol, 304 Incubation time of hybridization, 404 Incubators hybridization, 388 Initial velocity measurements in ISH, 518 Inosine use in oligomers, 224 Intensification autoradiograms, 283 Intensifying screen, 281 Interphase chromatin, 521 Interphase chromosomes, 496 Interphase chromosomes gene localization, 520 Intrastrand switching see triplex Introns see mRNA In-gel-hybridization see unblot

Iodination DNA or RNA, 369 IPTG inducer for P-galactosidase, 130 preparation, 152 ISH applications, 495 avoiding hybridization to RNA, 497 background, 532 biotinylated probes, 500 CCD image recording and analysis, 500 colony hybridization, 483 comparison ISH quantitation to capture assay, 517 cross-linking fixatives, 506 cryosectioning, 502 cryosectioning, details, 505 cytological procedures, 498, 502 denaturation efficiency, 514 details of prehybridization, 510 detection of probes, 515 different steps, 497 DIG-probes, 500 DNA prehybridization, 510 fixation and embedding, details, 504 fixation for DNA target, 511 fluoresceinated probes, 500 formaldehyde use as fixative, 507 gene localization, 520 hybridization and posthybridization, 512 hybridization procedures, 509 hybridization to DNA, 511 hybridization to RNA, 514 improvement accessibility of target, 511 improvement permeabilization, 511 in combination with immunohistochemistry, 518 initial velocity measurements, 518 interphase chromosomes, 496 lambda cosmid or YAC probes, 509 methacrylate sections, 502 multicolor labeling, 501

SUBJECT INDEX

negative chemography, 502 negative controls, 501 nonspecific adsorption probe to cell, 499 optimal fixation of tissues, 506 optimization, 497 overview, 495 PCR probes, 499 perfusion of mice, details, 504 permeabilization of tissues, 506 positive chemography, 502 positive controls, 501 posthybridization, 515 precipitant fixatives, 506 prehybridization treatments, 511 preparation cultured cells, details, 503 preparation metaphase chromosomes, details, 503 protease treatment, 507 retrospective studies, 509 RNA prehybridization in cryosections, 510 RNA prehybridization in cultured cells, 510 RNA prehybridization in paraffin sections, 510 sectioning and mounting of cells, 508 simultaneous denaturation probe and target, 514 synchronization cells, 521 theory, 496 tissue adhesion, 508 tissue morphology, 524 tissue preparation and requirements, 500 troubleshooting, 522 Isolation large-size DNA, 447 Isolation DNA for PCR, 115, 116 from animal cells, details, 103 from animal cells, large DNA, 102 from animal cells, very large DNA, 104

591

from archaeological specimens, 119 from bacteria, comparison methods, 97 from bacteria, details procedures, 96 from bacteria, rapid procedures, 98 from bacteria, with CTAJ3, 95 from bacteria, yield, 97 from biopsies, 102 from blood samples, 102, 116 from cells in monolayers, 102, 116 from embedded tissue, 119 from embedded tissue, details, 120 from fungal spores, 100 from fungal spores, CTAB method, 101 from plant cells, details, 106 from spheroplasted yeast, 99 from unspheroplasted yeast, 101 from yeast, 100 from yeast, approaches, 98 mitochondria1 DNA, 105 quality required, 94 rapid isolation for hybridization, 114-115 rapid methods, 116 separation from RNA on CsCI, 119 simultaneous with RNA, 118 Isolation RNA acid GuSCN/phenol/chloroform (AGPC), 111 bacterial RNA, 107 bacterial RNA, details procedures, 108 from mammalian cells, AGPC method, 110 from mammalian cells, GuSCN method, 110 from plant cells, 110, 113 from unspheroplasted yeast, 109 methods for mRNA, 113 paramagnetic beads for mRNA, 114 paramagnetic beads for poly(A)+RNA, 114 rapid methods, 117 rapid, from blood cells, 118 rapid, from tissue culture, 118 separation from DNA on CsCI, 119

592

HYBRIDIZATION WITH NUCLEIC ACID PROBES

simultaneous with DNA, 118 steps involved, 107 use of GuSCN, 107 Isopropanol see solvents Isopsoralen post-PCR sterilization, 199 Isothiocyanate structure and reaction, 326 Keyhole limpet hemocyanin, 325 Kinasing characteristics, 272 end-labeling, 354, 357 for S1 probes, 539 linkers or adaptors, 160 oligomers, 233 S1 probes, 540 Kinetics competing reactions, theory, 249-250 nucleation, 42 overview, 41 zippering, 42 Kinetics (see abo annealing constant) Kinetics see reassociation kinetics Kinetics of annealing, 14

Km for RTases, 157 importance in labeling, 329 importance in random priming, 340 K, or V,,, discrimination fidelity polymerase, 178 Labeling acridinium esters, 290, 293 chemical, 273 choice method, 269 efficiency in nick translation, 332 enzymatic, overview, 329 Eu-chelates, 295 FITC, 296 fluorochromes, 294

importance of K,, 329 importance of template concentrations, 329 oligomers, 230, 232-234 streptavidin or antibody with fluorochromes, 296 transamination, 294 uniform incorporation, 329 Labeling method choice, 271 Labeling see cDNA probes see end-labeling see fill-in reactions see fluorene labeling see in vitro transcription see kinasing see mercuration see nick translation see PCR labeling see photohaptenation see primer extension see psoralen see random priming see replacement synthesis see transamination Labels choice, 269 comparison, 271 criteria, 270 detectability, 271 direct primary, 269 enzymes, 298 indirect primary, 269 nature, 270 nonradioactive primary, 287 overview, 270 overview of nonradioactive, 287 primary, 269 secondary, 269 signal vs background, 271 stability, 270 Lac operon structure, 128

