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

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108 Figure 7.4 Cloning with an E. coli–yeast shuttle vector such as YEp13. Figure 7.5 Recombination between plasmid and chromosomal LEU2 genes can integrate YEp13 into yeast chromosomal DNA. After integration there are two copies of the LEU2 gene; usually one is functional, and the other mutated. Recombinant YEp13 molecules The desired recombinant tet R Mutated LEU2 LEU2 amp R Self-ligated vector Transform E. coli Transform yeast Yeast recombinant YEp13 LEU2 Recombination LEU2 LEU2 Plasmid DNA E. coli clones Ampicillin medium Purify DNA from several clones, identify correct molecule Minimal medium – no leucine Yeast chromosomal DNA in insertion of the entire plasmid into one of the yeast chromosomes (Figure 7.5). The plasmid may remain integrated, or a later recombination event may result in it being excised again. 7.1.4 Other types of yeast cloning vector Part I The Basic Principles of Gene Cloning and DNA Analysis In addition to YEps, there are several other types of cloning vector for use with S. cerevisiae. Two important ones are as follows: l Yeast integrative plasmids (YIps) are basically bacterial plasmids carrying a yeast gene. An example is YIp5, which is pBR322 with an inserted URA3 gene
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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
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
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Page 230: 98 Figure 6.10 The λ genetic map,
Page 234: 100 (a) Cloning with a λ replaceme
Page 238: 102 Figure 6.15 (a) A typical cosmi
Page 242: 104 capsid structure. Cosmid-type v
Page 246: 106 Figure 7.1 The yeast 2 µm plas
Page 252: Chapter 7 Cloning Vectors for Eukar
Page 288: Chapter 8 How to Obtain a Clone of
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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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