Analysis of Genes and Genomes

About this book

Analysis of Genes and Genomes is a clear introduction to the theoretical and practical basis 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.

Newer technologies such as DNA macro and macroarrays, proteomics and bioinformatics are introduced in later chapters helping students to analyse and understand the vast amounts of data that are now available through genome sequence and function projects.

Aimed at students with a basic knowledge of the molecular side of biology, this will be invaluable to those looking to better understand the complexities and capabilities of these important new technologies.

Contents

Preface
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

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