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 Part I The Basic Principles of Gene Cloning and DNA Analysis
  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
  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
     Gene organization in the λDNA molecule
     The linear and circular forms ofλDNA
     M13 - a filamentous phage
   2.2.3 Viruses as cloning vectors for other organisms
    Further reading
  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
     Removing contaminants by organic extraction and enzyme digestion
     Using ion-exchange chromatography to purify DNA from a cell extract
     Using silica to purify 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
     Alkaline denaturation
     Ethidium bromide-caesium chloride density gradient centrifugation
    3.2.3 Plasmid amplification
   3.3 Preparation of bacteriophage DNA
    3.3.1 Growth of cultures to obtain a high À titre
    3.3.2 Preparation of non-Iysogenic À phages
    3.3.3 Collection of phages from an infected culture
    3.3.4 Purification of DNA from À phage particles
    3.3.5 Purification of M13 DNA causes few problems
    Further reading
  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 11 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
     Separation of molecules by gel electrophoresis
     Visualizing DNA molecules in an agarose gel
    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 on to a blunt-ended molecule
     Linkers
     Adaptors
     Homopolymer tailing
    4.3.4 Blunt end ligation with a DNA topoisomerase
    Further reading
  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. co/i cells
    5.1.3 Selection for transformed cells
   5.2 Identification of recombinants
    5.2.1 Recombinant selection with pBR322: Insertional inactivation。f 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 ofλcloning vectors
    5.3.3 Phage infection is visualized as plaques。n an agar medium
   5.4 Identification of recombinant phages
    5.4.1 Insertional inactivation of a lacZ' gene carried by the phagevector
    5.4.2 Insertional inactivation of the À d gene
    5.4.3 Selection using the Spi phenotype
    5.4.4 Selection on the basis of À genome size
   5.5 Introduction of DNA into non-bacterial cells
    5.5.1 Transformation of individual cells
    5.5.2 Transformation of whole organisms
    Further reading
  6 Cloning Vectors for Escherichia co/i
   6.1 Cloning vectors based on E. co/i 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. co/i plasmid cloning vectors
     pUC8: A Lac selection plasmid
     pGEM3Z: In vitro transcription of cloned DNA
   6.2 Cloning vectors based on À bacteriophage
    6.2.1 Segments of the À genome can be deleted without impairing viability
    6.2.2 Natural selection was used to isolate modified À that lack certain restriction sites
    6.2.3 Insertion and replacement vectors
     Inse同ion vectors
     Replacement vectors
    6.2.4 Cloning experiments with À insertion or replacement vectors
    6.2.5 Long DNA fragments can be cloned using a cosmid
    6.2.6 À and other high-capacity vectors enable genomic libraries t。be constructed
   6.3 Cloning vectors for the 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
  7 Cloning Vectors for Eukaryotes
   7.1 Vectors for yeast and other fungi
    7.1 .1 Selectable markers for the 2μm plasmid
    7.1.2 Vectors based on the 2μm 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
     The structure and use of a YAC vector
     Applications for YAC vectors
    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
     Using the Ti plasmid to introduce new genes into a plant cell
     Production of transformed plants with the Ti plasmid
     The Ri plasmid
     Limitations of cloning with Agrobacterium plasmids
    7.2.2 Cloning genes in plants by direct gene transfer
     Direct gene transfer into the nucleus
     Transfer of genes into the chloroplast genome
    7.2.3 Attempts to use plant viruses as cloning vectors
     Caulimovirus vectors
     Geminivirus vectors
   7.3 Cloning vectors for animals
    7.3.1 Cloning vectors for insects
     P elements as cloning vectors for Drosophila
     Cloning vectors based on insect viruses
    7.3.2 Cloning in mammals
     Viruses as cloning vectors for mammals
     Gene cloning without a vector
    Further reading
