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From Genes to Genomes ffirs.indd ii 9/5/2007 11:07:35 AM From Genes to Genomes Second Edition ffirs.indd i 9/5/2007 11:07:35 AM ffirs.indd ii 9/5/2007 11:07:35 AM From Genes to Genomes Second Edition Concepts and Applications of DNA Technology Jeremy W. Dale and Malcolm von Schantz University of Sarrey, UK ffirs.indd iii 9/5/2007 11:07:35 AM Copyright © 2007 John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England Telephone (+44) 1243 779777 Email (for orders and customer service enquiries): [email protected] Visit our Home Page on www.wileyeurope.com or www.wiley.com All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except under the terms of the Copyright, Designs and Patents Act 1988 or under the terms of a licence issued by the Copyright Licensing Agency Ltd, 90 Tottenham Court Road, London W1T 4LP, UK, without the permission in writing of the Publisher. Requests to the Publisher should be addressed to the Permissions Department, John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex PO19 8SQ, England, or emailed to [email protected], or faxed to (+44) 1243 770620. Designations used by companies to distinguish their products are often claimed as trademarks. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The Publisher is not associated with any product or vendor mentioned in this book. This publication is designed to provide accurate and authoritative information in regard to the subject matter covered. It is sold on the understanding that the Publisher is not engaged in rendering professional services. If professional advice or other expert assistance is required, the services of a competent professional should be sought. Other Wiley Editorial Offi ces John Wiley & Sons Inc., 111 River Street, Hoboken, NJ 07030, USA Jossey-Bass, 989 Market Street, San Francisco, CA 94103-1741, USA Wiley-VCH Verlag GmbH, Boschstr. 12, D-69469 Weinheim, Germany John Wiley & Sons Australia Ltd, 33 Park Road, Milton, Queensland 4064, Australia John Wiley & Sons (Asia) Pte Ltd, 2 Clementi Loop #02-01, Jin Xing Distripark, Singapore 129809 John Wiley & Sons Canada Ltd, 6045 Freemont Blvd, Mississauga, Ontario, L5R 4J3 Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. Library of Congress Cataloging-in-Publication Data Dale, Jeremy. From genes to genomes : concepts and applications of DNA technology / Jeremy W. Dale and Malcolm von Schantz. – 2nd ed. p. ; cm. Includes bibliographical references and index. ISBN 978-0-470-01733-3 (cloth) 1. Genetic engineering. 2. DNA. 3. Genes. 4. Genomes. I. Schantz, Malcolm von. II. Title. [DNLM: 1. Genetic Engineering. 2. Cloning, Molecular. 3. DNA, Recombinant. 4. Genome. QU 450 D139f 2007] QH442.D35 2007 660.6′5 | – dc22 2007025153 British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library ISBN 978-0-470-01733-3 (HB) 978-0-470-01734-0 (PB) Typeset in 10.5 on 13 pt Times by SNP Best-set Typesetter Ltd., Hong Kong Printed and bound in Spain by Graphos SpA, Barcelona This book is printed on acid-free paper responsibly manufactured from sustainable forestry in which at least two trees are planted for each one used for paper production. ffirs.indd iv 9/5/2007 11:07:35 AM Contents Preface ix 1 Introduction 1 2 Basic molecular biology 7 2.1 Nucleic acid structure 7 2.2 What is a gene? 15 2.3 Information fl ow: gene expression 16 2.4 Gene structure and organization 21 3 How to clone a gene 25 3.1 What is cloning? 25 3.2 Overview of the procedures 26 3.3 Gene libraries 29 3.4 Hybridization 30 3.5 Polymerase chain reaction 32 3.6 Extraction and purifi cation of nucleic acids 33 3.7 Detection and quantitation of nucleic acids 36 3.8 Gel electrophoresis 37 4 Cutting and joining DNA 41 4.1 Restriction endonucleases 41 4.2 Ligation 47 4.3 Modifi cation of restriction fragment ends 53 4.4 Other ways of joining DNA molecules 57 5 Vectors 61 5.1 Plasmid vectors 61 5.2 Vectors based on the lambda bacteriophage 69 5.3 Cosmids 78 5.4 M13 vectors 79 ftoc.indd v 9/5/2007 11:08:45 AM vi CONTENTS 5.5 Expression vectors 81 5.6 Vectors for cloning and expression in eukaryotic cells 84 5.7 Supervectors: YACs and BACs 92 5.8 Summary 94 6 Genomic and cDNA libraries 95 6.1 Genomic libraries 96 6.2 Growing and storing libraries 105 6.3 cDNA libraries 106 6.4 Random, arrayed and ordered libraries 113 7 Finding the right clone 117 7.1 Screening libraries with gene probes 117 7.2 Screening expression libraries with antibodies 128 7.3 Subcloning 130 7.4 Characterization of plasmid clones 131 8 Polymerase chain reaction 135 