PCR Protocols

Methods in Molecular Biology

John M. Walker, SERIES EDITOR

15. PCR Protocols: Current Methods and Applications, edited by Bruce A. White, 1993

14. Glycoprotein Analysis in Biomedicine, edited by Elizabeth F. Hounsell, 1993

13. Protocols in Molecular Neurobiology, edited by Alan Longstaff and Patricia Revest, 1992

12. Pulsed-Field Gel Electrophoresis: Protocols, Methods, and Theories, edited by Margit Burmeister and Levy Ulunovsky, 1992

11. Practical Protein Chromatography, edited by Andrew Kenney and Susan Fowell, 1992

10. Immunochemical Protocols, edited by Margaret M. Manson, 1992

9. Protocols in Human Molecular Genetics, edited by Christopher G. Mathew, 1991

8. Practical Molecular Virology, edited by Mary K. L. Collins, 1991

7. Gene Transfer and Expression Protocols, edited by E. J. Murray, 1991

6. Plant Cell and Tissue Culture, edited by Jeffrey W. Pollard and John M. Walker, 1990

5. Animal Cell Culture, edited by Jeffi.ey W. Pollard and John M. Walker, 1990

4. New Nucleic Acid Techniques, edited by John M. Walker, 1988

3. New Protein Techniques, edited by John M. Walker, 1988

2. Nucleic Acids, edited by John M. Walker, 1984 1. Proteins, edited by John M. Walker, 1984

PCR Protocols Current Methods and Applications

Edited by

Bruce A. White University of Connecticut Health Center,

Farmington, CT

Humana Press Totowa, New Jersey

O 1993 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

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Library of Congress Cataloging in Publication Data

Main entry under title: Methods in molecular biology.

PCR protocols ; current methods and applications / edited by Bruce A. White. p. cm. - (Methods in molecular biology : 15)

Includes index. ISBN 0-89603-244-2 1. Polymerase chain reaction-Methodology. I. White, Bruce Alan.

11. Series: Methods in molecular biology (Totowa, NJ) ; 15. QP606.D46P363 1993 574.87'3224~20 92-34874

CIP

Preface

PCR has been successfully utilized in every facet of basic, clini- cal, and applied studies of the life sciences, and the impact that PCR has had on life science research is already staggering. Con- comitant with the essentially universal use of PCR has been the creative and explosive development of a wide range of PCR-based techniques and applications. These increasingly numerous proto- cols have each had the general effect of facilitating and accelerat- ing research. Because PCR technology is relatively easy and inexpensive, PCR applications are well within the reach of every research lab. In this sense, PCR has become the "equalizer" between "small" and "big" labs, since its use makes certain projects, especially those related to molecular cloning, now far more feasible for the small lab with a modest budget.

This new volume on PCR Protocols does not attempt the impossible task of representing all PCR-based protocols. Rather, it presents a range of protocols, both analytical and preparative, that provide a solid base of knowledge on the use of PCR in many com- mon research problems. The first six chapters provide some basic information on how to get started. Chapters 7-19 represent primarily analytical uses of PCR, both for simple DNA and RNA detection, as well as for more complex analyses of nucleic acid (e.g., DNA footprin ting, RNA splice site localization). The remaining chapters represent "synthetic," or preparative, uses of PCR. The use of PCR for aspects of cloning, including obtaining full-length cDNA sequence, sitedirected mutagenesis, and production of synthetic genes has been emphasized in these chapters. Some duplication of important topics (e.g., sequencing, cDNA cloning, the use of degenerate oli- gonucleotides, and sitedirected mutagenesis) has been introduced purposely to offer the reader several approaches to the same prob lem. As has been done in previous volumes of the Methods in Molecular Biology series, an emphasis has been placed on generally

vi Preface

applicable protocols. The description of specific experimental sys- tems has been deemphasized, and used only when the provision of a specific example is helpful. As part of the MkYuds in M k 1 . r - series, the chapters each include a "Notes" section, whose purpose is to provide a discussion of problems, tips, and alternatives. This type of discussion, not usually available in the original publications, should enhance the ability of the reader to get a procedure up and running, as well as increase experimenters' understanding of its strengths, limitations, and pitfalls. It is hoped that this collection of PCR protocols will be especially useful to young investigators, or to those new to PCR, by providing a knowledge base and encouraging the design of novel approaches and applications of PCR.

