Gene Cloning and DNA Analysis: An Introduction (Brown,Gene Cloning and DNA Analysis)

405 Pages·2006·20.53 MB·English

by  Terry A. Brown

,,-----------------,
--~--~--,--.- ----~"--'"
GENE CLONING
AND DNA ANAL I
An Introduction
A, ROW
Faculty of Life Sciences
University of Manchester
UK
Fifth Edition
'~( I~I Blackwell
Publishing
'f) 2001 bv BlackWell Ltd
1;;; 2006 T:A. Brown
Blackwell Publishing
Editorial offices: "
Blackwell Publishing Ltd, 9600 Road, Oxford OX4 2DQ, UK
Tel: ~A4 (0)1865 776868
Blackwell Publishmg Inc .. 350 Main Malden. MA 02148-5020, USA
Tel: +} '781 388 8250
Blackwell Publishing Asia Pty Ltd, 550 Swanston Street, Carltor:, Victoria Australia
TeL +61 (Oj3 8359 1011
The right of the Author to be identified the Author of this Work has been
accord-anee 'xith the and Patents Act 1988,
All rights reserved, No may be reproduced, stored in a retrieval
or transmitted, in !lny or electronic, mechanical,
nnm(1rr,fWlTW
otherwise, except as permitted Designs and Patents
prior of the publisher.
First, second and third edilions by Chapman & Hall 1S98 6r, o19t9 0L o1Q! G.:; E
"thomes (Pubiishers'l t"ff{()MA,.,
Fourth edition Blackwell Sci. ",Ii
Fifth edition '\' , • ~ N r, . " 8 H H. • A ,
RUAS",
j.,
lSBN-13: 978-14C51-1121-8 a: ....
ISB!'J-lO: 1-4051-1121-6 ).AT E, ". ...-. "" ,.-, ..... '\'q'~
", EsstON NO •. J,;,;:;,.: ...
u"'~"""u Da\[1 lIiIil~~'~'
an lIltmduction ! T,A. Brown. - 5th ed,
em,
DNA - Analysis.
572.8' - dc22
2005006606
A catalogue record for this litlr,;; is available from British Library
Set iIi JO!D Plfim:;s
by SN? Best-set Typesetter Ltd"
Print;:;d and bound ill Singapore
by Markollo PrilH Ivredia Pte Ltd
The publisher'~ polk\' i~ to use permanent from mills that operate a sustainable
policy, and which has r.(;:en manufactured nrf)"p,<.prI using acid-free and elementary
chlorine·frc·" practices, Fnrthermore, the ensures that the text paper and cover board
used have md acceptable C1iviron:me,nt,~1 accreditation standards,
For t<.lIther Illfurmation on BJack\vell 1>,,1'_1;,1-:_ visit our website:
wW'\Y.blackwellpubJishing,com
7 _7R g-me tM"W1f .....
Contents

Preface to the Fifth Edition, xv

Preface to the Fourth Edition, xvi

Preface to the Third Edition, xvii

Preface to the Second Edition, xviii

Preface to the First Edition, xx
p.~~t '1: ; The Basic Principles of Gene ClonEng and
" . ! t .
DNA. A~lysis~ 1

,:,; .
1 ,~TJ1yGene 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, 8

1.5.1 Gene isolation by cloning, 8

1.5.2 Gene isolation by PCR, 9

1.6 How to find your way through this book, 12

2 Vectors for Gene Cloning: Plasmids and Bacteriophages, 14

2.1 Plasmids. 14

2.1.1 Basic features of plasmids, 14

2.1.2 Size and copy number, 16

2.1.3 Conjugation and compatibility, 17

2.1.4 Plasmid classification, 18

2.1.5 Plasmids in organisms other than bacteria, 19

2.2 Bacteriophages, 19

2.2.1 Basic features of bacteriophages, 19

2.2.2 Lysogenic phages, 20

v
t'ir
Contents

Preface to the Fifth Edition, xv

Preface to the Fourth Edition, xvi

Preface to the Third Edition, xvii

Preface to the Second Edition, xviii

Preface to the First Edition, xx
t
I '( t
P~tt '1: The Basic Principles of Gene C~onB!l'!g and
DNA" " ~A n., a lysis, 1
; ~- ,; ;
1 ,yvliyGene 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, 8

