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

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68 Figure 4.25 The use of adaptors: (a) the actual structure of an adaptor, showing the modified 5′-OH terminus; (b) conversion of blunt ends to sticky ends through the attachment of adaptors. Figure 4.26 Homopolymer tailing: (a) synthesis of a homopolymer tail; (b) construction of a recombinant DNA molecule from a tailed vector plus tailed insert DNA; (c) repair of the recombinant DNA molecule. Part I The Basic Principles of Gene Cloning and DNA Analysis (a) The precise structure of an adaptor HO The modified 5’-OH terminus (a) Synthesis of a homopolymer tail (b) Ligation of homopolymer tails Vector - poly(dC) tails G (b) Ligation using adaptors OH HO HO OH 2– O3 P Blunt-ended molecule 3’ 5’ 5’ Terminal transferase + dCTP C CC C C C C C CC + A T C C C G G G GG G G GG G Insert DNA - poly(dG) tails G G C C HO 2– PO3 DNA ligase (c) The repair steps Nick G G G G C C C C C C C Discontinuity Klenow polymerase repairs the nick G G G G G G G C C C C C C C Ligase repairs the discontinuities OH Polynucleotide kinase HO Recombinant DNA molecule OH Adaptor HO OH 2– PO3 5’-P terminus 3’ CCCCCCCCCCC C G CCCC GGGG G G G C G G CCCC
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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
Page 118: 42 Figure 3.21 Collection of phage
Page 122: 44 Further reading FURTHER READING
Page 126: 46 Part I The Basic Principles of G
Page 130: 48 Figure 4.3 The reactions catalyz
Page 134: 50 Figure 4.6 (a) Alkaline phosphat
Page 138: 52 Figure 4.8 (a) Restriction of ph
Page 142: 54 Figure 4.9 (a) Production of blu
Page 146: 56 Table 4.2 A 10 × buffer suitabl
Page 150: 58 Figure 4.13 Visualizing DNA band
Page 154: 60 Figure 4.15 Using a restriction
Page 158: 62 Figure 4.17 The influence of DNA
Page 162: 64 Figure 4.20 (a) Ligating blunt e
Page 166: 66 Figure 4.23 Adaptors and the pot
Page 172: Chapter 4 Manipulation of Purified
Page 176: Chapter 4 Manipulation of Purified
Page 180: Chapter 5 Introduction of DNA into
Page 212: Chapter 6 Cloning Vectors for E. co
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Chapter 6 Cloning Vectors for E. co
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Chapter 7 Cloning Vectors for Eukar
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Chapter 8 How to Obtain a Clone of
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Chapter 9 The Polymerase Chain Reac
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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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