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

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 restriction-modification 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 RT-PCR 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

7.5 QuikChange ® mutagenesis 248

7.6 Creating random mutations in specific genes 250

7.7 Protein engineering 254

8 Protein production and purification 257

8.1 Expression in E. coli 258

8.1.1 The lac promoter 259

8.1.2 The tac promoter 259

8.1.3 The ¿P L promoter 260

8.1.4 The T7 expression system 261

8.2 Expression in yeast 265

8.2.1 Saccharomyces cerevisiae 265

8.2.1.1 The GAL system 266

8.2.1.2 The CUP1 system 268

8.2.2 Pichia pastoris 268

8.2.3 Schizosaccharomyces pombe 269

8.3 Expression in insect cells 269

8.4 Expression in higher-Eukaryotic cells 272

8.4.1 Tet-on/Tet-off system 272

8.5 Protein purification 275

8.5.1 The His-tag 276

8.5.2 The GST-tag 279

8.5.3 The MBP-tag 282

8.5.4 Impact 282

8.5.5 TAP-tagging 286

9 Genome sequencing projects 287

9.1 Genomic mapping 289

9.2 Genetic mapping 290

9.3 Physical mapping 293

9.4 Nucleotide sequencing 295

9.4.1 Manual DNA sequencing 296

9.4.2 Automated DNA sequencing 300

9.5 Genome sequencing 303

9.6 The human genome project 305

9.7 Finding genes 307

9.8 Gene assignment 309

9.9 Bioinformatics 311

10 Post-genome analysis 313

10.1 Global changes in gene expression 314

10.1.1 Differential display 315

10.1.2 Microarrays 317

10.1.3 ChIPs with everything 324

10.2 Protein function on a genome-wide scale 327

10.3 Knock-out analysis 327

10.4 Antisense and RNA interference (RNAi) 329

10.5 Genome-wide two-hybrid screens 333

10.6 Protein detection arrays 335

10.7 Structural genomics 335

11 Engineering plants 341

11.1 Cloning in plants 341

11.1.1 Agrobacterium tumefaciens 342

11.1.2 Direct nuclear transformation 347

11.1.3 Viral vectors 348

11.1.4 Chloroplast transformation 350

11.2 Commercial exploitation of plant transgenics 354

11.2.1 Delayed ripening 354

11.2.2 Insecticidal resistance 355

11.2.3 Herbicidal resistance 356

11.2.4 Viral resistance 357

11.2.5 Fungal resistance 358

11.2.6 Terminator technology 358

11.3 Ethics of genetically engineered crops 360

12 Engineering animal cells 361

12.1 Cell culture 361

12.2 Transfection of animal cells 362

12.2.1 Chemical transfection 363

12.2.2 Electroporation 364

12.2.3 Liposome-mediated transfection 364

12.2.4 Peptides 366

12.2.5 Direct DNA transfer 366

12.3 Viruses as vectors 367

12.3.1 SV 40 367

12.3.2 Adenovirus 369

12.3.3 Adeno-associated virus (AAV) 371

12.3.4 Retrovirus 372

12.4 Selectable markers and gene amplification in animal cells 375

12.5 Expressing genes in animal cells 378

13 Engineering animals 379

13.1 Pronuclear injection 381

13.2 Embryonic stem cells 384

13.3 Nuclear transfer 390

13.4 Gene therapy 396

13.5 Examples and potential of gene therapy 398

Glossary 401

Proteins 409

A. 1 409

A1. 2 410

A1. 3 411

Nobel prize winners 413

References 417

Index 459