1.5.1 Gene isolation by cloning. 8

1.5.2 Gene isolation by PCR 9

How to find your way through this book, 12

2 Vectors for Gene Cloning: Plasmids and Bacteriophages, 14

2.1 Plasmids, 14

2,1.1 Basic features of plasmids, 14

2,1.2 Size and copy number, 16

2.1.3 Conjugation and compatibility, 17

2.1.4 Plasmid classification,18
2.1.5 Plasmids in organisms other than bacteria, 19

2.2 Bacteriophages, 19

2.2,1 Basic features of bacteriophages, 19

2.2.2 Lysogenic phages, 20

v
Contents
4.2.2 Type II restriction endonucleases cut DNA at specific

nucleotide sequences, 64

4.2.3 Blunt ends and sticky ends, 64

4.2.4 The frequency of recognition sequences in a DNA

molecule, 65

Performing a restriction digest in the laboratory, 68

4.2.6 Analysing the result of restriction endonuclease

cleavage, 70

Separation of molecules by,gel electrophoresis, 70

Visualizing DNA molecules by staining a gel, 70

Visualizing DNA molecules by autoradiography, 72

4.2.7 Estimation of the sizes of DNA molecules, 74

4.2.8 :\lapping the positions of different restriction sites in a

DNA molecule. 76

4.3 Ligation joining DNA molecules together, 78

4.3.1 The mode of action of DNA ligase, 78

4.3.2 Sticky ends increase the efficiency of ligation, 78

4.3.3 Putting sticky ends onto a blunt-ended molecule, 80

Linkers, 80

Adaptors, 80

Producing sticky ends by homopolymer tailing, 84

5 Introduction of DNA into Living Cells, 87

, Transformation the uptake of DNA by bacterial cells, 90

5.1.1 Not all species of bacteria are equally efficient at DNA

uptake, 90

Preparation of competent E. coli cells, 90

5.1.3 Selection for transformed cells, 91

5.2 Identification of recombinants, 93

5.2.1 Recombinant selection with pBR322 insertional

inactivation of an antibiotic resistance gene, 93

5.2.2 Insertional inactivation does not always involve antibiotic

resistance, 95

5.3 Introduction of phage DNA into bacterial cells, 97

5.3.1 Transfection,98
5.3.2 In vitro packaging of A cloning vectors, 98

5.3.3 Phage infection is visualized as plaques on an agar

medium, 99

5.4 Identification of recombinant phages, 101

5.4.1 Insertional inactivation of a lacZ' gene carried by the

phage vector, 101

5.4.2 Insertional inactivation of the AcI 101

vii
Contents
5.4.3 Selection using the Spi phenotype, 103

5.4.4 Selection on the basis of 'A genome size, 103

5.5 Introduction of DNA into non-bacterial cells, 103

5.5.1 Transformation of individual cells, 103

5.5.2 Transformation of whole organisms, 106

6 Cloning Vectors for E. coli. 107

6.1 Cloning vectors based on E. coli plasmids, 108

6.1.1 1be nomenclature of plasmid cloning vectors, 108

6.1.2 The useful properties of pBR322, 108

6.1.3 The pedigree of pBR322, 109

6.1.4 More sophisticated E. coli plasmid cloning vectors, 109

pUC8 - a Lac selection plasmid, 111

pGEM3Z in vitro transcription of cloned DNA, 113

6.2 Cloning vectors based on M13 bacteriophage, 114

6.2.1 Development of the M13 cloning vectors, 115

M13mp7 - symmetrical cloning sites, 115

More complex M13 vectors, 118

6.2.2 Hybrid plasmid-M13 vectors, 120

6.3 Cloning vectors based on A bacteriophage, 120

6.3.1 Segments of the A genome can be deleted without

impairing viability, 122

6.3.2 Natural selection can be used to isolate modified A that

lack certain restriction sites, 123

6.3.3 Insertion and replacement vectors, 123

Insertion vectors, 123

Replacement vectors, 124

6.3.4 Cloning experiments with Ainsertion or replacement

vectors. 126

6.3.5 Long DNA fragments can be cloned using a cosmid, 127

6.4 A and other high capacity vectors enable genomic libraries to be

constructed, 129

6.5 Vectors for other bacteria. 130

7 Cloning Vectors for Eukaryotes, 132

7.1 Vectors for yeast and other fungi, 132

7.1.1 Selectable markers for the 2 J.lm plasmid, 132

