POGA01 12/8/05 8:41 AM Page i Principles of Gene Manipulation and Genomics ·· POGA01 12/8/05 8:41 AM Page ii ·· POGA01 12/8/05 8:41 AM Page iii Principles of Gene Manipulation and Genomics SEVENTH EDITION S.B. Primrose and R.M. Twyman ·· POGA01 12/8/05 8:41 AM Page iv © 2006 Blackwell Publishing BLACKWELL PUBLISHING 350 Main Street, Malden, MA 02148-5020, USA 9600 Garsington Road, Oxford OX4 2DQ, UK 550 Swanston Street, Carlton, Victoria 3053, Australia The rights of Sandy Primrose and Richard Twyman to be identified as the Authors of this Work have been asserted in accordance with the UK 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. This material was originally published in two separate volumes: Principles of Gene Manipulation, 6th edition (2001) and Principles of Genetic Analysis and Genomics, 3rdedition (2003). First published 1980 Second edition published 1981 Third edition published 1985 Fourth edition published 1989 Fifth edition published 1994 Sixth edition published 2001 Seventh edition published 2006 1 2006 Library of Congress Cataloging-in-Publication Data Primrose, S.B. Principles of gene manipulation and genomics / S.B. Primrose and R.M. Twyman.—7th ed. p. ; cm. Rev. ed. of: Principles of gene manipulation. 6th ed. 2001 and: Principles of genome analysis and genomics / Sandy B. Primrose, Richard M. Twyman. 3rd ed. 2003. Includes bibliographical references and index. ISBN 1-4051-3544-1 (pbk. : alk. paper) 1. Genetic engineering. 2. Genomics. 3. Gene mapping. 4. Nucleotide sequence. [DNLM: 1. Genetic Engineering. 2. Base Sequence. 3. Chromosome Mapping. 4. DNA, Recombinant. 5. Genomics. QH 442 P952pa 2006] I. Twyman, Richard M. II. Primrose, S.B. Principles of gene manipulation. III. Primrose, S. B. Principles of genome analysis and genomics. IV. Title. QH442.O42 2006 660.6′5—dc22 2005018202 A catalogue record for this title is available from the British Library. Set in 10/12.5pt Photina by Graphicraft Limited, Hong Kong Printed and bound in the United Kingdom by TJ International, Padstow, Cornwall, UK The publisher’s policy is to use permanent paper from mills that operate a sustainable forestry policy, and which has been manufactured from pulp processed using acid-free and elementary chlorine-free practices. Furthermore, the publisher ensures that the text paper and cover board used have met acceptable environmental accreditation standards. For further information on Blackwell Publishing, visit our website: www.blackwellpublishing.com ·· POGA01 12/8/05 8:41 AM Page v Contents Preface, xviii Southern blotting is the method used to Abbreviations, xx transfer DNA from agarose gels to membranes so that the compositional properties of the 1 Gene manipulation in the DNA can be analyzed, 18 post-genomics era, 1 Northern blotting is a variant of Southern Introduction, 1 blotting that is used for RNA analysis, 19 Gene manipulation involves the creation Western blotting is used to transfer proteins and cloning of recombinant DNA, 1 from acrylamide gels to membranes, 19 Recombinant DNA has opened new horizons A number of techniques have been devised in medicine, 3 to speed up and simplify the blotting Mapping and sequencing technologies formed process, 24 a crucial link between gene manipulation The ability to transform E. coliwith DNA is an and genomics, 4 essential prerequisite for most experiments on The genomics era began in earnest in 1995 gene manipulation, 24 with the complete sequencing of a Electroporation is a means of introducing DNA bacterial genome, 6 into cells without making them competent for Genome sequencing greatly increases our transformation, 25 understanding of basic biology, 7 The ability to transform organisms other The post-genomics era aims at the complete than E. coliwith recombinant DNA enables characterization of cells at all levels, 7 genes to be studied in different host Recombinant DNA technology and genomics backgrounds, 25 form the foundation of the biotechnology The polymerase chain reaction (PCR) has industry, 8 revolutionized the way that biologists Outline of the rest of the book, 8 manipulate and analyze DNA, 26 The principle of the PCR is exceedingly Part I Fundamental Techniques of Gene simple, 27 Manipulation RT-PCR enables the sequences on a mRNA molecule to be amplified as DNA, 28 2 Basic techniques, 15 The basic PCR is not efficient at amplifying Introduction, 15 long DNA fragments, 28 Three technical problems had to be solved The success of a PCR experiment is very before in vitrogene manipulation was possible dependent on the choice of experimental on a routine basis, 15 variables, 29 A number of basic techniques are common By using special instrumentation it is possible to most gene-cloning experiments, 15 to make the PCR quantitative, 