Gene Cloning and DNA Analysis PDF

GENE CLONING AND DNA ANALYSIS 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 This edition first published 2010, © 2010, 2006 by T.A. Brown First, second and third e...

GENE CLONING AND DNA ANALYSIS 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 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. Registered office: John Wiley & Sons Ltd, The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK Editorial offices: 9600 Garsington Road, Oxford, OX4 2DQ, UK The Atrium, Southern Gate, Chichester, West Sussex, PO19 8SQ, UK 111 River Street, Hoboken, NJ 07030-5774, USA For details of our global editorial offices, for customer services and for information about how to apply for permission to reuse the copyright material in this book please see our website at www.wiley.com/wiley-blackwell The right of the author to be identified as the author of this work has been asserted in accordance with the Copyright, Designs and Patents Act 1988. 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 or otherwise, except as permitted by the UK Copyright, Designs and Patents Act 1988, without the prior permission of the publisher. Wiley also publishes its books in a variety of electronic formats. Some content that appears in print may not be available in electronic books. 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. Library of Congress Cataloguing-in-Publication Data Brown, T.A. (Terence A.) Gene cloning and DNA analysis : an introduction / T.A. Brown.—6th ed. p. cm. 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  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  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 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 Contents ix 4.3 Ligation—joining DNA molecules together 63 4.3.1 The mode of action of DNA ligase 63 4.3.2 Sticky ends increase the efficiency of ligation 64 4.3.3 Putting sticky ends onto a blunt-ended molecule 64 Linkers 64 Adaptors 65 Producing sticky ends by homopolymer tailing 67 4.3.4 Blunt end ligation with a DNA topoisomerase 69 5 Introduction of DNA into Living Cells 72 5.1 Transformation—the uptake of DNA by bacterial cells 74 5.1.1 Not all species of bacteria are equally efficient at DNA uptake 74 5.1.2 Preparation of competent E. coli cells 75 5.1.3 Selection for transformed cells 75 5.2 Identification of recombinants 76 5.2.1 Recombinant selection with pBR322—insertional inactivation of an antibiotic resistance gene 77 5.2.2 Insertional inactivation does not always involve antibiotic resistance 79 5.3 Introduction of phage DNA into bacterial cells 81 5.3.1 Transfection 81 5.3.2 In vitro packaging of 2 cloning vectors 81 5.3.3 Phage infection is visualized as plaques on an agar medium 81 5.4 Identification of recombinant phages 83 5.4.1 Insertional inactivation of a lacZ′ gene carried by the phage vector 83 5.4.2 Insertional inactivation of the 2 cI gene 83 5.4.3 Selection using the Spi phenotype 83 5.4.4 Selection on the basis of 2 genome size 84 5.5 Introduction of DNA into non-bacterial cells 85 5.5.1 Transformation of individual cells 85 5.5.2 Transformation of whole organisms 85 6 Cloning Vectors for E. coli 88 6.1 Cloning vectors based on E. coli plasmids 89 6.1.1 The nomenclature of plasmid cloning vectors 89 6.1.2 The useful properties of pBR322 89 6.1.3 The pedigree of pBR322 90 6.1.4 More sophisticated E. coli plasmid cloning vectors 90 pUC8—a Lac selection plasmid 92 pGEM3Z—in vitro transcription of cloned DNA 93 6.2 Cloning vectors based on M13 bacteriophage 94 6.2.1 How to construct a phage cloning vector 94 6.2.2 Hybrid plasmid–M13 vectors 96 x Contents 6.3 Cloning vectors based on 8 bacteriophage 97 6.3.1 Segments of the 2 genome can be deleted without impairing viability 98 6.3.2 Natural selection can be used to isolate modified 2 that lack certain restriction sites 98 6.3.3 Insertion and replacement vectors 98 Insertion vectors 99 Replacement vectors 100 6.3.4 Cloning experiments with 2 insertion or replacement vectors 100 6.3.5 Long DNA fragments can be cloned using a cosmid 101 6.4 8 and other high-capacity vectors enable genomic libraries to be constructed 102 6.5 Vectors for other bacteria 104 7 Cloning Vectors for Eukaryotes 105 7.1 Vectors for yeast and other fungi 105 7.1.1 Selectable markers for the 2 3m plasmid 106 7.1.2 Vectors based on the 2 3m plasmid—yeast episomal plasmids 106 7.1.3 A YEp may insert into yeast chromosomal DNA 107 7.1.4 Other types of yeast cloning vector 108 7.1.5 Artificial chromosomes can be used to clone long pieces of DNA in yeast 110 The structure and use of a YAC vector 110 Applications for YAC vectors 111 7.1.6 Vectors for other yeasts and fungi 112 7.2 Cloning vectors for higher plants 112 7.2.1 Agrobacterium tumefaciens—nature’s smallest genetic engineer 113 Using the Ti plasmid to introduce new genes into a plant cell 113 Production of transformed plants with the Ti plasmid 115 The Ri plasmid 117 Limitations of cloning with Agrobacterium plasmids 117 7.2.2 Cloning genes in plants by direct gene transfer 118 Direct gene transfer into the nucleus 118 Transfer of genes into the chloroplast genome 119 7.2.3 Attempts to use plant viruses as cloning vectors 119 Caulimovirus vectors 120 Geminivirus vectors 120 7.3 Cloning vectors for animals 120 7.3.1 Cloning vectors for insects 121 P elements as cloning vectors for Drosophila 121 Cloning vectors based on insect viruses 122 7.3.2 Cloning in mammals 122 Viruses as cloning vectors for mammals 123 Gene cloning without a vector 124 Contents xi 8 How to Obtain a Clone of a Specific Gene 126 8.1 The problem of selection 126 8.1.1 There are two basic strategies for obtaining the clone you want 127 8.2 Direct selection 128 8.2.1 Marker rescue extends the scope of direct selection 129 8.2.2 The scope and limitations of marker rescue 130 8.3 Identification of a clone from a gene library 131 8.3.1 Gene libraries 131 8.3.2 Not all genes are expressed at the same time 131 8.3.3 mRNA can be cloned as complementary DNA 133 8.4 Methods for clone identification 133 8.4.1 Complementary nucleic acid strands hybridize to each other 133 8.4.2 Colony and plaque hybridization probing 133 Labeling with a radioactive marker 136 Non-radioactive labeling 137 8.4.3 Examples of the practical use of hybridization probing 137 Abundancy probing to analyse a cDNA library 137 Oligonucleotide probes for genes whose translation products have been characterized 138 Heterologous probing allows related genes to be identified 141 Southern hybridization enables a specific restriction fragment containing a gene to be identified 142 8.4.4 Identification methods based on detection of the translation product of the cloned gene 144 Antibodies are required for immunological detection methods 144 Using a purified antibody to detect protein in recombinant colonies 145 The problem of gene expression 146 9 The Polymerase Chain Reaction 147 9.1 The polymerase chain reaction in outline 147 9.2 PCR in more detail 149 9.2.1 Designing the oligonucleotide primers for a PCR 149 9.2.2 Working out the correct temperatures to use 152 9.3 After the PCR: studying PCR products 153 9.3.1 Gel electrophoresis of PCR products 154 9.3.2 Cloning PCR products 154 9.3.3 Problems with the error rate of Taq polymerase 157 9.4 Real-time PCR enables the amount of starting material to be quantified 158 9.4.1 Carrying out a quantitative PCR experiment 159 9.4.2 Real-time PCR can also quantify RNA 160 xii Contents Part II The Applications of Gene Cloning and DNA Analysis in Research 163 10 Sequencing Genes and Genomes 165 10.1 The methodology for DNA sequencing 165 10.1.1 Chain termination DNA sequencing 166 Chain termination sequencing in outline 166 Not all DNA polymerases can be used for sequencing 168 Chain termination sequencing requires a single-stranded DNA template 169 The primer determines the region of the template DNA that will be sequenced 169 10.1.2 Pyrosequencing 171 Pyrosequencing involves detection of pulses of chemiluminescence 171 Massively parallel pyrosequencing 171 10.2 How to sequence a genome 173 10.2.1 The shotgun approach to genome sequencing 174 The Haemophilus influenzae genome sequencing project 174 Problems with shotgun sequencing 176 10.2.2 The clone contig approach 177 Clone contig assembly by chromosome