Analysis of Genes and Genomes

This beautifully illustrated textbook provides a clear guide
to the tools and techniques of genetic engineering, gene
cloning and molecular biology. All aspects of genetic
engineering in the post-genomic era are covered, beginning with the
basics of DNA structure and DNA metabolism. Using an example-driven
approach, the fundamentals of creating mutations in DNA, cloning in
bacteria, yeast, plants and animals are all clearly presented.

Strong emphasis is placed on the latest, post genomic
technologies including DNA macro and microarrays, genome-wide two
hybrid analysis, proteomics and bioinformatics.

• A modern post-genome era introduction to key techniques used in
  genetic engineering.

• An example driven past-to-present approach to allow the
  experiments of today to be placed in an historical context

• The book is beautifully illustrated in full-colour
  throughout.

• Associated website including updates, additional content and
  illusions

Autorentext

Richard J. Reece is the author of Analysis of *Genes and Genomes, published by Wiley.

Zusammenfassung
This beautifully illustrated textbook provides a clear guide to the tools and techniques of genetic engineering, gene cloning and molecular biology. All aspects of genetic engineering in the post-genomic era are covered, beginning with the basics of DNA structure and DNA metabolism. Using an example-driven approach, the fundamentals of creating mutations in DNA, cloning in bacteria, yeast, plants and animals are all clearly presented. Strong emphasis is placed on the latest, post genomic technologies including DNA macro and microarrays, genome-wide two hybrid analysis, proteomics and bioinformatics.

• A modern post-genome era introduction to key techniques used in genetic engineering.
• An example driven past-to-present approach to allow the experiments of today to be placed in an historical context
• The book is beautifully illustrated in full-colour throughout.
• Associated website including updates, additional content and illusions

Inhalt

Preface xiii

Acknowledgements xv

Abbreviations and acronyms xvii

1 DNA: Structure and function 1

1.1 Nucleic acid is the material of heredity 2

1.2 Structure of nucleic acids 7

1.3 The double helix 11

1.3.1 The antiparallel helix 12

1.3.2 Base pairs and stacking 14

1.3.3 Gaining access to information with the double helix without breaking it apart 16

1.3.4 Hydrogen bonding 17

1.4 Reversible denaturing of DNA 18

1.5 Structure of DNA in the cell 21

1.6 The eukaryotic nucleosome 24

1.7 The replication of DNA 28

1.8 DNA polymerases 31

1.9 The replication process 33

1.10 Recombination 37

1.11 Genes and genomes 39

1.12 Genes within a genome 40

1.13 Transcription 43

1.13.1 Transcription in prokaryotes 43

1.13.2 Transcription in eukaryotes 46

1.14 RNA processing 54

1.14.1 RNA splicing 55

1.14.2 Alternative splicing 58

1.15 Translation 59

2 Basic techniques in gene analysis 65

2.1 Restriction enzymes 66

2.1.1 Types of restrictionmodification system 70

2.1.2 Other modification systems 72

2.1.3 How do type II restriction enzymes work? 74

2.2 Joining DNA molecules 76

2.3 The basics of cloning 78

2.4 Bacterial transformation 84

2.4.1 Chemical transformation 86

2.4.2 Electroporation 87

2.4.3 Gene gun 88

2.5 Gel electrophoresis 88

2.5.1 Polyacrylamide gels 89

2.5.2 Agarose gels 89

2.5.3 Pulsed-field gel electrophoresis 95

2.6 Nucleic acid blotting 98

2.6.1 Southern blotting 100

2.6.2 The compass points of blotting 102

2.7 DNA purification 103

3 Vectors 109

3.1 Plasmids 112

3.1.1 pBR 322 116

3.1.2 pUC plasmids 119

3.2 Selectable markers 122

3.3 vectors 126

3.4 Cosmid vectors 135

3.5 M13 vectors 137

3.6 Phagemids 140

3.7 Artificial chromosomes 142

3.7.1 YACs 143

3.7.2 PACs 146

3.7.3 BACs 148

3.7.4 HACs 149

4 Polymerase chain reaction 153

4.1 PCR reaction conditions 159

4.2 Thermostable DNA polymerases 162

4.3 Template DNA 164

4.4 Oligonucleotide primers 165

4.4.1 Synthesis of oligonucleotide primers 167

4.5 Primer mismatches 169

4.6 PCR in the diagnosis of genetic disease 173

4.7 Cloning PCR products 175

4.8 RTPCR 177

4.9 Real-time PCR 179

4.10 Applications of PCR 181

5 Cloning a gene 183

5.1 Genomic libraries 185

5.2 cDNA libraries 191

5.3 Directional cDNA cloning 196

5.4 PCR based libraries 199

5.5 Subtraction libraries 200

5.6 Library construction in the post-genome era 204

6 Gene identification 205

6.1 Screening by nucleic acid hybridization 206

6.2 Immunoscreening 211

6.3 Screening by function 216

6.4 Screening by interaction 217

6.5 Phage display 218

6.6 Two-hybrid screening 218

6.6.1 Problems, and some solutions, with two-hybrid screening 225

6.7 Other interaction screens variations on a theme 228

6.7.1 One hybrid 229

6.7.2 Three hybrid 229

6.7.3 Reverse two hybrid 229

7 Creating mutations 231

7.1 Creating specific DNA changes using primer extension mutagenesis 233

7.2 Strand selection methods 237

7.2.1 Phosphorothioate strand selection 237

7.2.2 dut ung (or Kunkel) strand selection 238

7.3 Cassette mutagenesis 240

7.4 PCR based mutagenesis 241 &lt...