30 Gel electrophoresis is used to separate There are a number of different ways of different nucleic acid molecules on the basis generating fluorescence in quantitative PCR of their size, 16 reactions, 31 Blotting is used to transfer nucleic acids It is now possible to amplify whole genomes as from gels to membranes for further analysis, 18 well as gene segments, 34 ·· POGA01 12/8/05 8:41 AM Page vi vi CONTENTS 3 Cutting and joining DNA molecules, 36 The host range of plasmids is determined by Cutting DNA molecules, 36 the replication proteins that they encode, 57 Understanding the biological basis of host- The number of copies of a plasmid in a cell controlled restriction and modification of varies between plasmids and is determined by bacteriophage DNA led to the identification of the regulatory mechanisms controlling restriction endonucleases, 36 replication, 57 Four different types of restriction and The stable maintenance of plasmids in modification (R-M) system have been cells requires a specific partitioning recognized but only one is widely used in gene mechanism, 59 manipulation, 37 Plasmids with similar replication and The naming of restriction endonucleases partitioning systems cannot be maintained in provides information about their source, 39 the same cell, 59 Restriction enzymes cut DNA at sites of The purification of plasmid DNA, 59 rotational symmetry and different enzymes Good plasmid cloning vehicles share a number recognize different sequences, 39 of desirable features, 61 The G+C content of a DNA molecule affects its pBR322 is an early example of a widely used, susceptibility to different restriction purpose-built cloning vector, 62 endonucleases, 41 Example of the use of plasmid pBR322 as a Simple DNA manipulations can convert vector: isolation of DNA fragments which a site for one restriction enzyme into a site carry promoters, 64 for another enzyme, 41 A large number of improved vectors have Methylation can reduce the susceptibility been derived from pBR322, 64 of DNA to cleavage by restriction Bacteriophage λλ, 66 endonucleases and the efficiency The genetic organization of bacteriophage λ of DNA transformation, 42 favors its subjugation as a vector, 66 It is important to eliminate restriction systems Bacteriophage λhas sophisticated control in E. colistrains used as hosts for recombinant circuits, 66 DNA, 43 There are two basic types of phage λ The success of a cloning experiment is vectors: insertional vectors and critically dependent on the quality of any replacement vectors, 69 restriction enzymes that are used, 43 A number of phage λvectors with improved Joining DNA molecules, 44 properties have been described, 69 The enzyme DNA ligase is the key to joining By packaging DNA into phage λin vitroit is DNA molecules in vitro, 44 possible to eliminate the need for competent Adaptors and linkers are short double- cells of E. coli, 70 stranded DNA molecules that permit different DNA cloning with single-stranded DNA cleavage sites to be interconnected, 48 vectors, 71 Homopolymer tailing is a general method for Filamentous bacteriophages have a number of joining DNA molecules that has special unique properties that make them suitable as uses, 49 vectors, 72 Special methods are often required if DNA Vectors with single-stranded DNA genomes produced by PCR amplification is to be have specialist uses, 72 cloned, 49 Phage M13 has been modified to make it a DNA molecules can be joined without DNA better vector, 72 ligase, 50 Amplified DNA can be cloned using in vitro 5 Cosmids, phasmids, and other advanced recombination, 50 vectors, 75 Introduction, 75 4 Basic biology of plasmid and phage Vectors for cloning large fragments of vectors, 55 DNA, 75 Plasmid biology and simple plasmid Cosmids are plasmids that can be packaged vectors, 55 into bacteriophage λparticles, 75 ·· POGA01 12/8/05 8:41 AM Page vii Contents vii BACs and PACs are vectors that can carry Fragment libraries can be prepared from much larger fragments of DNA than cosmids material that is unsuitable for conventional because they do not have packaging library cloning, 102 constraints, 76 Complementary DNA (cDNA) libraries are Recombinogenic engineering generated by the reverse transcription of (recombineering) simplifies the cloning of mRNA, 102 DNA, particularly with high-molecular- cDNA is representative of the mRNA weight constructs, 79 population, and therefore reflects mRNA A number of factors govern the choice of levels and the diversity of splice isoforms in vector for cloning large fragments of DNA, 81 particular tissues, 102 Specialist-purpose vectors, 81 The first stage of cDNA library construction is M13-based vectors can be used to make the synthesis of double-stranded DNA using single-stranded DNA