walking 177 Rapid methods for clone contig assembly 178 Clone contig assembly by sequence tagged site content analysis 179 10.2.3 Using a map to aid sequence assembly 180 Genetic maps 180 Physical maps 181 The importance of a map in sequence assembly 183 11 Studying Gene Expression and Function 185 11.1 Studying the RNA transcript of a gene 186 11.1.1 Detecting the presence of a transcript and determining its nucleotide sequence 186 11.1.2 Transcript mapping by hybridization between gene and RNA 188 11.1.3 Transcript analysis by primer extension 190 11.1.4 Transcript analysis by PCR 191 11.2 Studying the regulation of gene expression 192 11.2.1 Identifying protein binding sites on a DNA molecule 193 Gel retardation of DNA–protein complexes 193 Footprinting with DNase I 194 Modification interference assays 194 11.2.2 Identifying control sequences by deletion analysis 197 Reporter genes 197 Carrying out a deletion analysis 198 Contents xiii 11.3 Identifying and studying the translation product of a cloned gene 199 11.3.1 HRT and HART can identify the translation product of a cloned gene 199 11.3.2 Analysis of proteins by in vitro mutagenesis 200 Different types of in vitro mutagenesis techniques 202 Using an oligonucleotide to create a point mutation in a cloned gene 203 Other methods of creating a point mutation in a cloned gene 204 The potential of in vitro mutagenesis 205 12 Studying Genomes 207 12.1 Genome annotation 207 12.1.1 Identifying the genes in a genome sequence 208 Searching for open reading frames 208 Simple ORF scans are less effective at locating genes in eukaryotic genomes 209 Gene location is aided by homology searching 210 Comparing the sequences of related genomes 211 12.1.2 Determining the function of an unknown gene 212 Assigning gene function by experimental analysis requires a reverse approach to genetics 212 Specific genes can be inactivated by homologous recombination 213 12.2 Studies of the transcriptome and proteome 214 12.2.1 Studying the transcriptome 215 Studying a transcriptome by sequence analysis 215 Studying transcriptomes by microarray or chip analysis 215 12.2.2 Studying the proteome 217 Separating the proteins in a proteome 217 Identifying the individual proteins after separation 218 12.2.3 Studying protein–protein interactions 220 Phage display 220 The yeast two hybrid system 220 Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology 223 13 Production of Protein from Cloned Genes 225 13.1 Special vectors for expression of foreign genes in E. coli 227 13.1.1 The promoter is the critical component of an expression vector 228 The promoter must be chosen with care 228 Examples of promoters used in expression vectors 231 13.1.2 Cassettes and gene fusions 232 xiv Contents 13.2 General problems with the production of recombinant protein in E. coli 234 13.2.1 Problems resulting from the sequence of the foreign gene 235 13.2.2 Problems caused by E. coli 236 13.3 Production of recombinant protein by eukaryotic cells 237 13.3.1 Recombinant protein from yeast and filamentous fungi 237 Saccharomyces cerevisiae as the host for recombinant protein synthesis 237 Other yeasts and fungi 238 13.3.2 Using animal cells for recombinant protein production 239 Protein production in mammalian cells 239 Protein production in insect cells 240 13.3.3 Pharming—recombinant protein from live animals and plants 241 Pharming in animals 241 Recombinant proteins from plants 242 Ethical concerns raised by pharming 243 14 Gene Cloning and DNA Analysis in Medicine 245 14.1 Production of recombinant pharmaceuticals 245 14.1.1 Recombinant insulin 246 Synthesis and expression of artificial insulin genes 247 14.1.2 Synthesis of human growth hormones in E. coli 247 14.1.3 Recombinant factor VIII 249 14.1.4 Synthesis of other recombinant human proteins 251 14.1.5 Recombinant vaccines 252 Producing vaccines as recombinant proteins 252 Recombinant vaccines in transgenic plants 253 Live recombinant virus vaccines 253 14.2 Identification of genes responsible for human diseases 255 14.2.1 How to identify a gene for a genetic disease 256 Locating the approximate position of the gene in the human genome 256 Identification of candidates for the disease gene 258 14.3 Gene therapy 259 14.3.1 Gene therapy for inherited diseases 259 14.3.2 Gene therapy and cancer 260 14.3.3 The ethical issues raised by gene therapy 262 15 Gene Cloning and DNA Analysis in Agriculture 264 15.1 The gene addition approach to plant genetic engineering 265 15.1.1 Plants that make their own insecticides 265 The 1-endotoxins of Bacillus thuringiensis 265 Cloning a 1-endotoxin gene in maize 266 Cloning 1-endotoxin genes in chloroplasts 268 Countering insect resistance to 1-endotoxin crops 269 Contents xv 15.1.2 Herbicide resistant crops 270 “Roundup Ready” crops 271 A new generation of glyphosate resistant crops 272 15.1.3 Other gene addition projects 273 15.2 Gene subtraction 274 15.2.1 Antisense RNA and the engineering of fruit ripening in tomato 274 Using antisense RNA to inactivate the polygalacturonase gene 274 Using antisense RNA to inactivate ethylene synthesis 276 15.2.2 Other examples of the use of antisense RNA in plant genetic engineering 276 15.3 Problems with genetically modified plants 277 15.3.1 Safety concerns with selectable markers 277 15.3.2 The terminator technology 278 15.3.3 The possibility of harmful effects on the environment 279 16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology 282 16.1 DNA analysis in the identification of crime suspects 283 16.1.1 Genetic fingerprinting by hybridization probing 283 16.1.2 DNA profiling by PCR of short tandem repeats 283 16.2 Studying kinship by DNA profiling 286 16.2.1 Related individuals have similar DNA profiles 286 16.2.2 DNA profiling and the remains of the Romanovs 286 STR analysis of the Romanov bones 286 Mitochondrial DNA was used to link the Romanov skeletons with living relatives 287 The missing children 289 16.3 Sex identification by DNA analysis 289 16.3.1 PCRs directed at Y chromosome-specific sequences 289 16.3.2 PCR of the amelogenin gene 290 16.4 Archaeogenetics—using DNA to study human prehistory 291 16.4.1 The origins of modern humans 291 DNA analysis has challenged the multiregional hypothesis 291 DNA analysis shows that Neanderthals are not the ancestors of modern Europeans 293 16.4.2 DNA can also be used to study prehistoric human migrations 294 The spread of agriculture into Europe 294 Using mitochondrial DNA to study past human migrations into Europe 294 Glossary 298 Index 312 Companion website available at www.wiley.com/go/brown/cloning  Preface to the Sixth Edition PREFACE TO THE SIXTH EDITION uring the four years since publication of the Fifth Edition of Gene Cloning and DNA D Analysis: An Introduction there have been important advances in DNA sequencing technology, in particular the widespread adoption of high throughput approaches based on pyrosequencing. Inclusion of these new techniques in the Sixth Edition has prompted me to completely rewrite the material on DNA sequencing and to place all the relevant information—both on the methodology itself and its application to genome sequencing —into a single chapter. This has enabled me to devote another entire chapter to the post-sequencing methods used to study genomes. The result is, I hope, a more balanced treatment of the various aspects of genomics and post-genomics than I had managed in previous editions. A second important development of the last few years has been the introduction of real-time PCR as a means of quantifying the amount of a particular DNA sequence pre- sent in a preparation. This technique is now described as part of Chapter 9. Elsewhere, I have made various additions, such as inclusion of topoisomerase-based methods for blunt end ligation in Chapter 4, and generally tidied up parts of chapters that had become slightly unwieldy due to the cumulative effects of modifications made over the 25 years since the First Edition of this book. The Sixth Edition is almost twice as long as the First, but retains the philosophy of that original edition. It is still an introductory text that begins at the beginning and does not assume that the reader has any prior knowledge of the techniques used to study genes and genomes. I would like to thank Nigel Balmforth and Andy Slade at Wiley-Blackwell for help- ing me to make this new edition a reality. As always I must also thank my wife Keri for the unending support that she has given to me in my decision to use up evenings and weekends writing this and other books. T.A. Brown Faculty of Life Sciences University of Manchester xvi PART I The Basic Principles of Gene Cloning and DNA Analysis 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 Chapter 1 Why Gene Cloning and DNA Analysis are Important Chapter contents CHAPTER CONTENTS 1.1 The early development of genetics 1.2 The advent of gene cloning and the polymerase chain reaction 1.3 What is gene cloning? 