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

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178 Figure 10.13 Chromosome walking. Figure 10.14 F2 A1 H7 G6 I4 Building up a clone contig by a clone fingerprinting technique. Part II The Applications of Gene Cloning and DNA Analysis in Research B4 A1 H7 B4 I4 (a) Probe the library with clone A1 1 2 3 4 5 6 ABCDEFGH I J (b) Probe the library with clone I4 1 2 3 4 5 6 ABCDEFGH I J I4 F2 F2 B4 G6 A1 B4 B4 Overlaps identified by clone fingerprinting Deduced clone contig A1 I4 A1 F2 used as a hybridization probe against all the other clones in the library (Figure 10.13a). Those clones that give hybridization signals are ones that overlap with the probe. One of these overlapping clones is now labeled and a second round of probing carried out. More hybridization signals are seen, some of these indicating additional overlaps (Figure 10.13b). Gradually the clone contig is built up in a step-by-step fashion. But this is a laborious process and is only attempted when the contig is for a short chromosome and so involves relatively few clones, or when the aim is to close one or more small gaps between contigs that have been built up by more rapid methods. Rapid methods for clone contig assembly The weakness of chromosome walking is that it begins at a fixed starting point and builds up the clone contig step by step, and hence slowly, from that fixed point. The more rapid techniques for clone contig assembly do not use a fixed starting point and instead aim to identify pairs of overlapping clones: when enough overlapping pairs have been identified the contig is revealed (Figure 10.14). The various techniques that can be used to identify overlaps are collectively known as clone fingerprinting. Clone fingerprinting is based on the identification of sequence features that are shared by a pair of clones. The simplest approach is to digest each clone with one or more restriction endonucleases and to look for pairs of clones that share restriction fragments of the same size, excluding those fragments that derive from the vector rather than I4
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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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Page 342: 154 Figure 9.9 Calculating the T m
Page 346: 156 Figure 9.13 5’ 3’ A G A C T
Page 350: 158 Figure 9.16 Double-stranded PCR
Page 354: 160 Figure 9.18 Hybridization of a
Page 360: PART II The Applications of Gene Cl
Page 366: 166 termination sequencing has gain
Page 370: 168 Figure 10.3 Reading the sequenc
Page 374: 170 Figure 10.5 The basis to therma
Page 378: 172 Figure 10.7 Pyrosequencing. Fig
Page 382: 174 Figure 10.9 Part II The Applica
Page 386: 176 Figure 10.11 Using oligonucleot
Page 392: Chapter 10 Sequencing Genes and Gen
Page 404: Chapter 11 Studying Gene Expression
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Chapter 11 Studying Gene Expression
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Chapter 11 Studying Gene Expression
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Chapter 12 Studying Genomes Chapter
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Chapter 12 Studying Genomes 209 Fig
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Chapter 12 Studying Genomes 213 (a)
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Chapter 12 Studying Genomes 215 12.
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Chapter 12 Studying Genomes 217 C G
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PART III The Applications of Gene C
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226 Figure 13.1 Two different syste
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228 Figure 13.3 The three most impo
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230 Figure 13.6 Strong and weak pro
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232 Part III The Applications of Ge
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234 Figure 13.12 Fusion protein bou
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236 organism has a bias toward pref
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238 Figure 13.16 Part III The Appli
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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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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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