I am indebted to Jennifer Swanson for her superb secretarial skilis. I also wish to thank John Walker and Humana Press for their assistance and support in putting this volume together. Finally, I want to express my appreciation to the contributing authors, who displayed a remarkable degree of enthusiasm for this volume and provided such excellent material for it.

Contents Preface .................................................................................................................. v Contributors ....................................................................................................... i x

Polymerase Chain Reaction: Bask h l o c o k , Beverly C. Delidow, John P. Lynch, John J. Peluso,

............................................................. and Bruce A. m i t e 1 Selection of Primers for Polymerase Chain Reaction,

........................................................................ Wojciech Rydtlik 31 Direct Radioactive Labeling of Polymerase Chain Reaction Products,

.......................................... Tim McDaniel and Stephen J. Meltrer 41 Use of Arithmetic Polymerase Chain Reaction for Synthesis

of Single-Stranded Probes for S1 Nuclease Assays, &.a Aganual and Bruce A. White ........................................... 4 5

Nonradioactive Labeling of Polymerase Chain Reaction Products, Udo Reischl, Riidiger Ruger, and Christoph Kmler ...................... .51

Quantitation and Purification of Polymerase Chain Reaction Products by High-Performance Liquid Chromatography,

Elena D. Katx ............................................................................ 63 Use of Polymerase Chain Reaction for Screening Transgenic Mice,

Shixhong Chen and G h A. Evans ................................................ 75 Polymerase Chain Reaction Analysis of DNA from Paraffin-

Embedded Tissue, Matthius Volkenandt, Adam P. Dicker, Renato Fanin,

Debabrata Banerjee, Anthony Albino, and Joseph R Bertino ..... 81 The Use of Polymerase Chain Reaction for Chromosome

Assignment, MidtaelJaye and Cmig A. Dionne ............................................... 89

Mapping MHC Class I1 Genes and Disease-Susceptibility: Use of Polymerase Chain Reaction and Dot Hybridization

for Human h k o c y t e A n t i p Allele Typing, ............................................... Stephen C. Bain and John A. Todd 95

The Use of the Polymerase Chain Reaction and the Detection of Amplified Products,

Robert C. Allen and Bruce Budowle ........................................ 113 Determination of Loss of Heterozygosity Using Polymerase

Chain Reaction, Stephen J. Meltxer ..................................................................... 129

vii

... U L L Z Contents

Direct Sequencing of Polymerase Chain Reaction Products, StephenJ. Mel&er ...................................................................... 1 3 7

Manual and Automated Direct Sequencing of Product Generated by the Polymerase Chain Reaction,

Adam P. Dicker, Matthias V o l b n d t , and Joseph R Bertim ......... 143 Genomic Footprinting by Ligation Mediated Polymerase

Chain Reaction, Gerd P. &zifer and Arthur D. Riggs ........................................... 153

RNA Template-Specific Polymerase Chain Reaction (RSPCR): A Modijication of RNA-PCR that Dramatically Reduces the Frequency of False Positives,

Alan R. Shuldiner, Riccurdo Pgfetti, and Jesse Roth ..................... 169 Quantitative Measurement of Relative Gene Expression

in Human Tumors, Tdncro Horikoshi, Kathleen Danenbetg, Matthias Volkenandt,

Thomas Stadlbauet; and Peter V. Dunenberg ...................... 177 Identification of Alternatively Spliced mRNAs and Localization

of 5' Ends by Polymerase Chain Reaction Amplification, ....... Qmg-Ming Giang, Louise T. mow, and Thomas R. Broker 189

