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PCR Cloning Protocols Springer
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METHODS IN MOLECULAR BIOLOGY,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 J rgen 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. by John 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. by William 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 G rard 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. by Barry 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,METHODS IN MOLECULAR BIOLOGY.
PCR Cloning,Second Edition,Bing Yuan Chen,Harry W Janes. Rutgers University,New Brunswick NJ,Humana Press Totowa New Jersey. 2002 Humana Press Inc,999 Riverview Drive Suite 208. Totowa New Jersey 07512,www humanapress 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, All papers comments opinions conclusions or recommendations are those of the author s and do not.
necessarily reflect the views of the publisher,This publication is printed on acid free paper. ANSI Z39 48 1984 American Standards Institute Permanence of Paper for Printed Library Materials. Cover illustration The Subtracted cDNA amplification of PBMCs stimulated with PHA See Fig 2 on page 107. Cover design by Patricia F Cleary,Production Editor Mark J Breaugh. 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 973 256 1699 Fax 973 256 8341. E mail humana humanapr com Website http humanapress com. Photocopy Authorization Policy, Authorization to photocopy items for internal or personal use or the internal or personal use of specific. clients is granted by Humana Press Inc provided that the base fee of US 10 00 per copy plus US 00 25. per page is paid directly to the Copyright Clearance Center at 222 Rosewood Drive Danvers MA 01923. For those organizations that have been granted a photocopy license from the CCC a separate system of. payment has been arranged and is acceptable to Humana Press Inc The fee code for users of the Transactional. Reporting Service is 0 89603 969 2 02 10 00 00 25, Printed in the United States of America 10 9 8 7 6 5 4 3 2 1. Library of Congress Cataloging in Publication Data. Main entry under title Methods in molecular biology. PCR cloning protocols second edition edited by Bing Yuan Chen and Harry W Janes 2nd ed. p cm Methods in molecular biology 192,Includes bibliographical references and index.
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. 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. 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,Contributors xi.
PART I PERFORMING AND 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. 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. J zsef G l and Mikl s K lm n 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 MUTAGENESIS AND 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 CONSTRUCTION AND 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 h 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,Contributors. JYRKI T AATSINKI Institute of Dentistry University of Oulu Finland. JIRI ADAMEC Mayo Clinic and Foundation Rochester MN. ANNA MARIE AGUINALDO Division of Chemistry and Chemical Engineering. California Institute of Technology Pasadena CA, ABEBE AKALU Institute for Genetic Medicine USC School of Medicine. Los Angeles CA, FRANCES ARNOLD Division of Chemistry and Chemical Engineering. California Institute of Technology Pasadena CA, SAILEN BARIK Department of Biochemistry and Molecular Biology University.
of South Alabama Mobile AL, YUPING BI Institute of Plant Biotechnology Shangdong Academy. of Agricultural Sciences Jinan China,DAVID E BIRCH Roche Molecular Systems Alameda CA. HAROLD BURGER Wadsworth Center Albany NY, XUETAO CAO Department of Immunology Second Military Medical. University Shanghai China, MARTINA CELERIN Department of Biology Indiana University Bloomington IN. BING YUAN CHEN Department of Plant Science Rutgers University New. Brunswick NJ, GUO JUN CHEN F Hoffmann La Roche Basel Switzerland.
STEVE CHEN NetOsprey Inc Berkeley CA, KRISTIN T CHUN Department of Pediatrics Indiana University School. of Medicine Indianapolis IN, SILVIA DONINI Istituto di Ricerca Cesare Serono Rome Italy. COLETTE DUEZ Centre D Ing nierie des Prot ines Universit de Li ge. Liege Belgium, JEAN DUSART Centre D Ing nierie des Prot ines Universit de Li ge. Liege Belgium,GUOWEI FANG Wadsworth Center Albany NY. JAMES A L FENTON Department of Molecular Oncology University of Leeds. STEVE FORGET Sainte Justine Hospital Research Center Montreal Canada. J ZSEF G L Institute for Biotechnology Bay Zolt n Foundation for Applied. Research Szeged Hungary,xii Contributors,ERNEST A GOULD CEH Oxford Oxford UK.
TAMARA S GRITSUN CEH Oxford Oxford UK, F PETER GUENGERICH Department of Biochemistry and Center in Molecular. Toxicology Vanderbilt University School of Medicine Nashville TN. BAOTAI GUO Institute of Plant Biotechnology Laiyang Agricultural College. Shandong China, SARAH JANE GURR Department of Plant Sciences University of Oxford. LEI HE PhenoGenomics Corp Bothell WA,JENS HEROLD SWITCH Biotech AG Martinsried Germany. SHENG HE HUANG Department of Pediatrics University of Southern California. Los Angeles CA, XIN HUANG Department of Immunology Second Military Medical University. Shanghai China, ERIC KA WAI HUI Department of Microbiology Immunology and Molecular.