SUBJECT INDEX

Lac1 repressor in lac operon, 129 LacZ gene alpha complementation, 128 alpha fragments, 128 detection P-galactosidase, 128 omega fragments, 128 Lambda packaging, 528 Lambda 2001 handling, 151 properties, 146 Lambda 2001 (see also lambda vectors) Lambda DASH properties, 146 Lambda DASH (see also lambda vectors) Lambda exonuclease use in replacement synthesis, 346 Lambda gt properties, 146 Lambda gt (see also lamda vectors) Lambda vectors Charon, 144 Chi replacement vectors, 144 Chi sites and recBC host, 144 deletion nonessential DNA, 143 dephosphorylation DNA, 149 EMBL, 144 exonuclease V/recBC nuclease, 144 flow chart cosmid cloning, 145 flow chart lambda cloning, 145 handling, 147 in vitro packaging, 150 isolation lambda arms, 148 ligation, 149 plating, 147 production and titration, 150 purification, 148 size selection with EDTA, 144 small-scale preparation, 147 spi selection, 144

593

stuffer DNA, 144 substitution vectors, 148 transfer of plaques, 147 widely-used vectors, 146 @-mode replication, 143 Lambda ZAP,529, 534 Lambda ZAP excision of plasmids, 476 properties, 146 Lambda ZAP (see also lambda vectors) Latex beads hydrazide-functionalized, 384 Law of mass action, 7 LCR application in oligonucleotide ligation assay, 212 background, 214 concepts, 211 flow chart, 212 improving signal/noise ratio, 215 multiplex, 215 principles, 169 procedures, 214 prospects, 214 thermostable ligase, 211 Length see probes Libraries amplification, 485 in colony hybridization, 478 phage, 485 screening, 73 Library maximum randomness, 476 optimal digestion target DNA, 476 presence of sequence of interest, 477 underrepresentation of clones, 476 Library (see also cDNA) Ligase chain reaction see LCR Ligation blunt ends, 163

594

HYBRIDIZATION WITH NUCLEIC ACID PROBES

circularization and length of DNA, 201 compatible sites, 163 DNA procedures, 163 in absence of ATP (before PCR), 200 macromolecular crowding, 166 optimum DNA concentrations, 166 Liquid hybridization see solution hybridization Lorist vectors see cosmid vectors Low ratio subtractive hybridization see subtractive Low-abundance, 531 Luminol nonuniform luminescence, 320 reaction, 319 Luminol substrates use, 31 7 Lysogeny frequency of, 149 phage, 143 Lysozyme see enzymes Lyticase, 447

M13 vectors alpha-complementation, 153 comparison with phagemids, 154 general properties, 153 handling, 154 plating, 152 preparation stock, 152 RF molecules, 154 storage, 152 widely-used vectors, 153 M13K07 helper phage, 155 Mammalian cDNA libray complexity, 24 Mammalian genomes, 25 Maximum specific activity, 275

Melting, 2 Melting temperature see T, Membranes cellophane, 386 choice of pore size, 377 different types, 377 formats, 379 handling, 476 hybrid selection, 533 hybridization kinetics, 58-59 nitrocellulose vs reinforced nitrocellulose, 380 salt concentration and immobilization, 3 79 shrinking of nitrocellulose, 479 suppliers and popularity, 378 wetting, 397 (didadvantages, 379 Membranes see charged nylon see individual types of membranes see nitrocellulose see nylon see paper see PVDF Mercurated nucleic acid/SH-ligands, 323 Mercuration characteristics, 273 nucleic acid (details), 370 Metaphase chromosomes banding, 522 condensation, 521 G-banding (Giemsa), 522 interpretation distance, 521 preparation, 503 problems banding, 523 quinacrine staining, 522 Methylases dam gene, 126 dam-host, 127 dcm gene, 126 flanking sequence of target, 127 hsdM gene, 126-127

SUBJECT INDEX

Methylene blue details of RNA staining, 469 Methylene blue staining effect on hybridization, 468 Methylmercuric hydroxide characteristics, 449 inactivation, 449 use, 449 waste disposal, 449 Methylumbelliferyl for P-galactosidase, 304-305 Methyl-C hypermutability, 25 Membrane-bound kinetics cross-hybridization, 62 cross/specific hybridization, 64 discriminatory power probes, 62 examples of calculations, 63 overview, 62 summary of important parameters, 65 theory, 250-253 Michaelis-Menten constant, 301 Michaelis-Menten kinetics enzymes, 301-302 P-galactosidase, 304 Michaelis-Menten parameters T7 RNA polymerase, 352 Microtiter plates use in hybridization, 385 Microtitre plate methods, 481 Microtomes carryover, 202 Mismatches allowed in hybridization, 57 effect on annealing rate, 44 fatal in PCR, 186 impact on acridinium ester probes, 292 in PCR, 175 in primers, 183 oligomer hybridization, 223 Mitochondria] DNA isolation, 105 Mitogen stimulation, 521

595

Mixed phase hybridization see solid-phase hybridization Mobility DNA and agarose concentration, 442 in different buffers, 442 in different potential gradients, 442 in presence of ethidium bromide, 442 Mobility RNA compared to DNA, 450 Modified nucleosides use in oligomers, 225 Modified nucleotides structure, 288 Monosomies, 500 Morphology preservation, 512 requirements, 496 mRNA, 15 mRNA abundance, 525 characterization by S1 analysis, 535, 538 degradation in hybrid selection, 534 detection with RPA, 544 hybrid selection, 533 intron/exon mapping using RNase H, 543 ISH, 496 isolation methods, 113 purification see isolation RNA quantification by S1 analysis, 538 quantitative analysis in ISH, 517 quantitative S1 analysis, 541 S1 analysis of 3' and 5' ends, 538 S1 analysis of introns and exons, 538 S1 mapping, 540 tissue-specific expression, 72 ultracentrifugation, 534 Multigene families, 543 Multigene families membrane hybridization kinetics, 62 problems in RPA, 550

