Gene Cloning and DNA Analysis: An Introduction

Cover
Title Page
Copyright Page
Contents in Brief
Contents
Preface to the Eighth Edition
Part I The Basic Principles of Gene Cloning and DNA Analysis
	Chapter 1 Why Gene Cloning and DNA Analysis are Important
		1.1 The early development of genetics
		1.2 The advent of gene cloning and the polymerase chain reaction
		1.3 What is gene cloning?
		1.4 What is PCR?
		1.5 Why gene cloning and PCR are so important
			1.5.1 Obtaining a pure sample of a gene by cloning
			1.5.2 PCR can also be used to purify a gene
		1.6 How to find your way through this book
		Further reading
	Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages
		2.1 Plasmids
			2.1.1 Size and copy number
			2.1.2 Conjugation and compatibility
			2.1.3 Plasmid classification
			2.1.4 Plasmids in organisms other than bacteria
		2.2 Bacteriophages
			2.2.1 The phage infection cycle
			2.2.2 Lysogenic phages
			2.2.3 Viruses as cloning vectors for other organisms
		Further reading
	Chapter 3 Purification of DNA from Living Cells
		3.1 Preparation of total cell DNA
			3.1.1 Growing and harvesting a bacterial culture
			3.1.2 Preparation of a cell extract
			3.1.3 Purification of DNA from a cell extract
			3.1.4 Concentration of DNA samples
			3.1.5 Measurement of DNA concentration
			3.1.6 Other methods for the preparation of total cell DNA
		3.2 Preparation of plasmid DNA
			3.2.1 Separation on the basis of size
			3.2.2 Separation on the basis of conformation
			3.2.3 Plasmid amplification
		3.3 Preparation of bacteriophage DNA
			3.3.1 Growth of cultures to obtain a high  titre
			3.3.2 Preparation of non-lysogenic  phages
			3.3.3 Collection of phages from an infected  culture
			3.3.4 Purification of DNA from  phage particles
			3.3.5 Purification of M13 DNA causes few problems
		Further reading
	Chapter 4 Manipulation of Purified DNA
		4.1 The range of DNA manipulative enzymes
			4.1.1 Nucleases
			4.1.2 Ligases
			4.1.3 Polymerases
			4.1.4 DNA modifying enzymes
		4.2 Enzymes for cutting DNA – restriction endonucleases
			4.2.1 The discovery and function of restriction endonucleases
			4.2.2 Type II restriction endonucleases cut DNA at specific nucleotide sequences
			4.2.3 Blunt ends and sticky ends
			4.2.4 The frequency of recognition sequences in a DNA molecule
			4.2.5 Performing a restriction digest in the laboratory
			4.2.6 Analysing the result of restriction endonuclease cleavage
			4.2.7 Estimation of the sizes of DNA molecules
			4.2.8 Mapping the positions of different restriction sites in a DNA molecule
			4.2.9 Special gel electrophoresis methods for separating larger molecules
		4.3 Ligation – joining DNA molecules together
			4.3.1 The mode of action of DNA ligase
			4.3.2 Sticky ends increase the efficiency of ligation
			4.3.3 Putting sticky ends onto a blunt-ended molecule
			4.3.4 Blunt-end ligation with a DNA topoisomerase
		Further reading
	Chapter 5 Introduction of DNA into Living Cells
		5.1 Transformation – the uptake of DNA by bacterial cells
			5.1.1 Not all species of bacteria are equally efficient at DNA uptake
			5.1.2 Preparation of competent E. coli cells
			5.1.3 Selection for transformed cells
		5.2 Identification of recombinants
			5.2.1 Recombinant selection with pBR322 – insertional inactivation of an antibiotic resistance gene
			5.2.2 Insertional inactivation does not always involve antibiotic resistance
		5.3 Introduction of phage DNA into bacterial cells
			5.3.1 Transfection
			5.3.2 In vitro packaging of  cloning vectors
			5.3.3 Phage infection is visualized as plaques on an agar medium
			5.3.4 Identification of recombinant phages
		5.4 Introduction of DNA into non-bacterial cells
			5.4.1 Transformation of individual cells
			5.4.2 Transformation of whole organisms
		Further reading
	Chapter 6 Cloning Vectors for E. coli
		6.1 Cloning vectors based on E. coli plasmids
			6.1.1 The nomenclature of plasmid cloning vectors
			6.1.2 The useful properties of pBR322
			6.1.3 The pedigree of pBR322
			6.1.4 More sophisticated E. coli plasmid cloning vectors
