Table Of ContentM E T H O D S I N M O L E C U L A R B I O L O G YTM
John M. Walker, SERIES EDITOR
210.MHC Protocols, edited by Stephen H. Powis and Robert W. 181.Genomic Imprinting: Methods and Protocols, edited by
Vaughan, 2003 Andrew Ward, 2002
209.Transgenic Mouse Methods and Protocols, edited by Marten 180.Transgenesis Techniques, 2nd ed.: Principles and Protocols,
Hofker and Jan van Deursen, 2002 edited by Alan R. Clarke, 2002
208.Peptide Nucleic Acids: Methods and Protocols, edited by 179.Gene Probes: Principles and Protocols, edited by Marilena
Peter E. Nielsen, 2002 Aquino de Muro and Ralph Rapley, 2002
207.Human Antibodies for Cancer Therapy: Reviews and Protocols. 178.`Antibody Phage Display: Methods and Protocols, edited by
edited by Martin Welschof and Jürgen Krauss, 2002 Philippa M. O’Brien and Robert Aitken, 2001
206.Endothelin Protocols, edited by Janet J. Maguire and Anthony 177.Two-Hybrid Systems: Methods and Protocols, edited by Paul
P. Davenport, 2002 N. MacDonald, 2001
205.E. coli Gene Expression Protocols, edited by Peter E. 176.Steroid Receptor Methods: Protocols and Assays, edited by
Vaillancourt, 2002 Benjamin A. Lieberman, 2001
204.Molecular Cytogenetics: Methods and Protocols, edited by 175.Genomics Protocols, edited by Michael P. Starkey and
Yao-Shan Fan, 2002 Ramnath Elaswarapu, 2001
203.In Situ Detection of DNA Damage: Methods and Protocols, 174.Epstein-Barr Virus Protocols, edited by Joanna B. Wilson
edited by Vladimir V. Didenko, 2002 and Gerhard H. W. May, 2001
202.Thyroid Hormone Receptors: Methods and Protocols, edited 173.Calcium-Binding Protein Protocols, Volume 2: Methods and
by Aria Baniahmad, 2002 Techniques,edited by Hans J. Vogel, 2001
201.Combinatorial Library Methods and Protocols, edited by 172.Calcium-Binding Protein Protocols, Volume 1: Reviews and
Lisa B. English, 2002 Case Histories, edited by Hans J. Vogel, 2001
200.DNA Methylation Protocols, edited by Ken I. Mills and Bernie 171.Proteoglycan Protocols, edited by Renato V. Iozzo, 2001
H, Ramsahoye, 2002 170.DNA Arrays: Methods and Protocols, edited by Jang B.
199.Liposome Methods and Protocols, edited by Subhash C. Basu Rampal, 2001
and Manju Basu, 2002 169.Neurotrophin Protocols, edited by Robert A. Rush, 2001
198.Neural Stem Cells: Methods and Protocols, edited by Tanja 168.Protein Structure, Stability, and Folding, edited by Kenneth
Zigova, Juan R. Sanchez-Ramos, and Paul R. Sanberg, 2002 P. Murphy, 2001
197.Mitochondrial DNA: Methods and Protocols, edited by William 167.DNA Sequencing Protocols, Second Edition, edited by Colin
C. Copeland, 2002 A. Graham and Alison J. M. Hill, 2001
196.Oxidants and Antioxidants: Ultrastructural and Molecular 166. Immunotoxin Methods and Protocols, edited by Walter A. Hall, 2001
Biology Protocols, edited by Donald Armstrong, 2002 165.SV40 Protocols, edited by Leda Raptis, 2001
195.Quantitative Trait Loci: Methods and Protocols,edited by 164.Kinesin Protocols, edited by Isabelle Vernos, 2001
Nicola J. Camp and Angela Cox, 2002 163.Capillary Electrophoresis of Nucleic Acids, Volume 2:
194.Posttranslational Modifications of Proteins: Tools for Func- Practical Applications of Capillary Electrophoresis, edited by
tional Proteomics,edited by Christoph Kannicht, 2002 Keith R. Mitchelson and Jing Cheng, 2001
193.RT-PCR Protocols, edited by Joseph O’Connell, 2002 162.Capillary Electrophoresis of Nucleic Acids, Volume 1:
192.PCR Cloning Protocols, 2nd ed., edited by Bing-Yuan Chen Introduction to the Capillary Electrophoresis of Nucleic Acids,
and Harry W. Janes, 2002 edited by Keith R. Mitchelson and Jing Cheng, 2001
191.Telomeres and Telomerase:Methods and Protocols, edited 161.Cytoskeleton Methods and Protocols, edited by Ray H. Gavin, 2001
byJohn A. Double and Michael J. Thompson, 2002 160.Nuclease Methods and Protocols, edited by Catherine H.
