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Искусственные генетические системы. Том 1 - Патрушев Л.И.

Патрушев Л.И. Искусственные генетические системы. Том 1 — М.: Наука, 2004. — 256 c.
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6.3.10. Methylation-specific PCR.............................. 218
6.3.11. Immuno-PCR ........................................... 220
6.3.12. Quantitative (real-time) PCR ......................... 223
6.4. Amplification of sequences with unknown primary structure 233
6.4.1. Use of PCR for walking from a known region of the
genome into flanking unknown sequences (singlesided PCR).............................................. 233
6.4.2. Reducing DNA complexity with PCR (RAPD method) 237
6.5. Total genome amplification methods ........................... 239
6.6. Alternative methods for nucleic acid amplification in vitro 241
6.6.1. Ligase chain reaction ................................. 241
6.6.2. Transcription-based isothermal amplification of
nucleic acids........................................... 243
6.6.3. Rolling circle amplification .......................... 250
Summary. The strategy for new gene isolation .............................. 251
Literature ................................................................ 254
Part II
PROTEIN ENGINEERING
Introduction .............................................................. 273
Chapter 1. Rational protein design and redesign............................ 279
1.1. Designing proteins and enzymes................................ 279
1.1.1. The energy requirements for new protein construction 279
1.1.2. Choosing a three-dimensional structure ................ 281
1.1.3. Combinatorial problem of inverse protein design ....... 281
1.1.4. Designing protein surface ............................. 282
1.1.5. Applications of rational design methods ............... 282
1.1.6. Molecular fitness landscape in protein sequence space 284
1.2. Methods for site-directed mutagenesis......................... 286
1.2.1. Generation of deletions and insertions ................ 287
1.2.2. Oligonucleotide-based mutagenesis...................... 290
1.2.3. Overlap extension PCR.................................. 294
1.2.4. Megaprimers in site-directed mutagenesis .............. 298
1.2.5. Nonsense suppressors in site-directed mutagenesis ..... 302
1.3. Chemo-enzymatic synthesis in establishing semisynthetic polypeptides: expressed protein ligation........ 306
1.3.1. Combining chemical synthesis and enzymatic semisynthesis in designing new polypeptides ......................... 307
1.3.2. Protein splicing and /ra^s-splicing in peptide ligation 312
1.3.3. Applications of expressed protein ligation methods for
protein engineering .................................... 316
Chapter 2. Directed protein evolution...................................... 319
2.1. Combinatorial libraries of polynucleotide sequences........... 321
2.2. Random mutagenesis methods.................................... 323
2.2.1. Chemical mutagenesis................................... 324
2.2.2. Error-prone DNA synthesis ............................. 326
2.2.3. Random shuffling of homologous and nonhomologous
gene regions .......................................... 327
2.3. Screening and selection of proteins with required properties ........................................................... 332
2.3.1. Phage display ......................................... 333
2.3.2. Cell-surface display................................... 349
2.3.3. Ribosomal display and mRNA display........... 351
2.3.4. AMiybrid systems as a tools for protein research....... 355
Chapter 3. Advances in protein engineering ............................... 369
3.1. Chemical modifications of proteins........................... 369
3.1.1. Artificial protein crosslinking........................ 370
3.1.2. Optimization of enzymes for functioning in nonaqueous
solvents............................................... 372
3.1.3. Molecular imprinting .................................. 374
3.2. Hybrid proteins ............................................. 375
3.2.1. Hybrid enzymes ........................................ 376
3.2.2. Green fluorescent protein (GFP) and its analogs as a
reporters ............................................. 382
3.2.3. Hybrid toxins ......................................... 392
3.3. Extremozymes................................................. 397
3.3.1. Extremophiles and their enzymes........................ 397
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