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

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234 Figure 13.12 Fusion protein bound to glutathione via the glutathione-S-transferase component Glutathione molecules attached to the surface of the bead Add glutathione-S-transferase fusion protein Glutathioneagarose beads Wash with buffer G G Agarose bead G G G G G G G G The use of affinity chromatography to purify a glutathione-S-transferase fusion protein. Figure 13.13 Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology One method for the recovery of the foreign polypeptide from a fusion protein. The methionine residue at the fusion junction must be the only one present in the entire polypeptide: if others are present cyanogen bromide will cleave the fusion protein into more than two fragments. E. coli peptide Methionine residue Elute with glutathione Cleavage specifically at the methionine residue Pure fusion protein Animal or plant protein Treat with cyanogen bromide specifically at methionine residues (Figure 13.13). Alternatively, enzymes such as thrombin (which cleaves adjacent to arginine residues) or factor Xa (which cuts after the arginine of Gly–Arg) can be used. The important consideration is that recognition sequences for the cleavage agent must not occur within the recombinant protein. 13.2 General problems with the production of recombinant protein in E. coli Despite the development of sophisticated expression vectors, there are still numerous difficulties associated with the production of protein from foreign genes cloned in E. coli. These problems can be grouped into two categories: those that are due to the sequence of the foreign gene, and those that are due to the limitations of E. coli as a host for recombinant protein synthesis.
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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
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178 Figure 10.13 Chromosome walking
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180 Figure 10.16 Part II The Applic
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182 Figure 10.20 Fluorescent in sit
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184 s Part II The Applications of G
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186 Figure 11.1 Some fundamentals o
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188 Figure 11.3 The 5′ end of an
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190 Figure 11.6 5' 3' RNA transcrip
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192 Figure 11.8 Possible positions
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194 Figure 11.11 A bound protein pr
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196 Figure 11.14 A modification int
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198 Table 11.1 Figure 11.16 A repor
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200 Pure mRNA Labelled translation
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202 Figure 11.22 (a) Restriction fr
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204 Part II The Applications of Gen
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206 Part II The Applications of Gen
Page 450: 208 as molecular biology in silico,
Page 454: 210 Figure 12.4 Part II The Applica
Page 458: 212 Figure 12.7 Using comparisons b
Page 462: 214 Figure 12.10 The use of a yeast
Page 466: 216 Figure 12.11 Serial analysis of
Page 470: 218 Load the protein sample Figure
Page 474: 220 Figure 12.16 Analyzing two prot
Page 478: 222 Part II The Applications of Gen
Page 484: Chapter 13 Production of Protein fr
Page 506: 236 organism has a bias toward pref
Page 510: 238 Figure 13.16 Part III The Appli
Page 514: 240 Figure 13.18 Crystalline inclus
Page 518: 242 Figure 13.20 Recombinant protei
Page 522: 244 s Part III The Applications of
Page 526: 246 14.1.1 Recombinant insulin Figu
Page 530: 248 Figure 14.2 The synthesis of re
Page 534: 250 Figure 14.5 (a) The factor VIII
Page 538: 252 Figure 14.7 Part III The Applic
Page 542: 254 Figure 14.8 Part III The Applic
Page 546: 256 Part III The Applications of Ge
Page 550: 258 Figure 14.10 Part III The Appli
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260 Figure 14.11 Differentiation of
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262 Part III The Applications of Ge
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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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288 Figure 16.5 Part III The Applic
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290 Figure 16.6 Sex identification
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292 Figure 16.8 Part III The Applic
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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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