In Vitro Mutagenesis Protocols

Methods in Molecular Biology John M. Walker, SERIES EDITOR

60. Protein NMR Protocols, edited by David G. Reid. 1996 59. Protein Purification Protocols, edited by Shawn Doonan,

1996

38. Basic DNA and RNA Protocols, edited by Adrian J. Harwood 1996

57. In Vitro Mutagenesis Protocols, edited by Michael fC. Trower, 1996

56. CrystaliograpWc Methods and Protocols, edited by Chris­topher Jones, Barbara Mulloy, and Mark Sanderson, 1996

55. Plant Cell Electroporation and Electrofuslon Protocols, edited by Jac A. Nickoloff, 1995

54. YAC Protocols, edited by David Markie, 1995 53. Yeast Protocols: Methods in Cell and Molecular Biology,

edited by Ivor H. Evans, 1996 52. Capillary Electrophoresis: Principles, Instrumentation,

and Applications, edited by Kevin D. Altria, 1996 51. Antibody Engineering Protocols, edited by Sudhir Paul,

1995 50. Species Diagnostics Protocols: PCR and Other Nucleic

Acid Methods, edited by Justin P. Clapp, 1996 49. Plant Gene Transfer and Expression Protocols,

edited by Heddwyn Jones, 1995 48. Animal Cell Electroporation and Electrofuslon Proto­

cols, edited by Jac A. Nickoloff, 1995 47. Electroporation Protocols for Microorganisms, edited by

Jac A. Nickoloff, 1995 46. Diagnostic Bacteriology Protocols, edited by Jenny

Howard and David M. Whitcombe, 1995 45. Monoclonal Antibody Protocols, edited by William C.

Davis, 1995 44. AgrobttCUrium Protocols, edited by Kevan M. A. Garlland

and Michael R. Davey, 1995 43. In Vitro Toxicity Testing Protocols, edited by Sheila

O'Hare and Chris K. Atterwill, 1995 42. ELISA: Theory and Practiccby John R. Crowther. 1995 41. Signal Transduction Protocols, edited by David A. Kendall

and Stephen J Hill, 1995 40. Protein Stability and Folding: Theory and Practice,

edited by Bret A. Shirley, 1995 39. Baculovirus Expression Protocols, edited by Christopher

D. Richardson, 1995 38. Cryoprescrvation and Freeze-Drying Protocols,

edited by John G. Day and Mark R. McLellan, 1995 37. In Vitro Transcription and Translation Protocols,

edited by Martin J. Tymms, 1995 36. Peptide Analysis Protocols, edited by Ben M. Dunn and

Michael W. Pennington, 1994 35. Peptide Synthesis Protocols, edited by Michael W.

Pennington and Ben M. Dunn, 1994 34. Immunocytochemical Methods and Protocols, edited by

Lorette C. Javois, 1994 33. In Situ Hybridization Protocols, edited by K. H. Andy

Choo, 1994 32. Basic Protein and Peptide Protocols, edited by John M.

Walker, 1994

31. Protocols for Gene Analysis, edited by Adrian J. Harwood, 1994

30. DNA-Protein Interactions, edited by G. GeoffKneale, 1994 29. Chromosome Analysis Protocols, edited by John R.

Gosden, 1994 28. Protocols for Nucleic Acid Analysis by Nonradioactive

Probes, edited by Peter G. Isaac, 1994 27. Biomembrane Protocols: //. Architecture and Function,

edited by John M. Graham and Joan A. Higgins, 1994 26. Protocols for Oligonucleotide Conjugates: Synthesis and

Analytical Techniques, ediied by Sudhir Agrawal, 1994 25. Computer Analysis of Sequence Data: Part 11, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 24. Computer Analysis of Sequence Data: Part I, edited by

Annette M. Griffin and Hugh G. Griffin, 1994 23. DNA Sequencing Protocols, edited by Hugh G. Griffin and