  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
     Not all genes are expressed at the same time
     mRNA can be cloned as complementary DNA
   8.4 Methods for clone identification
    8.4.1 Complementary nudeic acid strands hybridize to each other
    8.4.2 Colony and plaque hybridization probing
     Labelling with a radioactive marker
     Non-radioactive labelling
    8.4.3 Examples of the practical use of hybridization probing
     Abundancy probing to analyse a cDNA library
     Oligonucleotide probes for genes whose translation products have been characterized
     Heterologous probing allows related genes to be identified
     Southern hybridization enables a specific restriction fragment containing a gene to be identified
    8.4.4 Identification methods based on detection of the translation product of the cloned gene
     Antibodies are required for immunological detection methods
     Using a purified antibody to detect protein in recombinant colonies
     The problem of gene expression
    Further reading
  9 The Polymerase Chain Reaction
   9.1 PCR in outline
   9.2 PCR in more detail
    9.2.1 Designing the oligonudeotide 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
    9.3.3 Problems with the error rate of Taq polymerase
   9.4 Real-time PCR enables the amount of starting material to be quantified
    9.4.1 Carrying out a quantitative PCR experiment
    9.4.2 Real-time PCR can also quantify RNA
    Further reading
 Part 11 The Applications of Gene Cloning and DNA Analysis in Research
  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 Preparation of a next-generation sequencing library
     DNA fragmentation
     Immobilization of the library
     Amplification of the library
    10.2.2 Next-generation sequencing methods
     Reversible terminator sequencing
     Pyrosequencing
    10.2.3 Third-generation sequencing
    10.2.4 Directing next-generation sequencing at specific sets of genes
   10.3 How to sequence a genome
    10.3.1 Shotgun sequencing of prokaryotic genomes
     Shotgun sequencing of the Haemophi/us influenza genome
     Shotgun sequencing of other prokaryotic genomes
    10.3.2 Sequencing of eukaryotic genomes
     The hierarchical shotgun approach
     Shotgun sequencing of eukaryotic genomes
     What do we mean by 'genome sequence'?
    Further reading
  11 Studying Gene Expression and Function
   11.1 Studying the RNA transcript of a gene
    11 .1.1 Detecting the presence of a transcript and determining its nucleotide sequence
    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
     Gel retardation of DNA-protein complexes
     Footprinting with DNase I
     Modification interference assays
    11.2.2 Identifying control sequences by deletion analysis
     Reporter genes
     Carrying out a 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 ;n v;tro mutagenesis
     Different types of ;n v;tro mutagenesis techniques
     Using an oligonucleotide to create a point mutation in a cloned gene
     Other methods of creating a point mutation in a cloned gene
     The potential of ;n v;tro mutagenesis
    Further reading
  12 Studying Genomes
   12.1 Genome annotation
    12.1.1 Identifying the genes in a genome sequence
     Searching for open reading frames
     Simple ORF scans are less effective at locating genes in eukaryotic genomes
     Gene location is aided by homology searching
     Comparing the sequences of related genomes
     Identifying the binding sites for regulatory proteins in a genome sequence
    12.1 .2 Determining the function of an unknown gene
     Assigning gene function by experimental analysis requires a reverse approach to genetics
     Specific genes can be inactivated by homologous recombination
   12.2 Studies of the transcriptome and proteome
    12.2.1 Studying the transcriptome
     Studying transcriptomes by microarray or chip analysis
     Studying a transcriptome by SAGE
     Sequencing a transcriptome by RNA-seq
     Advantages of the different methods for transcriptome analysis
    12.2.2 Studying the proteome
     Separating the proteins in a proteome
     Identifying the individual proteins after separation
    12.2.3 Studying protein-protein interactions
     Phage display
     The yeast two-hybrid system
    Further reading
 Part 111 The Applications of Gene Cloning and DNA Analysis in Biotechnology
