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

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Chapter 6 Cloning Vectors for E. coli 101 Figure 6.14 (a) Cloning with circular λ DNA cos EcoRI EcoRI EcoRI λ arms EcoRI EcoRI New DNA cos EcoRI EcoRI, ligate cos cos λ insertion factor – circular form (b) Cloning with linear λ DNA Ligate cos Recombinant molecule cos Infect E. coli EcoRI Catenane Recombinant λ New DNA EcoRI cos Transfect E. coli cos in vitro packaging mix Different strategies for cloning with a λ vector. (a) Using the circular form of λ as a plasmid. (b) Using left and right arms of the λ genome, plus in vitro packaging, to achieve a greater number of recombinant plaques. be constructed by mixing together the DNA to be cloned with the vector arms (Figure 6.14b). Ligation results in several molecular arrangements, including catenanes comprising left arm–DNA–right arm repeated many times (Figure 6.14b). If the inserted DNA is the correct size, then the cos sites that separate these structures will be the right distance apart for in vitro packaging (p. 81). Recombinant phage are therefore produced in the test tube and can be used to infect an E. coli culture. This strategy, in particular the use of in vitro packaging, results in a large number of recombinant plaques. 6.3.5 Long DNA fragments can be cloned using a cosmid The final and most sophisticated type of e-based vector is the cosmid. Cosmids are hybrids between a phage DNA molecule and a bacterial plasmid, and their design centers on the fact that the enzymes that package the e DNA molecule into the phage protein coat need only the cos sites in order to function (p. 21). The in vitro packaging reaction works not only with e genomes, but also with any molecule that carries cos sites separated by 37–52 kb of DNA. A cosmid is basically a plasmid that carries a cos site (Figure 6.15a). It also needs a selectable marker, such as the ampicillin resistance gene, and a plasmid origin of replication, as cosmids lack all the e genes and so do not produce plaques. Instead colonies are formed on selective media, just as with a plasmid vector.
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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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Page 186: 76 Figure 5.4 Normal E. coli cell (
Page 190: 78 Figure 5.8 (b) Replica plating W
Page 194: 80 Figure 5.10 (a) The role of the
Page 198: 82 Figure 5.11 (a) A single-strain
Page 202: 84 Figure 5.13 Strategies for the s
Page 206: 86 Figure 5.14 Figure 5.15 (a) Prec
Page 210: 88 Chapter 6 Cloning Vectors for E.
Page 214: 90 during purification. pBR322 is 4
Page 218: 92 (a) pUC8 (b) Restriction sites i
Page 222: 94 Figure 6.5 The M13 genome, showi
Page 226: 96 not totally disrupt the lacZ′
Page 230: 98 Figure 6.10 The λ genetic map,
Page 234: 100 (a) Cloning with a λ replaceme
Page 240: Chapter 6 Cloning Vectors for E. co
Page 244: 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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