Annette M. Griffin, 199} 22. Microscopy, Optical Spectroscopy, and Macroscopic

Techniques, edited by Christopher Jones, Barbara Mulloy, and Adrian H. Thomas, 1993

21. Protocols in Molecular Parasitology, edited by John E. Hyde, 1993

20. Protocols for Oligonucleotides and Analogs: Synthesis and Properties, edited by Sudhir Agrawal, 1993

19. Biomembrane Protocols: /. Isolation and Analysis, edited by John M. Graham and Joan A. Higgins, 1993

18. Transgenesis Techniques: Principles and Protocols, edited by David Murphy and David A. Carter, 1993

17. Spectroscopic Methods and Analyses: NMR, Mass Spec­trometry, and Metalloprotein Techniques, edited by Chris­topher Jones, Barbara Mulloy, and Adrian H. Thomas, 1993

16. Enzymes of Molecular Biology, edited by Michael M. Burrell, 1993

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

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

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

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

11. Practical Protein Chromatography, edited by Andrew Kenney and Susan Powell. 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: Viral Vectors for Gene Expression, edited by Mary K. L. Collins, 1991

7. Gene Transfer and Expression Protocols, edited by Edward 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 Jeffrey W. Pollard and John M. Walker, 1990

Methods in Molecular Biology • 57

In Vitro Mutagenesis Protocols

Edited by

Michael K. Troiver Glaxo Research and Development Ltd., Glaxo-Wellcome

Medicines Research Centre, Stevenage, Hertfordshire, UK

Humana Press Totowa, New Jersey

Dedication To my wife, Diana,

and my children, Isabella, Florence, and Sebastian

© 1996 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512

For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 201-256-1699; Fax: 201-256-8341; E-mail: humana@interramp.com

All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology- is a trademark of The Humana Press Inc.

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This publication is printed on acid-free paper. <S> ANSi Z39.48-1984 (American Standards Institute) Permanence of Paper for Printed Library Materials.

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Preface

In Vitro Mutagenesis Protocols is about mutagenesis. Not mutagen­esis in which living cells are exposed to cocktails of chemicals or doses of damaging radiation, but rather a more refined approach in which spe­cific DNA sequences are targeted for alteration in vitro as designated by the investigator. This facility has been brought about by the advent of recombinant DNA technology, the arrival of which has provided extraordinarily powerful tools for the manipulation of DNA. Such tools have empowered us with the capability to readily engineer defined-target DNA sequences, providing unprecedented opportunities to study gene regulation and to probe structure/function relationships in proteins. This technology has also enabled us to revise DNA sequences for other purposes such as vector construction.

In Vitro Mutagenesis Protocols was designed to bring together a wide and varied array of specific mutagenesis protocols for both site-directed and random mutagenesis into a single-source volume. The book is informally divided into two parts, the first on protocols for site-directed mutagenesis, incorporating a number of methods based on strand selection, amber stop codon suppression, gapped-duplex formation, solid-phase technology, triple-helix formation, the ligase chain reac­tion, and a host of polymerase chain reaction-based procedures includ­ing splicing by overlap extension and the megaprimer technique. The second is devoted to random mutagenic approaches encompassing pro­tocols, many in combination with the polymerase chain reaction, based on degenerate oligonucleotides, cassette mutagenesis, linker-scanning mutagenesis, chemical mutagenesis, nested-deletion mutagenesis, the infidelity of Taq DNA polymerase, and a DNA repair mechanism-defi­cient strain of Escherichia coll In my role as editor I have striven to ensure that the chapters are understandable to the informed nonspecial-ist molecular biologist and that each protocol details extensive practical

vi Preface

information to ensure that already competent scientists can utilize the methodology described at their own laboratory benches.

The revolutionary impact that in vitro mutagenesis has made in our understanding of the biological universe cannot be understated. This technology will continue to play a critical role in research labora­tories around the world, whether it is for identifying altered protein prop­erties and activities crucial to the development of the biotechnology industry or for unraveling the biological roles of the wealth of genes of unknown function currently being isolated from the human genome and those of other model organisms. It is clear that the need for efficient, rapid, and practical in vitro mutagenesis protocols has never been greater. It is therefore my fervent hope and wish that the time, effort, and sacri­fices taken in creating this volume of protocols will be rewarded through satisfying the thirst of those scientists eager to drink from this particu­lar foimtain of knowledge!