suitable for mRNA as the template, 105 sequencing, 81 Obtaining full-length cDNA for cloning can be Expression vectors enable a cloned gene to be a challenge, 107 placed under the control of a promoter that The PCR can be used as an alternative to functions in E. coli, 81 cDNA cloning, 110 Specialist vectors have been developed that Full-length cDNA cloning is facilitated by the facilitate the production of RNA probes and rapid amplification of cDNA ends (RACE), 111 interfering RNA, 82 Many different strategies are available Vectors with strong, controllable promoters for library screening, 111 are used to maximize synthesis of cloned gene Both genomic and cDNA libraries can be products, 85 screened by hybridization, 111 Purification of a cloned gene product can be Probes are designed to maximize the chances facilitated by use of purification tags, 87 of recovering the desired clone, 113 Vectors are available that promote The PCR can be used as an alternative to solubilization of expressed proteins, 92 hybridization for the screening of genomic Proteins that are synthesized with signal and cDNA libraries, 115 sequences are exported from the cell, 93 More diverse strategies are available for the The Gateway®system is a highly efficient screening of expression libraries, 116 method for transferring DNA fragments to a Immunological screening uses specific large number of different vectors, 94 antibodies to detect expressed gene products, 116 Putting it all together: vectors with Southwestern and northwestern screening are combinations of features, 94 used to detect clones encoding nucleic acid binding proteins, 117 6 Gene-cloning strategies, 96 Functional cloning exploits the biochemical or Introduction, 96 physiological activity of the gene product, 119 Genomic DNA libraries are generated Positional cloning is used when there is no by fragmenting the genome and cloning biological information about a gene, but its overlapping fragments in vectors, 97 position can be mapped relative to other genes The first genomic libraries were cloned in or markers, 121 simple plasmid and phage vectors, 97 Difference cloning exploits differences in More sophisticated vectors have been the abundance of particular DNA developed to facilitate genomic library fragments, 121 construction, 99 Library-based approaches may involve Genomic libraries for higher eukaryotes differential screening or the creation of are usually constructed using high- subtracted libraries enriched for differentially capacity vectors, 101 represented clones, 122 The PCR can be used as an alternative to Differentially expressed genes can also be genomic DNA cloning, 101 identified using PCR-based methods, 122 Long PCR uses a mixture of enzymes to amplify Representational difference analysis is a PCR- long DNA templates, 102 based subtractive-cloning procedure, 124 ·· POGA01 12/8/05 8:41 AM Page viii viii CONTENTS 7 Sequencing genes and short stretches Altered proteins can be produced by of DNA, 126 inserting unusual amino acids during The commonest method of DNA sequencing protein synthesis, 147 is Sanger sequencing (also known as chain- Phage display can be used to facilitate the terminator or dideoxy sequencing), 126 selection of mutant peptides, 148 The original Sanger method has been greatly Cell-surface display is a more versatile improved by a number of experimental alternative to phage display, 149 modifications, 128 Protein engineering, 150 It is possible to automate DNA sequencing by A number of different methods of gene replacing radioactive labels with fluorescent shuffling have been developed, 153 labels, 130 Chimeric proteins can be produced in the DNA sequencing throughput can be greatly absence of gene homology, 154 increased by replacing slab gels with capillary array electrophoresis, 131 9 Bioinformatics, 157 The accuracy of automated DNA sequencing Introduction, 157 can be determined with basecalling Databases are required to store and algorithms, 131 cross-reference large biological Different strategies are required depending datasets, 158 on the complexity of the DNA to be The primary nucleotide sequence databases sequenced, 132 are repositories for annotated nucleotide Alternatives to Sanger sequencing have been sequence data, 158 developed and are particularly useful for SWISS-PROT and TrEMBL are databases of resequencing of DNA, 134 annotated protein sequences, 158 Pyrosequencing permits sequence analysis The Protein Databank is the main repository in real time, 134 for protein structural information, 160 It is possible to sequence DNA by Secondary sequence databases pull out hybridization using microarrays, 136 common features of protein sequences Massively parallel signature sequencing and structures, 160 can be used