Utilization of Polymerase Chain Reaction for Clonal Analysis of Gene Expression,

..................................... Alice L. Mtsell and Lawrence B. Schook 199 Sequencing DNA Amplified Directly from a Bacterial Colony,

Martin A. Hofmann and David A. Brian .................................... 205 Use of Polymerase Chain Reaction to Screen Phage Libraries,

Lei Yu and Laum J. Bloem ........................................................ 21 1 Molecular Cloning of Polymerase Chain Reaction Fragments

with Cohesive Ends, Beverly C. Delidow ................................................................... 21 7

Rapid (Ligase-Free) Subcloning of Polymerase Chain Reaction Products,

Alan R Shuldiner and Kkith Tanner ............................................. 229 Use of Polymerase Chain Reaction for Making Recombinant

Constructs, Dough H. Jaes and Stanlq C. Winistotfer .................................. 241

In Vitro Recombination and Mutagenesis of DNA: SOEing Together Tailm-Made Genes,

..................................................................... Robert M. Horton 251 Use of Polymerase Chain Reaction for the Rapid Construction

of Synthetic Genes, Pahick J. Dillon and Cmig A. Rosen ........................................ 263

Recombinant Circle Polymerase Chain Reaction for Site-Directed Mutagenesis,

...................................................................... Dough H. Jones 269

Contents ix

Site-Directed Mutagenesis by Double Polymerase Chain Reaction: Megapnmer Method,

Sailen Bank ...................................................................... 2 7 7 Generation of a Polymerase Chain Reaction Renewable Source

of Subtractive cDNA, W. Midrael Kuehl and James Bat* ........................................ 2 8 7

PCR-Based Full-Length cDNA Cloning Utilizing the Universal- Adaptor/Specific DOS Primer-Pair Strategy,

David L. Cooper and Namyana R Zsola ..................................... 305 Use of Degenerate Oligonucleotide Primers and the Polymerase

Chain Reaction to Clone Gene Family Members, Gregory M. Preston ................................................................. -31 7

Single Specific Primer-Polymerase Chain Reaction (SSP-PCR) and Genome Walking,

Venkatakrishna ShyQmala and Giovanna Ferro-Luzri Ames .......... 339 cDNA Cloning by Inverse Polymerase Chain Reaction,

Sheng-He Huang, mun-Hua Wu, Bing Chi, and John Hotcenbetg ....................................................... 3 4 9

Amplification of Gene Ends from Gene Libraries by Polymerase Chain Reaction with Single-Sided Specificity,

Sheng-He Huang, Ambrose Y. Jong, Wu Yang, and John Holcenbetg ....................................................... 3 5 7

Anchoring a Defined Sequence to the 5' Ends of mRNAs: The Bolt to Clone Rare Full Length mRNAs and Generate M A Libraries from a F m Cells,

Jean Baptiste Dumas Milne Edwards, Jacques Delort, and Jacques Mallet ....................................................... 3 6 5

................................................................................................................ Index 387

Contributors

PUJA AGARWAL Department ofAnatomy, University of Connecticut Health Center, Famington, CT

ANTHONY ALBINO Mamia l Sloan-Ketten'ng Cancer Center, New Y d ROBERT C. ALLEN Department of Pathology and Laboratmy Medicine,

Medical University of South Carolina, Charleston, SC GIOVANNA FERRO-LUZZI AMES Department of Molecular and Cell Biology,

University of California, Berkeley, CA STEPHEN C. BAIN University of Oxford, John Radclzfle Hospital,

Headington, Oxford, UK DEBABRATA BANERJEE Memorial Sban-Keth'ng Cancer Center, New York SAILEN BARIK Department o f Molecular Biology, The Cleveland Clinic