Michael K. Trower

Contents

Preface v

Contributors xi

Ch. 1. Site-Directed Mutagenesis Using Positive Antibiotic Selection, Richard N. Bohnsack 1

Ch. 2. In Vitro Site-Directed Mutagenesis Using the Unique Restriction Site Elimination (USE) Method,

LiZhu J3 Ch. 3. Site-Directed Mutagenesis Using Double-Stranded Plasmid DNA

Templates, Jeffrey Braman, Carol Papworth, and Alan Greener 31

Ch. 4. Site-Directed Mutagenesis Using a Uracil-Containing Phagemid Template,

Christian Hagemeier 45 Ch. 5. Oligonucleotide-Directed Mutagenesis Using an Improved

Phosphorothioate Approach, Susan J. Dale and Ian R. Felix 55

Ch. 6. Analysis of Point Mutations by Use of Amber Stop Codon Suppression,

Scott A. Lesley 65 Ch. 7. A Simple Method for Site-Directed Mutagenesis

with Double-Stranded Plasmid DNA, Derhsing Lai and Sidney Pestka 75

Ch. 8. Double-Stranded DNA Site-Directed Mutagenesis, Stephane Viville 87

Ch. 9. Solid-Phase In Vitro Mutagenesis Using a Plasmid DNA Template, Roy Edward 97

Ch. 10. Targeted Mutagenesis Mediated by the Triple Helix Formation, Peter M. Glazer, Gan Wang, Pamela A. Havre,

andEdwardJ. Gunther 109 Ch. 11. A Universal Nested Deletion Method Using an Arbitrary Primer

and Elimination of a Unique Restriction Site, LiZhu and Ann E. Holtz 119

Ch. 12. Ordered Deletions Using Exonuclease III, Denise Clark and Steven Henikoff 139

vii

via Contents

Ch. 13. Ligase Chain Reaction for Site-Directed In Vitro Mutagenesis, Gerard J. A. Rouwendal, Emit J. H. Wolbert, Lute-Harm Zwiers,

and Jan Springer 149 Ch. 14. PCR-Based Site-Directed Mutagenesis,

Atsushi Shlmada 157 Ch. 15. In Vitro Recombination and Mutagenesis by Overlap

Extension PCR, Robert J. Pougulis, Abbe N. Vallejo, and Larry R. Pease 167

Ch. 16. Site-Directed Mutagenesis Using Overlap Extension PCR, AshokAiyar, YanXiang, and Jonathan Leis 177

Ch. 17. Modificationof the Overlap Extension Method for Extensive Mutagenesis on the Same Template,

Ivan Mikaelian and Alain Sergeant 193 Ch. 18. Site-Directed Mutagenesis In Vitro by Megaprimer PCR,

Sailen Barik 203 Ch. 19. Using PCR for Rapid Site-Specific Mutagenesis in Large Plasmids,

BrynmorA. Watkins and Marvin S. Reitz, Jr. 217 Ch. 20. PCR-Assisted Mutagenesis for Site-Directed Insertion/Deletion

of Large DNA Segments, Daniel C. Tessier and David Y. Thomas 229

Ch. 21. Site-Directed Mutagenesis Using a Rapid PCR-Based Method, Gina L. Costa, John C. Bauer, Barbara McGowan, Mila Angert,

and Michael P. Weiner 239 Ch. 22. A Simple Method to Introduce Internal Deletions or Mutations

into Any Position of a Target DNA Sequence, Marjana Tomic-Canic, Franfoise Bernerd,

and Miroslav Blumenberg 249 Ch. 23. A Simple Method for Site-Specific Mutagenesis that Leaves

the Rest of the Template Unaltered, Marjana Tomic-Canic, Ivana Sunjevaric,

and Miroslav Blumenberg 259 Ch. 24. Muhiple Site-Directed Mutagenesis,

Kolari S. Bhat 269 Ch. 25. Construction of Linker-Scanning Mutations by Oligonucleotide

