Principles of Gene Manipulation and Genomics

667 Pages·2005·5.94 MB·English

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Principles of Gene Manipulation
and Genomics
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Principles of Gene
Manipulation and
Genomics
SEVENTH EDITION
S.B. Primrose and R.M. Twyman
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© 2006 Blackwell Publishing
BLACKWELL PUBLISHING
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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.
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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
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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
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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
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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
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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
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