9781405181730_1_pre.qxd 1/11/10 10:23 Page iv 9781405181730_1_pre.qxd 1/11/10 10:23 Page i GENE CLONING AND DNA ANALYSIS 9781405181730_1_pre.qxd 1/11/10 10:23 Page ii 9781405181730_1_pre.qxd 1/11/10 10:23 Page iii GENE CLONING AND DNA ANALYSIS An Introduction T.A. BROWN Faculty of Life Sciences University of Manchester Manchester Sixth Edition A John Wiley & Sons, Ltd., Publication 9781405181730_1_pre.qxd 1/11/10 10:23 Page iv This edition first published 2010, © 2010, 2006 by T.A. Brown First, second and third editions published by Chapman & Hall 1986, 1990, 1995 Fourth and fifth editions published by Blackwell Publishing Ltd 2001, 2006 Blackwell Publishing was acquired by John Wiley & Sons in February 2007. Blackwell’s publishing program has been merged with Wiley’s global Scientific, Technical and Medical business to form Wiley-Blackwell. 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ISBN 978-1-4051-8173-0 (pbk. : alk. paper) – ISBN 978-1-4443-3407-4 (hbk. : alk. paper) 1. Molecular cloning. 2. Nucleotide sequence. 3. DNA—Analysis. I. Title. QH442.2.B76 2010 572.8′633—dc22 2009038739 ISBN: 9781405181730 (paperback) and 9781444334074 (hardback) A catalog record for this book is available from the British Library. Set in 10/12pt Classical Garamond by Graphicraft Limited, Hong Kong Printed in Malaysia 1 2010 9781405181730_1_pre.qxd 1/11/10 10:23 Page v Brief Contents BRIEF CONTENTS Preface to the Sixth Edition xvi Part I The Basic Principles of Gene Cloning and DNA Analysis 1 1 Why Gene Cloning and DNA Analysis are Important 3 2 Vectors for Gene Cloning: Plasmids and Bacteriophages 13 3 Purification of DNA from Living Cells 25 4 Manipulation of Purified DNA 45 5 Introduction of DNA into Living Cells 72 6 Cloning Vectors for E. coli 88 7 Cloning Vectors for Eukaryotes 105 8 How to Obtain a Clone of a Specific Gene 126 9 The Polymerase Chain Reaction 147 Part II The Applications of Gene Cloning and DNA Analysis in Research 163 10 Sequencing Genes and Genomes 165 11 Studying Gene Expression and Function 185 12 Studying Genomes 207 Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology 223 13 Production of Protein from Cloned Genes 225 14 Gene Cloning and DNA Analysis in Medicine 245 15 Gene Cloning and DNA Analysis in Agriculture 264 16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology 282 Glossary 298 Index 312 Companion website available at www.wiley.com/go/brown/cloning v 9781405181730_1_pre.qxd 1/11/10 10:23 Page vi 9781405181730_1_pre.qxd 1/11/10 10:23 Page vii Contents CONTENTS Preface to the Sixth Edition xvi Part I The Basic Principles of Gene Cloning and DNA Analysis 1 1 Why Gene Cloning and DNA Analysis are Important 3 1.1 The early development of genetics 3 1.2 The advent of gene cloning and the polymerase chain reaction 4 1.3 What is gene cloning? 5 1.4 What is PCR? 6 1.5 Why gene cloning and PCR are so important 7 1.5.1 Obtaining a pure sample of a gene by cloning 7 1.5.2 PCR can also be used to purify a gene 9 1.6 How to find your way through this book 11 2 Vectors for Gene Cloning: Plasmids and Bacteriophages 13 2.1 Plasmids 13 2.1.1 Size and copy number 15 2.1.2 Conjugation and compatibility 16 2.1.3 Plasmid classification 16 2.1.4 Plasmids in organisms other than bacteria 17 2.2 Bacteriophages 17 2.2.1 The phage infection cycle 18 2.2.2 Lysogenic phages 19 Gene organization in the 2 DNA molecule 19 The linear and circular forms of 2 DNA 19 M13—a filamentous phage 22 2.2.3 Viruses as cloning vectors for other organisms 24 vii 9781405181730_1_pre.qxd 1/11/10 10:23 Page viii viii Contents 3 Purification of DNA from Living Cells 25 3.1 Preparation of total cell DNA 25 3.1.1 Growing and harvesting a bacterial culture 26 3.1.2 Preparation of a cell extract 28 3.1.3 Purification of DNA from a cell extract 29 Removing contaminants by organic extraction and enzyme digestion 29 Using ion-exchange chromatography to purify DNA from a cell extract 30 3.1.4 Concentration of DNA samples 30 3.1.5 Measurement of DNA concentration 31 3.1.6 Other methods for the preparation of total cell DNA 32 3.2 Preparation of plasmid DNA 33 3.2.1 Separation on the basis of size 35 3.2.2 Separation on the basis of conformation 36 Alkaline denaturation 36 Ethidium bromide–caesium chloride density gradient centrifugation 36 3.2.3 Plasmid amplification 39 3.3 Preparation of bacteriophage DNA 39 3.3.1 Growth of cultures to obtain a high 2 titer 40 3.3.2 Preparation of non-lysogenic 2 phages 40 3.3.3 Collection of phages from an infected culture 42 3.3.4 Purification of DNA from 2 phage particles 42 3.3.5 Purification of M13 DNA causes few problems 43 4 Manipulation of Purified DNA 45 4.1 The range of DNA manipulative enzymes 46 4.1.1 Nucleases 46 4.1.2 Ligases 47 4.1.3 Polymerases 48 4.1.4 DNA modifying enzymes 49 4.2 Enzymes for cutting DNA—restriction endonucleases 50 4.2.1 The discovery and function of restriction endonucleases 51 4.2.2 Type II restriction endonucleases cut DNA at specific nucleotide sequences 52 4.2.3 Blunt ends and sticky ends 53 4.2.4 The frequency of recognition sequences in a DNA molecule 53 4.2.5 Performing a restriction digest in the laboratory 54 4.2.6 Analysing the result of restriction endonuclease cleavage 56 Separation of molecules by gel electrophoresis 57 Visualizing DNA molecules in an agarose gel 58 4.2.7 Estimation of the sizes of DNA molecules 58 4.2.8 Mapping the positions of different restriction sites in a DNA molecule 59 4.2.9 Special gel electrophoresis methods for separating larger molecules 60