WIXLIB

cDNA Library Protocols

METHODSIN M O L E C U L A RBIOLOGY John M. Walker, SERIES EDITOR43.71. PRINS and In Situ PCR Protocols, edited by John R.Gosden, 199770. Sequence Data Analysis Guidebook, edited by Simon R. 42.41.Swindell, 199769. cDNA Library Protocols, edited by Ian G. Cowelland Caroline Austin, 199740.68. Gene Isolation and Mapping Protocols, edited by39.Jacqueline Boullwood, 199767. PCR Cloning Protocols: From Molecular Cloning toGenetic Engineering, edited by Bruce A. White, 199738.66. Epitope Mapping Protocols, edited by Glenn E. Morris, 199665. PCR Sequencing Protocols, edited by Ralph Rapley. 1996 37.64. Protein Sequencing Protocols, edited by Bryan J. Smith,36.199663. Recombinant Proteins: Detection and Isolation Protocols,35.edited by SocjtyS. Tuan. 199662. Recombinant Gene Expression Protocols, edited by Rocky34.S Tuan. 199661. Protein and Peptide Analysis by Mass Spectrometry,33.edited by John R. Chapman, 199660. Protein NMR Protocols, edited by David G. Reid, 199659. Protein Purincation Protocols, edited by Shawn Doonan. 32.199658. Basic DNA and RNA Protocols, edited by Adrian J.31.Harwood, 199657. In Vitro Mutagenesis Protocols, edited by Michael K.30.Trower, 199629.56. Crystailographic Methods and Protocols, edited by Christopher Jones, Barbara Mulloy, and Mark Sanderson, 1996 28.55. Plant Cell Electroporation and Electrofusion Protocols,edited hyJac A. Nickoloff, 199527.54. VAC Protocols, edited by David Markie, 199553. Yeast Protocols: Methods in Cell and Molecular Biology, 26.edited by Ivor H. Evans, 199652. Capillary Electrophoresis: Principles, Instrumentation, 25.and Applications, edited by Kevin D. Altria, 199651. Antibody Engineering Protocols, edited by 5u( Air?au/, 1995 24.50. Species Diagnostics Protocols: PCR and Other NucleicAcid Methods, edited by ynsd'n P. Clapp, 199623.49. Plant Gene Transfer and Expression Protocols, edited byHeddwyn Jones, 199522.48. Animal Cell Electroporation and Electrofusion Protocols,edited by Jac A. Nickoloff, 199547. Electroporation Protocols for Microorganisms, edited by21.Jac A. Nickoloff, 199546. Diagnostic Bacteriology Protocols, edited by Jenny20.Howard and David M. Whitcombe, 199545. Monoclonal Antibody Protocols, edited by William C. 19.Davis, 199544. Agrobttclerium Protocols, edited by Kevan M. A. Gartland 18.and Michael R. Davey, 1995In Vitro Toxicity Testing Protocols, edited by SheilaO'Hare and Chris K. Atterwill. 1995ELISA: Theory and Practice, by John R. Crowther, 1995Signal Transduction Protocols, edited by David A. Kendalland Stephen J Hill, 1995Protein Stability and Folding: Theory and Practice,ed\Mby Bret A. Shirley, 1995Baculovirus Expression Protocols, edited by ChristopherD. Richardson, 1995Cryopreservation and Freeze-Drying Protocols,edited by John G. Day and Mark R. McLellan, 1995In Vitro Transcription and Translation Protocols,edited by Martin J. Tymms. 1995Peptide Analysis Protocols, edited by Ben M. Dunn andMichael W. Pennington. 1994Peptide Synthesis Protocols, edited by Michael W.Pennington and Ben M. Dunn, 1994Immunocytochemical Methods and Protocols, edited byLorette C. Javois, 1994In Situ Hybridization Protocols, edited by K. H AndyChoo. 1994Basic Protein and Peptide Protocols, edited by John M.Walker. 1994Protocols for Gene Analysis, edited by Adrian J. Harwood,1994DNA-Protein Interactions, edited by G. Geoff Kneale, 1994Chromosome Analysis Protocols, edited by John R.Gosden, 1994Protocols for Nucleic Acid Analysis by NonradioactiveProbes, edited by Peter G. Isaac. 1994Biomembrane Protocols: //. Architecture and Function.edited by John M. Graham and Joan A. Higgins. 1994Protocols for Oligonucleotide Conjugates: Synthesis andAnalytical Techniques, edited by Sudhir Agrawal. 1994Computer Analysis of Sequence Data: Part II. edited byAnnette M. Griffin and Hugh G. Griffin, 1994Computer Analysis of Sequence Data: Part /, edited byAnnette M. Griffin and Hugh G. Griffin, 1994DNA Sequencing Protocols, edited by Hugh G. Griffin andAnnette M. Griffin, 1993Microscopy, Optical Spectroscopy, and MacroscopicTechniques, edited by Christopher Jones, Barbara Mulloy,and Adrian H. Thomas, 1993Protocols in Molecular Parasitology, edited by John E.Hyde, 1993Protocols for Oligonucleotides and Analogs: Synthesis andProperties, edited by Sudhir Agrawal, 1993Biomembrane Protocols: /. Isolation and Analysis,edited by John M. Graham and Joan A. Higgins, 1993Transgenesis Techniques: Principles and Protocols,eiiled by David Murphy and David A. Carter, 1993

METHODSIN M O L E C U L A RBIOLOGYcDNA LibraryProtocolsEdited byIan G. Cowelland Caroline A. AustinUniversity of Newcastle, UKHumana Press i Totowa, New Jersey