		6.2 Cloning vectors based on  bacteriophage
			6.2.1 Natural selection was used to isolate modified  that lack certain restriction sites
			6.2.2 Segments of the  genome can be deleted without impairing viability
			6.2.3 Insertion and replacement vectors
			Replacement vectors
			6.2.4 Cloning experiments with  insertion or replacement vectors
			6.2.5 Long DNA fragments can be cloned using a cosmid
			6.2.6  and other high-capacity vectors enable genomic libraries to be constructed
		6.3 Cloning vectors for synthesis of single-stranded DNA
			6.3.1 Vectors based on M13 bacteriophage
			6.3.2 Hybrid plasmid–M13 vectors
		6.4 Vectors for other bacteria
		Further reading
	Chapter 7 Cloning Vectors for Eukaryotes
		7.1 Vectors for yeast and other fungi
			7.1.1 Selectable markers for the 2 μm plasmid
			7.1.2 Vectors based on the 2 μm plasmid – yeast episomal plasmids
			7.1.3 A YEp may insert into yeast chromosomal DNA
			7.1.4 Other types of yeast cloning vector
			7.1.5 Artificial chromosomes can be used to clone long pieces of DNA in yeast
			7.1.6 Vectors for other yeasts and fungi
		7.2 Cloning vectors for higher plants
			7.2.1 Agrobacterium tumefaciens – nature’s smallest genetic engineer
			7.2.2 Cloning genes in plants by direct gene transfer
			7.2.3 Attempts to use plant viruses as cloning vectors
		7.3 Cloning vectors for animals
			7.3.1 Cloning vectors for insects
			7.3.2 Cloning in mammals
		Further reading
	Chapter 8 How to Obtain a Clone of a Specific Gene
		8.1 The problem of selection
			8.1.1 There are two basic strategies for obtaining the clone you want
		8.2 Direct selection
			8.2.1 Marker rescue extends the scope of direct selection
			8.2.2 The scope and limitations of marker rescue
		8.3 Identification of a clone from a gene library
			8.3.1 Gene libraries
		8.4 Methods for clone identification
			8.4.1 Complementary nucleic acid strands hybridize to each other
			8.4.2 Colony and plaque hybridization probing
			8.4.3 Examples of the practical use of hybridization probing
			8.4.4 Identification methods based on detection of the translation product of the cloned gene
		Further reading
	Chapter 9 The Polymerase Chain Reaction
		9.1 PCR in outline
		9.2 PCR in more detail
			9.2.1 Designing the oligonucleotide primers for a PCR
			9.2.2 Working out the correct temperatures to use
		9.3 After the PCR: studying PCR products
			9.3.1 Gel electrophoresis of PCR products
			9.3.2 Cloning PCR products
		9.4 Real-time PCR
			9.4.1 Carrying out a real-time PCR experiment
			9.4.2 Real-time PCR enables the amount of starting material to be quantified
			9.4.3 Melting curve analysis enables point mutations to be identified
		Further reading
Part II The Applications of Gene Cloning and DNA Analysis in Research
	Chapter 10 Sequencing Genes and Genomes
		10.1 Chain-termination DNA sequencing
			10.1.1 Chain-termination sequencing in outline
			10.1.2 Not all DNA polymerases can be used for sequencing
			10.1.3 Chain-termination sequencing with Taq polymerase
			10.1.4 Limitations of chain-termination sequencing
		10.2 Next-generation sequencing
			10.2.1 Preparing a library for an Illumina sequencing experiment
			10.2.2 The sequencing phase of an Illumina experiment
			10.2.3 Ion semiconductor sequencing
			10.2.4 Third-generation sequencing
			10.2.5 Next-generation sequencing without a DNA polymerase
			10.2.6 Directing next-generation sequencing at specific sets of genes
		10.3 How to sequence a genome
			10.3.1 Shotgun sequencing of prokaryotic genomes
			10.3.2 Sequencing of eukaryotic genomes
		Further reading
	Chapter 11 Studying Gene Expression and Function
		11.1 Studying the RNA transcript of a gene
			11.1.1 Detecting the presence of a transcript in an RNA sample
			11.1.2 Transcript mapping by hybridization between gene and RNA
			11.1.3 Transcript analysis by primer extension
			11.1.4 Transcript analysis by PCR
		11.2 Studying the regulation of gene expression
			11.2.1 Identifying protein binding sites on a DNA molecule
			11.2.2 Identifying control sequences by deletion analysis
