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| Preface | |
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| From Genes to Genomes | |
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| Introduction | |
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| Basic molecular biology | |
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| The DNA backbone | |
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| The base pairs | |
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| RNA structure | |
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| Nucleic acid synthesis | |
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| Coiling and supercoilin | |
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| What is a gene? | |
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| Information flow: gene expression | |
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| Transcription | |
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| Translation | |
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| Gene structure and organisation | |
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| Operons | |
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| Exons and introns | |
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| Refinements of the model | |
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| How to Clone a Gene | |
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| What is cloning? | |
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| Overview of the procedures | |
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| Extraction and purification of nucleic acids | |
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| Breaking up cells and tissues | |
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| Alkaline denaturation | |
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| Column purification | |
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| Detection and quantitation of nucleic acids | |
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| Gel electrophoresis | |
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| Analytical gel electrophoresis | |
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| Preparative gel electrophoresis | |
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| Restriction endonucleases | |
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| Specificity | |
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| Sticky and blunt ends | |
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| Ligation | |
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| Optimising ligation conditions | |
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| Preventing unwanted ligation: alkaline phosphatase and double digests | |
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| Other ways of joining DNA fragments | |
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| Modification of restriction fragment ends | |
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| Linkers and adaptors | |
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| Homopolymer tailing | |
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| Plasmid vectors | |
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| Plasmid replication | |
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| Cloning sites | |
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| Selectable markers | |
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| Insertional inactivation | |
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| Transformation | |
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| Vectors based on the lambda bacteriophage | |
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| Lambda biology | |
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| In vitro packaging | |
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| Insertion vectors | |
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| Replacement vectors | |
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| Cosmids | |
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| Supervectors: YACs and BACs | |
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| Summary | |
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| Genomic and cDNA Libraries | |
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| Genomic libraries | |
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| Partial digests | |
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| Choice of vectors | |
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| Construction and evaluation of a genomic library | |
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| Growing and storing libraries | |
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| cDNA libraries | |
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| Isolation of mRNA | |
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| cDNA synthesis | |
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| Bacterial cDNA | |
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| Screening libraries with gene probes | |
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| Hybridization | |
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| Labelling probes | |
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| Steps in a hybridization experiment | |
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| Screening procedure | |
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| Probe selection and generation | |
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| Screening expression libraries with antibodies | |
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| Characterization of plasmid clones | |
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| Southern blots | |
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| PCR and sequence analysis | |
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| Polymerase Chain Reaction (PCR) | |
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| The PCR reaction | |
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| PCR in practice | |
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| Optimisation of the PCR reaction | |
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| Primer design | |
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| Analysis of PCR products | |
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| Contamination | |
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| Cloning PCR products | |
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| Long-range PCR | |
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| Reverse-transcription PCR | |
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| Quantitative and real-time PCR | |
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| SYBR Green | |
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| TaqMan | |
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| Molecular beacons | |
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| Applications of PCR | |
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| Probes and other modified products | |
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| PCR cloning strategies | |
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| Analysis of recombinant clones and rare events | |
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| Diagnostic applications | |
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| Sequencing a Cloned Gene | |
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| DNA sequencing | |
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| Principles of DNA sequencing | |
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| Automated sequencing | |
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| Extending the sequence | |
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| Shotgun sequencing; contig assembly | |
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| Databank entries and annotation | |
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| Sequence analysis | |
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| Identification of coding region | |
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| Expression signals | |
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| Sequence comparisons | |
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| DNA sequences | |
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| Protein sequence comparisons | |
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| Sequence alignments: Clustal | |
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| Protein structure | |
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| Structure predictions | |
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| Protein motifs and domains | |
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| Confirming gene function | |
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| Allelic replacement and gene knockouts | |
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| Complementation | |
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| Analysis of Gene Expression | |
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| Analysing transcription | |
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| Northern blots | |
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| Reverse transcription-PCR | |
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| In situ hybridization | |
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| Methods for studying the promoter | |
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| Locating the promoter | |
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| Reporter genes | |
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| Regulatory elements and DNA-binding proteins | |
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| Yeast one-hybrid assays | |
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| DNase I footprinting | |
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| Gel retardation assays | |
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| Chromatin immunoprecipitation (ChIP) | |
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| Translational analysis | |
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| Western blots | |
