Gene Cloning and DNA Analysis: An Introduction, Sixth Edition ...

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Chapter 11 Studying Gene Expression and Function 203 Figure 11.23 (a) The oligonucleotide (b) Complementary strand synthesis (c) Isolation of phage carrying the mutation Phage carrying the normal gene Gene in M13 vector Plaques Oligonucleotide Plate onto agar One method for oligonucleotide-directed mutagenesis. gly ala asn leu met GGA GCT AAT TTA ATG CCT CGA ATA AAT TAC gly ala tyr leu met Non-complementary mismatch DNA polymerase dNTPs Plaque hybridization Probe with labelled oligonucleotide Normal gene sequence Oligonucleotide Entire complementary strand is synthesized Phage carrying the mutated gene Plaques containing the mutated gene Using an oligonucleotide to create a point mutation in a cloned gene There are a number of different ways of carrying out oligonucleotide-directed mutagenesis; we will consider one of the simplest of these methods. The gene to be mutated must be obtained in a single-stranded form and so is generally cloned into an M13 vector. The single-stranded DNA is purified and the region to be mutated identified by DNA sequencing. A short oligonucleotide is then synthesized, complementary to the relevant region, but containing the desired nucleotide alteration (Figure 11.23a). Despite this mismatch the oligonucleotide will anneal to the single-stranded DNA and act as a primer for complementary strand synthesis by a DNA polymerase (Figure 11.23b). This
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GENE CLONING AND DNA ANALYSIS
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This edition first published 2010,
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Contents CONTENTS Preface to the Si
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Contents ix 4.3 Ligation—joining
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Contents xi 8 How to Obtain a Clone
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Contents xiii 11.3 Identifying and
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Contents xv 15.1.2 Herbicide resist
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PART I The Basic Principles of Gene
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4 Part I The Basic Principles of Ge
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6 1.4 What is PCR? Figure 1.2 The b
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8 Figure 1.3 Cloning allows individ
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10 Figure 1.5 Gene isolation by PCR
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12 Further reading FURTHER READING
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14 Figure 2.1 Plasmids: independent
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16 Figure 2.4 Plasmid transfer by c
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18 Figure 2.6 Capsid components Pha
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20 Figure 2.7 λ phage particle att
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22 (a) The linear form of the λ DN
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24 Part I The Basic Principles of G
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26 Bacterial culture Table 3.1 Cent
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28 Bacterial culture Figure 3.3 Har
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30 Figure 3.6 Removal of protein co
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32 Figure 3.8 Collecting DNA by eth
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34 Figure 3.10 DNA purification by
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36 Figure 3.12 Two conformations of
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38 Figure 3.15 Partial unwinding of
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40 Figure 3.18 Preparation of a pha
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42 Figure 3.21 Collection of phage
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44 Further reading FURTHER READING
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48 Figure 4.3 The reactions catalyz
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50 Figure 4.6 (a) Alkaline phosphat
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52 Figure 4.8 (a) Restriction of ph
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54 Figure 4.9 (a) Production of blu
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56 Table 4.2 A 10 × buffer suitabl
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58 Figure 4.13 Visualizing DNA band
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60 Figure 4.15 Using a restriction
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62 Figure 4.17 The influence of DNA
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64 Figure 4.20 (a) Ligating blunt e
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66 Figure 4.23 Adaptors and the pot
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68 Figure 4.25 The use of adaptors:
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70 Figure 4.28 (a) The ends of the
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72 Chapter 5 Introduction of DNA in
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76 Figure 5.4 Normal E. coli cell (
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78 Figure 5.8 (b) Replica plating W
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80 Figure 5.10 (a) The role of the
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82 Figure 5.11 (a) A single-strain
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84 Figure 5.13 Strategies for the s
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86 Figure 5.14 Figure 5.15 (a) Prec
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88 Chapter 6 Cloning Vectors for E.