		11.3 Identifying and studying the translation product of a cloned gene
			11.3.1 HRT and HART can identify the translation product of a cloned gene
			11.3.2 Analysis of proteins by in vitro mutagenesis
		Further reading
	Chapter 12 Studying Genomes
		12.1 Locating the genes in a genome sequence
			12.1.1 Locating protein-coding genes by scanning a genome sequence
			12.1.2 Gene location is aided by homology searching
			12.1.3 Locating genes for noncoding RNA transcripts
			12.1.4 Identifying the binding sites for regulatory proteins in a genome sequence
		12.2 Determining the function of an unknown gene
			12.2.1 Assigning gene functions by computer analysis
			12.2.2 Assigning gene function by experimental analysis
		12.3 Genome browsers
		Further reading
	Chapter 13 Studying Transcriptomes and Proteomes
		13.1 Studying transcriptomes
			13.1.1 Studying transcriptomes by microarray or chip analysis
			13.1.2 Studying transcriptomes by RNA sequencing
		13.2 Studying proteomes
			13.2.1 Protein profiling
			13.2.2 Studying protein–protein interactions
		Further reading
Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology
	Chapter 14 Production of Protein from Cloned Genes
		14.1 Special vectors for expression of foreign genes in E. coli
			14.1.1 The promoter is the critical component of an expression vector
			14.1.2 Cassettes and gene fusions
		14.2 General problems with the production of recombinant protein in E. coli
			14.2.1 Problems resulting from the sequence of the foreign gene
			14.2.2 Problems caused by E. coli
		14.3 Production of recombinant protein by eukaryotic cells
			14.3.1 Recombinant protein from yeast and filamentous fungi
			14.3.2 Using animal cells for recombinant protein production
			14.3.3 Pharming – recombinant protein from live animals and plants
		Further reading
	Chapter 15 Gene Cloning and DNA Analysis in Medicine
		15.1 Production of recombinant pharmaceuticals
			15.1.1 Recombinant insulin
			15.1.2 Synthesis of human growth hormones in E. coli
			15.1.3 Recombinant factor VIII
			15.1.4 Synthesis of other recombinant human proteins
			15.1.5 Recombinant vaccines
		15.2 Identification of genes responsible for human diseases
			15.2.1 How to identify a gene for a genetic disease
			15.2.2 Genetic typing of disease mutations
		15.3 Gene therapy
			15.3.1 Gene therapy for inherited diseases
			15.3.2 Gene therapy and cancer
			15.3.3 The ethical issues raised by gene therapy
		Further reading
	Chapter 16 Gene Cloning and DNA Analysis in Agriculture
		16.1 The gene addition approach to plant genetic engineering
			16.1.1 Plants that make their own insecticides
			16.1.2 Herbicide-resistant crops
			16.1.3 Improving the nutritional quality of plants by gene addition
			16.1.4 Other gene addition projects
		16.2 Gene subtraction
			16.2.1 Antisense RNA and the engineering of fruit ripening in tomato
			16.2.2 Other examples of the use of antisense RNA in plant genetic engineering
		16.3 Gene editing with a programmable nuclease
			16.3.1 Gene editing of phytoene desaturase in rice
			16.3.2 Editing of multiple genes in a single plant
			16.3.3 Future developments in gene editing of plants
		16.4 Are GM plants harmful to human health and the environment?
			16.4.1 Safety concerns with selectable markers
			16.4.2 The possibility of harmful effects on the environment
		Further reading
	Chapter 17 Gene Cloning and DNA Analysis in Forensic Science and Archaeology
		17.1 DNA analysis in the identification of crime suspects
			17.1.1 Genetic fingerprinting by hybridization probing
			17.1.2 DNA profiling by PCR of short tandem repeats
		17.2 Studying kinship by DNA profiling
			17.2.1 Related individuals have similar DNA profiles
			17.2.2 DNA profiling and the remains of the Romanovs
		17.3 Sex identification by DNA analysis
			17.3.1 PCRs directed at Y chromosome-specific sequences
			17.3.2 PCR of the amelogenin gene
		17.4 Archaeogenetics – using DNA to study human prehistory
			17.4.1 The origins of modern humans
			17.4.2 DNA can also be used to study prehistoric human migrations
		Further reading
Glossary
EULA