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

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Chapter 8 How to Obtain a Clone of a Specific Gene 135 (a) An unstable hybrid (b) A stable hybrid (c) A DNA–RNA hybrid Figure 8.9 Short non-complementary regions do not affect overall stability DNA is bound by the sugar–phosphate backbone DNA RNA (a) Transfer colonies to nitrocellulose or nylon Nitrocellulose/nylon membrane (b) Degrade cells, purify DNA Bacteria (c) Probe with labelled DNA Alkali + protease (d) The resulting autoradiograph Unpaired bases DNA * * * * * * * Wash * * * * Non-specific binding * Specific binding Figure 8.8 Nucleic acid hybridization. (a) An unstable hybrid molecule formed between two non-homologous DNA strands. (b) A stable hybrid formed between two complementary strands. (c) A DNA–RNA hybrid, such as may be formed between a gene and its transcript. Bacteria attached to the membrane 80°C for 2 hours or ultraviolet irradiation Apply X-ray film Positive hybridization DNA bound to the membrane X-ray film Colony hybridization probing. In this example, the probe is labelled with a radioactive marker and hybridization detected by autoradiography, but other types of label and detection system can also be used.
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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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Page 262: 114 Figure 7.10 The Ti plasmid and
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Page 270: 118 Figure 7.15 Direct gene transfe
Page 274: 120 Caulimovirus vectors Although o
Page 278: 122 Figure 7.18 Cloning in Drosophi
Page 282: 124 cause the death of the mouse ce
Page 286: 126 Chapter 8 How to Obtain a Clone
Page 290: 128 Figure 8.2 The basic strategies
Page 294: 130 Figure 8.4 (a) Construction of
Page 298: 132 Figure 8.5 Preparation of a gen
Page 302: 134 Figure 8.7 One possible scheme
Page 308: Chapter 8 How to Obtain a Clone of
Page 328: Chapter 9 The Polymerase Chain Reac
Page 332: Chapter 10 The Polymerase Chain Rea
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Chapter 10 The Polymerase Chain Rea
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Chapter 10 Sequencing Genes and Gen
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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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