190.High Throughput Screening: Methods and Protocols, edited Schein, 2001
byWilliam P. Janzen, 2002 159.Amino Acid Analysis Protocols, edited by Catherine Cooper,
189.GTPase Protocols: The RAS Superfamily, edited by Edward Nicole Packer, and Keith Williams, 2001
J. Manser and Thomas Leung, 2002 158.Gene Knockoout Protocols, edited by Martin J. Tymms and
188.Epithelial Cell Culture Protocols, edited by Clare Wise, 2002 Ismail Kola, 2001
187.PCR Mutation Detection Protocols, edited by Bimal D. M. 157.Mycotoxin Protocols, edited by Mary W. Trucksess and Albert
Theophilus and Ralph Rapley, 2002 E. Pohland, 2001
186.Oxidative Stress and Antioxidant Protocols, edited by 156.Antigen Processing and Presentation Protocols, edited by
Donald Armstrong, 2002 Joyce C. Solheim, 2001
185.Embryonic Stem Cells: Methods and Protocols, edited by 155.Adipose Tissue Protocols, edited by Gérard Ailhaud, 2000
Kursad Turksen, 2002 154.Connexin Methods and Protocols, edited by Roberto Bruzzone
184.Biostatistical Methods, edited by Stephen W. Looney, 2002 and Christian Giaume, 2001
183.Green Fluorescent Protein: Applications and Protocols, edited 153.Neuropeptide Y Protocols, edited by Ambikaipakan
byBarry W. Hicks, 2002 Balasubramaniam, 2000
182.In Vitro Mutagenesis Protocols, 2nd ed., edited by Jeff 152.DNA Repair Protocols: Prokaryotic Systems, edited by Patrick
Braman, 2002 Vaughan, 2000
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
PCR Cloning
Protocols
Second Edition
Edited by
Bing-Yuan Chen
and
Harry W. Janes
Rutgers University,
New Brunswick, NJ
Humana Press Totowa, New Jersey
© 2002 Humana Press Inc.
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PCR cloning protocols: second edition / edited by Bing-Yuan Chen and Harry W. Janes.--2nd ed.
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ISBN 0-89603-969-2 (hb : alk. paper) -- ISBN 0-89603-973-0 (comb. : alk. paper)
1. Molecular cloning--Laboratory manuals. 2. Polymerase chain reaction--Laboratory
manuals. I. Chen, Bing-Yuan. II. Janes, Harry W. III. Methods in molecular biology
(Clifton, N.J.) ; v. 192
QH442.2 .P37 2002
572.8'6--dc21
2001039702
Preface
PCR is probably the single most important methodological invention in
molecular biology to date. Since its conception in the mid-1980s, it has rapidly
become a routine procedure in every molecular biology laboratory for identify-
ing and manipulating genetic material, from cloning, sequencing, mutagenesis,
to diagnostic research and genetic analysis. What’s astounding about this inven-
tion is that new and innovative applications of PCR have been generated with
stunning regularity; its potential has shown no signs of leveling off. New
applications for PCR are literally transforming molecular biology. In the post-
genomic era, PCR has especially become the method of choice to clone existing
genes and generate a wide array of new genes by mutagenesis and/or recombina-
tion within the genes of interest. The fast and easy availability of these genes is
essential for the study of functional genomics, gene expression, protein struc-
ture–function relationships, protein–protein interactions, protein engineering, and
molecular evolution.
PCR Cloning Protocols was prepared in response to the need to have an
up-to-date compilation of proven protocols for PCR cloning and mutagenesis. It
builds upon the best-selling first edition, PCR Cloning Protocols: From Molecu-
lar Cloning to Genetic Engineering, a book in the Methods in Molecular Biol-
ogy™ series published in 1997. We divided the new edition into five parts. Part
I.Performing and Optimizing PCR, contains basic PCR methodology, includ-
ing PCR optimization and computer programs for PCR primer design and analy-
sis, as well as novel variations for cloning genes of particular characteristics or
origins, emphasizing long-distance PCR and GC-rich template amplification.
