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Die fünfte Auflage des renommierten Standardwerkes wurde deutlich erweitert: Sie enthält neue Beiträge zu glyphosatresistenten Pflanzen, zu Vervielfältigungstechniken wie Klonen und PCR in der Archäologie sowie zu ethischen Aspekten genmanipulierten Saatguts und der Gentherapie.
Die fünfte Auflage des renommierten Standardwerkes wurde deutlich erweitert: Sie enthält neue Beiträge zu glyphosatresistenten Pflanzen, zu Vervielfältigungstechniken wie Klonen und PCR in der Archäologie sowie zu ethischen Aspekten genmanipulierten Saatguts und der Gentherapie.
Produktdetails
- Produktdetails
- Verlag: Blackwell Publishing Ltd., Oxford / Wiley & Sons
- 5th ed.
- Seitenzahl: 386
- Englisch
- Abmessung: 245mm
- Gewicht: 968g
- ISBN-13: 9781405111218
- ISBN-10: 1405111216
- Artikelnr.: 20832143
- Herstellerkennzeichnung Die Herstellerinformationen sind derzeit nicht verfügbar.
- Verlag: Blackwell Publishing Ltd., Oxford / Wiley & Sons
- 5th ed.
- Seitenzahl: 386
- Englisch
- Abmessung: 245mm
- Gewicht: 968g
- ISBN-13: 9781405111218
- ISBN-10: 1405111216
- Artikelnr.: 20832143
- Herstellerkennzeichnung Die Herstellerinformationen sind derzeit nicht verfügbar.
Terry Brown, University of Manchester, Faculty of Life Sciences, UK.
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
1.6 How to find your way through this book
Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages
2.1 Plasmids
2.2 Bacteriophages
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
Chapter 3 Purification of DNA from Living Cells
3.1 Preparation of total cell DNA
Removing contaminants by organic extraction and enzyme digestion
Using ion-exchange chromatography to purify DNA from a cell extract
3.2 Preparation of plasmid DNA
Alkaline denaturation
Ethidium bromide-caesium chloride density gradient centrifugation
3.3 Preparation of bacteriophage DNA
Chapter 4 Manipulation of Purified DNA
4.1 The range of DNA manipulative enzymes
4.2 Enzymes for cutting DNA - restriction endonucleases
Separation of molecules by gel electrophoresis
Visualizing DNA molecules by staining a gel
Visualizing DNA molecules by autoradiography
4.3 Ligation - joining DNA molecules together
Linkers
Adaptors
Chapter 5 Introduction of DNA into Living Cells
5.1 Transformation - the uptake of DNA by bacterial cells
5.2 Identification of recombinants
resistance gene
5.3 Introduction of phage DNA into bacterial cells
5.4 Identification of recombinant phages
5.5 Introduction of DNA into non-bacterial cells
Producing sticky ends by homopolymer tailing
Chapter 6 Cloning Vectors for E. coli
6.1 Cloning vectors based on E. coli plasmids
pUC8 - a Lac selection plasmid
pGEM3Z - in vitro transcription of cloned DNA
6.2 Cloning vectors based on M13 bacteriophage
M13mp7 - symmetrical cloning sites
More complex M13 vectors
6.3 Cloning vectors based on l bacteriophage
sites
Insertion vectors
Replacement vectors
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
The structure and use of a YAC vector
Applications for YAC vectors
7.2 Cloning vectors for higher plants
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
Direct gene transfer into the nucleus
Transfer of genes into the chloroplast genome
Caulimovirus vectors
Geminivirus vectors
7.3 Cloning vectors for animals
P elements as cloning vectors for Drosophila
Cloning vectors based on insect viruses
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
8.2 Direct selection
8.3 Identification of a clone from a gene library
8.4 Methods for clone identification
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
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
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
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
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
Chapter 11 Studying Gene Expression and Function
11.1 Studying the transcript of a cloned gene
Northern hybridization
Reverse transcription-PCR (RT-PCR)
Rapid amplification of cDNA ends (RACE)
RNA sequencing
11.2 Studying the regulation of gene expression
Gel retardation of DNA-protein complexes
Footprinting with DNase I
Modification interference assays
Reporter genes
Carrying out a deletion analysis
11.3 Identifying and studying the translation product of a cloned gene
