Analysis of Genes and Genomes

Analysis of Genes and Genomes......Page 3
Contents......Page 9
Preface......Page 15
Acknowledgements......Page 17
Abbreviations and acronyms......Page 19
1 DNA: Structure and function......Page 23
1.1 Nucleic acid is the material of heredity......Page 24
1.2 Structure of nucleic acids......Page 29
1.3 The double helix......Page 33
1.3.1 The antiparallel helix......Page 34
1.3.2 Base pairs and stacking......Page 36
1.3.3 Gaining access to information with the double helix without breaking it apart......Page 38
1.3.4 Hydrogen bonding......Page 39
1.4 Reversible denaturing of DNA......Page 40
1.5 Structure of DNA in the cell......Page 43
1.6 The eukaryotic nucleosome......Page 46
1.7 The replication of DNA......Page 50
1.8 DNA polymerases......Page 53
1.9 The replication process......Page 55
1.10 Recombination......Page 59
1.11 Genes and genomes......Page 61
1.12 Genes within a genome......Page 62
1.13.1 Transcription in prokaryotes......Page 65
1.13.2 Transcription in eukaryotes......Page 68
1.14 RNA processing......Page 76
1.14.1 RNA splicing......Page 77
1.14.2 Alternative splicing......Page 80
1.15 Translation......Page 81
2 Basic techniques in gene analysis......Page 87
2.1 Restriction enzymes......Page 88
2.1.1 Types of restriction–modification system......Page 92
2.1.2 Other modification systems......Page 94
2.1.3 How do type II restriction enzymes work?......Page 96
2.2 Joining DNA molecules......Page 98
2.3 The basics of cloning......Page 100
2.4 Bacterial transformation......Page 106
2.4.1 Chemical transformation......Page 108
2.4.2 Electroporation......Page 109
2.5 Gel electrophoresis......Page 110
2.5.2 Agarose gels......Page 111
2.5.3 Pulsed-field gel electrophoresis......Page 117
2.6 Nucleic acid blotting......Page 120
2.6.1 Southern blotting......Page 122
2.6.2 The compass points of blotting......Page 124
2.7 DNA purification......Page 125
3 Vectors......Page 131
3.1 Plasmids......Page 134
3.1.1 pBR322......Page 138
3.1.2 pUC plasmids......Page 141
3.2 Selectable markers......Page 144
3.3 λ vectors......Page 148
3.4 Cosmid vectors......Page 157
3.5 M13 vectors......Page 159
3.6 Phagemids......Page 162
3.7.1 YACs......Page 165
3.7.2 PACs......Page 168
3.7.3 BACs......Page 170
3.7.4 HACs......Page 171
4 Polymerase chain reaction......Page 175
4.1 PCR reaction conditions......Page 181
4.2 Thermostable DNA polymerases......Page 184
4.3 Template DNA......Page 186
4.4 Oligonucleotide primers......Page 187
4.4.1 Synthesis of oligonucleotide primers......Page 189
4.5 Primer mismatches......Page 191
4.6 PCR in the diagnosis of genetic disease......Page 195
4.7 Cloning PCR products......Page 197
4.8 RT–PCR......Page 199
4.9 Real-time PCR......Page 201
4.10 Applications of PCR......Page 203
5 Cloning a gene......Page 205
5.1 Genomic libraries......Page 207
5.2 cDNA libraries......Page 213
5.3 Directional cDNA cloning......Page 218
5.4 PCR based libraries......Page 220
5.5 Subtraction libraries......Page 222
5.6 Library construction in the post-genome era......Page 226
6 Gene identification......Page 227
6.1 Screening by nucleic acid hybridization......Page 228
6.2 Immunoscreening......Page 233
6.3 Screening by function......Page 238
6.4 Screening by interaction......Page 239
6.6 Two-hybrid screening......Page 240
6.6.1 Problems, and some solutions, with two-hybrid screening......Page 247