596

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Multiplex advantages, 196 LCR, 215 minisatellites, 196 optimization, 196 Mung bean nuclease in S1 analysis, 539 Naphthol AS phosphates reaction with diazonium salt, 308 use with diazonium salts, 307 NASBA see IAS Negative chemography, 523 Negative predictive value, 8 Networking effect of accelerators, 406 Nick translation, 16 Nick translation analysis probe by electrophoresis, 333 calibration by electrophoresis, 444 characteristics, 272 conventional, simultaneous method, 331 DNase I stock solutions, 330 efficiency of incorporation, 335 E. coli polymerase I, 330 graphic representation, 333 large-scale, sequential method, 332 nonradioactive labels, 335 on solid phase, 333 principles, 330 quick, sequential method, 331 radiolabeling, 334 snap-back structures, 330 stability of enzymes used, 332 stopping, 332 suitable probe length, 330 Nicking by DNase in presence of ethidium bromide, 201 Nitroaryl halides structure and reaction, 326 Nitrocellulose binding characteristics, 380 characteristics, 377

details of blotting, 394 immobilization nucleic acid, 393 stability in TMAC, 408 (disladvantages, 379 Nitrocellulose membrane rendering transparent, 281 Nitrocellulose membranes drawbacks in transfer, 453 Nonradioactive labels see individual labels Nonradioactive probes adaptations, 411 concentrations, 405 detection, 411 Nonsense suppressors in vectors, 133 some strains E. coli, 133 Nonspecific binding in capture assays, 423 primer PCR, 176 Nonspecific duplices in S1 analysis, 541 Nonspecific hybridization, 525 Nonspecific interactions probes, 7 Nonspecific staining avidin, 324 Nonspecific sticking of denatured or ss DNA, 14 Nonspecific targets, 5 Nonspecifically retained probe QP-RS, 217 Nonspecifically-retained probes, 6 Nonspecificity comparison with background, 5 Nonspecific-signal reduction methods, 6 Northern blots use of RNA probes, 473 Northern blotting 32P-probe, 467 comparison to RPA, 544 critical parameters, 468 details acridine orange staining, 469 details methylene blue staining, 469 DIG-labeled probes, 467

SUBJECT INDEX

downward alkaline transfer, 469 efficiency of transfer, 468 flow chart, 438 oligomer probes, 470 optimized method, 469 posthybridization, 470 principles, 437, 467 use of acridine orange stain, 468 use of methylene blue stain, 468 (prelhybridization, 468 Northern prints, 484 Northern transfer, 15 Nuclear track emulsion, 515 Nuclear track emulsion see autoradiography Nucleation constant definition and theory, 243 Nucleation rate effect ionic strength, 45 effect pH and GC content, 45 effect viscosity, 45 influence of temperature, 45 Nucleic acid backbone, 19, 277, 555 base stacking, 27 chemical stability, 221 complexity, 21 expected complementary sequence, 23 frequency of complementarity F(c), 22-23 hydrogen bonding, 27 immobilization, 393 labeling with enzymes, 320 polarity, 20 probability of sequence, 21 random coil-duplex transition, 26 stacking, 21 p-anomers, 20 Nucleic acid hybridization definition, 1 Nucleic acid sequence-based amplification see IAS

597

Nucleic acid see duplex Null alleles, 496 NuSieve see electrophoresis Nylon alkaline immobilization nucleic acid, 381 binding characteristics, 380 details of blotting, 394 (disladvantages, 379 Nylon beads oligomer coated, 384 Nylon membranes advantages, 462 Nylon use in transfer, 453 N-hydoxysuccimide ester structure and reaction, 326 Oligomer mismatches, 29 specific sequence on paramagnetic beads, 382-383 Oligomer duplex stability during wash, 38, 40 during wash, example, 41 examples thermodynamic calculations, 39-40 impact salt concentration, 35 in TMAC or TEAC, 35 in TMAC prediction, 35 relation to sequence, 35 retention during wash, theory, 240 theory thermodynamics, 237-239 thermodynamic prediction, 36 thermodynamic stability, 37-38 Oligomers automated synthesis, 224 chemical modification, 233 chemistry of synthesis, 225 codon usage in different organisms, 226 codon usage in different tissues, 227 coupling efficiencies, 224

598

HYBRIDIZATION WITH NUCLEIC ACID PROBES

CpG intercodon links, underrepresentation, 223 deprotection, 230 electrophilic linkers, 235 end-labeling by fill-in reaction, 358 enzymatic modification, 233 fill-in reactions, 234-235 flow chart of synthesis, 229 guessmers, 223 helix-random coil transition, 26 incorporation aminoalkyl phosphoramidites, 232 incorporation modified nucleosides, 231 intercalating reagents, 236 kinasing, 233 labeling, 230 mismatches and hybridization, 223 modification internucleoside phosphate, 232 modified nucleosides, 225 modified, hybridization properties, 235-236 nucleotide mixture at 2 positions in codon, 223 number required to compensate degeneracy, 223 phosphoramidites, 224 protected, substrate for labeling, 232 purification, 229 purification on polyacrylamide gel, 231 reaction NHS esters with amines, 234 reaction with isothiocyanates, 234 reverse phase HPLC, 229 selection sequence, 222 steric modifications and hybridization, 236 types of oligomer probes, 222 use of inosine, 224 “n 1” sequences, 225 “n - 1” sequences, 225 pg relation to pmol, 233 Oligonucleotide probe complexity, 23

