Principles of Gene Manipulation: Sixth Edition
Multiple Choice Questions New edition of Principles of Genome Analysis and Genomics
First genome of multicellular organism to be sequenced
First plant genome to be sequenced
Biotechnology: the new industry
Box one point one The birth of an industry
The central role of Escherichia coli
Outline of the rest of the book
Getting DNA into bacteria Cloning in Gram-negative bacteria Cloning in Gram-positive bacteria
CHAPTER TWO Basic techniques
The solutions: basic techniques
Agarose gel electrophoresis
Box two point one Hybridization of nucleic acids on membranes
Detergents and blocking agents
Box two point one continued
Box two point two The principles of autoradiography
Box two point two continued
Alternative blotting techniques
Transformation of E. coli
Transformation of other organisms
The polymerase chain reaction
Key factors affecting the PCR
Real-time quantitative PCR
CHAPTER THREE Cutting and joining DNA molecules
Host-controlled restriction and modification
Types of restriction and modification system
Number and size of restriction fragments
Variations on cutting and joining DNA molecules
The Dam and Dcm methylases of E. coli
The importance of eliminating restriction systems in E. coli strains used as hosts for recombinant molecules
The importance of enzyme quality
Polynucleotide kinase, ATP
Joining polymerase chain reaction products
Incorporation of extra sequence at the five prime end of a primer into amplified DNA
Joining DNA molecules without DNA ligase
Basic biology of plasmid and phage vectors
Partitioning and segregative stability of plasmids
Incompatibility of plasmids
The purification of plasmid DNA
Desirable properties of plasmid cloning vehicles
pBR three hundred twenty-two, a purpose-built cloning vehicle
Example of the use of plasmid pBR three two two as a vector: isolation of DNA fragments which carry promoters
Improved vectors derived from pBR three two two
Promoters and control circuits
Box four point one Bacteriophage A: its important place in molecular biology and recombinant DNA technology
Improved phage-two vectors
Packaging phage-two DNA in vitro
DNA cloning with single-stranded DNA vectors
The biology of the filamentous coliphages
Why use single-stranded vectors?
Development of filamentous phage vectors
Cosmids, phasmids and other advanced vectors
Vectors for cloning large fragments of DNA
BACs and PACs as alternatives to cosmids
Specialist-purpose vectors
Vectors for making RNA probes
Vectors for maximizing protein synthesis
Vectors to facilitate protein purification
Box five point one Optimizing translation
Vectors to promote solubilization of expressed proteins
Vectors to promote protein export
Putting it all together: vectors with combinations of features
CHAPTER Six Cloning strategies
Protein-ligand interactions
Development of two replacement vectors for genomic library construction
Genomic libraries in high-capacity vectors
P C R as an alternative to genomic D N A cloning
Box 6.1 Phage-A vectors for cDNA cloning and expression
Preparation of cDNA for library construction
Development of cDNA cloning strategies
PCR as an alternative to cDNA cloning
Rapid amplification of cDNA ends
Expressed sequence tags for high-throughput genome research
Sequence-dependent screening
Box six point three Gridded arrayed hybridization reference libraries
Box 6.4 A landmark publication. Identification of the cystic fibrosis gene by chromosome walking and jumping
Screening expression libraries (expression cloning)
Insert cDNA at EcoRI site of Agt eleven or in polylinker region of AZAP
Screening with alternative ligands
Screening by functional complementation
Screening by 'gain of function'
Difference cloning with DNA libraries
Enrichment for differences - subtractive cloning
Box six point five Differential screening with DNA chips
Difference cloning by PCR
Box six point six A landmark publication. Subtraction cloning of the human Duchenne muscular dystrophy gene
Enrichment for differences - representational difference analysis
CHAPTER 7 Sequencing and mutagenesis
Modifications of chain-terminator sequencing
Genomic Sequence Databases
Sequence Search and Retrieval
Forward and Reverse Translation
Protein Analysis Utilities
Changing genes: site-directed mutagenesis
Primer extension: the single-primer method
Deficiencies of the single-primer method
PCR methods of site-directed mutagenesis
Selection of mutant peptides by phage and phasmid display
Directed mutation in vivo
Cloning in bacteria other than Escherichia coli
Introducing DNA into bacterial cells
Box eight point one Transforming Bacillus subtilis with plasmid DNA
Transformation by plasmid rescue
Box eight point one continued
Transformation of protoplasts
Maintenance of recombinant DNA in new hosts
Integration of recombinant DNA
Cloning in Gram-negative bacteria other than E. coli
Vectors derived from the IncQ-group plasmid RSF1010
Vectors derived from the IncP-group plasmids
Vectors derived from the IncW plasmid Sa
Vectors derived from pBBR1
Cloning in Gram-positive bacteria
Vectors for cloning in Bacillus subtilis and other low-GC organisms
The influence of mode of replication: vectors derived from pAMB1
Transcription and translation
Controlled expression in B. subtilis and other low-GC hosts
Secretion vectors for low-GC bacteria
Vectors for systematic gene inactivation
Cloning in streptomycetes
Vectors for streptomycetes
Homoeologous recombination
Cloning in Saccharomyces cerevisiae and other fungi
Introducing DNA into fungi
The fate of DNA introduced into fungi
Plasmid vectors for use in fungi
Yeast replicating plasmids
Yeast centromere plasmids
Yeast artificial chromosomes
Choice of vector for cloning
Plasmid construction by homologous recombination in yeast
Expression of cloned genes
Overexpression of proteins in fungi
Box nine point one Galactose metabolism and its control in Saccharomyces cerevisiae
Polylinkers for C-terminal tagged vectors.
Detecting protein-protein interactions.
Box nine point two Variations of the two-hybrid system.