6.7 Other interaction screens – variations on a theme......Page 250
6.7.3 Reverse two hybrid......Page 251
7 Creating mutations......Page 253
7.1 Creating specific DNA changes using primer extension mutagenesis......Page 255
7.2.1 Phosphorothioate strand selection......Page 259
7.2.2 dut(-) ung(-) (or Kunkel) strand selection......Page 260
7.3 Cassette mutagenesis......Page 262
7.4 PCR based mutagenesis......Page 263
7.5 QuikChange(®) mutagenesis......Page 270
7.6 Creating random mutations in specific genes......Page 272
7.7 Protein engineering......Page 276
8 Protein production and purification......Page 279
8.1 Expression in E. coli......Page 280
8.1.2 The tac promoter......Page 281
8.1.3 The λP(L) promoter......Page 282
8.1.4 The T7 expression system......Page 283
8.2.1 Saccharomyces cerevisiae......Page 287
8.2.1.1 The GAL system......Page 288
8.2.2 Pichia pastoris......Page 290
8.3 Expression in insect cells......Page 291
8.4.1 Tet-on/Tet-off system......Page 294
8.5 Protein purification......Page 297
8.5.1 The His-tag......Page 298
8.5.2 The GST-tag......Page 301
8.5.4 IMPACT......Page 304
8.5.5 TAP-tagging......Page 308
9 Genome sequencing projects......Page 309
9.1 Genomic mapping......Page 311
9.2 Genetic mapping......Page 312
9.3 Physical mapping......Page 315
9.4 Nucleotide sequencing......Page 317
9.4.1 Manual DNA sequencing......Page 318
9.4.2 Automated DNA sequencing......Page 322
9.5 Genome sequencing......Page 325
9.6 The human genome project......Page 327
9.7 Finding genes......Page 329
9.8 Gene assignment......Page 331
9.9 Bioinformatics......Page 333
10 Post-genome analysis......Page 335
10.1 Global changes in gene expression......Page 336
10.1.1 Differential display......Page 337
10.1.2 Microarrays......Page 339
10.1.3 ChIPs with everything......Page 346
10.3 Knock-out analysis......Page 349
10.4 Antisense and RNA interference (RNAi)......Page 351
10.5 Genome-wide two-hybrid screens......Page 355
10.7 Structural genomics......Page 357
11.1 Cloning in plants......Page 363
11.1.1 Agrobacterium tumefaciens......Page 364
11.1.2 Direct nuclear transformation......Page 369
11.1.3 Viral vectors......Page 370
11.1.4 Chloroplast transformation......Page 372
11.2.1 Delayed ripening......Page 376
11.2.2 Insecticidal resistance......Page 377
11.2.3 Herbicidal resistance......Page 378
11.2.4 Viral resistance......Page 379
11.2.6 Terminator technology......Page 380
11.3 Ethics of genetically engineered crops......Page 382
12.1 Cell culture......Page 383
12.2 Transfection of animal cells......Page 384
12.2.1 Chemical transfection......Page 385
12.2.3 Liposome-mediated transfection......Page 386
12.2.5 Direct DNA transfer......Page 388
12.3.1 SV40......Page 389
12.3.2 Adenovirus......Page 391
12.3.3 Adeno-associated virus (AAV)......Page 393
12.3.4 Retrovirus......Page 394
12.4 Selectable markers and gene amplification in animal cells......Page 397
12.5 Expressing genes in animal cells......Page 400
13 Engineering animals......Page 401
13.1 Pronuclear injection......Page 403
13.2 Embryonic stem cells......Page 406
13.3 Nuclear transfer......Page 412
13.4 Gene therapy......Page 418
13.5 Examples and potential of gene therapy......Page 420
Glossary......Page 423
A1.1......Page 431
A1.2......Page 432
A1.3......Page 433
Nobel prize winners......Page 435
References......Page 439
Index......Page 481