+

Oligonucleotide probes for Southern blots, 455 Oligonucleotide synthesis, 15 Oligonucleotides see oligomers Optical density, 3 Optimization ISH pretreatments, 511 o-dianisidine (ODA), 310 Palidromic sequences in primers, 184 Palindromic DNA immobilization, 462 Paper binding characteristics, 380 cyanuric chloride-activated, 380 diazotized, 380 (didadvantages, 379 Paraffin wax instead of mineral oil in PCR, 189 Paramagnetic beads, 533 Paramagnetic beads characteristics, 381 coated with specific oligomer, 382, 383 coating with avidin, 383 coating with streptavidin, 381 isolation mRNA, 114 sandwich assays, 427 synthesis of specific oligomers, 383 triplex affinity capture, 553 use in hybrid selection, 535 Parameters hybridization, reassociation kinetics, 52-53 Particulate solid phase see paramagnetic, nylon, latex pBluescript vectors see vectors pBR322 vectors see vectors PCR, 7 PCR amount of primers, 192

SUBJECT INDEX

amplifcation from embedded tissues, 20 1 amplification adjacent sequences, 199 Ampliwax, 189 ATP interference, 200 buffers RTase and Taq DNA polymerase, 188 capture hybridization, 177 carryover by microtomes, 202 cloning, 203 controls, 193 co-amplification see multiplex criteria to maintain probe specificity, 348 critical parameters, 191 cross-contamination, 198 dependence on fixatives used for embedding, 202 drawbacks, 177 efficiency incorporation nonradioactive labels, 348 efficiency of replication, 174 extension time, 193, 195 human minisatellites, 175 increasing specificity, 191 isopsoralen post-PCR sterilization, 199 labeling by random priming, 339, 341 linear/exponential amplification, 174 long targets, 192 maximum size of products, 195 MgC1, concentration optimum, 188 minisatellite DNA, 192 mismatches, 175 mismatching, 191 nonradioactive probes, 177 nonspecific binding primer, 176 of GC-rich segments, 193-194 of repeated DNA, 192 of RNA, 190 on DNA isolated from embedded specimens, 120 on recombinant vectors, 202 paraffin wax instead of mineral oil, 189

599

post-PCR sterilization, 198 pre-PCR sterilization with UNGase, 198 primer appendages, 176 primer mismatches, 29 principles, 168 probability of mismatching, 176 probe labeling, 177 production ss probes, 205 purification products, 197 rapid DNA isolation, 115-116 ratio of primers for ss probes, 205 reaction volumes, 189 retrospective analysis, 201 reverse transcription, 190 schematic diagram, 173-174 secondary structures in template, 193 special vectors for cloning, 203 stability dGTP, 188 standard procedure, 187 strategy amplification adjacent sequences, 200 synthesis nonradioactive probes, 347 template re-annealing, 175 template/primer competition, 175 theoretical details, 172 thermostable DNA polymerase (Fsu), 179 thermostable DNA polymerase (Pfu), 180 thermostable DNA polymerase (Replinase), 180 thermostable DNA polymerase (Taq), 172, 177 thermostable DNA polymerase (Tth), 172, 180 thermostable DNA polymerase (Vent), 180 thermostable DNA polymerases, use, 177 thermostable RTase (Taq), 172 to establish contiguity of clones, 203 use of 7-deaza-2'-deoxyguanosine,193 use of aliphatic amides, 194

600

HYBRIDIZATION WITH NUCLEIC ACID PROBES

use of ‘hot-start’, 189 Vent or Pfu DNA polymerase, 194 ways to produce ss probes, 206 PCR labeling characteristics, 272 PCR (see also primers) PCR (see also Taq DNA polymerase) PEG 6000 comparison to dextran sulfate, 407 properties as accelerator, 406 Peptide nucleic acid (PNA) chimerae, 555 Permeabilization improved by Triton X-100, 511 of tissues for ISH, 506 Peroxidase see POase Phage vectors general properties, 143 lysogenic infections, 143 Phagemid vectors cloning, 155 factors for consistency, 155 principles, 154 production helper phage, 155 production ss DNA, 155 rolling-circle replication, 154 Phagemids, 534 Phenol effect on T, and annealing, 46 Phenol extraction biotin or DIG probes, 335 Phenol see solvents Phosphor-intensifying screen, 281 Photoactivable haptens see photohaptenation Photobiotin see photohaptenation Photodigoxigenin see photohaptenation Photohaptenation characteristics, 273 drawbacks, 364

overview, 363 preparation photohapten, 364 spacer used, 364 Photohaptens structure, 288 Photolithography, 556 Placental RNase inhibitors see RNA Plant cells isolation RNA, 113 isolation RNA, 110 Plaque hybridization, 15 Plaque hybridization background, 486 characteristics, 475 details methods, 487 DIG probes, 488 essential steps, 475 hybridization, 488 nonradioactive probes, 490 plaque size, 486 preference membranes, 486 radioactive probes, 489 selection and picking clones, 492 use, 485 use of DIG probes, 491 useful modifications, 486 Plaque lift hybridization see plaque hybridization Plaque size, 486 Plaques required to obtain given clone, 486 Plasmids, 3 Plasmids bacterial conjugation, 122 chloramphenicol-amplifiable,484 for transcription in vitro, 349 in eukaryotes, 482 properties, 135 purification, 140-142 under relaxed control, 122 Plasmids (see also vectors) Plating

601

SUBJECT INDEX

M13-transformed E. coli cells, 165 transformed E. coli cells, 164 POase catalysis, 314 catalytic reaction mechanism, 311 chemiluminescence, 290 chromogenic detection, 310 conjugation, 323 crosslinking to DNA, 367 hydrogen donors, 310 imidazole enhancement of catalysis, 312 inhibition of endogenous, 289 luminol substrates, 318 optimal peroxide concentration, 317 properties, 303 purification, 304 sensitivity to contaminants, 303 substrate/chromogen ratio, 314 use of luminol, 31 9 Pocket blotting, 463 Poisson distribution clones in library, 477 grains autoradiogram, 285 of nicks in DNA, 201 radioisotope decay, 274 radioisotopes over strands, 276 Polyacrylamide depolymerization, 466 Polyclonal antisera antibodies to bacteria, 490 Polyethylene bags hybridization, 388 Polylinkers in vectors, advantages, 137 Polymerase chain reaction see PCR Polymerization advantage in enzyme detection, 299 APase, 299 Poly(A) RNA purification see isolation RNA