7.1.2 Vectors based on the 2 J.lm plasmid yeast episomal

plasmids, 133

7.1.3 A YEp may insert into yeast chromosomal DNA, 135

7.1.4 Other types of yeast cloning vector, 135

7.1.5 Artificial chromosomes can be used to clone long pieces

of DNA in yeast, 138

viii
•__=2.'.7_7.·.·._______________________________ .._.. . ..
l
.tA.. .._ ....A..V.W.i NAYI_.__l* .!~-.,....."".,.-£.~-
Contents
The structure and use of a YAC vector. 138

Applications for YAC vectors, 140

7.1.6 Vectors for other yeasts and fungi, 140

7.2 Cloning vectors for higher plants, 141

7.2.1 Agrobacterium rumefaciens nature's smallest genetic

engineer, 141

Using the Ti plasmid to introduce new genes into a

plant cell, 142

Production of transformedpl'<lnts with the Ti plasmid,

145

The Ri plasmid, 145

Limitations of cloning with Agrobacterium plasmids,

146

7.2.2 Cloning genes in plants by direct gene transfer, 147

Direct gene transfer into the nucleus, 147

Transfer of genes into the chloroplast genome, 149

7.2.3 Attempts to use plant viruses as cloning vectors, 150

Caulimovirus vectors, 150

Geminivirus vectors. 150

7.3 Cloning vectors for animals, 151

7.3.1 Cloning vectors for insects, 151

P elements as cloning vectors for Drosophila, 151

Cloning vectors based on insect viruses, 153

7.3.2 Cloning in mammals, 153

Cloning vectors for mammals, 153

Gene cloning without a vector, 155

8 How to Obtain a Clone of a Specific Gene, 158

8.1 The problem of selection, 158

8.1.1 There are two basic strategies for obtaining the clone you

want, 158

8.2 Direct selection. 159

8.2.1 Marker rescue extends the scope of direct selection, 161

8.2.2 The scope and limitations of marker rescue. 163

8.3 Identification of a clone from a gene library, 163

8.3.1 Gene libraries. 163

8.3.2 l'·Jot all genes are expressed at the same time, 164

8.3.3 mRNA can be cloned as complementary DNA, 166

8,4 Methods for clone identification. 166

8.4.1 Complementary nucleic acid strands hybridize to each

other, 166

8.4.2 Colony and plaque hybridization probing, 168

8.4.3 Examples of the practical use of hybridization probing, 172

ix
Contents
Abundancy probing to analyse a cDNA library, 172

Oligonucleotide probes for genes whose translation

products have been characterized, 172

Heterologous probing allows related genes to be

identified, 176

8,4,4 Identification methods based on detection of the

translation product of the cloned gene, 177

Antibodies are required for immunological detection

methods, 177

Using a purified antibody to detect protein in

recombinant colonies, 178

'The problem of gene expression, 178

9 The Polymerase Chain Reaction, 181

9.1 The polymerase chain reaction in outline, 181

9.2 PCR in more detail, 184

9.2.1 Designing the oligonucleotide primers for a PCR, 184

9.2.2 Working out the correct temperatures to use, 186

9.2.3 After the PCR: studying PCR products, 189

Gel electrophoresis of PCR products, 189

Cloning PCR products, 190

9.3 Problems with the error rate of Taq polymerase, 193

Part The Applications of Gene Cloning and DNA

A.nalysis in Research, 197

10 Studying Gene Location and Structure, 199

10.1 How to study the location of a gene, 199

10.1.1 Locating the position of a gene on a small DNA

molecule, 199

10.1.2 Locating the position of a gene on a large DNA

molecule, 202

Separating chromosomes by gel electrophoresis, 203

In situ hybridization to visualize the position of a gene

on a eukaryotic chromosome, 205

10.2 DNA sequencing - working out the structure of a gene, 207

10.2.1 The Sanger-Coulson method chain-terminating

nucleotides, 207

The primer, 207

Synthesis of the complementary strand, 207

Four separate reactions result in four families of

terminated strands, 209

Reading the DNA sequence from the autoradiograph,

209

x