PART 1 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 Gene isolation by cloning
1.5.2 Gene isolation by PCR
1.6 How to find your way through this book
Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages
2.1 Plasmids
2.1.1 Basic features of plasmids
2.1.2 Size and copy number
2.1.3 Conjugation and compatibility
2.1.4 Plasmid classification
2.1.5 Plasmids in organisms other than bacteria
2.2 Bacteriophages
2.2.1 Basic features of bacteriophages
2.2.2 Lysogenic phages
Gene organization in l DNA molecule
The linear and circular forms of l DNA
M13 - a filamentous phage
The attraction of M13 as a cloning vector
2.2.3 Viruses as cloning vectors for other organisms
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
Removing contaminants by organic extraction and enzyme digestion
Using ion-exchange chromatography 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 l titre
3.3.2 Preparation of non-lysogenic l phages
3.3.3 Collection of phages from an infected culture
3.3.4 Purification of DNA from l phage particles
3.3.5 Purification of M13 DNA causes few problems
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
4.1.5 Topoisomerases
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
Separation of molecules by gel electrophoresis
Visualizing DNA molecules by staining a gel
Visualizing DNA molecules by autoradiography
4.2.7 Estimation of the sizes of DNA molecules
4.2.8 Mapping the positions of different restriction sites in a DNA molecule
4.3 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
Linkers
Adaptors
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 l cloning vectors
5.3.3 Phage infection is visualized as plaques on an agar medium
5.4 Identification of recombinant phages
5.4.1 Insertional inactivation of a lacZ´ gene carried by the phage vector
5.4.2 Insertional inactivation of the l cI gene
5.4.3 Selection using the Spi phenotype
5.4.4 Selection on the basis of l 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
Producing sticky ends by homopolymer tailing
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
pUC8 - a Lac selection plasmid
pGEM3Z - in vitro transcription of cloned DNA
6.2 Cloning vectors based on M13 bacteriophage
6.2.1 Development of the M13 cloning vectors
M13mp7 - symmetrical cloning sites
More complex M13 vectors
6.2.2 Hybrid plasmid-M13 vectors
6.3 Cloning vectors based on l bacteriophage
6.3.1 Segments of the l genome can be deleted without impairing viability
6.3.2 Natural selection can be used to isolate modified l that lack certain restriction
sites
6.3.3 Insertion and replacement vectors
Insertion vectors
Replacement vectors
6.3.4 Cloning experiments with l insertion or replacement vectors
6.3.5 Long DNA fragments can be cloned using a cosmid
6.4 l and other high capacity vectors enable genomic libraries to be constructed
6.5 Vectors for other bacteria
Chapter 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 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
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
Cloning vectors for mammals
Gene cloning without a vector
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
8.3.2 Not all genes are expressed at the same time
8.3.3 mRNA can be cloned as complementary DNA
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
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
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
Chapter 9 The Polymerase Chain Reaction
9.1 The polymerase chain reaction 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
9.2.3 After the PCR: studying PCR products
Gel electrophoresis of PCR products
Cloning PCR products
9.3 Problems with the error rate of Taq polymerase
PART 2 THE APPLICATIONS OF GENE CLONING AND DNA ANALYSIS IN RESEARCH
Chapter 10 Studying Gene Location and Structure
10.1 How to study the location of a gene
10.1.1 Locating the position of a gene on a small DNA molecule
10.1.2 Locating the position of a gene on a large DNA molecule
Separating chromosomes by gel electrophoresis
In situ hybridization to visualize the position of a gene on a eukaryotic
chromosome
10.2 DNA sequencing - working out the structure of a gene
10.2.1 The Sanger-Coulson method - chain-terminating nucleotides
The primer
Synthesis of the complementary strand
Four separate reactions result in four families of terminated strands
Reading the DNA sequence from the autoradiograph
Not all DNA polymerases can be used for sequencing
10.2.2 Automated DNA sequencing
10.2.3 Sequencing PCR products
10.2.4 The Maxam-Gilbert method - chemical degradation of DNA
10.2.5 Building up a long DNA sequence
10.2.6 The achievements of DNA sequencing
Chapter 11 Studying Gene Expression and Function
11.1 Studying the transcript of a cloned gene
11.1.1 Electron microscopy of nucleic acid molecules
11.1.2 Analysis of DNA-RNA hybrids by nuclease treatment
11.1.3 Transcript analysis by primer extension
11.1.4 Other techniques for studying RNA transcripts
Northern hybridization
Reverse transcription-PCR (RT-PCR)
Rapid amplification of cDNA ends (RACE)
RNA sequencing
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 in vitro mutagenesis
Different types of in vitro 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 in vitro mutagenesis
11.3.3 Studying protein-protein interactions
Phage display
The yeast two hybrid system
Chapter 12 Studying Genomes
12.1 Genomics - how to sequence a genome
12.1.1 The shotgun approach to genome sequencing
The H. influenzae genome sequencing project
Problems with shotgun cloning
12.1.2 The clone contig approach
Clone contig assembly by chromosome walking
Rapid methods for clone contig assembly
Clone contig assembly by sequence tagged site content analysis
12.1.3 Using a map to aid sequence assembly
Genetic maps
Physical maps
The importance of a map in sequence assembly
12.2 Post-genomics - trying to understand a genome sequence
12.2.1 Identifying the genes in a genome sequence
Searching for open reading frames
Distinguishing real genes from chance ORFs
12.2.2 Determining the function of an unknown gene
12.3 Studies of the transcriptome and proteome
12.3.1 Studying the transcriptome
12.3.2 Studying the proteome
PART 3 THE APPLICATIONS OF GENE CLONING AND DNA ANALYSIS IN BIOTECHNOLOGY
Chapter 13 Production of Protein from Cloned Genes
13.1 Special vectors for expression of foreign genes in E. coli
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. coli
13.2.1 Problems resulting from the sequence of the foreign gene
13.2.2 Problems caused by E. coli
13.3 Production of recombinant protein by eukaryotic cells
13.3.1 Recombinant protein from yeast 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
Chapter 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. coli
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
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
Chapter 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 d-endotoxins of Bacillus thuringiensis
Cloning a d-endotoxin gene in maize
Cloning d-endotoxin genes in chloroplasts
Countering insect resistance to d-endotoxin crops
15.1.2 Herbicide resistant crops
'Roundup Ready' crops
A new generation of glyphosate resistant crops
15.1.2 Other gene addition projects
15.2 Gene subtraction
15.2.1 The principle behind antisense technology
15.2.2 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
15.2.3 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
Chapter 16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology
16.1 DNA analysis in the identification of crime suspects
16.1.1 Genetic fingerprinting 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
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 evolution
16.4.1 The origins of modern humans
DNA analysis has challenged the multiregional hypothesis
DNA analysis shows that Neanderthals are not the ancestors of modern
Europeans
16.4.2 DNA can also be used to study prehistoric human migrations
The spread of agriculture into Europe
Using mitochondrial DNA to study past human migrations into Europe
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