+

+

Poly(A) -capture subtractive hybridization, 529 Porex absorbent filters use in hybridization, 386 Positive predictive value, 8 Positive pressure transfer, 463 Postamplification caution, 171 Posthybridization in ISH, 515 washes, 407 washes nonradioactive probes, 408 washes/stringency (details), 4/39 Post-PCR sterilization approaches, 198 with isopsoralen, 199 Power supplies for pulsed field, 446 Precipitant fixatives for ISH, 506 Precipitation nucleic acid, 88, 89 nucleic acid (procedure), 90 Precision, 3 Precision graphic representation, 11 Precision of test results, 9 Preflashing of films, 280 Prehybridization in hybrid selection, 534 in ISH, 510 solutions and conditions, 402 Prehybridization solutions use, 391 Prevalence, 8, 9, 10 Pre-amplification caution, 170 Pre-PCR sterilization with UNGase, 198 Primary decomposition, radioisotopes, 277 Primer extension characteristics, 272

602

HYBRIDIZATION WITH NUCLEIC ACID PROBES

downstream priming, 342 graphic representation, 343 on ss templates, 341 upstream priming, 342 Primers appendages, 204 background, 185 fatal mismatches, 186 guessmers, 185 length, 184 mismatches, 183 palindromic sequences, 184 primer-dimer, 184 rules for selection, 183 stabilities, 185 sticky, 183 T,, 184 universal, 186 use of neutral bases, 185 with high Tm,191 Primer-dimer PCR artefact, 184 Probability sequences of interest in library, 477 Probability of sequence see nucleic acid Probe definition, 2 length in random priming, 338 suitable length and nick translation, 330 Probe concentrations in ISH, 509, 514 Probe length impact on hybridization, 5 Probe penetration in ultrastructural ISH, 519 Probes acridinium ester labeling, 290 advantages of RNA, 349 applications, 20 biological see biological probes chemical labeling, 361 chemical stability, 277

choice of polarity for detection of DNA, 513 complexity, effect on annealing rate, 47 criteria of labels, 270 detection in ISH, 515 fluorochrome labeling, 294 GC content, 24 labeling, 16 labeling by PCR, 177 labeling density enzymes, 299 labeling with enzymes, 320 labeling with protein A, 320 length, effect on annealing rate, 47 maximum vs optimum specific activity, 275 optimal length for ISH, 524 overview fluorochrome labels, 293 principle acridinium ester use, 291 production nucleic acid, 121 purification after random priming, 341 purification tailed probes, 358 quantification, 269 RNA, 16 secondary structures, 550 simultaneous detection in ISH, 516 ss, produced by PCR, 205 types of, 269 universal see universal probes use of acridinium ester, 290 Probes overview of production, 121 Promoters specific for RNA polymerases, 16 Protease treatment in ISH, 507, 509 Protection assay acridinium ester probes, 292 Protein A instead of anti-IgG antibody, 327 Proteinase E see enzymes Proteinase K see enzymes Proteolytic enzymes see enzymes Psoralen

SUBJECT INDEX

biotinylated, 365 characteristics, 273 crosslinking, 366 graphic representation, 366 overview, 365 pUC vectors see vectors, 138 Pulsed-field gel electrophoresis, 440 Pulsed-field gel electrophoresis see FIGE, CHEF, TAFE Purification oligomers, 229, 231 Purification DNA by gel electrophoresis, 540 Purines alkylation, 221 PVDF details of blotting, 394 suppliers, 378 (didadvantages, 381

QP-replication systems see QP-RS Qp-RS amplification, 218 cloning in Qp-vectors, 217 kinetics of reaction, 218 midivariant RNA, 217 nonspecifically retained probe, 217 principles, 216 quantification, 218 reverse target capture hybridization, 218 use of QP-replicase, 216 ‘quick’ blots hybridization, 414

Radioactivity counting efficiency, 273 dosimetry, 273-274, 286

603

half-life, 274 intensifying screens, 278 maximum of probes, 274 optimum of probes, 274 optimum specific activity, 275 principles, 273 rate of disintegration, 274 relative biological effectiveness, 274 safety considerations, 286-287 spills, 388 strand breakage, 276 8-radiation, 273, 278, 283, 286 units, 273 waste disposal, 287 Radioisotopes in ISH, 499 32P, 273 32P for optimization in ISH, 524 32P in I S H , 499 32P probes in Northern, 467 35Sin I S H , 499 3H in I S H , 499 characteristics (Table), 279 concentrations in commercial preparations, 332 detection limit, 278 detection limit on film, 279 half-life, 279 incorporation, 275 labeling methods, 279 stability, 277 typical specific activity, 279 Radiolabel choice, 278 Radiolabeling by nick translation, 334 by random priming, 338 chemical labeling, 369 end-labeling, 358 Radionuclides see radioisotopes Random priming details radiolabeling, 338 graphic representation, 337

604

HYBRIDIZATION WITH NUCLEIC ACID PROBES

nonradioactive labels, 339 on solid phase, 338 purification probes, 340 radiolabeling, 338 rate of incorporation, 337 solid phase, 340 specific activity obtained, 337 use of Klenow, 337 using PCR, 339, 341 using (aminohexyl)dATP, 340 Rate of renaturation see hybridization rDNA PCR, 202 Reannealing, 2-3 Reassociation, 2 Reassociation definition, 1 Reassociation kinetics annealing rate, 49 Cot curves, 50 Cot value, 49 calculation half-time, 51 concatenation effects, 60 determination, 48 diffusion rate, 59 diffusion-limited, 60-61 hydroxyapatite, 48 nucleation-limited, 59-61 parameters and equations, 52-53 theory, 244 viscosity effects, 61 Reassociation kinetics (see also membrane hybridization kinetics) Rec systems recA, 132 recB, 132 recC, 132 recognition by gam-/+, 133 sbcB, 132 schematic diagram, 133 SOS repair, 132 umuC, 132