Genetics University of California Los Angeles Los Angeles CA. HARRY W JANES Department of Plant Science Rutgers University New. Brunswick NJ, AMBROSE Y JONG Department of Pediatrics University of Southern California. Los Angeles CA, MIKL S K LM N Institute for Biotechnology Bay Zolt n Foundation. for Applied Research Szeged Hungary, DONGHAK KIM Department of Biochemistry and Center in Molecular. Toxicology Vanderbilt University School of Medicine Nashville TN. MICHAEL W KING Department of Biochemistry and Molecular Biology. Indiana University School of Medicine Terre Haute IN. LORI A KOLMODIN Roche Molecular Systems Pleasanton CA. FRANCO LACCONE Institute of Human Genetics University of Goettingen. Goettingen Germany, MICHAEL LARDELLI Department of Molecular Biosciences Adelaide. University Australia, DARIO E LEHOUX Health and Life Sciences Research Center Universit.
Laval Sainte Foy Qu bec Canada, ROGER C LEVESQUE Health and Life Sciences Research Center Universit. Laval Sainte Foy Qu bec Canada, DE PEI LIU Chinese Academy of Medical Sciences and Peking Union Medical. College Beijing China, SZECHENG J LO Institute of Microbiology and Immunology National Yang Ming. University Taipei Taiwan ROC,Contributors xiii, VINCENT J J MARTIN Department of Chemical Engineering University. of California Berkeley CA, RENATO MASTRANGELI Istituto di Ricerca Cesare Serono Rome Italy.
MICHAEL V MIKHAILOV CEH Oxford Oxford UK, T ERIK MIRKOV Department of Plant Pathology and Microbiology. The Texas A M University Agricultural Experiment Station Weslaco TX. WILLIAM W MOHN Department of Microbiology and Immunology. University of British Columbia Vancouver Canada,TIMOTHY MORAN Wadsworth Center Albany NY. ALAIN MOREAU Sainte Justine Hospital Research Center Montreal Canada. GARETH J MORGAN Department of Molecular Oncology University of Leeds. BRETT A NEILAN School of Microbiology and Immunology the University. of New South Wales Sydney Australia, HARUHIKO OHASHI Nagoya National Hospital Nagoya Japan. JAMES C PIERCE University of the Sciences in Philadelphia Philadelphia PA. GUY PRATT Department of Molecular Oncology University of Leeds Leeds UK. JUERGEN K V REICHARDT Institute for Genetic Medicine USC School. of Medicine Los Angeles CA, KENNETH H ROUX Department of Biological Science Florida State University. Tallahassee FL, BINZHANG SHEN Department of Molecular Biology Massachusetts General.
Hospital Boston MA, STUART G SIDDELL Institute of Virology and Immunology University. of W rzburg W rzburg Germany,RYAN SMITH PhenoGenomics Corp Bothell WA. ANNA T ANG Department of Pathology University of Southern California. Los Angeles CA, TORU TAKUMI Osaka Bioscience Institute Osaka Japan. VOLKER THIEL Institute of Virology and Immunology University of W rzburg. W rzburg Germany, DANIEL TILLETT School of Microbiology and Immunology University. of New South Wales Sydney Australia, TERUAKI TOZAKI Department of Molecular Genetics Laboratory of Racing.
Chemistry Utsunomiya Tochigi Japan,ALOISE VISOSKY Wadsworth Center Albany NY. DA SHEN WANG Sainte Justine Hospital Research Center Montreal Canada. KAI WANG PhenoGenomics Corp Bothell WA, PO CHING WANG Department of Medicine National Yang Ming University. Taipei Taiwan ROC,xiv Contributors,BARBARA WEISER Wadsworth Center Albany NY. HUA YANG WU Department of Pediatricsx University of Southern California. Los Angeles CA, ZHONG NAN YANG Department of Plant Pathology and Microbiology. The Texas A M University Agricultural Experiment Station Weslaco TX. YUJI YASUKOCHI National Institute of Agrobiological Sciences Ibaraki Japan. ZHENGLONG YUAN Department of Immunology Second Military Medical. University Shanghai China, ZIGUO ZHANG Department of Plant Sciences University of Oxford Oxford UK.
JINBAO ZHU Department of Genetics and Plant Breeding China Agricultural.

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