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

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Chapter 15 Gene Cloning and DNA Analysis in Agriculture 267 Figure 15.2 (a) Synthesis of an artificial δ-endotoxin gene Artificial gene 1 648 Preferred codons and GC content for maize (b) Attachment of a promoter and polyadenylation signal Promoter sequence (c) PCR analysis of mature plants 1 2 3 1 1155 B. thuringiensis gene Polyadenylation sequence 1. DNA size markers 2. Result of PCR with DNA from a transformed plant 3. Result of PCR with DNA from a non-transformed plant Important steps in the procedure used to obtain genetically engineered maize plants expressing an artificial δ-endotoxin gene. maize plants to synthesize c-endotoxin was made by plant biotechnologists in 1993, working with the CryIA(b) version of the toxin. The CryIA(b) protein is 1155 amino acids in length, with the toxic activity residing in the segment from amino acids 29–607. Rather than isolating the natural gene, a shortened version containing the first 648 codons was made by artificial gene synthesis. This strategy enabled modifications to be introduced into the gene to improve its expression in maize plants. For example, the codons that were used in the artificial gene were those known to be preferred by maize, and the overall GC content of the gene was set at 65%, compared with the 38% GC content of the native bacterial version of the gene (Figure 15.2a). The artificial gene was ligated into a cassette vector (p. 232) between a promoter and polyadenylation signal from cauliflower mosaic virus (Figure 15.2b), and introduced into maize embryos by bombardment with DNA-coated microprojectiles (p. 85). The embryos were grown into mature plants, and transformants identified by PCR analysis of DNA extracts, using primers specific for a segment of the artificial gene (Figure 15.2c). The next step was to use an immunological test to determine if c-endotoxin was being synthesized by the transformed plants. The results showed that the artificial gene was indeed active, but that the amounts of c-endotoxin being produced varied from plant to plant, from about 250–1750 ng of toxin per mg of total protein. These
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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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Page 516: Chapter 13 Production of Protein fr
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Page 524: Chapter 14 Gene Cloning and DNA Ana
Page 570: 268 Figure 15.3 Positional effects.
Page 574: 270 (a) Production of two toxins (c
Page 578: 272 (a) Detoxification of glyphosat
Page 582: 274 are being produced by transferr
Page 586: 276 Figure 15.10 The differences in
Page 590: 278 Figure 15.11 DNA excision by th
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
Page 610: 288 Figure 16.5 Part III The Applic
Page 614: 290 Figure 16.6 Sex identification
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292 Figure 16.8 Part III The Applic
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294 Part III The Applications of Ge
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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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Gene Cloning and DNA Analysis: An Introduction, Sixth Edition ...

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