From genes to genomes : concepts and applications of DNA technology

Front Cover
Title Page
Copyright Page
CONTENTS
Preface
1 From Genes to Genomes
	1.1 Introduction
	1.2 Basic molecular biology
		1.2.1 The DNA backbone
		1.2.2 The base pairs
		1.2.3 RNA structure
		1.2.4 Nucleic acid synthesis
		1.2.5 Coiling and supercoilin
	1.3 What is a gene?
	1.4 Information flow: gene expression
		1.4.1 Transcription
		1.4.2 Translation
	1.5 Gene structure and organisation
		1.5.1 Operons
		1.5.2 Exons and introns
	1.6 Refinements of the model
2 How to Clone a Gene
	2.1 What is cloning?
	2.2 Overview of the procedures
	2.3 Extraction and purification of nucleic acids
		2.3.1 Breaking up cells and tissues
		2.3.2 Alkaline denaturation
		2.3.3 Column purification
	2.4 Detection and quantitation of nucleic acids
	2.5 Gel electrophoresis
		2.5.1 Analytical gel electrophoresis
		2.5.2 Preparative gel electrophoresis
	2.6 Restriction endonucleases
		2.6.1 Specificity
		2.6.2 Sticky and blunt ends
	2.7 Ligation
		2.7.1 Optimising ligation conditions
		2.7.2 Preventing unwanted ligation: alkaline phosphatase and double digests
		2.7.3 Other ways of joining DNA fragments
	2.8 Modification of restriction fragment ends
		2.8.1 Linkers and adaptors
		2.8.2 Homopolymer tailing
	2.9 Plasmid vectors
		2.9.1 Plasmid replication
		2.9.2 Cloning sites
		2.9.3 Selectable markers
		2.9.4 Insertional inactivation
		2.9.5 Transformation
	2.10 Vectors based on the lambda bacteriophage
		2.10.1 Lambda biology
		2.10.2 In vitro packaging
		2.10.3 Insertion vectors
		2.10.4 Replacement vectors
	2.11 Cosmids
	2.12 Supervectors: YACs and BACs
	2.13 Summary
3 Genomic and cDNA Libraries
	3.1 Genomic libraries
		3.1.1 Partial digests
		3.1.2 Choice of vectors
		3.1.3 Construction and evaluation of a genomic library
	3.2 Growing and storing libraries
	3.3 cDNA libraries
		3.3.1 Isolation of mRNA
		3.3.2 cDNA synthesis
		3.3.3 Bacterial cDNA
	3.4 Screening libraries with gene probes
		3.4.1 Hybridization
		3.4.2 Labelling probes
		3.4.3 Steps in a hybridization experiment
		3.4.4 Screening procedure
		3.4.5 Probe selection and generation
	3.5 Screening expression libraries with antibodies
	3.6 Characterization of plasmid clones
		3.6.1 Southern blots
		3.6.2 PCR and sequence analysis
4 Polymerase Chain Reaction (PCR)
	4.1 The PCR reaction
	4.2 PCR in practice
		4.2.1 Optimisation of the PCR reaction
		4.2.2 Primer design
		4.2.3 Analysis of PCR products
		4.2.4 Contamination
	4.3 Cloning PCR products
	4.4 Long-range PCR
	4.5 Reverse-transcription PCR
	4.6 Quantitative and real-time PCR
		4.6.1 SYBR Green
		4.6.2 TaqMan
		4.6.3 Molecular beacons
	4.7 Applications of PCR
		4.7.1 Probes and other modified products
		4.7.2 PCR cloning strategies
		4.7.3 Analysis of recombinant clones and rare events
		4.7.4 Diagnostic applications
5 Sequencing a Cloned Gene
	5.1 DNA sequencing
		5.1.1 Principles of DNA sequencing
		5.1.2 Automated sequencing
		5.1.3 Extending the sequence
		5.1.4 Shotgun sequencing; contig assembly
	5.2 Databank entries and annotation
	5.3 Sequence analysis
		5.3.1 Identification of coding region
		5.3.2 Expression signals
	5.4 Sequence comparisons
		5.4.1 DNA sequences
		5.4.2 Protein sequence comparisons
		5.4.3 Sequence alignments: Clustal
	5.5 Protein structure
		5.5.1 Structure predictions
		5.5.2 Protein motifs and domains
	5.6 Confirming gene function
		5.6.1 Allelic replacement and gene knockouts
		5.6.2 Complementation
6 Analysis of Gene Expression
	6.1 Analysing transcription
		6.1.1 Northern blots
		6.1.2 Reverse transcription-PCR
		6.1.3 In situ hybridization
	6.2 Methods for studying the promoter
		6.2.1 Locating the promoter
		6.2.2 Reporter genes
	6.3 Regulatory elements and DNA-binding proteins
		6.3.1 Yeast one-hybrid assays
		6.3.2 DNase I footprinting
		6.3.3 Gel retardation assays
		6.3.4 Chromatin immunoprecipitation (ChIP)
	6.4 Translational analysis
		6.4.1 Western blots
		6.4.2 Immunocytochemistry and immunohistochemistry
7 Products from Native and Manipulated Cloned Genes