1.4 What is PCR? 1.5 Why gene cloning and PCR are so important 1.6 How to find you way through this book In the middle of the 19th century, Gregor Mendel formulated a set of rules to explain the inheritance of biological characteristics. The basic assumption of these rules is that each heritable property of an organism is controlled by a factor, called a gene, that is a physical particle present somewhere in the cell. The rediscovery of Mendel’s laws in 1900 marks the birth of genetics, the science aimed at understanding what these genes are and exactly how they work. 1.1 The early development of genetics For the first 30 years of its life this new science grew at an astonishing rate. The idea that genes reside on chromosomes was proposed by W. Sutton in 1903, and received experimental backing from T.H. Morgan in 1910. Morgan and his colleagues then developed the techniques for gene mapping, and by 1922 had produced a comprehen- sive analysis of the relative positions of over 2000 genes on the 4 chromosomes of the fruit fly, Drosophila melanogaster. Despite the brilliance of these classical genetic studies, there was no real under- standing of the molecular nature of the gene until the 1940s. Indeed, it was not until Gene Cloning and DNA Analysis: An Introduction. 6th edition. By T.A. Brown. Published 2010 by 3 Blackwell Publishing. 4 Part I The Basic Principles of Gene Cloning and DNA Analysis the experiments of Avery, MacLeod, and McCarty in 1944, and of Hershey and Chase in 1952, that anyone believed that deoxyribonucleic acid (DNA) is the genetic material: up until then it was widely thought that genes were made of protein. The discovery of the role of DNA was a tremendous stimulus to genetic research, and many famous biologists (Delbrück, Chargaff, Crick, and Monod were among the most influential) contributed to the second great age of genetics. In the 14 years between 1952 and 1966, the structure of DNA was elucidated, the genetic code cracked, and the processes of transcription and translation described. 1.2 The advent of gene cloning and the polymerase chain reaction These years of activity and discovery were followed by a lull, a period of anticlimax when it seemed to some molecular biologists (as the new generation of geneticists styled themselves) that there was little of fundamental importance that was not understood. In truth there was a frustration that the experimental techniques of the late 1960s were not sophisticated enough to allow the gene to be studied in any greater detail. Then in the years 1971–1973 genetic research was thrown back into gear by what at the time was described as a revolution in experimental biology. A whole new method- ology was developed, enabling previously impossible experiments to be planned and carried out, if not with ease, then at least with success. These methods, referred to as recombinant DNA technology or genetic engineering, and having at their core the pro- cess of gene cloning, sparked another great age of genetics. They led to rapid and efficient DNA sequencing techniques that enabled the structures of individual genes to be determined, reaching a culmination at the turn of the century with the massive genome sequencing projects, including the human project which was completed in 2000. They led to procedures for studying the regulation of individual genes, which have allowed molecular biologists to understand how aberrations in gene activity can result in human diseases such as cancer. The techniques spawned modern biotechnology, which puts genes to work in production of proteins and other compounds needed in medicine and industrial processes. During the 1980s, when the excitement engendered by the gene cloning revolution was at its height, it hardly seemed possible that another, equally novel and equally revolutionary process was just around the corner. According to DNA folklore, Kary Mullis invented the polymerase chain reaction (PCR) during a drive along the coast of California one evening in 1985. His brainwave was an exquisitely simple technique that acts as a perfect complement to gene cloning. PCR has made easier many of the techniques that were possible but difficult to carry out when gene cloning was used on its own. It has extended the range of DNA analysis and enabled molecular biology to find new applications in areas of endeavor outside of its traditional range of medicine, agri- culture, and biotechnology. Archaeogenetics, molecular ecology, and DNA forensics are just three of the new disciplines that have become possible as a direct consequence of the invention of PCR, enabling molecular biologists to ask questions about human evolution and the impact of environmental change on the biosphere, and to bring their powerful tools to bear in the fight against crime. Forty years have passed since the dawn- ing of the age of gene cloning, but we are still riding the rollercoaster and there is no end to the excitement in sight. Chapter 1 Why Gene Cloning and DNA Analysis are Important 5 Construction of a recombinant DNA molecule Figure 1.1 The basic steps in gene cloning. + Recombinant DNA molecule Vector Fragment of DNA + Bacterium Bacterium Transport into the carrying host cell recombinant DNA molecule Multiplication of recombinant DNA molecule Division of host cell Numerous cell divisions resulting in a clone Bacterial colonies growing on solid medium 1.3 What is gene cloning? What exactly is gene cloning? The easiest way to answer this question is to follow through the steps in a gene cloning experiment (Figure 1.1): 1 A fragment of DNA, containing the gene to be cloned, is inserted into a circular DNA molecule called a vector, to produce a recombinant DNA molecule. 2 The vector transports the gene into a host cell, which is usually a bacterium, although other types of living cell can be used. 3 Within the host cell the vector multiplies, producing numerous identical copies, not only of itself but also of the gene that it carries. 4 When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place. 5 After a large number of cell divisions, a colony, or clone, of identical host cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule; the gene carried by the recombinant molecule is now said to be cloned. 6 Part I The Basic Principles of Gene Cloning and DNA Analysis 1.4 What is PCR? The polymerase chain reaction is very different from gene cloning. Rather than a series of manipulations involving living cells, PCR is carried out in a single test tube simply by mixing DNA with a set of reagents and placing the tube in a thermal cycler, a piece of equipment that enables the mixture to be incubated at a series of temperatures that are varied in a preprogrammed manner. The basic steps in a PCR experiment are as follows (Figure 1.2): Template DNA Figure 1.2 3’ 5’ The basic steps in the polymerase chain reaction. 5’ 3’ Denaturation of the template DNA at 94°C 3’ 5’ 5’ 3’ Annealing of the oligonucleotide primers at 50–60°C 3’ 5’ 5’ Primers 5’ 5’ 3’ Synthesis of new DNA at 74°C 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ 3’ 5’ 5’ 3’ Repeat the cycle 25–30 times Chapter 1 Why Gene Cloning and DNA Analysis are Important 7 1 The mixture is heated to 94°C, at which temperature the hydrogen bonds that hold together the two strands of the double-stranded DNA molecule are broken, causing the molecule to denature. 2 The mixture is cooled down to 50–60°C. The two strands of each molecule could join back together at this temperature, but most do not because the mixture contains a large excess of short DNA molecules, called oligonucleotides or primers, which anneal to the DNA molecules at specific positions. 