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: [email protected]
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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