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

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116 (a) Wound infection by recombinant A. tumefaciens (b) Transformation of cultured plant cells Inoculate with recombinant A. tumefaciens Plant cell suspension Figure 7.13 Apply recombinant bacteria Plant cell Transformed callus Bacterium Part I The Basic Principles of Gene Cloning and DNA Analysis Cloned gene only present in the crown gall Plate onto solid medium Transfer to medium with different growth hormone balance Shoots form Plant into soil Transformed plant Transformation of plant cells by recombinant A. tumefaciens. (a) Infection of a wound: transformed plant cells are present only in the crown gall. (b) Transformation of a cell suspension: all the cells in the resulting plant are transformed. isolate transformants. A mature plant regenerated from transformed cells will contain the cloned gene in every cell and will pass the cloned gene to its offspring. However, regeneration of a transformed plant can occur only if the Ti vector has been “disarmed” so that the transformed cells do not display cancerous properties. Disarming is possible because the cancer genes, all of which lie in the T-DNA, are not needed for the infection process, infectivity being controlled mainly by the virulence region of the Ti plasmid. In fact, the only parts of the T-DNA that are involved in infection are two 25 bp repeat sequences found at the left and right borders of the region integrated into the plant DNA. Any DNA placed between these two repeat sequences will be treated as “T-DNA” and transferred to the plant. It is therefore possible to remove all the cancer genes from the normal T-DNA, and replace them with an entirely new set of genes, without disturbing the infection process. A number of disarmed Ti cloning vectors are now available, a typical example being the binary vector pBIN19 (Figure 7.14). The left and right T-DNA borders present in this vector flank a copy of the lacZ′ gene, containing a number of cloning sites, and a kanamycin resistance gene that functions after integration of the vector sequences into the plant chromosome. As with a yeast shuttle vector (p. 106), the initial manipulations
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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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82 Figure 5.11 (a) A single-strain
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84 Figure 5.13 Strategies for the s
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86 Figure 5.14 Figure 5.15 (a) Prec
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88 Chapter 6 Cloning Vectors for E.
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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
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Page 242: 104 capsid structure. Cosmid-type v
Page 246: 106 Figure 7.1 The yeast 2 µm plas
Page 250: 108 Figure 7.4 Cloning with an E. c
Page 254: 110 Figure 7.7 Chromosome structure
Page 258: 112 for instance to examine the seg
Page 262: 114 Figure 7.10 The Ti plasmid and
Page 268: 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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