8.1 The PCR reaction 136 8.2 PCR in practice 140 8.3 Cloning PCR products 144 8.4 Long-range PCR 146 8.5 Reverse-transcription PCR 146 8.6 Rapid amplifi cation of cDNA ends 147 8.7 Quantitative PCR 149 8.8 Applications of PCR 154 9 Characterization of a cloned gene 159 9.1 DNA sequencing 159 9.2 Databank entries and annotation 167 9.3 Sequence analysis 173 9.4 Sequence comparisons 178 9.5 Protein structure 190 9.6 Confi rming gene function 196 10 Analysis of gene expression 201 10.1 Analysing transcription 201 10.2 Methods for studying the promoter 209 10.3 Regulatory elements and DNA-binding proteins 213 10.4 Translational analysis 217 ftoc.indd vi 9/5/2007 11:08:45 AM CONTENTS vii 11 Products from native and manipulated cloned genes 221 11.1 Factors affecting expression of cloned genes 222 11.2 Expression of cloned genes in bacteria 227 11.3 Expression in eukaryotic host cells 235 11.4 Adding tags and signals 239 11.5 In vitro mutagenesis 242 11.6 Vaccines 247 12 Genomic analysis 251 12.1 Genome sequencing 251 12.2 Analysis and annotation 261 12.3 Comparing genomes 269 12.4 Genome browsers 271 12.5 Relating genes and functions: genetic and physical maps 273 12.6 Transposon mutagenesis and other screening techniques 276 12.7 Conclusion 285 13 Analysis of genetic variation 287 13.1 Single nucleotide polymorphisms 288 13.2 Larger-scale variations 291 13.3 Other methods for studying variation 293 13.4 Human genetic diseases 300 13.5 Molecular phylogeny 305 14 Post-genomic analysis 313 14.1 Analysing transcription; transcriptomes 313 14.2 Array-based methods 319 14.3 Translational analysis; proteomics 326 14.4 Post-translational analysis: protein interactions 329 14.5 Integrative studies; systems biology 332 15 Modifying organisms; transgenics 333 15.1 Modifi cation of bacteria and viruses: live vaccines 333 15.2 Transgenesis and cloning 337 15.3 Animal transgenesis 338 15.4 Applications of transgenic animals 347 15.5 Transgenic plants and their applications 351 Glossary 355 Bibliography 373 Index 375 ftoc.indd vii 9/5/2007 11:08:45 AM ftoc.indd viii 9/5/2007 11:08:45 AM Preface The fi rst edition of this book was published in 2002. At that time the revolu- tion in molecular biology, with gene cloning at its heart, had already pro- gressed to encompass a variety of other techniques and advances, especially genome sequencing, the polymerase chain reaction and microarray technol- ogy. This has now developed still further, to the extent that many researchers no longer need sophisticated techniques to clone and identify specifi c genes. They can simply screen the genome sequence for the relevant gene, and (if needed) amplify it, using PCR. The changes we have made in this edition represent an attempt to refl ect this development – especially by enhancing the treatment of bioinformatics. At the same time, we are conscious of the need to keep the book accessible to our target audience, which means not allowing it to expand much beyond its original size. Therefore, some topics have had to go. However, we have tried not to throw out the baby with the bathwater. Many of the older techniques still have a role to play – especially for those who are working with organisms whose genomes have not yet been sequenced. We have also retained a description of some techniques which, although now used less frequently, are either signifi cant in understanding the development of the subject or play a useful role in understanding underlying concepts. The main title of the book, From Genes to Genomes, is derived from the progress of this revolution. It signifi es the move from the early focus on the isolation and identifi cation of specifi c genes to the exciting advances that have been made possible by the sequencing of complete genomes. This has in turn spawned a whole new range of technologies (post-genomics) that are designed for genome-wide analysis of gene structure and expression. In this edition, we have strengthened this theme, by dealing fi rst with methods that are applicable to individual genes before moving on to genome-wide concepts. Dealing only with the techniques, without the applications, would be rather dry. Some of the applications are obvious – recombinant product formation, genetic diagnosis, transgenic plants and animals, and so on – and we have fpref.indd ix 9/5/2007 11:08:08 AM x PREFACE attempted to introduce these to give you a fl avour of the advances that con- tinue to be made, but at the same time without burdening you with excessive detail.
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