Analysis of Genes and Genomes

Acknowledgements.

Dedication.

1. DNA: structure and function.

1.1 Nucleic acid is the material of heredity.

1.2 Structure of nucleic acids.

1.3 The double helix.

1.4 Reversible denaturing of DNA.

1.5 Structure of DNA in the cell.

1.6 The eukaryotic nucleosome.

1.7 The replication of DNA.

1.8 DNA polymerases.

1.9 The replication process.

1.10 Recombination.

1.11 Genes and genomes.

1.12 Genes within a genome.

1.13 Transcription.

1.14 RNA processing.

1.15 Translation.

2. Basic techniques in gene analysis.

2.1 Restriction enzymes.

2.2 Joining DNA molecules.

2.3 The basics of cloning.

2.4 Bacterial transformation.

2.5 Gel electrophoresis.

2.6 Nucleic acid blotting.

2.7 DNA purification.

3. Vectors.

3.1 Plasmids.

3.2 Selectable markers.

3.3 l Vectors.

3.4 Cosmid vectors.

3.5 M1 3 vectors.

3.6 Phagemids.

3.7 Artificial chromosomes.

4. Polymerase chain reaction.

4.1 PCR reaction conditions.

4.2 Thermostable DNA polymerases.

4.3 Template DNA.

4.4 Oligonucleotide primers.

4.5 Primer mismatches.

4.6 PCR in the diagnosis of genetic disease.

4.7 Cloning PCR products.

4.8 RT-PCR.

4.9 Real-time PCR.

4.10 Applications of PCR.

5. Cloning a gene.

5.1 Genomic libraries.

5.2 cDNA libraries.

5.3 Directional cDNA cloning.

5.4 PCR-based libraries.

5.5 Subtraction libraries.

5.6 Library construction in the post-genome era.

6. Gene identification.

6.1 Screening by nucleic acid hybridization.

6.2 Immunoscreening.

6.3 Screening by function.

6.4 Screening by interaction.

6.5 Phage display.

6.6 Two-hybrid screening.

6.7 Other interaction screens – variations on a theme.

7. Creating mutations.

7.1 Creating specific mutations.

7.2 Primer extension mutagenesis.

7.3 Strand selection methods.

7.4 Cassette mutagenesis.

7.5 PCR-based mutagenesis.

7.6 QuikChange® mutagenesis.

7.7 Creating random mutations in specific genes.

7.8 Protein engineering.

8. Protein production and purification.

8.1 Expression in E. coli.

8.2 Expression in yeast.

8.3 Expression in insect cells.

8.4 Expression in higher eukaryotic cells.

8.5 Protein purification.

9. Genome sequencing projects.

9.1 Genomic mapping.

9.2 Genetic mapping.

9.3 Physical mapping.

9.4 Nucleotide sequencing.

9.5 Genome sequencing.

9.6 The Human Genome Project.

9.7 Finding genes.

9.8 Gene assignment.

9.9 Bioinformatics.

10. Post-genome analysis.

10.1 Global changes in gene expression.

10.2 Protein function on a genome-wide scale.

10.3 Knock-out analysis.

10.4 Antisense and RNA interference (RNAi).

10.5 Genome-wide two-hybrid screens.

10.6 Protein-detection arrays.

10.7 Structural genomics.

11. Engineering plants.

11.1 Cloning in plants.

11.2 Commercial exploitation of plant transgenics.

11.3 Ethics of genetically engineered crops.

12. Engineering animal cells.

12.1 Cell culture.

12.2 Transfection of animal cells.

12.3 Viruses as vectors.

12.4 Selectable markers and gene amplification in animal cells.

12.5 Expressing genes in animal cells.

13. Engineering animals.

13.1 Pronuclear injection.

13.2 Embryonic stem cells.

13.3 Nuclear transfer.

13.4 Gene therapy.

13.5 Examples and potential of gene therapy.

Glossary.

Appendices.

Nobel prize winners.

References.

Index.