Foundation, Cleveland, OH JAMES B A ~ Y NCI-Na y Medical Oncology, Bethesda, MD JOSEPH R. BERTINO Come11 University Graduate School of Medial Sciences

and the Laboratmy of Molecular Pharmacology, Memorial Sban- Kettering Cancer Center, New Yo&

LAURA J. BLOEM Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN

DAVID A. BRIAN Department of Microbiology, University of Tennessee, Knom'lle, T N

THOMAS R. BROKER Department of Biochemistry, University of Rochester School of Medicine, Rochester, NY

BRUCE BUDOWLE Formic Science Research and Training Center, Federal Bureau of Investigation Academy, Quantico, VA

BING CAI Division of Medical Genetics, University of S o u t h California, Childrens Hospital of Los Angeles, CA

SHIZHONG CHEN Molecular Genetics Laboratory, Center for Human Genome Research, The Salk Institute for Biological Sciences, Sun Diego, CA

CHENGMING CHIANG Depai-tment of Biochemist7y, University of Rochester School ofhledicine, Rochester, NY (Present address: Laboratory of Biochemistry and Molecular Biology, T h Rockefeller Univemvemty, New Yo&)

xii Contributors

LOUISE T. CHOW Department of Biochemisty, University of Rochster School of Medicine, Rochstq, AY

DAVID L. COOPER Department ofPathology, University ofPittsburgh School of Medicine, Pittsburgh, PA

KATHLEEN DANENBERG Kenneth Norris Jr. Cancer Cancer Hospital and Research Institute, University of S o u t h Califmia Comfnehensive Cancer Center, Los Angela, CA

PETER V. DANENBERG Xenneth N h s JT. Cancer Cancer Hospital and h e a r c h Institute, University of S o u t h California Comfnehensive Cancer Cenw, Los Angeles, CA

BEVERLY C. DELIDOW Department of Anatomy, University of Connecticut Health htq, Farmington, CT

JACQUES DELORT Laboratoire de Neurobiologie Cellulaire et Moleculaire, Gifsur Yvette, Cedex, France (Present address: Howard Hughs Medical Institute, University of Utah, Salt Lalze City, UT) P. DICKER Cornell University Graduate School of Medical Sciences

and the Laboratmy of Molecular Pharmacology, Memorial Sloan- Xeth'ng Cancer Center, New Yo?*

PATRICK J. DILLON D e p a ~ t ~ t of Gem Regulation, Roche Institute of Molecular Biology, Nuthy, NJ

CRAIG A. DIONNE Rhone-Poulenc Rmer Central Research, Collegm'lle, PA JEAN BAPTISTE DUMAS MILNE EDWARDS Labmatoire de Neurobiologie

Cellulaire el Moleculaire, Gif sur Yvette, Cedex, France GLEN A. EVANS Molecular Genetics Labmatmy, Center f m Human Genome

Research, The Salk Institute for Biological Scienm, Sun Diego, CA RENATO FANIN Memorial Sloan-&ttering Cancer Centq, N m Yorlz MARTIN A. HOFMANN Department of Microbiology, University o f Tennessee,

Knoxville, TN JOHN HOLCENBERG Division of Hernatology-Oncology, Department of

Pediat~ics, University of S o u t h California, Childrens Hospital of Los A ngeles, CA

TETSURO HORIKOSHI Kenneth N017i.s Jr. Canncer Cancer Hospital and Research Institute, University of Southern California Comfnehensive Cancer Center, Los Angeles, CA

ROBERT M. HORTON Depa~iment of Biochemist7y, College of Biological Sciences, University of Minnesota, St. Paul, MN

SHENG-HE HUANG Diwkion ofI-Iernatowcology, Dt$wtment ofpediatn'cs, University of S o u t h Cah&niu, ChiIdm Hosplpltul o f h Angeles, CA

... Contributors X L L Z

NARAYANA R. ISOLA Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, PA

MICHAEL JAYE RhonePoulenc Rorer Central Research, Collegm'lle, PA DOUGLAS H . JONES Department of Pediatrics, College of Medicine,

