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

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216 Figure 12.11 Serial analysis of gene expression (SAGE). Figure 12.12 Microarray analysis. The microarray shown here has been hybridized to two different cDNA preparations, each labelled with a fluorescent marker. The clones which hybridize with the cDNAs are identified by confocal microscopy. Part II The Applications of Gene Cloning and DNA Analysis in Research 5’ mRNA cRNA 3’ 5’ 3’ TTTT Oligo (dT) strand Discard the end fragment Hybridize with cDNA samples Hybridization detected by confocal microscopy Cellulose bead Add RNA Collect, ligate to other fragments, sequence Microarray TTTT AAAA Convert to doublestranded cDNA Digest with AluI TTTT AAAA TTTT AAAA Ligate linker containing BamFI sequence TTTT AAAA Digest with BamFI TTTT AAAA Hybridization signals examining the microarrays by confocal microscopy. Those spots that gave a signal indicated genes that were active under the conditions being studied, and the intensities of the hybridization signals revealed the relative amounts of the mRNAs in the transcriptome. Changes in gene expression, when the yeast were transferred to different growth conditions (e.g., oxygen starvation), could be monitored by repeating the experiment with a second cDNA preparation. Microarrays are now being used to monitor changes in the transcriptomes of many organisms. In some cases the strategy is the same as used with yeast, the microarray representing all the genes in the genome, but this is possible only for those organisms that
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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
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 442: 204 Part II The Applications of Gen
Page 446: 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 468: Chapter 12 Studying Genomes 217 C G
Page 472: Chapter 12 Studying Genomes 219 Fig
Page 476: Chapter 12 Studying Genomes 221 Fig
Page 480: PART III The Applications of Gene C
Page 486: 226 Figure 13.1 Two different syste
Page 490: 228 Figure 13.3 The three most impo
Page 494: 230 Figure 13.6 Strong and weak pro
Page 498: 232 Part III The Applications of Ge
Page 502: 234 Figure 13.12 Fusion protein bou
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
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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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256 Part III The Applications of Ge
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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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