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| Immunocytochemistry and immunohistochemistry | |
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| Products from Native and Manipulated Cloned Genes | |
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| Factors affecting expression of cloned genes | |
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| Transcription | |
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| Translation initiation | |
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| Codon usage | |
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| Nature of the protein product | |
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| Expression of cloned genes in bacteria | |
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| Transcriptional fusions | |
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| Stability: conditional expression | |
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| Expression of lethal genes | |
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| Translational fusions | |
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| Yeast systems | |
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| Cloning vectors for yeasts | |
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| Yeast expression systems | |
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| Expression in insect cells: baculovirus systems | |
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| Mammalian cells | |
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| Cloning vectors for mammalian cells | |
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| Expression in mammalian cells | |
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| Adding tags and signals | |
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| Tagged proteins | |
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| Secretion signals | |
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| In vitro mutagenesis | |
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| Site-directed mutagenesis | |
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| Synthetic genes | |
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| Assembly PCR | |
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| Synthetic genomes | |
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| Protein engineering | |
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| Vaccines | |
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| Subunit vaccines | |
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| DNA vaccines | |
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| Genomic Analysis | |
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| Overview of genome sequencing | |
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| Strategies | |
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| Next generation sequencing (NGS) | |
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| Pyrosequencing (454) | |
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| SOLiD sequencing (Applied Biosystems) | |
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| Bridge amplification sequencing (Solexa/Ilumina) | |
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| Other technologies | |
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| De novo sequence assembly | |
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| Repetitive elements and gaps | |
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| Analysis and annotation | |
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| Identification of ORFs | |
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| Identification of the function of genes and their products | |
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| Other features of nucleic acid sequences | |
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| Comparing genomes | |
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| BLAST | |
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| Synteny | |
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| Genome browsers | |
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| Relating genes and functions: genetic and physical maps | |
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| Linkage analysis | |
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| Ordered libraries and chromosome walking | |
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| Transposon mutagenesis and other screening techniques | |
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| Transposition in bacteria | |
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| Transposition in Drosophila | |
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| Transposition in other organisms | |
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| Signature-tagged mutagenesis | |
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| Gene knockouts, gene knockdowns and gene silencing | |
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| Metagenomics | |
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| Conclusion | |
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| Analysis of Genetic Variation | |
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| Single nucleotide polymorphisms | |
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| Direct sequencing | |
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| SNP arrays | |
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| Larger scale variations | |
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| Microarrays and indels | |
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| Other methods for studying variation | |
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| Genomic Southern blot analysis: restriction fragment length polymorphisms (RFLPs) | |
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| VNTR and microsatellites | |
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| Pulsed-field gel electrophoresis | |
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| Human genetic variation: relating phenotype to genotype | |
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| Linkage analysis | |
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| Genome-wide association studies (GWAS) | |
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| Database resources | |
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| Genetic diagnosis | |
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| Molecular phylogeny | |
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| Methods for constructing trees | |
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| Post-Genomic Analysis | |
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| Analysing transcription: transcriptomes | |
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| Differential screening | |
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| Other methods: transposons and reporters | |
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| Array-based methods | |
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| Expressed sequence tag (EST) arrays | |
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| PCR product arrays | |
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| Synthetic oligonucleotide arrays | |
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| Important factors in array hybridization | |
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| Transcriptome sequencing | |
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| Translational analysis: proteomics | |
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| Two-dimensional electrophoresis | |
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| Mass spectrometry | |
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| Post-translational analysis: protein interactions | |
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| Two-hybrid screening | |
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| Phage display libraries | |
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| Epigenetics | |
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| Integrative studies: systems biology | |
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| Metabolomic analysis | |
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| Pathway analysis and systems biology | |
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| Modifying Organisms: Transgenics | |
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| Transgenesis and cloning | |
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| Common species used for transgenesis | |
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| Control of transgene expression | |
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| Animal transgenesis | |
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| Basic methods | |
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| Direct injection | |
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| Retroviral vectors | |
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| Embryonic stem cell technology | |
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| Gene knockouts | |
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| Gene knock-down technology: RNA interference | |
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| Gene knock-in technology | |
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| Applications of transgenic animals | |
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| Disease prevention and treatment | |
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| Live vaccine production: modification of bacteria and viruses | |
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| Gene therapy | |
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| Viral vectors for gene therapy | |
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| Transgenic plants and their applications | |
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| Introducing foreign genes | |
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| Gene subtraction | |
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| Applications | |
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| Transgenics: a coda 353 Glossary | |
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| Bibliography | |
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| Index | |