Principles of Gene Manipulation and Genomics Principles of Gene Manipulation and Genomics S EVENTH EDITION S. Twyman © 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, 3rd edition (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. Principles of gene manipulation and genomics / S. of: Principles of gene manipulation.
2001 and: Principles of genome analysis and genomics / Sandy B. Includes bibliographical references and index. Principles of gene manipulation. Principles of genome analysis and genomics.6′5—dc22 2005018202 A catalogue record for this title is available from the British Library.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.com 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. coli with 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. coli with 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 vitro gene 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 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 λ , 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 λ has sophisticated control in E.
coli strains 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 λ vectors 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 λ in vitro it 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 λ particles, 75 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.