Ligation, Grace M. Hobson, Patricia P. Harlow,

and Pamela A. Benfield 279 Ch. 26. Construction ofLinker-Scanning Mutations Using PCR,

Patricia P. Harlow, Grace M. Hobson, and Pamela A. Benfield 287

Ch. 27. Use of Codon Cassette Mutagenesis for Saturation Mutagenesis, DeenaM. Kegler-Ebo, Glenda W. Polack, and Daniel DiMaio 297

Contents ix

Ch. 28. Saturation Mutagenesis by Mutagenic Oligonucleotide-Directed PCR Amplification (Mod-PCR),

Lillian W. Chiang 311 Ch. 29. Random Mutagenesis of Short Target DNA Sequences via PCR

with Degenerate Oligonucleotides, Frank Kirchhoff and Ronald C. Desrosiers 323

Ch. 30. Random Sequence Mutagenesis for the Generation of Active Enz5̂ mes,

Margaret E. Black and Lawrence A. Loeb 535 Ch. 31. Random Mutagenesis by Using Mixtures of dNTP and dITP in PCR,

Oscar P. Kuipers 351 Ch. 32. PCR-Mediated Chemical Mutagenesis,

Donald J. Roufa 357 Ch. 33. Oligonucleotide-Directed Random Mutagenesis Using the

Phosphorothioate Method, Susan J. Dale and Maxine Belfield 369

Ch. 34. An Efficient Random Mutagenesis Technique Using an E. colt Mutator Strain,

Alan Greener, Marie Callahan, and Bruce Jerpseth 375 Index 387

Contributors

MiLA ANGERT • Stratagene Inc.. La Jolla, CA AsHOK AiYAR • Department of Biochemistry, School of Medicine,

Case Western Reserve University, Cleveland, OH SAILEN BARIK • Department of Biochemistry and Molecular Biology,

School of Medicine, University of South Alabama, Mobile, AL JOHN C . BAUER • Stratagene Inc., La Jolla, CA MAXINE BELFIELD • Amersham International, Buckinghamshire, UK PAMELA A. BENFIELD • Research and Development, DuPont Merck,

Wilmington, DE FRANCOISE BERNERD • L 'Oreal Laboratories, Clichy, France KoLARi S. BHAT • Department of Cell Biology, Vanderbilt University,

Nashville, TN MARGARET E . BLACK • Department of Pathology, School of Medicine,

University of Washington, Seattle, WA MiROSLAV BLUMENBERG • Department of Dermatology, New York

University Medical Center, New York, NY RICHARD N . BOHNSACK • Department of Biochemistry, Medical College

of Wisconsin, Milwaukee, WI JEFFREY BRAMAN • Stratagene Inc., La Jolla, CA MARIE CALLAHAN • Stratagene Inc., La Jolla, CA LILLIAN W . CHIANG • Department of Neurobiology, Stanford University

School of Medicine, Stanford, CA DENISE CLARK • Fred Hutchinson Cancer Research Center, Howard

Hughes Medical Institute, Seattle, WA GiNA L. COSTA • Stratagene Inc., La Jolla, CA SUSAN J. DALE • Amersham International, Buckinghamshire, UK RONALD C . DESROSIERS • New England Regional Primate Research

Center, Harvard Medical School, Southboro, MA DANIEL DIMAIO • Department of Genetics, Yale University School

of Medicine, New Haven, CT

xi

xii Contributors

ROY EDWARD • Dynal Ltd., Wirral, UK IAN R. FELIX • Amersham International, Buckinghamshire, UK PETER M . GLAZER • Department of Therapeutic Radiology, Yale

University School of Medicine, New Haven, CT ALAN GREENER • Stratagene Inc., La Jolla, CA EDWARD J. GUNTHER • Department of Therapeutic Radiology, Yale