Part II. Cloning PCR Products, presents both conventional and novel enzyme-
free and restriction site-free procedures to clone PCR products into various vec-
tors, either directionally or non-directionally. Part III. Mutagenesis and
Recombination, addresses the use of PCR to facilitate DNA mutagenesis and
recombination in various innovative approaches to generate a wide array of
mutants. Part IV. Cloning Unknown Neighboring DNA, contains a compre-
hensive collection of protocols to fulfill the frequent and challenging task of
cloning uncharacterized DNA flanking a known DNA fragment. Finally, Part V.
Library Construction and Screening, addresses particular applications of PCR
in library and sublibrary generation and screening. Each part also contains an
overview, which summarizes the current methods available and their underlying
v
vi Preface
strategies, advantages, and disadvantages for that particular topic. These reviews
are especially helpful to new researchers to orient themselves with the field and
to guide them to choose a procedure that is most suitable for their experiments.
We hope that PCR Cloning Protocols will provide readily reproducible
laboratory protocols that researchers in the field will follow closely and thereby
increase their success rate in their experiments.
We are indebted to Mirah Riben for her superb help during the editing of
the book. We also thank Prof. John M. Walker, the series editor, for his help,
advice, and guidance.
Bing-Yuan Chen
Harry W. Janes
Contents
Preface .............................................................................................................v
Contributors.....................................................................................................xi
PART I. PERFORMINGAND OPTIMIZING PCR
1 Polymerase Chain Reaction: Basic Principles and Routine Practice
Lori A. Kolmodin and David E. Birch..................................................3
2 Computer Programs for PCR Primer Design and Analysis
Bing-Yuan Chen, Harry W. Janes, and Steve Chen........................19
3 Single-Step PCR Optimization
Using Touchdown and Stepdown PCR Programming
Kenneth H. Roux..................................................................................31
4 XL PCR Amplification of Long Targets from Genomic DNA
Lori A. Kolmodin..................................................................................37
5 Coupled One-Step Reverse Transcription and Polymerase Chain
Reaction Procedure for Cloning Large cDNA Fragments
Jyrki T. Aatsinki...................................................................................53
6 Long Distance Reverse-Transcription PCR
Volker Thiel, Jens Herold, and Stuart G. Siddell.............................59
7 Increasing PCR Sensitivity for Amplification
from Paraffin-Embedded Tissues
Abebe Akalu and Juergen K. V. Reichardt.......................................67
8 GC-Rich Template Amplification by Inverse PCR:
DNA Polymerase and Solvent Effects
Alain Moreau, Da Shen Wang, Steve Forget, Colette Duez,
and Jean Dusart...............................................................................75
9 PCR Procedure for the Isolation of Trinucleotide Repeats
Teruaki Tozaki......................................................................................81
10 Methylation-Specific PCR
Haruhiko Ohashi..................................................................................91
vii
viii Contents
11 Direct Cloning of Full-Length Cell Differentially Expressed Genes
by Multiple Rounds of Subtractive Hybridization
Based on Long-Distance PCR and Magnetic Beads
Xin Huang, Zhenglong Yuan, and Xuetao Cao................................99
PART II. CLONING PCR PRODUCTS
12 Cloning PCR Products: An Overview
Baotai Guo and Yuping Bi................................................................111
13 Using T4 DNA Polymerase to Generate Clonable PCR Products
Kai Wang.............................................................................................121
14 Enzyme-Free Cloning of PCR Products
and Fusion Protein Expression
Brett A. Neilan and Daniel Tillett.....................................................125
15 Directional Restriction Site-Free Insertion of PCR Products
into Vectors
Guo Jun Chen....................................................................................133
16 Autosticky PCR:
Directional Cloning of PCR Products with Preformed 5' Overhangs
József Gál and Miklós Kálmán.........................................................141
17 A Rapid and Simple Procedure for Direct Cloning
of PCR Products into Baculoviruses
Tamara S. Gritsun, Michael V. Mikhailov,
and Ernest A. Gould......................................................................153
PART III. MUTAGENESISAND RECOMBINATION