	7.1 Factors affecting expression of cloned genes
		7.1.1 Transcription
		7.1.2 Translation initiation
		7.1.3 Codon usage
		7.1.4 Nature of the protein product
	7.2 Expression of cloned genes in bacteria
		7.2.1 Transcriptional fusions
		7.2.2 Stability: conditional expression
		7.2.3 Expression of lethal genes
		7.2.4 Translational fusions
	7.3 Yeast systems
		7.3.1 Cloning vectors for yeasts
		7.3.2 Yeast expression systems
	7.4 Expression in insect cells: baculovirus systems
	7.5 Mammalian cells
		7.5.1 Cloning vectors for mammalian cells
		7.5.2 Expression in mammalian cells
	7.6 Adding tags and signals
		7.6.1 Tagged proteins
		7.6.2 Secretion signals
	7.7 In vitro mutagenesis
		7.7.1 Site-directed mutagenesis
		7.7.2 Synthetic genes
		7.7.3 Assembly PCR
		7.7.4 Synthetic genomes
		7.7.5 Protein engineering
	7.8 Vaccines
		7.8.1 Subunit vaccines
		7.8.2 DNA vaccines
8 Genomic Analysis
	8.1 Overview of genome sequencing
		8.1.1 Strategies
	8.2 Next generation sequencing (NGS)
		8.2.1 Pyrosequencing (454)
		8.2.2 SOLiD sequencing (Applied Biosystems)
		8.2.3 Bridge amplification sequencing (Solexa/Ilumina)
		8.2.4 Other technologies
	8.3 De novo sequence assembly
		8.3.1 Repetitive elements and gaps
	8.4 Analysis and annotation
		8.4.1 Identification of ORFs
		8.4.2 Identification of the function of genes and their products
		8.4.3 Other features of nucleic acid sequences
	8.5 Comparing genomes
		8.5.1 BLAST
		8.5.2 Synteny
	8.6 Genome browsers
	8.7 Relating genes and functions: genetic and physical maps
		8.7.1 Linkage analysis
		8.7.2 Ordered libraries and chromosome walking
	8.8 Transposon mutagenesis and other screening techniques
		8.8.1 Transposition in bacteria
		8.8.2 Transposition in Drosophila
		8.8.3 Transposition in other organisms
		8.8.4 Signature-tagged mutagenesis
	8.9 Gene knockouts, gene knockdowns and gene silencing
	8.10 Metagenomics
	8.11 Conclusion
9 Analysis of Genetic Variation
	9.1 Single nucleotide polymorphisms
		9.1.1 Direct sequencing
		9.1.2 SNP arrays
	9.2 Larger scale variations
		9.2.1 Microarrays and indels
	9.3 Other methods for studying variation
		9.3.1 Genomic Southern blot analysis: restriction fragment length polymorphisms (RFLPs)
		9.3.2 VNTR and microsatellites
		9.3.3 Pulsed-field gel electrophoresis
	9.4 Human genetic variation: relating phenotype to genotype
		9.4.1 Linkage analysis
		9.4.2 Genome-wide association studies (GWAS)
		9.4.3 Database resources
		9.4.4 Genetic diagnosis
	9.5 Molecular phylogeny
		9.5.1 Methods for constructing trees
10 Post-Genomic Analysis
	10.1 Analysing transcription: transcriptomes
		10.1.1 Differential screening
		10.1.2 Other methods: transposons and reporters
	10.2 Array-based methods
		10.2.1 Expressed sequence tag (EST) arrays
		10.2.2 PCR product arrays
		10.2.3 Synthetic oligonucleotide arrays
		10.2.4 Important factors in array hybridization
	10.3 Transcriptome sequencing
	10.4 Translational analysis: proteomics
		10.4.1 Two-dimensional electrophoresis
		10.4.2 Mass spectrometry
	10.5 Post-translational analysis: protein interactions
		10.5.1 Two-hybrid screening
		10.5.2 Phage display libraries
	10.6 Epigenetics
	10.7 Integrative studies: systems biology
		10.7.1 Metabolomic analysis
		10.7.2 Pathway analysis and systems biology
11 Modifying Organisms: Transgenics
	11.1 Transgenesis and cloning
		11.1.1 Common species used for transgenesis
		11.1.2 Control of transgene expression
	11.2 Animal transgenesis
		11.2.1 Basic methods
		11.2.2 Direct injection
		11.2.3 Retroviral vectors
		11.2.4 Embryonic stem cell technology
		11.2.5 Gene knockouts
		11.2.6 Gene knock-down technology: RNA interference
		11.2.7 Gene knock-in technology
	11.3 Applications of transgenic animals
	11.4 Disease prevention and treatment
		11.4.1 Live vaccine production: modification of bacteria and viruses
		11.4.2 Gene therapy
		11.4.3 Viral vectors for gene therapy
	11.5 Transgenic plants and their applications
		11.5.1 Introducing foreign genes
		11.5.2 Gene subtraction
		11.5.3 Applications
	11.6 Transgenics: a coda
Glossary
Bibliography
INDEX