University School of Medicine, New Haven, CT CHRISTIAN HAGEMEIER • Laboratory of Molecular Biology, Department

of Pediatrics, Humboldt University, Berlin, Germany PATRICIA P. HARLOW • Research and Development, DuPont Merck,

Wilmington, DE PAMELA A. HAVRE • Department of Therapeutic Radiology, Yale

University School of Medicine, New Haven, CT STEVEN HENIKOFF • Fred Hutchinson Cancer Research Center, Howard

Hughes Medical Institute, Seattle, WA GRACE M . HOBSON • Research and Development, DuPont Merck,

Wilmington, DE ANN E . HOLTZ • Clontech Laboratories, Palo Alto, CA BRUCE JERPSETH • Stratagene Inc., La Jolla, CA DEENA M . KEGLER-EBO • Department of Genetics, Yale University

School of Medicine, New Haven, CT FRANK KIRCHHOFF • Virology Institute, Erlangen-Nurnberg University,

Erlangen, Germany OSCAR P. KUIPERS • Netherlands Institute for Dairy Research, Ede,

The Netherlands DERHSING LAI • Department of Molecular Genetics and Microbiology,

University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ

JONATHAN LEIS • Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH

SCOTT A. LESLEY • Promega Corp., Madison, WI LAWRENCE A. LOEB • Department of Pathology, School of Medicine,

University of Washington, Seattle, WA BARBARA MCGOWAN • Stratagene Inc., La Jolla, CA IVAN MIKAELIAN • MRC-LMB, Cambridge, UK CAROL PAPWORTH • Stratagene Inc., La Jolla, CA

Contributors xiii

LARRY R. PEASE • Department of Immunology, Mayo Clinic, Rochester, MN SIDNEY PESTKA • Department of Molecular Genetics and Microbiology,

University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, NJ

GLENDA W . POLACK • Department of Genetics, Yale University School of Medicine, New Haven, CT

ROBERT J. POUGULIS • Department of Immunology, Mayo Clinic, Rochester, MN

MARVIN S. REITZ, JR • Laboratory of Tumor Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD

DONALD J. ROUFA • Center for Basic Cancer Research, Division of Biology, Kansas State University, Manhattan, KS

GERARD J. A. ROUWENDAL • Agrotechnological Research Institute, Wageningen, The Netherlands

ALAIN SERGEANT • Ecole Normale Superieure de Lyon, France ATSUSHI SHIMADA • Department of Molecular Pathology, Takara Shuzo Co.,

Shiga, Japan JAN SPRINGER • Agrotechnological Research Institute, Wageningen,

The Netherlands IvANA SUNJEVARIC • Department of Genetics and Development, Columbia

University, New York, NY DANIEL C . TESSIER • National Research Council of Canada,

Biotechnology Research Institute, Montreal, Quebec, Canada DAVID Y . THOMAS • National Research Council of Canada,

Biotechnology Research Institute, Montreal, Quebec, Canada MARJANA TOMIC-CANIC • Department of Dermatology, New York

University Medical Center, New York, NY ABBE N . VALLEJO • Department of Immunology, Mayo Clinic,

Rochester, MN STEPHANE VIVILLE • Institute for Genetic and Molecular and Cellular

Biology, Louis Pasteur University, Strasboug, France GAN WANG • Department of Therapeutic Radiology, Yale University

School of Medicine, New Haven, CT BRYNMOR A . WATKINS • Laboratory of Tumor Cell Biology, National

Cancer Institute, National Institutes of Health, Bethesda, MD MICHAEL P. WEINER • Glaxo Inc., NC

xiv Contributors

EMIL J. H. WoLBERT • Agrotechnologicol Research Institute, Wageningen, The Netherlands

YAN XIANG • Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, OH

LI ZHU • Clontech Laboratories, Palo Alto, CA LUTE-HARM ZWIERS • Agrotechnologicol Research Institute, Wageningen,

The Netherlands