Recognition systems overview, 321 primary labels, 270 secondary labels, 270 Recognition systems (see also individual systems) Recombinant DNA see rDNA Recombination repair see Rec systems Recombination-based assay extension cDNA, 74 Reduction background, 491 Reference accuracy, 9 Reliability of negativity definition, 6 Reliability of positivity definition, 6 Reliability of test negatives, 8 Reliability of test positives, 8 Reliability of test results calculation, 9 Renaturation, 2-3, 26 Renaturation kinetics see kinetics of annealing Renaturation rate, 26 Repeat DNA, 14 Repetitive sequences, 525 Repetitive sequences interference, 532 Replacement synthesis activity exonuclease 111, 347 characteristics, 272 details T4 polymerase methods, 345 exo 111-Klenow method, 345 exonuclease-polymerase activity, 342 graphic representation, 344 lambda exonuclease methods, 346 T4 polymerase, 344 use of BIO-11-dUTP, 346 Replacement vectors see lambda vectors

SUBJECT INDEX

Replacement vectors see vectors Replicas microtitre plate methods, 482 platers, 482 plating, 481 preparation, 478 problems, 482 Reprobing see stripping Restriction compatible sites for ligation, 163 DNA in aggregates, 455 DNA, procedures, 162 Restriction enzymes, 15 Restriction systems mcrA, 127 mcrB, 127 mrr, 127 Restriction-modification systems classification, 125 EcoB, 126 EcoK, 126 Retardation coefficient, 441-442 Retrospective studies in ISH, 509 Reverse capture assays differential elution, 425 optimization, 426 QP-replicatable probes, 424 Reverse capture target assays details, 424 Reverse hybridization principles, 406 Reverse Southern analysis, 470 Reverse transcriptase MMLV vs AMV, 531 Reverse transcription AMV vs MMLV, 353 in PCR, 190 RFLPs, 439 RFLPs analysis, 73

605

RGE principles, 446 variants, 446 Rhodamine comparison to other fluorochromes, 293 Rhodamine labeling details, 296 Rhodamines structure, 294 RNA, 353 Superscript (cloned MMLV), 353 RNA 3’-end labeling, 359 aggregates, 531 denaturation, 449 denaturation with formaldehyde, 449 denaturation with glyoxal/dimethylsulfoxide, 449 denaturation with methylmercuric hydroxide, 449 details of blotting, 395 electrophoresis, 449 extraction from cells, 393 PCR, 190 precautions during extraction, 92 precipitation according to size, 91 purification see isolation RNA, 107 stability, 221 use of DEPC, 93 use of placental RNase inhibitors, 93 RNA (see also nucleic acid) RNA extraction simultaneous with RPA, 549 RNA polymerase from E. coli, 16 RNA polymerases from bacteriophages, 16 RNase contamination, 450 denaturation, 546 digestion, 548 in blocking reagents, 389

606

HYBRIDIZATION WITH NUCLEIC ACID PROBES

RNase A, 548 RNase decontamination with DEPC, 452 RNase protection assays see RPA, 535 RNase T1,548 RNase/TCA assay, 431 Roentgen see radioactivity units Rotating gel electrophoresis see RGE RPA, 535 RPA analysis protected RNA, 548 comparison to S1 analysis, 544 critical parameters, 549 digestion unhybridized RNA, 547 hybridization of RNA probe, 546 interpretation, 549 preparation of RNA probes, 545 principles, 543 short vs long probes, 545 troubleshooting, 549 rRNA sizes, 450

S oxidation of %, 499 use in nick translation, 334 S1 analysis alternative enzymes, 539 comparison to RPA, 544 controls, 542 critical parameters, 542 goals, 537 graphic representation, 536 kinetics of hybridization, 537 methods, 539 preparation of probes, 539 principles, 535 quantification of mRNA, 538 quantitative, 541 S1 buffer, 540 S1 mapping, 270

S1 mapping artifacts, 543 confirmation with RNase H, 543 mRNA, 540 S1 nuclease digestion unhybridized sequences, 542 in solution hybridization, 430, 433 Safety considerations radioactivity, 286 Sandwich assay comparison to capture hybridization, 418 detectability and sensitivity, 418 kinetics of hybridization, 418 Sandwich assays anti-nucleic acid antibodies, 429 details, 428 detection, 427 DNA targets, 427 immunocapture, 427 paramagnetic beads, 427 principles, 426 RNA targets, 427 use in hybrid selection, 535 Sandwich hybridization graphic representation, 417 Satellite DNA and PCR, 175 in PCR, 192 Satellite DNA see repeat DNA Scavengers radicals, 277 SDS use in hybridization solutions, 403 SeaKem see electrophoresis Secondary decomposition, radioisotopes, 277

Secondary structures in PCR, 193 Second-order rate constant, 14 Selection of primers for PCR, 183

SUBJECT INDEX

Selection of clones clone or plaque hybridization, 492 Self-sustained sequence replication see IAS Semi-solution hybridization advantages, 416 biosensors, 418 separation systems, 417 Sensitivity as used by clinicians, 5 definition, 3-4 graphic representation, 7 heteroscedasticity, 3 of label, 6 vs detectability, 295, 432 Sephacryl S-500 diazotization and coupling oligomeirs, 384 Sievert see radioactivity units Signal/noise ratio in ISH, 515

SINES use in fingerprinting, 472 Single-copy gene detection in Southern blots, 454 Single-copy genes detection with enzymes, 299 Single-copy sequences, 532 Single-gene detection with 32P probes, 278 Single-strand binding protein accelerator of hybridization, 407 use in crosslinking, 367 Size markers see electrophoresis Slides slide immunoenzymatic assay, 387 use in hybridization, 387 Snap-back structures in nick translation, 330