18 PCR Approaches to DNA Mutagenesis and Recombination:
An Overview
Binzhang Shen...................................................................................167
19 In-Frame Cloning of Synthetic Genes Using PCR Inserts
James C. Pierce.................................................................................175
20 Megaprimer PCR
Sailen Barik........................................................................................189
21 PCR-Mediated Recombination:
A General Method Applied to Construct Chimeric Infectious
Molecular Clones
Guowei Fang, Barbara Weiser, Aloise Visosky, Timothy Moran,
and Harold Burger.........................................................................197
22 PCR Method for Generating Multiple Mutations at Adjacent Sites
Jiri Adamec.........................................................................................207
Contents ix
23 A Fast Polymerase Chain Reaction-Mediated Strategy for Introducing
Repeat Expansions into CAG-Repeat Containing Genes
Franco Laccone.................................................................................217
24 PCR Screening in Signature-Tagged Mutagenesis of Essential Genes
Dario E. Lehoux and Roger C. Levesque.......................................225
25 Staggered Extension Process (StEP) In Vitro Recombination
Anna Marie Aguinaldo and Frances Arnold...................................235
26 Random Mutagenesis by Whole-Plasmid PCR Amplification
Donghak Kim and F. Peter Guengerich..........................................241
PART IV. CLONING UNKNOWN NEIGHBORING DNA
27 PCR-Based Strategies to Clone Unknown DNA Regions
from Known Foreign Integrants: An Overview
Eric Ka-Wai Hui, Po-Ching Wang, and Szecheng J. Lo................249
28 Long Distance Vectorette PCR (LDV PCR)
James A. L. Fenton, Guy Pratt, and Gareth J. Morgan.................275
29 Nonspecific, Nested Suppression PCR Method
for Isolation of Unknown Flanking DNA (“Cold-Start Method”)
Michael Lardelli..................................................................................285
30 Inverse PCR: cDNA Cloning
Sheng-He Huang................................................................................293
31 Inverse PCR: Genomic DNA Cloning
Ambrose Y. Jong, Anna T’ang, De-Pei Liu,
and Sheng-He Huang....................................................................301
32 Gene Cloning and Expression Profiling by Rapid Amplification
of Gene Inserts with Universal Vector Primers
Sheng-He Huang, Hua-Yang Wu, and Ambrose Y. Jong..............309
33 The Isolation of DNA Sequences Flanking Tn5 Transposon Insertions
by Inverse PCR
Vincent J. J. Martin and William W. Mohn......................................315
34 Rapid Amplification of Genomic DNA Sequences Tagged
by Insertional Mutagenesis
Martina Celerin and Kristin T. Chun................................................325
35 Isolation of Large Terminal Sequences of BAC Inserts Based
on Double-Restriction-Enzyme Digestion Followed
by Anchored PCR
Zhong-Nan Yang and T. Erik Mirkov...............................................337
x Contents
36 A “Step Down” PCR-Based Technique for Walking
Into and the Subsequent Direct Sequence Analysis
of Flanking Genomic DNA
Ziguo Zhang and Sarah Jane Gurr..................................................343
PART V. LIBRARY CONSTRUCTIONAND SCREENING
37 Use of PCR in Library Screening: An Overview
Jinbao Zhu..........................................................................................353
38 Cloning of Homologous Genes by Gene-Capture PCR
Renato Mastrangeli and Silvia Donini.............................................359
39 Rapid and Nonradioactive Screening of Recombinant Libraries by PCR
Michael W. King.................................................................................377
40 Rapid cDNA Cloning by PCR Screening (RC-PCR)
Toru Takumi.......................................................................................385
41 Generation and PCR Screening of Bacteriophage λ Sublibraries
Enriched for Rare Clones (the “Sublibrary Method”)
Michael Lardelli..................................................................................391
42 PCR-Based Screening for Bacterial Artificial Chromosome Libraries
Yuji Yasukochi...................................................................................401
43 A 384-Well Microtiter-Plate-Based Template Preparation
and Sequencing Method
Lei He and Kai Wang.........................................................................411
44 A Microtiter-Plate-Based High Throughput PCR Product
Purification Method
Ryan Smith and Kai Wang................................................................417
Index............................................................................................................423