University of Iowa, Iowa City, IA AMBROSE Y. JONG Division of Hematology-Oncology, Departments of

Pediatrics and Microbiology, University of Southern Cal;fmia, Childrens Hospital of Los Angeles, CA

ELENA D. KA~Z Biotechnology Department, Perkin-Elmer, N m a k , CT CHRISTOPH KESSLER Department of Molecular Genetics, Boehringer

Mannheim GmbFI, Penzberg/Obb., Germany W. MICHAEL KUEHL N U - N a y Medical Oncology, Bethesda, MD JOHN P. LYNCH Department of Anatomy, University of Connecticut Health

Center, Farmington, CT JACQUES MALLET Labmatoire de Neurobiologie Cellulaire et Moleculaire,

Gzfsur Yvelte, Cedex, France TIM MCDANIEL University of Maryland Hospital, Baltimore, MD STEPHEN J . MELTZER University of Maryland EIospztal, Baltimore, MD JOHN J. PELUSO Departmmt ofOb/Gyn, University of Connecticut Health

Center, Famington, CT RICCARDO P E ~ Johns IIopkins University School of Medicine

and National Institute on Aging, National Institutes of Health, Department of Medicine, Baltimore, MD

GERD P. PFEIFER Department of Biology, Beckman Raearch Institute of the City of Hope, Duarte, CA

GREGORY M. P ~ O N Departments of Medicine and Cell Biology/Anatomy, Johns Hopkins University School of Medicine, Baltimore, MD

UDO REISCHL Department of Molecular Genetics, Boehringer Mannheim GmbH, Penzberg/Obb., Germany

ARTHUR D. RIGGS Department o f Biology, Beckman Research Institute of the City of Hope, Duarte, CA

CRAIG A. ROSEN Department of Gene Regulation, Roche Institute of Molecular Biology, Nutley, NJ

JESSE ROTH Johns IIopkins University School of Medicine and National Institute on Aging, National Institutes of Health, Department of Medicine, Bakimore, MD

RUDIGER RUGER Department of Molecular Genetics, Boehringer Mannheim GmbH, Penzberg/Obb., Germany

xiv Contributors

WOJCIECH RYCHLIK National Biosciences, Plymouth, MN LAWRENCE B. SCHOOK Laboratmy of Molecular Immunology, Department

of Animal Scienm, University of Illinois, Urbana, IL ALAN R. SHULDINER Johns Hopkins University School of Medicine and

National Institute on Aging, National Institutes $Health, Department of Medicine, Baltimore, MD

VENKATAKRISHNA SHYAMALA Chiron Corporation, EmeryvilZe, GI THOMAS STADLBAUER Xenneth Nmris Jr. Cancer Cancer Hospital and

Research Institute, University of Southern California Comprehensive Cancer Center, Los Angeles, CA

KEITH TANNER Johns Hopkins University School $Medicine, Department of Medicine, Baltimore, MD

JOHN A. TODD University of Oxford, John Radclzffe Hospital, Headington, Oxford, UK

MATTHIAS VOLKENANDT Department ofDmtology, University of Munich,

-ny BRUCE A. WHITE Department ofAnatomy, University o f Connecticut

Health Center, Fannington, CT STANLEY C. WINISTORFER Department of Pediatrics, College o f Medicine,

University ofIowa, Iowa City, IA ALICE L. WITSELL Laboratory of Molecular Immunology, Department of

Animal Sciences, University of Illinois, Urbana, IL CHUN-HUA WU Division of fImatology-Oncology, Department o f Pediatrics,

University of Southern Califmnia, Childrens Hospital of Los Angeles, CA Wu YANG Department of Molecular Pharmacology, University of Southern

Calijiiia, Childrens Ilospital of Los Angeles, CA LEI YU Department o f Medical and Molecular Genetics, Indiana University

School of Medicine, Indianapolis, 1Rr