From Genes to Genomes: Concepts and Applications of DNA Technology, Second Edition

396 Pages·2007·2.72 MB·English

by  Jeremy W. Dale

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From Genes to Genomes
Second Edition
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From Genes to Genomes
Second Edition
Concepts and Applications of DNA Technology
Jeremy W. Dale and Malcolm von Schantz
University of Sarrey, UK
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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.
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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
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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
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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
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