3 The temperature is raised to 74°C. This is a good working temperature for the Taq DNA polymerase that is present in the mixture. We will learn more about DNA polymerases on p. 48. All we need to understand at this stage is that the Taq DNA polymerase attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA molecules, during this step of the PCR. Now we have four stands of DNA instead of the two that there were to start with. 4 The temperature is increased back to 94°C. The double-stranded DNA molecules, each of which consists of one strand of the original molecule and one new strand of DNA, denature into single strands. This begins a second cycle of denaturation–annealing–synthesis, at the end of which there are eight DNA strands. By repeating the cycle 30 times the double-stranded molecule that we began with is converted into over 130 million new double-stranded molecules, each one a copy of the region of the starting molecule delineated by the annealing sites of the two primers. 1.5 Why gene cloning and PCR are so important As you can see from Figures 1.1 and 1.2, gene cloning and PCR are relatively straight- forward procedures. Why, then, have they assumed such importance in biology? The answer is largely because both techniques can provide a pure sample of an individual gene, separated from all the other genes in the cell. 1.5.1 Obtaining a pure sample of a gene by cloning To understand exactly how cloning can provide a pure sample of a gene, consider the basic experiment from Figure 1.1, but drawn in a slightly different way (Figure 1.3). In this example the DNA fragment to be cloned is one member of a mixture of many different fragments, each carrying a different gene or part of a gene. This mixture could indeed be the entire genetic complement of an organism—a human, for instance. Each of these fragments becomes inserted into a different vector molecule to produce a family of recombinant DNA molecules, one of which carries the gene of interest. Usu- ally only one recombinant DNA molecule is transported into any single host cell, so that although the final set of clones may contain many different recombinant DNA molecules, each individual clone contains multiple copies of just one molecule. The gene is now separated away from all the other genes in the original mixture, and its specific features can be studied in detail. In practice, the key to the success or failure of a gene cloning experiment is the abil- ity to identify the particular clone of interest from the many different ones that are obtained. If we consider the genome of the bacterium Escherichia coli, which contains 8 Part I The Basic Principles of Gene Cloning and DNA Analysis Figure 1.3 Cloning allows individual fragments of DNA to be purified. + Vectors DNA fragments Construct recombinant DNA molecules Each carries a different fragment Introduce into bacteria Plate out Each colony contains multiple copies of just one recombinant DNA molecule just over 4000 different genes, we might at first despair of being able to find just one gene among all the possible clones (Figure 1.4). The problem becomes even more over- whelming when we remember that bacteria are relatively simple organisms and that the human genome contains about five times as many genes. However, as explained in Chapter 8, a variety of different strategies can be used to ensure that the correct gene can be obtained at the end of the cloning experiment. Some of these strategies involve modifications to the basic cloning procedure, so that only cells containing the desired recombinant DNA molecule can divide and the clone of interest is automatically selected. Other methods involve techniques that enable the desired clone to be identified from a mixture of lots of different clones. Once a gene has been cloned there is almost no limit to the information that can be obtained about its structure and expression. The availability of cloned material has stimulated the development of analytical methods for studying genes, with new tech- niques being introduced all the time. Methods for studying the structure and expression of a cloned gene are described in Chapters 10 and 11, respectively. Chapter 1 Why Gene Cloning and DNA Analysis are Important 9 Figure 1.4 pyrF dnaL The problem of selection. cysB trpE trpD A very small part of the E.coli genome trpC trpB tonB trpA opp aroT The gene to be cloned trpB pyrF dnaL opp tonB trpC trpD trpE aroT cysB trpA ? trpA How can we select or identify just one gene? 1.5.2 PCR can also be used to purify a gene The polymerase chain reaction can also be used to obtain a pure sample of a gene. This is because the region of the starting DNA molecule that is copied during PCR is the segment whose boundaries are marked by the annealing positions of the two oligonu- cleotide primers. If the primers anneal either side of the gene of interest, many copies of that gene will be synthesized (Figure 1.5). The outcome is the same as with a gene cloning experiment, although the problem of selection does not arise because the desired gene is automatically “selected” as a result of the positions at which the primers anneal. A PCR experiment can be completed in a few hours, whereas it takes weeks if not months to obtain a gene by cloning. Why then is gene cloning still used? This is because PCR has two limitations: l In order for the primers to anneal to the correct positions, either side of the gene of interest, the sequences of these annealing sites must be known. It is easy to synthesize a primer with a predetermined sequence (see p. 139), but if the sequences of the annealing sites are unknown then the appropriate primers cannot be made. This means that PCR cannot be used to isolate genes that have not been studied before—that has to be done by cloning. 10 Part I The Basic Principles of Gene Cloning and DNA Analysis Figure 1.5 Gene isolation by PCR. pyrF dnaL cysB trpE trpD trpC trpB tonB trpA opp aroT Polymerase chain reaction trpA trpA trp A A trp trpA trpA trpA × several million l There is a limit to the length of DNA sequence that can be copied by PCR. Five kilobases (kb) can be copied fairly easily, and segments up to forty kb can be dealt with by using specialized techniques, but this is shorter than the lengths of many genes, especially those of humans and other vertebrates. Cloning must be used if an intact version of a long gene is required. Gene cloning is therefore the only way of isolating long genes or those that have never been studied before. But PCR still has many important applications. For example, even if the sequence of a gene is not known, it may still be possible to determine the appropriate sequences for a pair of primers, based on what is known about the sequence of the equivalent gene in a different organism. A gene that has been isolated and sequenced from, say, mouse could therefore be used to design a pair of primers for isolation of the equivalent gene from humans. In addition, there are many applications where it is necessary to isolate or detect genes whose sequences are already known. A PCR of human globin genes, for example, is used to test for the presence of mutations that might cause the blood disease called thalassaemia. Design of appropriate primers for this PCR is easy because the sequences of the human globin genes are known. After the PCR, the gene copies are sequenced or studied in some other way to determine if any of the thalassaemia mutations are present. Another clinical application of PCR involves the use of primers specific for the DNA of a disease-causing virus. A positive result indicates that a sample contains the virus and that the person who provided the sample should undergo treatment to prevent onset of the disease. The polymerase chain reaction is tremendously sensitive: a carefully set up reaction yields detectable amounts of DNA, even if there is just one DNA molecule in the starting mixture. This means that the technique can detect viruses at the earliest stages of an infection, increasing the chances of treatment being successful. This great sensitivity means that PCR can also be used with DNA from forensic material such as hairs and dried bloodstains or even from the bones of long-dead humans (Chapter 16). Chapter 1 Why Gene Cloning and DNA Analysis are