Protein-peptide interactions.
Reverse two-hybrid system.
Identifying genes encoding particular cellular activities
Determining functions associated with particular genes
CHAPTER 10 Gene transfer to animal cells
Overview of gene-transfer strategies
DNA-mediated transformation
DNA/calcium phosphate coprecipitate method
Other chemical transfection methods
Phospholipids as gene-delivery vehicles
Transformation with non-replicating DNA
Cotransformation and selection of stable transformants
Other selectable markers for animal cells
Selectable markers and transgene amplification
Non-replicating plasmid vectors for transient transformation
Transformation with replicon vectors
Stable transformation with BK and BPV replicons
Stable transformation with EBV replicons
Gene transfer by viral transduction
Strategies for vector construction
Sindbis virus and Semliki forest virus (alphaviruses)
Vaccinia and other poxvirus vectors
Transgenic mice (Chapter 11)
High-level transient expression
Summary of expression systems for animal cells
The inclusion of a polyadenylation signal
The removal of unnecessary untranslated sequence
Optimization of the transgene for translational efficiency
The incorporation of a targeting signal
CHAPTER eleven Genetic manipulation of animals
Genetic manipulation of mammals
Pronuclear microinjection
Gene targeting with ES cells
Design of targeting vectors
Introducing subtle mutations
Intrachromosomal recombination
Applications of genetically modified mice
Yeast artificial chromosome transgenic mice
Incorporating dominantly acting transcriptional control elements
Using boundary elements or matrix attachment regions
Using large genomic transgenes
Dominantly acting transgenes (transgene rescue)
Site-specific integration
Applications of gene targeting
The injection of sperm heads directly into the cytoplasm of the egg (intracytoplasmic sperm injection
Nuclear transfer technology
DNA transfer to other vertebrates
Xenopus oocytes as a heterologous expression system
Xenopus oocytes for functional expression cloning
Extraction Affinity chromatography
Transient gene expression in Xenopus embryos
DNA transfer to invertebrates
Development of P-element vectors for gene transfer
Structure of P-element derivative: Carnegie twenty
CHAPTER TWELVE Gene transfer to plants
Plant callus and cell culture
Regeneration of fertile plants
Overview of gene-transfer strategies
Agitation in liquid media
Agrobacterium-mediated transformation
Tumour-inducing (Ti) plasmids
Genes required (in trans) for transfer
Disarmed Ti-plasmid derivatives as plant vectors
Prototype disarmed Ti vectors
Other components of the expression vector
Box twelve point two Selectable markers for plants
Box twelve point two continued
A simple experimental procedure for Agrobacterium-mediated transformation
Agrobacterium and monocots
High-capacity binary vectors
Agrobacterium rhizogenes and Ri plasmids
Direct DNA transfer to plants
Other direct DNA-transfer methods
Chloroplast transformation
DNA viruses as expression vectors
RNA viruses as expression vectors
CHAPTER thirteen Advances in transgenic technology
Inducible expression systems
Endogenous inducible promoters
Recombinant inducible systems
The lac and tet repressor systems
The tet activator and reverse activator systems
Heterologous use of steroids: Drosophila ecdysone in mammals and mammalian glucocorticoids in plants
Chemically induced dimerization
Inducible protein activity
Applications of site-specific recombination
Site-specific recombination
Box 13.1 Visible marker genes
Green fluorescent protein
Site-specific deletion of transgene sequences
Site-specific transgene activation and switching
Site-specific transgene integration
Cre-mediated conditional mutants in mice
Further transgenic strategies for gene inhibition
Gene inhibition at the RNA level
Position-dependent silencing and sequence-dependent silencing
Homology-dependent gene silencing
Post-transcriptional gene silencing and RNA interference as a common mechanism - the evidence
Gene inhibition at the protein level
Dominant-negative mutants
Transgenic technology for functional genomics
Vectors for insertional mutagenesis and tagging
Enhancer traps in Drosophila
Enhancer traps in other species
Gene traps and functional genomics
Box 13.3 Internal ribosome entry sites
Function-specific trapping
Applications of recombinant DNA technology
Theme 1: Nucleic acid sequences as diagnostic tools
Detection of sequences at the gross level
Comparative sequence analysis: single-nucleotide polymorphisms
Variable number tandem repeat polymorphisms
Forensic applications of V N T R s
Theme 2: New drugs and new therapies for genetic diseases
Transgenic animals and plants as bioreactors: 'pharming'
Transgenic animals as models of human disease
Box 14.2 Xenotransplantation
Gene transfer to humans - gene therapy
Gene-augmentation therapy for recessive diseases
Gene-therapy strategies for cancer
Theme Three: Combating infectious disease
Recombinant bacterial vaccines
Recombinant viruses as vaccines
Selecting targets for new antimicrobial agents
In vivo expression technology
Differential fluorescence induction
Signature-tagged mutagenesis
Theme 4: Protein engineering
Improving therapeutic proteins with single amino acid changes
Box fourteen point three. Oxidation-resistant variants of alpha one antitrypsin.
Improving enzymes: subtilisin as a paradigm for protein engineering.
Methods for engineering proteins: the rational approach.
Protein engineering through directed evolution.
Gene families as aids to protein engineering
Theme Five: Metabolic engineering
Designed overproduction of phenylalanine
New routes to small molecules
Combinatorial biosynthesis
Engineering metabolic control over recombinant pathways
Metabolic engineering in plant cells
Theme 6: Plant breeding in the twenty-first century
Improving agronomic traits
Basis of engineered resistance to herbicide
Resistance to microbial pathogens
Resistance to insects and other pests
Modification of production traits
Starch and oil modification
Improving vitamin and mineral content
Epilogue: from genes to genomes