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
Phage display
The yeast two hybrid system
Chapter 12 Studying Genomes
12.1 Genomics - how to sequence a genome
The H. influenzae genome sequencing project
Problems with shotgun cloning
Clone contig assembly by chromosome walking
Rapid methods for clone contig assembly
Clone contig assembly by sequence tagged site content analysis
Genetic maps
Physical maps
The importance of a map in sequence assembly
12.2 Post-genomics - trying to understand a genome sequence
Searching for open reading frames
Distinguishing real genes from chance ORFs
12.3 Studies of the transcriptome and 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
The promoter must be chosen with care
Examples of promoters used in expression vectors
13.2 General problems with the production of recombinant protein in E. coli
13.3 Production of recombinant protein by eukaryotic cells
Saccharomyces cerevisiae as the host for recombinant protein synthesis
Other yeasts and fungi
Protein production in mammalian cells
Protein production in insect cells
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
Synthesis and expression of artificial insulin genes
Producing vaccines as recombinant proteins
Recombinant vaccines in transgenic plants
Live recombinant virus vaccines
14.2 Identification of genes responsible for human diseases
Locating the approximate position of the gene in the human genome
Identification of candidates for the disease gene
14.3 Gene therapy
Chapter 15 Gene Cloning and DNA Analysis in Agriculture
15.1 The gene addition approach to plant genetic engineering
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
'Roundup Ready' crops
A new generation of glyphosate resistant crops
15.2 Gene subtraction
Using antisense RNA to inactivate the polygalacturonase gene
Using antisense RNA to inactivate ethylene synthesis
15.3 Problems with genetically modified plants
Chapter 16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology
16.1 DNA analysis in the identification of crime suspects
16.2 Studying kinship by DNA profiling
STR analysis of the Romanov bones
The missing children
16.3 Sex identification by DNA analysis
16.4 Archaeogenetics - using DNA to study human evolution
DNA analysis has challenged the multiregional hypothesis
DNA analysis shows that Neanderthals are not the ancestors of modern
Europeans
The spread of agriculture into Europe
Using mitochondrial DNA to study past human migrations into Europe
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
1.6 How to find your way through this book
Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages
2.1 Plasmids
2.2 Bacteriophages
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
Chapter 3 Purification of DNA from Living Cells
3.1 Preparation of total cell DNA
Removing contaminants by organic extraction and enzyme digestion
Using ion-exchange chromatography to purify DNA from a cell extract
3.2 Preparation of plasmid DNA
Alkaline denaturation
Ethidium bromide-caesium chloride density gradient centrifugation
3.3 Preparation of bacteriophage DNA
Chapter 4 Manipulation of Purified DNA
4.1 The range of DNA manipulative enzymes
4.2 Enzymes for cutting DNA - restriction endonucleases
Separation of molecules by gel electrophoresis
Visualizing DNA molecules by staining a gel
Visualizing DNA molecules by autoradiography
4.3 Ligation - joining DNA molecules together
Linkers
Adaptors
Chapter 5 Introduction of DNA into Living Cells
5.1 Transformation - the uptake of DNA by bacterial cells
5.2 Identification of recombinants
resistance gene
5.3 Introduction of phage DNA into bacterial cells
5.4 Identification of recombinant phages
5.5 Introduction of DNA into non-bacterial cells
Producing sticky ends by homopolymer tailing
Chapter 6 Cloning Vectors for E. coli
6.1 Cloning vectors based on E. coli plasmids
pUC8 - a Lac selection plasmid
pGEM3Z - in vitro transcription of cloned DNA
6.2 Cloning vectors based on M13 bacteriophage
M13mp7 - symmetrical cloning sites
More complex M13 vectors
6.3 Cloning vectors based on l bacteriophage
sites
Insertion vectors
Replacement vectors
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
The structure and use of a YAC vector
Applications for YAC vectors
7.2 Cloning vectors for higher plants
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