  13 Production of Protein from Cloned Genes
   13.1 Special vectors for the expression of foreign genes in E. co/i
    13.1.1 The promoter is the critical component of an expression vector
     The promoter must be chosen with care
     Examples of promoters used in expression vectors
    13.1.2 Cassettes and gene fusions
   13.2 General problems with the production of recombinant protein in E. co/i
    13.2.1 Problems resulting from the sequence of the foreign gene
    13.2.2 Problems caused by E. co/i
   13.3 Production of recombinant protein by eukaryotic cells
    13.3.1 Recombinant protein from yeasts and filamentous fungi
     Saccharomyces cerevisiae as the host for recombinant protein synthesis
     Other yeasts and fungi
    13.3.2 Using animal cells for recombinant protein production
     Protein production in mammalian cells
     Protein production in insect cells
    13.3.3 Pharming: Recombinant protein from live animals and plants
     Pharming in animals
     Recombinant proteins from plants
     Ethical concerns raised by pharming
    Further reading
  14 Gene Cloning and DNA Analysis in Medicine
   14.1 Production of recombinant pharmaceuticals
    14.1.1 Recombinant insulin
     Synthesis and expression of artificial insulin genes
    14.1.2 Synthesis of human growth hormones in E. co/i
    14.1.3 Recombinant factor VIII
    14.1.4 Synthesis of other recombinant human proteins
    14.1.5 Recombinant vaccines
     Producing vaccines as recombinant proteins
     Recombinant vaccines in transgenic plants
     Live recombinant virus vaccines
   14.2 Identification of genes responsible for human diseases
    14.2.1 How to identify a gene for a genetic disease
     Locating the approximate position of the gene in the human genome
     Linkage analysis of the human BRCA 1 gene
     Identification of candidates for the disease gene
    14.3 Gene therapy
     14.3.1 Gene therapy for inherited diseases
     14.3.2 Gene therapy and cancer
     14.3.3 The ethical issues raised by gene therapy
    Further reading
  15 Gene Cloning and DNA Analysis in Agriculture
   15.1 The gene addition approach to plant genetic engineering
    15.1.1 Plants that make their own insecticides
     The δ-endotoxins of 8acillus thuringiensis
     Cloning a δ-endotoxin gene in maize
     Cloningδ-endotoxin genes in chloroplasts
     Countering insect resistance to õ-endotoxin crops
    15.1.2 Herbicide-resistant crops
     'Roundup Ready' crops
     A new generation of glyphosate-resistant crops
    15.1 .3 Other gene addition projects
   15 . 2 Gene subtraction
    15.2.1 Antisense RNA and the engineering of fruit ripening in tomato
     Using antisense RNA to inactivate the polygalacturonase gene
     Using antisense RNA to inactivate ethylene synthesis
    15.2.2 Other examples of the use of antisense RNA in plant genetic engineering
   15.3 Problems with genetically modified plants
    15.3.1 Safety concerns with selectable markers
    15.3.2 The terminator technology
    15.3.3 The possibility of harmful effects on the environment
    Further reading
  16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology
   16.1 DNA analysis in the identification of crime suspects
    16.1.1 Geneticfingerprinting by hybridization probing
    16.1 .2 DNA profiling by PCR of short tandem repeats
   16.2 Studying kinship by DNA profiling
    16.2.1 Related individuals have similar DNA profiles
    16.2.2 DNA profiling and the remains of the Romanovs
     STR analysis of the Romanov bones
     Mitochondrial DNA was used to link the Romanov skeletons with living relatives
     The missing children
   16.3 Sex identification by DNA analysis
    16.3.1 PCRs directed at Y chromosome-specific sequences
    16.3.2 PCR of the amelogenin gene
   16.4 Archaeogenetics: Using DNA to study human prehistory
    16.4.1 The origins of modern humans
     DNA analysis has challenged the multiregional hypothesis
     DNA analysis shows that Neanderthals are not the direct ancestors of modern Europeans
     The Neanderthal genome sequence suggests there was interbreeding with H. sap;ens
    16.4.2 DNA can also be used to study prehistoric human migrations
     Modern humans may have migrated from Ethiopia t。Arabia
     Colonization of the New World
    Further reading
 Glossary
 lndex