Important 11 1.6 How to find your way through this book This book explains how gene cloning, PCR and other DNA analysis techniques are carried out and describes the applications of these techniques in modern biology. The applications are covered in the second and third parts of the book. Part II describes how genes and genomes are studied and Part III gives accounts of the broader applica- tions of gene cloning and PCR in biotechnology, medicine, agriculture, and forensic science. In Part I we deal with the basic principles. Most of the nine chapters are devoted to gene cloning because this technique is more complicated than PCR. When you have understood how cloning is carried out you will have understood many of the basic principles of how DNA is analyzed. In Chapter 2 we look at the central component of a gene cloning experiment—the vector—which transports the gene into the host cell and is responsible for its replication. To act as a cloning vector a DNA molecule must be capable of entering a host cell and, once inside, replicating to produce multiple copies of itself. Two naturally occurring types of DNA molecule satisfy these requirements: l Plasmids, which are small circles of DNA found in bacteria and some other organisms. Plasmids can replicate independently of the host cell chromosome. l Virus chromosomes, in particular the chromosomes of bacteriophages, which are viruses that specifically infect bacteria. During infection the bacteriophage DNA molecule is injected into the host cell where it undergoes replication. Chapter 3 describes how DNA is purified from living cells—both the DNA that will be cloned and the vector DNA—and Chapter 4 covers the various techniques for handling purified DNA molecules in the laboratory. There are many such techniques, but two are particularly important in gene cloning. These are the ability to cut the vector at a specific point and then to repair it in such a way that the gene is inserted (see Figure 1.1). These and other DNA manipulations were developed as an offshoot of basic research into DNA synthesis and modification in living cells, and most of the manipulations make use of purified enzymes. The properties of these enzymes, and the way they are used in DNA studies, are described in Chapter 4. Once a recombinant DNA molecule has been constructed, it must be introduced into the host cell so that replication can take place. Transport into the host cell makes use of natural processes for uptake of plasmid and viral DNA molecules. These processes and the ways they are utilized in gene cloning are described in Chapter 5, and the most important types of cloning vector are introduced, and their uses examined, in Chapters 6 and 7. To conclude the coverage of gene cloning, in Chapter 8 we investi- gate the problem of selection (see Figure 1.4), before returning in Chapter 9 to a more detailed description of PCR and its related techniques. 12 Part I The Basic Principles of Gene Cloning and DNA Analysis Further reading FURTHER READING Blackman, K. (2001) The advent of genetic engineering. Trends in Biochemical Science, 26, 268–270. [An account of the early days of gene cloning.] Brock, T.D. (1990) The Emergence of Bacterial Genetics. Cold Spring Harbor Laboratory Press, New York. [Details the discovery of plasmids and bacteriophages.] Brown, T.A. (2006) Genomes, 3rd edn. Garland Science, Oxford. [An introduction to modern genetics and molecular biology.] Cherfas, J. (1982) Man Made Life. Blackwell, Oxford. [A history of the early years of genetic engineering.] Judson, H.F. (1979) The Eighth Day of Creation. Penguin Science, London. [A very readable account of the development of molecular biology in the years before the gene cloning revolution.] Mullis, K.B. (1990) The unusual origins of the polymerase chain reaction. Scientific American, 262(4), 56–65. [An entertaining account of how PCR was invented.] Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages Chapter contents CHAPTER CONTENTS 2.1 Plasmids 2.2 Bacteriophages A DNA molecule needs to display several features to be able to act as a vector for gene cloning. Most importantly it must be able to replicate within the host cell, so that numerous copies of the recombinant DNA molecule can be produced and passed to the daughter cells. A cloning vector also needs to be relatively small, ideally less than 10 kb in size, as large molecules tend to break down during purification, and are also more difficult to manipulate. Two kinds of DNA molecule that satisfy these criteria can be found in bacterial cells: plasmids and bacteriophage chromosomes. 2.1 Plasmids Plasmids are circular molecules of DNA that lead an independent existence in the bacterial cell (Figure 2.1). Plasmids almost always carry one or more genes, and often these genes are responsible for a useful characteristic displayed by the host bacterium. For example, the ability to survive in normally toxic concentrations of antibiotics such as chloramphenicol or ampicillin is often due to the presence in the bacterium of a plasmid carrying antibiotic resistance genes. In the laboratory, antibiotic resistance is often used as a selectable marker to ensure that bacteria in a culture contain a particu- lar plasmid (Figure 2.2). Most plasmids possess at least one DNA sequence that can act as an origin of replica- tion, so they are able to multiply within the cell independently of the main bacterial Gene Cloning and DNA Analysis: An Introduction. 6th edition. By T.A. Brown. Published 2010 by 13 Blackwell Publishing. 14 Part I The Basic Principles of Gene Cloning and DNA Analysis Figure 2.1 Plasmids Plasmids Plasmids: independent genetic elements found in bacterial cells. Bacterial chromosome Figure 2.2 Ampicillin The use of antibiotic resistance as a selectable marker resistance for a plasmid. RP4 (top) carries genes for resistance to RP4 ampicillin, tetracycline and kanamycin. Only those E. coli cells that contain RP4 (or a related plasmid) are able to Kanamycin Tetracycline survive and grow in a medium that contains toxic resistance resistance amounts of one or more of these antibiotics. Cell with plasmid Cell without E. coli cells, some plasmid containing RP4 Normal growth Growth medium medium – +50 µg/ml no antibiotic tetracycline All cells Only cells containing can grow RP4 can grow chromosome (Figure 2.3a). The smaller plasmids make use of the host cell’s own DNA replicative enzymes in order to make copies of themselves, whereas some of the larger ones carry genes that code for special enzymes that are specific for plasmid replication. A few types of plasmid are also able to replicate by inserting themselves into the bac- terial chromosome (Figure 2.3b). These integrative plasmids or episomes may be stably maintained in this form through numerous cell divisions, but always at some stage exist as independent elements. Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages 15 (a) Non-integrative plasmid Plasmids Cell division Bacterial chromosome (b) Episome Plasmid Cell division Bacterial chromosome Chromosome carrying integrated plasmid Figure 2.3 Replication strategies for (a) a non-integrative plasmid, and (b) an episome. Table 2.1 Sizes of representative plasmids. SIZE NUCLEOTIDE LENGTH MOLECULAR MASS PLASMID (kb) (MDa) ORGANISM pUC8 2.1 1.8 E. coli ColE1 6.4 4.2 E. coli RP4 54.0 36.0 Pseudomonas and others F 95.0 63.0 E. coli TOL 117.0 78.0 Pseudomonas putida pTiAch5 213.0 142.0 Agrobacterium tumefaciens 2.1.1 Size and copy number The size and copy number of a plasmid are particularly important as far as cloning is concerned. We have already mentioned the relevance of plasmid size and stated that less than 10 kb is desirable for a cloning vector. Plasmids range from about 1.0 kb for the smallest to over 250 kb for the largest plasmids (Table 2.1), so only a few are useful for cloning purposes. However, as we will see in Chapter 7, larger plasmids can be adapted for cloning under some circumstances. The copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell. The factors that control copy number are not well understood. Some plasmids, especially the larger ones, are stringent and have a 16 Part I The Basic Principles of Gene Cloning and DNA Analysis Figure 2.4 Donor cell Conjugative Recipient cell plasmid Plasmid transfer by conjugation between bacterial cells. The donor and recipient cells attach to each other by a pilus, a hollow appendage present on the surface of the donor cell. A copy of the plasmid is then passed to the recipient cell. Transfer is thought to occur through the pilus, but this has not been proven and transfer by some other means (e.g. directly across the bacterial cell walls) remains a possibility. Pilus low copy number of perhaps just one or two per cell; others, called relaxed plasmids, are present in multiple copies of 50 or more per cell. Generally speaking, a useful cloning vector needs to be present in the cell in multiple copies so that large quantities of the recombinant DNA molecule can be obtained. 