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

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Chapter 15 Gene Cloning and DNA Analysis in Agriculture 279 (a) The RIP gene RIP gene (b) The terminator system Embryo promoter Promoter becomes active Ribosome inactivating protein Blocking DNA Cre Cre Functional RIP gene created Growth of mature plants Tetracycline promoter Cre recombinase Ribosome inactivating protein is synthesized Blocks protein synthesis Cre recombinase gene Tetracycline Figure 15.12 The terminator technology. (a) The RIP gene codes for a protein that blocks protein synthesis. (b) The system that is used to allow first generation seeds to be produced. How are the first generation seeds, those sold to farmers, obtained? To begin with, the RIP gene is non-functional because it is disrupted by a segment of non-RIP DNA (Figure 15.12b). However, this DNA is flanked by the 34 bp recognition sequences for the Cre recombinase. In these plants the gene for the Cre recombinase is placed under control of a promoter that is switched on by tetracycline. Once seeds have been obtained, the supplier activates the Cre recombinase by placing the seeds in a tetracycline solution. This removes the blocking DNA from the RIP gene, which becomes functional but remains silent until its own promoter becomes active during embryogenesis. 15.3.3 The possibility of harmful effects on the environment A second area of concern regarding genetically modified plants is that their new gene combinations might harm the environment in some way. These concerns have to be addressed individually for each type of GM crop, as different engineered genes might have different impacts. We will examine the work that has been carried out to assess whether it is possible that herbicide resistant plants, one of the two examples of gene addition that we studied earlier in this chapter, can have a harmful effect. As these are the most widely grown GM crops, they have been subject to some of the most comprehensive environmental studies. In particular, in 1999, the UK Government commissioned an independent investigation into how herbicide resistant crops, whose growth in the UK was not at that time permitted, might affect the abundance and diversity of farmland wildlife.
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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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208 as molecular biology in silico,
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210 Figure 12.4 Part II The Applica
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212 Figure 12.7 Using comparisons b
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214 Figure 12.10 The use of a yeast
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216 Figure 12.11 Serial analysis of
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218 Load the protein sample Figure
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220 Figure 12.16 Analyzing two prot
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Chapter 13 Production of Protein fr
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Chapter 14 Gene Cloning and DNA Ana
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Page 540: Chapter 14 Gene Cloning and DNA Ana
Page 594: 280 Part III The Applications of Ge
Page 598: 282 Chapter 16 Gene Cloning and DNA
Page 602: 284 Figure 16.1 Genetic fingerprint
Page 606: 286 Figure 16.3 Inheritance of STR
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Page 618: 292 Figure 16.8 Part III The Applic
Page 622: 294 Part III The Applications of Ge
Page 626: 296 Figure 16.11 Origin of agricult
Page 630: 298 Glossary GLOSSARY 2 Fm circle A
Page 634: 300 Glossary Chromosome walking A t
Page 638: 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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