In Vitro Mutagenesis Protocols

305 Pages·2002·1.871 MB·English

by  Priya Handa, Swapna Thanedar, Umesh Varshney (auth.), Jeff Braman (eds.)

In Vitro Mutagenesis Protocols
huangzhiman 2002.12.18
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M e t h o d s i n M o l e c u l a r B I O L O G YTM
In Vitro Mutagenesis
Protocols
Second Edition
Edited by
Jeff Braman
Stratagene, La Jolla, CA
Humana Press Totowa, New Jersey
© 2002 Humana Press Inc.
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Mutagenesis,” by Tomoaki Matsuura, Tetsuya Yomo, and Itaru Urabe.
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Main entry under title: Methods in molecular biology™.
In vitro mutagenesis protocols/edited by Jeff Braman.—2nd ed.
p.cm.—(Methods in molecular biology; v. 182)
Includes bibliographical references and index.
ISBN 0-89603-910-2 (alk. paper)
1. Mutagenesis—Methodology. I. Braman, Jeff. II. Methods in molecular biology
(Totowa, N.J.); v. 182.
QH465.A1 15 2001
660.6'5—dc21 2001039227
Dedication
To Barbara, Ryan, Emily, Rebecca, Michael, Colin, and Connor
Jeff Braman, PhD
v

Preface
In vitro mutagenesis is a major tool used by molecular biologists to make
connections between nucleotide sequence and sequence function. In the post-
genome era, in vitro mutagenesis is being used to establish the function of
components of the proteome. There has never been a more exciting and criti-
cal time for molecular biologists to master the use of efficient and reliable in
vitro mutagenesis protocols.
Anyone skilled in the use of tools will tell you that a well-equipped toolbox
is essential for solving the myriad problems encountered in the practice of
their art. Likewise, molecular biologists require an arsenal of reliable tools
appropriate to solve complex problems they encounter. In Vitro Mutagenesis
Protocols is intended to represent such a toolbox. Chapter authors were cho-
sen because their protocols (tools) have been published in reputable, peer-
reviewed journals. Their chapters focus on improvements to conventional
site-directed mutagenesis, including a chapter on chemical site-directed
mutagenesis, PCR-based mutagenesis and modifications thereto allowing high
throughput mutagenesis experiments, and mutagenesis based on gene disrup-
tion (both in vitro and in situ based). Last, but certainly not least, a section of
chapters is devoted to the subject of accelerated protein evolution relying on
in vitro evolution, gene shuffling, and random mutagenesis.
I trust that these protocols will be successful in your hands and allow you to
quickly reach the point of analyzing the results for inclusion in the discussion
section of your own peer reviewed journal article. I am indebted to Humana
Press representatives Craig Adams and Professor John Walker for guiding me
through the process of editing this book and to the many good scientists from Mas-
sachusetts to California who took the time to help me with my own research.
Jeff Braman, PhD
vii

Contents
Dedication.........................................................................................................v
Preface ...........................................................................................................vii
Contributors...................................................................................................xiii
1 Rapid and Reliable Site-Directed Mutagenesis
Using Kunkel's Approach
Priya Handa, Swapna Thanedar,
and Umesh Varshney........................................................................1
2 Site-Directed Mutagenesis Using Altered
β-Lactamase Specificity
Christine A. Andrews and Scott A. Lesley.........................................7
3 Site-Directed Mutagenesis Facilitated by DpnI Selection
on Hemimethylated DNA
Fusheng Li and James I. Mullins.......................................................19
4 Multiple Site-Directed Mutagenesis In Vitro
Yang-Gyun Kim and Stefan Maas......................................................29
5 Two-Stage Polymerase Chain Reaction Protocol Allowing
Introduction of Multiple Mutations, Deletions, and Insertions,
Using QuikChange™ Site-Directed Mutagenesis
Wenyan Wang and Bruce A. Malcolm...............................................37
6 Efficient and Accurate Site-Directed Mutagenesis
of Large Plasmids
Susan A. Nadin-Davis.........................................................................45
7 Combining Site-Specific Chemical Modification with Site-Directed
Mutagenesis: Versatile Strategy to Move Beyond Structural
Limitations of 20 Natural Amino Acids Side Chains
in Protein Engineering
Grace DeSantis and J. Bryan Jones.................................................55
8 Site-Directed Mutagenesis Mediated by a Single Polymerase
Chain Reaction Product
Xueni Chen, Weimin Liu, Ileana Quinto,
and Giuseppe Scala........................................................................67
ix