2.1.2 Conjugation and compatibility Plasmids fall into two groups: conjugative and non-conjugative. Conjugative plasmids are characterized by the ability to promote sexual conjugation between bacterial cells (Figure 2.4), a process that can result in a conjugative plasmid spreading from one cell to all the other cells in a bacterial culture. Conjugation and plasmid transfer are con- trolled by a set of transfer or tra genes, which are present on conjugative plasmids but absent from the non-conjugative type. However, a non-conjugative plasmid may, under some circumstances, be cotransferred along with a conjugative plasmid when both are present in the same cell. Several different kinds of plasmid may be found in a single cell, including more than one different conjugative plasmid at any one time. In fact, cells of E. coli have been known to contain up to seven different plasmids at once. To be able to coexist in the same cell, different plasmids must be compatible. If two plasmids are incompatible then one or the other will be rapidly lost from the cell. Different types of plasmid can there- fore be assigned to different incompatibility groups on the basis of whether or not they can coexist, and plasmids from a single incompatibility group are often related to each other in various ways. The basis of incompatibility is not well understood, but events during plasmid replication are thought to underlie the phenomenon. 2.1.3 Plasmid classification The most useful classification of naturally occurring plasmids is based on the main char- acteristic coded by the plasmid genes. The five major types of plasmid according to this classification are as follows: l Fertility or F plasmids carry only tra genes and have no characteristic beyond the ability to promote conjugal transfer of plasmids. A well-known example is the F plasmid of E. coli. Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages 17 l Resistance or R plasmids carry genes conferring on the host bacterium resistance to one or more antibacterial agents, such as chloramphenicol, ampicillin, and mercury. R plasmids are very important in clinical microbiology as their spread through natural populations can have profound consequences in the treatment of bacterial infections. An example is RP4, which is commonly found in Pseudomonas, but also occurs in many other bacteria. l Col plasmids code for colicins, proteins that kill other bacteria. An example is ColE1 of E. coli. l Degradative plasmids allow the host bacterium to metabolize unusual molecules such as toluene and salicylic acid, an example being TOL of Pseudomonas putida. l Virulence plasmids confer pathogenicity on the host bacterium; these include the Ti plasmids of Agrobacterium tumefaciens, which induce crown gall disease on dicotyledonous plants. 2.1.4 Plasmids in organisms other than bacteria Although plasmids are widespread in bacteria they are by no means as common in other organisms. The best characterized eukaryotic plasmid is the 2 Fm circle that occurs in many strains of the yeast Saccharomyces cerevisiae. The discovery of the 2 fm plasmid was very fortuitous as it allowed the construction of cloning vectors for this very import- ant industrial organism (p. 105). However, the search for plasmids in other eukaryotes (such as filamentous fungi, plants and animals) has proved disappointing, and it is sus- pected that many higher organisms simply do not harbor plasmids within their cells. 2.2 Bacteriophages Bacteriophages, or phages as they are commonly known, are viruses that specifically infect bacteria. Like all viruses, phages are very simple in structure, consisting merely of a DNA (or occasionally ribonucleic acid (RNA)) molecule carrying a number of genes, including several for replication of the phage, surrounded by a protective coat or capsid made up of protein molecules (Figure 2.5). (a) Head-and-tail (b) Filamentous Figure 2.5 The two main types of phage structure: (a) head-and- Head Protein tail (e.g. λ); (b) filamentous (e.g. M13). (contains molecules DNA) (capsid) DNA molecule Tail 18 Part I The Basic Principles of Gene Cloning and DNA Analysis Phage particle DNA 1 The phage attaches to the bacterium and injects its DNA Phage DNA molecule 2 The phage DNA molecule is replicated 3 Capsid components are synthesized, new phage particles are assembled and released Capsid components Cell lysis New phage particle Figure 2.6 The general pattern of infection of a bacterial cell by a bacteriophage. 2.2.1 The phage infection cycle The general pattern of infection, which is the same for all types of phage, is a three-step process (Figure 2.6): 1 The phage particle attaches to the outside of the bacterium and injects its DNA chromosome into the cell. 2 The phage DNA molecule is replicated, usually by specific phage enzymes coded by genes in the phage chromosome. 3 Other phage genes direct synthesis of the protein components of the capsid, and new phage particles are assembled and released from the bacterium. With some phage types the entire infection cycle is completed very quickly, possibly in less than 20 minutes. This type of rapid infection is called a lytic cycle, as release of the new phage particles is associated with lysis of the bacterial cell. The characteristic feature of a lytic infection cycle is that phage DNA replication is immediately followed Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages 19 by synthesis of capsid proteins, and the phage DNA molecule is never maintained in a stable condition in the host cell. 2.2.2 Lysogenic phages In contrast to a lytic cycle, lysogenic infection is characterized by retention of the phage DNA molecule in the host bacterium, possibly for many thousands of cell divisions. With many lysogenic phages the phage DNA is inserted into the bacterial genome, in a manner similar to episomal insertion (see Figure 2.3b). The integrated form of the phage DNA (called the prophage) is quiescent, and a bacterium (referred to as a lysogen) that carries a prophage is usually physiologically indistinguishable from an uninfected cell. However, the prophage is eventually released from the host genome and the phage reverts to the lytic mode and lyses the cell. The infection cycle of lambda (E), a typical lysogenic phage of this type, is shown in Figure 2.7. A limited number of lysogenic phages follow a rather different infection cycle. When M13 or a related phage infects E. coli, new phage particles are continuously assembled and released from the cell. The M13 DNA is not integrated into the bacterial genome and does not become quiescent. With these phages, cell lysis never occurs, and the infected bacterium can continue to grow and divide, albeit at a slower rate than uninfected cells. Figure 2.8 shows the M13 infection cycle. Although there are many different varieties of bacteriophage, only e and M13 have found a major role as cloning vectors. We will now consider the properties of these two phages in more detail. Gene organization in the 5 DNA molecule e is a typical example of a head-and-tail phage (see Figure 2.5a). The DNA is contained in the polyhedral head structure and the tail serves to attach the phage to the bacterial surface and to inject the DNA into the cell (see Figure 2.7). The e DNA molecule is 49 kb in size and has been intensively studied by the tech- niques of gene mapping and DNA sequencing. As a result the positions and identities of all of the genes in the e DNA molecule are known (Figure 2.9). A feature of the e genetic map is that genes related in terms of function are clustered together in the