to monitor RNA Other databases cover a variety of useful abundance, 140 topics, 163 Methods are being developed for sequencing Sequence analysis is based on alignment single DNA molecules, 140 scores, 163 Algorithms for pairwise similarity searching 8 Changing genes: site-directed find the best alignment between pairs of mutagenesis and protein sequences, 164 engineering, 141 Multiple alignments allow important Introduction, 141 features of gene and protein families to be Primer extension (the single-primer method) identified, 166 is a simple method for site-directed Sequence analysis of genomic DNA mutation, 141 involves the de novoidentification of The single-primer method has a number of genes and other features, 166 deficiencies, 142 Genes in prokaryotic DNA can often be found Methods have been developed that by six-frame translation, 166 simplify the process of making all Algorithms have been developed that find possible amino acid substitutions at genes automatically, 168 a selected site, 143 Additional algorithms are necessary to find The PCR can be used for site-directed non-coding RNA genes and regulatory mutagenesis, 144 elements, 171 Methods are available to enable mutations to Several in silicomethods are available be introduced randomly throughout a target for the functional annotation of gene, 146 genes, 173 ·· POGA01 12/8/05 8:41 AM Page ix Contents ix Caution must be exercised when using Specialist vectors have been developed that purely in silicomethods to annotate permit controlled expression in B. subtilisand genomes, 175 other low-GC hosts, 194 Sequencing also provides new data for Vectors have been developed that facilitate molecular phylogenetics, 175 secretion of foreign proteins from B. subtilis, 195 Part II Manipulating DNA in Microbes, As an aid to understanding gene function in Plants, and Animals B. subtilis, vectors have been developed for directed gene inactivation, 195 10 Cloning in bacteria other than The mechanism whereby B. subtilisis Escherichia coli, 179 transformed with plasmid DNA facilitates the Introduction, 179 ordered assembly of dispersed genes, 196 Many bacteria are naturally competent A variety of different methods can be used to for transformation, 179 transform high-GC organisms such as the Recombinant DNA needs to replicate or be streptomycetes, 196 integrated into the chromosome in new Most of the vectors used with streptomycetes hosts, 183 are derivatives of endogenous plasmids and Recombinant DNA can integrate into the bacteriophages, 199 chromosome in different ways, 183 Cloning in Archaea, 200 Cloning in Gram-negative bacteria other than E. coli, 185 11 Cloning in Saccharomyces cerevisiaeand Vectors derived from the IncQ-group plasmid other fungi, 202 RSF1010 are not self-transmissible, 185 There are a number of reasons for cloning Mini-versions of the IncP-group plasmids have DNA in S.cerevisiae, 202 been developed as conjugative broad-host- Fungi are not naturally transformable and range vectors, 186 special methods are required to introduce Vectors derived from the broad-host-range exogenous DNA, 202 plasmid Sa are used mostly with Agrobacterium Exogenous DNA that is not carried on a vector tumefaciens, 187 can only be maintained by integration into a pBBR1 is another plasmid that has been chromosome, 203 used to develop broad-host-range cloning Different kinds of vector have been developed vectors, 188 for use in S.cerevisiae, 204 Cloned DNA can be shuttled between The availability of different kinds of vector high-copy-number and low-copy-number offers yeast geneticists great flexibility, 205 vectors, 188 Recombinogenic engineering can be Proper transcriptional analysis of a cloned used to move genes from one vector to gene requires that it is present on the another, 207 chromosome, 188 Yeast promoters are more complex than Cloning in Gram-positive bacteria, 189 bacterial promoters, 208 Many of the cloning vectors used with Promoter systems have been developed to Bacillus subtilisand other low-GC bacteria facilitate overexpression of recombinant are derived from plasmids found in proteins in yeast, 209 Staphylococcus aureus, 190 A number of specialist multi-purpose The mode of plasmid replication can vectors have been developed for use affect the stability of cloning vectors in in yeast, 211 B. subtilis, 191 Heterologous proteins can be synthesized Compared with E. coli, B. subtilishas additional as fusions for display on the cell surface of requirements for efficient transcription and yeast, 212 translation and this can prevent the expression The methylotrophic yeast Pichia pastorisis of genes from Gram-negative organisms in particularly suited to high-level expression ones that are Gram-positive, 194 of recombinant proteins, 212 ··