607

SOB preparation, 164 SOC preparation, 164 Solid phase, 5 Solid phase alternatives, 386 and enzyme inhibitors, 300 hybridization, 376 impact on enzyme kinetics, 300 limitations, 376 loss (desorption) of target, 376-377 merits and disadvantages, 375 microtiter plates, 385 nick translation, 333 particulate see paramagnetic, nylon, latex random priming on, 338, 340 unstirred layer, 300 Solid phase (see also membranes) Solid phase (see also mixed phase) Solution assays overview, 429 selective destruction free probe, 430 Solution hybridization, 14 Solution hybridization acridinium probes, 292 drawbacks, 41 7 Solvents alternatives to phenol, 84 chloroform, prevention foaming, 80 ethanol, 81 isopropanol, 81 phenol, DNA extraction, 90 phenol, equilibration, 80 phenol, oxidation, 80 phenol, storage, 80 phenol, use, 80 Somatic cell hybrids gene localization, 73 Southern blots, 532

608

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Southern blots hybrid selection, 533 probe labeling, 334 Southern blotting flow chart, 438 principles, 437 Southern transfer, 15 Southern transfer centrifugal transfer, 463 details pocket blotting, 465 details vacuum blotting, 464 electroblotting, 465 electroblotting polyacrylamide, 466 pocket blotting, 463 positive pressure transfer, 463 principles, 454 reverse, 470 small DNA fragments, 461 to diazotized membranes, 461 transfer ds DNA, 465 two types of nylon membranes, 461 vacuum transfer, 463 SP6 promter transcription in vitro, 349 Specific activity, 16 Specific activity calculation, 336 end-labeling, 358 for colony hybridization, 489 for ISH probes, 498 for plaque hybridization, 489 for Southern blots, 455 optimum in ISH, 515 required for RPA, 544 RPA probes, 546 Specific activity see radioactivity Specificity, 3 Specificity as used by clinicians, 5 comparison with discriminability, 5 definition, 4 graphic representation, 7 in PCR, 191 probes, 1

Spi selection see lambda vectors Spot hybridization see dot hybridization 3SR see IAS

ssc

formulation, 390 SSPE formulation and preparation, 390 Stability chaotropic salts and, 33 enzymes in nick translation, 332 equations, 31 impact formamide concentration, 31-33 impact GC content, 31-32 impact salt concentration, 31-33 in TMAC or TEAC, 33 oligomer duplex see oligomer duplex stability radioisotopes, 277 schematic representation, 34 storage labels, 270 Staining acridine orange in ISH, 507 of DNA with ethidium bromide, 446 of RNA prior to transfer, 453 of RNA with ethidium bromide, 452 Standard value see reference accuracy Strand displacement assays BCR, 215 D-loop formation, 433 graphic representation, 434 hybridization rate, 433 R-loop formation, 433 Strataclean, 548 Strataclean use DNA clean-up, 84 Streptavidin binding to different biotin labels, 335 coated microtiter plates, 535 coated paramagnetic beads, 535 coated to paramagnetic beads, 381

SUBJECT INDEX

comparison to antibodies, 335 comparison to avidin, 323 detection of biotinylated probes, 408 enzyme conjugation, 322 fluorochrome labeling, 296 for strand-specific purification, 206 inhibition by BLOTTO, 410 partitioning into organic solvents, 531 properties, 323 Stringency, 7 Stringency capture assys, 426 criterion, 408 in posthybridization, 407 open or relaxed, 408 posthybridization of oligomer probes, 410 relation to Tm,408 Stripping BCIP/NBT product, 309 from nylon, 415 from reinforced nitrocellulose, 415 in unblot format, 471 loss of target DNA, 415 naphthol AS products, 309 overview, 415 RNA probes from Northern blots, 473 Stuffer DNA see lambda vectors Subbing inclusion of DEPC, 505 with egg white, 508 with gelatin, 498 with poly-L-lysine, 498 Substitution vectors see lambda vectors Substrate BCIP/NBT, 305 diffusion constant, 301 inhibition, 300 nonspecific binding of probe to DAB, 519

609

structure methylumbelliferyl, 294 X-gal for P-galactosidase, 128 Substrate inhibition, 300 Substrates chemiluminescence, 289 chemiluminescent, 314-316 dioxetanes, 314 luminol, 31 7 methylumbelliferyl, 304, 305 POase, 310 Subtractive hybridization against poly(A)+RNA, 531 aims, 525 biotin-driver DNA, 528 cloning inhibition, 526 examples, 527 high ratio, 526, 529 low ratio, 526, 529, 531 low ratio flow chart, 530 Sulfhydryl ligands mercurated nucleic acids, 327 reaction with mercurated DNA, 370 synthesis, 328 Supercoiled DNA details of blotting, 396 electrophoretic mobility, 441 s u pp0rts for ISH, 508 Suppression hybridization aims, 525 in ISH, 509 Synchronization cells for ISH, 521 Synthesis oligomers, 229 Synthetic oligonucleotides see oligomers p-actin internal reference, 542 mRNA abundance, 546 probe, 547 p-actin mRNA use as standards, 473

610

HYBRIDIZATION WITH NUCLEIC ACID PROBES

P-galactosidase P-galactosidase (see also lacZ gene) IPTG, inducer lacZ gene, 130 Michaelis-Menten kinetics, 304 properties, 304 schematic diagram, 129 substrates, 304 turnover numbers, 304 X-gal substrate, 128

T3 promoter transcription in vitro, 349 T4 gene, 32 protein accelerator of hybridization, 407 T4 polymerase use in replacement synthesis, 345 T4 RNA ligase use in fluorochrome labeling, 295 T7 promoter transcription in vitro, 349 TAE see electrophoresis buffers TAFE principles, 446 Tailing cDNA, 160 characteristics, 272 drawbacks, 161 Taq DNA polymerase 3’ + 5’ exonuclease, 178 5’ 3‘ exonuclease activity, 181 alternatives, 180 and detergents, 181, 183 error frequency, 180 importance pH, 180 inhibition by blood, 181 inhibition by hemoglobin, 181 MgCI, concentration, 180 properties, 178 properties (Table), 182 transferase activity, 181, 204 Taq DNA polymerase reaction parameters, 179