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90 during purification. pBR322 is 4
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92 (a) pUC8 (b) Restriction sites i
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94 Figure 6.5 The M13 genome, showi
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96 not totally disrupt the lacZ′
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98 Figure 6.10 The λ genetic map,
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100 (a) Cloning with a λ replaceme
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102 Figure 6.15 (a) A typical cosmi
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104 capsid structure. Cosmid-type v
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106 Figure 7.1 The yeast 2 µm plas
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108 Figure 7.4 Cloning with an E. c
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110 Figure 7.7 Chromosome structure
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112 for instance to examine the seg
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114 Figure 7.10 The Ti plasmid and
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116 (a) Wound infection by recombin
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118 Figure 7.15 Direct gene transfe
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120 Caulimovirus vectors Although o
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122 Figure 7.18 Cloning in Drosophi
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124 cause the death of the mouse ce
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126 Chapter 8 How to Obtain a Clone
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128 Figure 8.2 The basic strategies
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130 Figure 8.4 (a) Construction of
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132 Figure 8.5 Preparation of a gen
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134 Figure 8.7 One possible scheme
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136 Figure 8.10 The structure of α
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138 Figure 8.12 Two methods for the
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140 Figure 8.15 A simplified scheme
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142 Figure 8.17 Heterologous probin
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144 skills (Figure 8.19b). The same
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146 The problem of gene expression
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148 Figure 9.1 Hybridization of the
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150 Figure 9.3 Figure 9.4 5’ 3’
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152 Figure 9.7 A typical temperatur
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154 Figure 9.9 Calculating the T m
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156 Figure 9.13 5’ 3’ A G A C T
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158 Figure 9.16 Double-stranded PCR
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160 Figure 9.18 Hybridization of a
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PART II The Applications of Gene Cl
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166 termination sequencing has gain
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168 Figure 10.3 Reading the sequenc
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170 Figure 10.5 The basis to therma
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172 Figure 10.7 Pyrosequencing. Fig
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174 Figure 10.9 Part II The Applica
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176 Figure 10.11 Using oligonucleot
Page 390: 178 Figure 10.13 Chromosome walking
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Page 398: 182 Figure 10.20 Fluorescent in sit
Page 402: 184 s Part II The Applications of G
Page 406: 186 Figure 11.1 Some fundamentals o
Page 410: 188 Figure 11.3 The 5′ end of an
Page 414: 190 Figure 11.6 5' 3' RNA transcrip
Page 418: 192 Figure 11.8 Possible positions
Page 422: 194 Figure 11.11 A bound protein pr
Page 426: 196 Figure 11.14 A modification int
Page 430: 198 Table 11.1 Figure 11.16 A repor
Page 434: 200 Pure mRNA Labelled translation
Page 438: 202 Figure 11.22 (a) Restriction fr
Page 444: Chapter 11 Studying Gene Expression
Page 448: Chapter 12 Studying Genomes Chapter
Page 452: Chapter 12 Studying Genomes 209 Fig
Page 460: Chapter 12 Studying Genomes 213 (a)
Page 464: Chapter 12 Studying Genomes 215 12.
Page 468: Chapter 12 Studying Genomes 217 C G
Page 480: PART III The Applications of Gene C
Page 486: 226 Figure 13.1 Two different syste
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228 Figure 13.3 The three most impo
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230 Figure 13.6 Strong and weak pro
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232 Part III The Applications of Ge
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234 Figure 13.12 Fusion protein bou
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236 organism has a bias toward pref
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238 Figure 13.16 Part III The Appli
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240 Figure 13.18 Crystalline inclus
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242 Figure 13.20 Recombinant protei
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244 s Part III The Applications of
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246 14.1.1 Recombinant insulin Figu
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248 Figure 14.2 The synthesis of re
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250 Figure 14.5 (a) The factor VIII
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252 Figure 14.7 Part III The Applic
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258 Figure 14.10 Part III The Appli
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260 Figure 14.11 Differentiation of
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264 Chapter 15 Gene Cloning and DNA
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266 Table 15.1 Part III The Applica
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268 Figure 15.3 Positional effects.
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270 (a) Production of two toxins (c
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272 (a) Detoxification of glyphosat
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274 are being produced by transferr
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276 Figure 15.10 The differences in
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278 Figure 15.11 DNA excision by th
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282 Chapter 16 Gene Cloning and DNA
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284 Figure 16.1 Genetic fingerprint
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286 Figure 16.3 Inheritance of STR
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290 Figure 16.6 Sex identification
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296 Figure 16.11 Origin of agricult
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298 Glossary GLOSSARY 2 Fm circle A
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300 Glossary Chromosome walking A t
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302 Glossary Ethanol precipitation
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304 Glossary In vitro mutagenesis A
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306 Glossary Nick translation The r
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308 Glossary Proteome The entire pr
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310 Glossary Single nucleotide poly
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312 INDEX Index (g = glossary, f =
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314 cloning vectors (continued) exp
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316 herpes simplex virus glycoprote
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318 polyethylene glycol, see PEG po
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320 T m , 153, 305g tobacco, 269 TO
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