genome. For example, all of the genes coding for components of the capsid are grouped together in the left-hand third of the molecule, and genes controlling integration of the prophage into the host genome are clustered in the middle of the molecule. Clustering of related genes is profoundly important for controlling expression of the e genome, as it allows genes to be switched on and off as a group rather than individually. Clustering is also important in the construction of e-based cloning vectors, as we will discover when we return to this topic in Chapter 6. The linear and circular forms of 5 DNA A second feature of e that turns out to be of importance in the construction of cloning vectors is the conformation of the DNA molecule. The molecule shown in Figure 2.9 is linear, with two free ends, and represents the DNA present in the phage head struc- ture. This linear molecule consists of two complementary strands of DNA, base-paired according to the Watson–Crick rules (that is, double-stranded DNA). However, at either end of the molecule is a short 12-nucleotide stretch in which the DNA is single-stranded (Figure 2.10a). The two single strands are complementary, and so can base pair with one another to form a circular, completely double-stranded molecule (Figure 2.10b). 20 Part I The Basic Principles of Gene Cloning and DNA Analysis λ phage particle attaches to an E.coli cell and injects its DNA Bacterial chromosome λ DNA circularizes λ DNA λ DNA integrates into the host chromosome Cell Cell division division A Induction: A λ DNA excises from the host chromosome B New phage particles are produced (see steps 2 and 3 of figure 2.6) B Figure 2.7 The lysogenic infection cycle of bacteriophage λ. Complementary single strands are often referred to as “sticky” ends or cohesive ends, because base pairing between them can “stick” together the two ends of a DNA molecule (or the ends of two different DNA molecules). The e cohesive ends are called the cos sites and they play two distinct roles during the e infection cycle. First, they allow the linear DNA molecule that is injected into the cell to be circularized, which is a neces- sary prerequisite for insertion into the bacterial genome (see Figure 2.7). Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages 21 M13 phage Pilus M13 DNA M13 phage attaches to a pilus on an E.coli cell and injects its DNA M13 DNA replication New M13 phages are continuously extruded M13 phages from an infected cell Infected cells continue to grow and divide Daughter cells continue to release M13 particles Figure 2.8 The infection cycle of bacteriophage M13. Lysis of the host Early regulation Late regulation DNA synthesis and excision Integration Capsid components b2 region and assembly (non-essential) AWB C D EFZUVGT H M LKI J int xis exo clll N cl cro OP Q SR 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 49 kb Figure 2.9 The λ genetic map, showing the positions of the important genes and the functions of the gene clusters. The second role of the cos sites is rather different, and comes into play after the prophage has excised from the host genome. At this stage a large number of new e DNA molecules are produced by the rolling circle mechanism of replication (Figure 2.10c), in which a continuous DNA strand is “rolled off” the template molecule. The result is a catenane consisting of a series of linear e genomes joined together at the cos sites. The role of the cos sites is now to act as recognition sequences for an endonuclease that cleaves the catenane at the cos sites, producing individual e genomes. This endonu- clease, which is the product of gene A on the e DNA molecule, creates the single- stranded sticky ends, and also acts in conjunction with other proteins to package each e genome into a phage head structure. The cleavage and packaging processes recognize just the cos sites and the DNA sequences to either side of them, so changing the structure of the internal regions of the e genome, for example by inserting new genes, has no effect on these events so long as the overall length of the e genome is not altered too greatly. 22 Part I The Basic Principles of Gene Cloning and DNA Analysis (a) The linear form of the λ DNA molecule Right cohesive end C C C GC C G C T G GA GG G C GG CG A CC T Left cohesive end (b) The circular form of cos site the λ DNA molecule CC G TG CG G A C C GGCA C C GC T GG (c) Replication and packaging of λ DNA cos cos cos Catenane ‘rolled off’ cos the λ DNA molecule 3 2 1 The gene A endonuclease cleaves the catenane at the cos sites Protein components of the capsid New phage particles are assembled Figure 2.10 The linear and circular forms of λ DNA. (a) The linear form, showing the left and right cohesive ends. (b) Base pairing between the cohesive ends results in the circular form of the molecule. (c) Rolling circle replication produces a catenane of new linear λ DNA molecules, which are individually packaged into phage heads as new λ particles are assembled. M13—a filamentous phage M13 is an example of a filamentous phage (see Figure 2.5b) and is completely different in structure from e. Furthermore, the M13 DNA molecule is much smaller than the e genome, being only 6407 nucleotides in length. It is circular and is unusual in that it consists entirely of single-stranded DNA. The smaller size of the M13 DNA molecule means that it has room for fewer genes than the e genome. This is possible because the M13 capsid is constructed from multiple copies of just three proteins (requiring only three genes), whereas synthesis of the e Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages 23 M13 particle Figure 2.11 injects DNA The M13 infection cycle, showing the different into cell Pilus types of DNA replication that occur. (a) After infection the single-stranded M13 DNA molecule (a) Injection of single- is converted into the double-stranded replicative stranded DNA into form (RF). (b) The RF replicates to produce the host cell, followed by synthesis of the multiple copies of itself. (c) Single-stranded second strand molecules are synthesized by rolling circle replication and used in the assembly of new Single-stranded Double-stranded DNA DNA - replicative M13 particles. form (RF) (b) Replication of the RF to produce new double-stranded molecules (c) Mature M13 phage are continuously produced RF replicates by rolling circle mechanism to produce linear single-stranded DNA Circularized DNA Mature phage particles head-and-tail structure involves over 15 different proteins. In addition, M13 follows a simpler infection cycle than e, and does not need genes for insertion into the host genome. Injection of an M13 DNA molecule into an E. coli cell occurs via the pilus, the structure that connects two cells during sexual conjugation (see Figure 2.4). Once inside the cell the single-stranded molecule acts as the template for synthesis of a comple- mentary strand, resulting in normal double-stranded DNA (Figure 2.11a). This molecule is not inserted into the bacterial genome, but instead replicates until over 100 copies are present in the cell (Figure 2.11b). When the bacterium divides, each daughter cell receives copies of the phage genome, which continues to replicate, thereby maintaining its overall numbers per cell. As shown in Figure 2.11c, new phage particles are continu- ously assembled and released, about 1000 new phages being produced during each generation of an infected cell. Several features of M13 make this phage attractive as a cloning vector. The genome is less than 10 kb in size, well within the range desirable for a potential vector. In addi- tion, the double-stranded replicative form (RF) of the M13 genome behaves very much like a plasmid, and can be treated as such for experimental purposes. It is easily prepared from a culture of infected E. coli cells (p. 43) and can be reintroduced by transfection (p. 81). Most importantly, genes cloned with an M13-based vector can be obtained in the form of single-stranded DNA. Single-stranded versions of cloned genes are useful for several techniques, notably DNA sequencing and in vitro mutagenesis (pp. 169 and 203). Cloning in an M13 vector is an easy and reliable way of obtaining single-stranded DNA for this type of work. M13 vectors are also used in phage display, a technique for identifying pairs of genes whose protein products interact with one another (p. 220). 