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

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134 Figure 8.7 One possible scheme for cDNA cloning. Poly(A) = polyadenosine, oligo(dT) = oligodeoxythymidine. Part I The Basic Principles of Gene Cloning and DNA Analysis (a) First strand synthesis 5’ 3’ AAAAA mRNA Poly(A) tail (b) RNA degradation (c) Second strand synthesis RNA fragments act as primers DNA (d) Ligation into a vector (e) Transform Anneal an oligo(dT) primer Reverse transcriptase DNA pol I AAAAA TTTTT TTTTT TTTTT AAAAA TTTTT AAAAA TTTTT Primer transferred to a nitrocellulose or nylon membrane (Figure 8.9a) and then treated to remove all contaminating material, leaving just DNA (Figure 8.9b). Usually this treatment also results in denaturation of the DNA molecules, so that the hydrogen bonds between individual strands in the double helix are broken. These single-stranded molecules can then be bound tightly to the membrane by a short period at 80°C if a nitrocellulose membrane is being used, or with a nylon membrane by ultraviolet irradiation. The molecules become attached to the membrane through their sugar–phosphate backbones, so the bases are free to pair with complementary nucleic acid molecules. RNA DNA RNase HI RNA fragments Double-stranded DNA Attach sticky ends, ligate cDNA cDNA clones Gliadin clone
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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
Page 250: 108 Figure 7.4 Cloning with an E. c
Page 254: 110 Figure 7.7 Chromosome structure
Page 258: 112 for instance to examine the seg
Page 262: 114 Figure 7.10 The Ti plasmid and
Page 266: 116 (a) Wound infection by recombin
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 304: 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 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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