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

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102 Figure 6.15 (a) A typical cosmid (b) Cloning with pJB8 amp R BamHI Circular pJB8 cos BamHI BamHI BamHI ori BamHI Restrict with BamHI cos amp New DNA cos R Recombinant cosmid DNA amp R λ particles pJB8 5.4 kb In vitro package cos Infect E. coli BamHI BamHI cos Linear pJB8 Ligate A typical cosmid and the way it is used to clone long fragments of DNA. Part I The Basic Principles of Gene Cloning and DNA Analysis λ DNA amp R Catenane BamHI BamHI New DNA Colonies containing circular recombinant pJB8 molecules Ampicillin medium A cloning experiment with a cosmid is carried out as follows (Figure 6.15b). The cosmid is opened at its unique restriction site and new DNA fragments inserted. These fragments are usually produced by partial digestion with a restriction endonuclease, as total digestion almost invariably results in fragments that are too small to be cloned with a cosmid. Ligation is carried out so that catenanes are formed. Providing the inserted DNA is the right size, in vitro packaging cleaves the cos sites and places the recombinant cosmids in mature phage particles. These e phage are then used to infect an E. coli culture, though of course plaques are not formed. Instead, infected cells are plated onto a selective medium and antibiotic-resistant colonies are grown. All colonies are recombinants, as non-recombinant linear cosmids are too small to be packaged into e heads. 6.4 8 and other high-capacity vectors enable genomic libraries to be constructed The main use of all e-based vectors is to clone DNA fragments that are too long to be handled by plasmid or M13 vectors. A replacement vector, such as eEMBL4, can carry up to 20 kb of new DNA, and some cosmids can manage fragments up to 40 kb. This
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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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Gene Cloning and DNA Analysis: An Introduction, Sixth Edition ...

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