24 Part I The Basic Principles of Gene Cloning and DNA Analysis 2.2.3 Viruses as cloning vectors for other organisms Most living organisms are infected by viruses and it is not surprising that there has been great interest in the possibility that viruses might be used as cloning vectors for higher organisms. This is especially important when it is remembered that plasmids are not commonly found in organisms other than bacteria and yeast. Several eukaryotic viruses have been employed as cloning vectors for specialized applications: for example, human adenoviruses are used in gene therapy (p. 259), baculoviruses are used to synthesize important pharmaceutical proteins in insect cells (p. 240), and caulimoviruses and geminiviruses have been used for cloning in plants (p. 120). These vectors are discussed more fully in Chapter 7. Further reading FURTHER READING Dale, J.W. & Park, S.T. (2004) Molecular Genetics of Bacteria, 4th edn. Wiley Blackwell, Chichester. [Provides a detailed description of plasmids and bacteriophages.] Willey, J., Sherwood, L. & Woolverton, C. (2007) Prescott’s Microbiology, 7th edn. McGraw Hill Higher Education, Maidenhead. [A good introduction to microbiology, including plasmids and phages.] Chapter 3 Purification of DNA from Living Cells Chapter contents CHAPTER CONTENTS 3.1 Preparation of total cell DNA 3.2 Preparation of plasmid DNA 3.3 Preparation of bacteriophage DNA The genetic engineer will, at different times, need to prepare at least three distinct kinds of DNA. First, total cell DNA will often be required as a source of material from which to obtain genes to be cloned. Total cell DNA may be DNA from a culture of bacteria, from a plant, from animal cells, or from any other type of organism that is being studied. It consists of the genomic DNA of the organism along with any additional DNA mole- cules, such as plasmids, that are present. The second type of DNA that will be required is pure plasmid DNA. Preparation of plasmid DNA from a culture of bacteria follows the same basic steps as purification of total cell DNA, with the crucial difference that at some stage the plasmid DNA must be separated from the main bulk of chromosomal DNA also present in the cell. Finally, phage DNA will be needed if a phage cloning vector is to be used. Phage DNA is generally prepared from bacteriophage particles rather than from infected cells, so there is no problem with contaminating bacterial DNA. However, special techniques are needed to remove the phage capsid. An exception is the double-stranded replicative form of M13, which is prepared from E. coli cells in the same way as a bacterial plasmid. 3.1 Preparation of total cell DNA The fundamentals of DNA preparation are most easily understood by first considering the simplest type of DNA purification procedure, that where the entire DNA complement Gene Cloning and DNA Analysis: An Introduction. 6th edition. By T.A. Brown. Published 2010 by 25 Blackwell Publishing. 26 Part I The Basic Principles of Gene Cloning and DNA Analysis Centrifugation Bacterial culture Pellet of cells Cell extract Pure DNA 1 2 3 4 A culture of bacteria is The cells are removed and The DNA is purified The DNA is grown and then harvested broken to give a cell extract from the cell extract concentrated Figure 3.1 The basic steps in preparation of total cell DNA from a culture of bacteria. of a bacterial cell is required. The modifications needed for plasmid and phage DNA preparation can then be described later. The procedure for total DNA preparation from a culture of bacterial cells can be divided into four stages (Figure 3.1): 1 A culture of bacteria is grown and then harvested. 2 The cells are broken open to release their contents. 3 This cell extract is treated to remove all components except the DNA. 4 The resulting DNA solution is concentrated. 3.1.1 Growing and harvesting a bacterial culture Most bacteria can be grown without too much difficulty in a liquid medium (broth culture). The culture medium must provide a balanced mixture of the essential nutrients at concentrations that will allow the bacteria to grow and divide efficiently. Two typical growth media are detailed in Table 3.1. M9 is an example of a defined medium in which all the components are known. This medium contains a mixture of inorganic nutrients to provide essential elements such as Table 3.1 The composition of two typical media for the growth of bacterial cultures. MEDIUM COMPONENT g/l OF MEDIUM M9 medium Na2HPO4 6.0 KH2PO4 3.0 NaCl 0.5 NH4Cl 1.0 MgSO4 0.5 Glucose 2.0 CaCl2 0.015 LB (Luria-Bertani medium) Tryptone 10.0 Yeast extract 5.0 NaCl 10.0 Chapter 3 Purification of DNA from Living Cells 27 nitrogen, magnesium, and calcium, as well as glucose to supply carbon and energy. In practice, additional growth factors such as trace elements and vitamins must be added to M9 before it will support bacterial growth. Precisely which supplements are needed depends on the species concerned. The second medium described in Table 3.1 is rather different. Luria-Bertani (LB) is a complex or undefined medium, meaning that the precise identity and quantity of its components are not known. This is because two of the ingredients, tryptone and yeast extract, are complicated mixtures of unknown chemical compounds. Tryptone in fact supplies amino acids and small peptides, while yeast extract (a dried preparation of partially digested yeast cells) provides the nitrogen requirements, along with sugars and inorganic and organic nutrients. Complex media such as LB need no further supple- mentation and support the growth of a wide range of bacterial species. Defined media must be used when the bacterial culture has to be grown under precisely controlled conditions. However, this is not necessary when the culture is being grown simply as a source of DNA, and under these circumstances a complex medium is appropriate. In LB medium at 37°C, aerated by shaking at 150–250 rpm on a rotary platform, E. coli cells divide once every 20 min or so until the culture reaches a maximum density of about 2–3 × 109 cells /ml. The growth of the culture can be mon- itored by reading the optical density (OD) at 600 nm (Figure 3.2), at which wavelength 1 OD unit corresponds to about 0.8 × 109 cells /ml. In order to prepare a cell extract, the bacteria must be obtained in as small a volume as possible. Harvesting is therefore performed by spinning the culture in a centrifuge (Figure 3.3). Fairly low centrifugation speeds will pellet the bacteria at the bottom of the centrifuge tube, allowing the culture medium to be poured off. Bacteria from a (a) Measurement of optical density Figure 3.2 Estimation of bacterial cell number by Incident light measurement of optical density (OD). (a) A at 600 nm Transmitted OD sample of the culture is placed in a glass cuvette wavelength light and light with a wavelength of 600 nm shone through. The amount of light that passes Detector through the culture is measured and the OD (also called the absorbance) calculated as: Bacterial culture contained intensity of transmitted light 1 OD unit = log10 in a 1 cm wide cuvette intensity of incident light The operation is performed with a (b) Estimation of cell number spectrophotometer. (b) The cell number from a calibration curve corresponding to the OD reading is calculated 2.5 from a calibration curve. This curve is plotted from the OD values of a series of cultures Optical density at 600 nm 2.0 of known cell density. For E. coli, 1 OD unit = 0.8 × 109 cells/ml. 1.5 1.0 0.5 0 0.4 0.8 1.2 1.6 2.0 Cell number (× 109) per ml 28 Part I The Basic Principles of Gene Cloning and DNA Analysis Spin at 8000 rpm for 10 minutes Bacterial culture Pellet of bacteria Centrifuge rotor Figure 3.3 Harvesting bacteria by centrifugation. 1000 ml culture at maximum cell density can then be resuspended into a volume of 10 ml or less. 3.1.2 Preparation of a cell extract The bacterial cell is enclosed in a cytoplasmic membrane and surrounded by a rigid cell wall. With some species, including E. coli, the cell wall may itself be enveloped by a second, outer membrane. All of these barriers have to be disrupted to release the cell components. Techniques for breaking open bacterial cells can