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

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254 Figure 14.8 Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology The rationale behind the potential use of a recombinant vaccinia virus. Vaccinia promoter Recombinant vaccinia genome Hepatitis B major surface antigen gene Inject recombinant vaccinia particles into the bloodstream Hepatitis B protein Vaccinia virus Immune system synthesizes antibodies against both vaccinia and hepatitis B Anti-hepatitis B Anti-vaccinia against the much more dangerous smallpox virus. The term “vaccine” comes from vaccinia; its use resulted in the worldwide eradication of smallpox in 1980. A more recent idea is that recombinant vaccinia viruses could be used as live vaccines against other diseases. If a gene coding for a virus coat protein, for example HBsAg, is ligated into the vaccinia genome under control of a vaccinia promoter, then the gene will be expressed (Figure 14.8). After injection into the bloodstream, replication of the recombinant virus results not only in new vaccinia particles, but also in significant quantities of the major surface antigen. Immunity against both smallpox and hepatitis B would result. This remarkable technique has considerable potential. Recombinant vaccinia viruses expressing a number of foreign genes have been constructed and shown to confer immunity against the relevant diseases in experimental animals (Table 14.2). The possibility of broad-spectrum vaccines is raised by the demonstration that a single recombinant vaccinia, expressing the genes for influenza virus hemagglutinin, HBsAg, and herpes simplex virus glycoprotein, confers immunity against each disease in monkeys. Studies of vaccinia viruses expressing the rabies glycoprotein have shown that deletion of the vaccinia gene for the enzyme thymidine kinase prevents the virus from replicating. This avoids the possibility that animals treated with the live vaccine will develop any form of cowpox, the disease caused by normal vaccinia. This particular live virus vaccine is now being used in rabies control in Europe and North America.
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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 13 Production of Protein fr
Page 492: Chapter 13 Production of Protein fr
Page 524: Chapter 14 Gene Cloning and DNA Ana
Page 546: 256 Part III The Applications of Ge
Page 550: 258 Figure 14.10 Part III The Appli
Page 554: 260 Figure 14.11 Differentiation of
Page 558: 262 Part III The Applications of Ge
Page 562: 264 Chapter 15 Gene Cloning and DNA
Page 566: 266 Table 15.1 Part III The Applica
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
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280 Part III The Applications of Ge
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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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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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