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

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Chapter 7 Cloning Vectors for Eukaryotes 107 Figure 7.2 (a) leu2 – yeast Chromosomes - no LEU2 gene (b) Using LEU2 as a selectable marker LEU2 Vector - carries correct LEU2 gene Transform yeast Using the LEU2 gene as a selectable marker in a yeast cloning experiment. amp R 10.7 kb LEU2 tet R ori pBR322 DNA 2 µm DNA Yeast chromosomal DNA leu2 – colonies Medium must contain leucine Only transformed cells can survive Minimal medium – no leucine Figure 7.3 A yeast episomal plasmid, YEp13. recombinant DNA is essential in order for the correct construct to be identified by, for example, DNA sequencing. The standard procedure when cloning in yeast is therefore to perform the initial cloning experiment with E. coli, and to select recombinants in this organism. Recombinant plasmids can then be purified, characterized, and the correct molecule introduced into yeast (Figure 7.4). 7.1.3 A YEp may insert into yeast chromosomal DNA The word “episomal” indicates that a YEp can replicate as an independent plasmid, but also implies that integration into one of the yeast chromosomes can occur (see the definition of “episome” on p. 14). Integration occurs because the gene carried on the vector as a selectable marker is very similar to the mutant version of the gene present in the yeast chromosomal DNA. With YEp13, for example, homologous recombination can occur between the plasmid LEU2 gene and the yeast mutant LEU2 gene, resulting
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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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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
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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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