-

TAS difference with IAS, 219 principles, 169, 219 procedures, 219 TJ3E see electrophoresis buffers Td oligomer duplex, prediction, 35 Tdr Duplex-retention temperature see duplex Template concentrations importance in labeling, 329 Tetramethylbenzidine (TMB), 310 Tetramethylrhodamine see rhodamine Texas Red structure, 294 Thermal stability, 27 Thermal stability nucleotide analogs, 27 oligomer duplex, 27 Thermal stability see stability, 31 Thermocycling automated, 171 manual, 171 variants of cyclers, 171 Thermodynamic parameters oligomer duplex stability, 37 Thermophilic protease see enzymes Thermostable DNA polymerase (Taq), 178 Thermostable DNA polymerases use, 168 Thermostable DNA polymerases (see also PCR) Thermostable RTase Tth DNA polymerase, 172 Time-resolved fluorescence see TRF Tissue printing colony hybridization, 483

SUBJECT INDEX

Tissue-specific gene expression, 483 T,,. 12

TIn definition, 26 difference poly- and oligonucleotide, 26 examples of calculations, 32 hybrid selection, 534 in LCR, 169 in PCR, 175 in TMAC, prediction, 35 of primers, 184 of tailed probes, 356 oligomer duplex, thermodynamic prediction, 37-38 optimal hybridization, 404 time-dependent equilibration, 26 T,, (see also stability) TMAC comparison to TEAB and TMAC, 408 stability nitrocellulose, 408 TMB use on membranes, 312 Transamination characteristics, 273 competition with deamination, 362 fluorochrome labeling, 294 graphic representation, 362 of RNA, 363 with ethylene diamine, 360 with hydrazide biotin, 363 Transcription, 16 Transcription in vitro for ISH, 499 in vitro for RPA, 545 'crosstalk' unrelated promoters, 549 Transcription in vitro convenient plasmids, 349 details using [32P]NTP, 352 details using [ ''SIUTP, 351 gel retardation assays, 352 graphic representation, 350 RNA polymerases, 349 use of DIG-UTP, 352

61 1

use of spermidine, 349 yield, 351 Transcription-based amplification system see TAS Transfer loss nucleic acid, 470 Transfer see capillary transfer Transfer buffers blotting, 396 Transfer DNA see Southern transfer Transfer procedures principles, 453 Transfer RNA see Northern transfer Transfer small fragments use of Trioxsalen, 466 Transformation competent E. coli cells, 164 E. coli, 137 Transgenic animals, 496 Transilluminators immobilization nucleic acid, 387 Transverse alternating field see TAFE TRF cycles, 297 principles, 297 special equipment, 297 Trichloroacetic acid formulation and preparation, 390 Trimethylpsoralen see Trioxsalen Trioxsalen improvement transfer small fragments, 466 Triple helices see triplex Triple helix basic types, 550 graphic representation, 551 intrastrand switching, 552 PNA, 555

612

HYBRIDIZATION WITH NUCLEIC ACID PROBES

Triplex affinity capture, 552 graphic representation, 66 in RecA system, 67 inhibition of DNA restriction, 554 stability, 34 stabilization, 552 strand switching, 68 stringency conditions, 552 structure, 65 types of, 65 Trisomies, 500 TRNA anticodon, 29 True negatives, 4 True positives, 4

Ts, oligomer duplex, prediction, 36 Tth DNA polymerase RTase activity, 181 Turbohybridization in ISH, 51 7 Tween-20 for background reduction, 405 T-vector PCR cloning, 203

Ultracentrifugation mRNA, 534 Ultrastructural ISH, 519 Unblot characteristics, 454 details, 471 drawbacks and advantages, 470 flow chart, 438 of RNA target, 472 short vs long probes, 472 stripping, 471 Universal primers for PCR, 186 Universal probe advantages, 372 two stage, 372

Universal probes detection M13 libraries, 412 from phagemids, 372 Uracil-N-glycosylase pre-PCR sterilization, 198 Urea compared to GuSCN, 432 USBioclean see glassmilk UV crosslinking optimzation, 398 use, 397 UV fixation RNA, 449 UV-crosslinking nucleic acid on membranes, 393 W-irradiators immobilization nucleic acid, 387 UV-shadowing purification of nucleic acid, 231

Vacuum manifold alternative, 396 Vacuum transfer, 463 Validity (statistical) of test results, 9 Vectors, 3 Vectors bacteriophages, 135 chloramphenicol amplification, 137-138 comparison of often-used, 139 cosmids, 136 CsCI-gradient purification, 142 essential elements, 122 fl-type origins of replication, 138 general properties, 136 handling, 138 multiple cloning site, 122 origin of replication, 136 pBR322, 137 PEG 8000, selective precipitation, 142 plasmids, 135 polylinkers, 139 polylinkers, advantages, 137

SUBJECT INDEX

613

promoters phage RNA polymerases, 138 purification, 138, 140-142 rapid purification for screening, 139 replacement vectors, 136 replicators, 137 selection systems, 122 supercoiled, purification, 142 transformation, 137 Viscosity macroscopic, 46 microscopic, 46

YAC, 532 YAC probes in ISH, 509 Yeast isolation of DNA, 98-99 isolation RNA, 109 spheroplasting, 100 Yeast artificial chromosome, 532 Yield transcription in vitro, 351

X-gal preparation, 152 substrate for P-galactosidase, 128 X-ray diffraction patterns, 12

Zippering, 2 Zymolase, 447 Zymolase use in DNA isolation from yeast, 98

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