1997 Humana Press Inc.999 Riverview Drive, Suite 208Totowa, New Jersey 07512All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted inany form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwisewithout written permission from the Publisher. Methods in Molecular Biology" is a trademark of TheHumana Press Inc.All authored papers, comments, opinions, conclusions, or recommendations are those of the author(s), anddo not necessarily reflect the views of the publisher.This publication is printed on acid-free paper. CS)ANSI Z39.48-1984 (American Standards InstiUite) Permanence of Paper for Printed Library Materials.For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contactHumana at the above address or at any of the following numbers: Tel: 201-256-1699; Fax: 201-256-8341;E-mail: humana@interramp.comPhotocopy Authorization Policy:Authorization to photocopy items for internal or personal use, or the internal or personal use of specificclients, is granted by Humana Press Inc., provided that the base fee of US 5.00 per copy, plus US 00.25per page, is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Dan vers, MA 01923.For those organizations that have been granted a photocopy license from the CCC, a separate system ofpayment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the TransactionalReporting Service is: [0-89603-383-X/97 5.00 00.25].Printed in the United States of America.1 0 9 8 7 6 5 4 3 2Library of Congress Cataloging in Publication DataMain entry under title:Methods in molecular biology".cDNA library protocols / edited by Ian G. Cowell and Caroline A. Austin.p. cm. — (Methods in molecular biology ; 69)Includes index.ISBN 0-89603-383-X (alk. paper)1. Gene libraries-Laboratory manuals. 2. DNA Laboratory manuals. 3. Antisense DNA Laboratory manuals. 4. Molecular cloning-Laboratory manuals. I. Cowell, Ian G. II. Austin,Caroline A. III. Series.[DNLM: 1. Gene Library. 2. DNA, Complementary. 3. Genetic Techniques. WlME9616JV.69 1997 / QH 442.4 C386 1997]QH442.4.C36 1997574.87'3282-dc20DNLM/DLCfor Library of Congress96-36127CIP

PrefaceThe first libraries of complementary DNA (cDNA) clones were constructed in the mid-to-late 1970s using RNA-dependent DNA polymerase(reverse transcriptase) to convert poly A* mRNA into double-stranded cDNAsuitable for insertion into prokaryotic vectors. Since then cDNA technologyhas become a fundamental tool for the molecular biologist and at the sametime some very significant advances have occurred in the methods for constructing and screening cDNA libraries.It is not the aim of cDNA Library Protocols to give a comprehensivereview of all cDNA library-based methodologies; instead we present a seriesof up-to-date protocols that together should give a good grounding of procedures associated with the construction and use of cDNA libraries. In decidingwhat to include, we endeavored to combine up-to-date versions of some of themost widely used protocols with some very usefiil newer techniques. cDNALibrary Protocols should therefore be especially useful to the investigator whois new to the use of cDNA libraries, but should also be of value to the moreexperienced worker.Chapters 1—5 concentrate on cDNA library construction and manipulation, Chapters 6 and 7 describe means of cloning difficult-to-obtain ends ofcDNAs, Chapters 8-18 give various approaches to the screening of cDNAlibraries, and the remaining chapters present methods of analysis of cDNAclones including details of how to analyze cDNA sequence data and how tomake use of the wealth of cDNA data emergingfromthe human genome project.In most cloning projects the library screening method to be employed will bethe most important factor in the cloning strategy. We have therefore includedprotocols for a wide range of approaches to screening cDNA libraries, including contemporary protocols for screening by hybridization of labeled DNAprobes to filter-bound cDNA clones (Chapters 9, 11, and 12), protocols forscreening expression libraries with different types of probe (Chapters 13-15),and some more specialized screening techniques (Chapters 16-18).Except where specific examples are useful, the protocols are presentedwith minimum reference to particular experimental systems. The format ofthe chapters will be familiar to those who have used other volumes in the

viPrefaceMethods in Molecular Biology series. Additional information that is not usually present in original publications, such as details of potential problems,troubleshooting tips, and alternative methods, are included in the Notes sections found at the end of each chapter.Once again, we hope that this volume will benefit the beginner and thecDNA aficionado alike. Finally, we would like to thank all of the contributingauthors for providing the material for this volume as well as John Walker andthe staff at Humana Press for their assistance in putting this volume together.Ian G. CowellCaroline A. Austin

ContentsPrefacevContributorsix1 cDNA Library Construction from Small Amounts of RNA UsingParamagnetic Beads and PCR,Kris N. Lambert and Valerie M. Wiliiamson/2 Increasing the Average Abundance of Low-Abundance cDNAsby Ordered Subdivision of cDNA Populations,David R. Sibson and Micl aei P. Starliey133 Isolation of Messenger RNA from Plant Tissues,Alison Dunn334 cDNA Library Construction for the Lambda ZAP -Based Vectors,Marjory A. Snead, Michelle A. Aiting-Mees,and Jay M. Short395 Clone Excision Methods for the Lambda ZAP -Based Vectors,Marjory A. Snead, Michelle A. Aiting-Mees,and Jay M. Short536 Using Rapid Amplification of cDNA Ends (RACE) to ObtainFull-Length cDNAs,Yue Zhang and Michael A. Frohman617 Inverse PCR Approach to Cloning cDNA Ends,Sheng-He Huang898 Cloning Gene Family Members Using the Polymerase ChainReaction with Degenerate Oligonucleotide Primers,Gregory M. Preston979 Subtractive Hybridization for the Isolation of Differentially ExpressedGenes Using Magnetic Beads,Hans-Christian Aasheim, Ton Logtenberg,and Frank Larsen11510 Preparation of Competent Cells for High-Efficiency PlasmidTransformation of Escherichia coli,Yasuhiko Nakata, Xiaoren Tang, and Kazunari K. Yokoyama . 129VII