Direct gene transfer into the nucleus
Transfer of genes into the chloroplast genome
Caulimovirus vectors
Geminivirus vectors
7.3 Cloning vectors for animals
P elements as cloning vectors for Drosophila
Cloning vectors based on insect viruses
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
8.2 Direct selection
8.3 Identification of a clone from a gene library
8.4 Methods for clone identification
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
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
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
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
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
Chapter 11 Studying Gene Expression and Function
11.1 Studying the transcript of a cloned gene
Northern hybridization
Reverse transcription-PCR (RT-PCR)
Rapid amplification of cDNA ends (RACE)
RNA sequencing
11.2 Studying the regulation of gene expression
Gel retardation of DNA-protein complexes
Footprinting with DNase I
Modification interference assays
Reporter genes
Carrying out a deletion analysis
11.3 Identifying and studying the translation product of a cloned gene
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
Phage display
The yeast two hybrid system
Chapter 12 Studying Genomes
12.1 Genomics - how to sequence a genome
The H. influenzae genome sequencing project
Problems with shotgun cloning
Clone contig assembly by chromosome walking
Rapid methods for clone contig assembly
Clone contig assembly by sequence tagged site content analysis
Genetic maps
Physical maps
The importance of a map in sequence assembly
12.2 Post-genomics - trying to understand a genome sequence
Searching for open reading frames
Distinguishing real genes from chance ORFs
12.3 Studies of the transcriptome and 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
The promoter must be chosen with care
Examples of promoters used in expression vectors
13.2 General problems with the production of recombinant protein in E. coli
13.3 Production of recombinant protein by eukaryotic cells
Saccharomyces cerevisiae as the host for recombinant protein synthesis
Other yeasts and fungi
Protein production in mammalian cells
Protein production in insect cells
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
Synthesis and expression of artificial insulin genes
Producing vaccines as recombinant proteins
Recombinant vaccines in transgenic plants
Live recombinant virus vaccines
14.2 Identification of genes responsible for human diseases
Locating the approximate position of the gene in the human genome
Identification of candidates for the disease gene
14.3 Gene therapy
Chapter 15 Gene Cloning and DNA Analysis in Agriculture
15.1 The gene addition approach to plant genetic engineering
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
'Roundup Ready' crops
A new generation of glyphosate resistant crops
15.2 Gene subtraction
Using antisense RNA to inactivate the polygalacturonase gene
Using antisense RNA to inactivate ethylene synthesis
15.3 Problems with genetically modified plants
Chapter 16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology
16.1 DNA analysis in the identification of crime suspects
16.2 Studying kinship by DNA profiling
STR analysis of the Romanov bones
The missing children
16.3 Sex identification by DNA analysis
16.4 Archaeogenetics - using DNA to study human evolution
DNA analysis has challenged the multiregional hypothesis
DNA analysis shows that Neanderthals are not the ancestors of modern
Europeans
The spread of agriculture into Europe
Using mitochondrial DNA to study past human migrations into Europe
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
1.6 How to find your way through this book
Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages
2.1 Plasmids
2.2 Bacteriophages
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
Chapter 3 Purification of DNA from Living Cells
3.1 Preparation of total cell DNA
Removing contaminants by organic extraction and enzyme digestion
Using ion-exchange chromatography to purify DNA from a cell extract
3.2 Preparation of plasmid DNA
Alkaline denaturation
Ethidium bromide-caesium chloride density gradient centrifugation
3.3 Preparation of bacteriophage DNA
Chapter 4 Manipulation of Purified DNA
4.1 The range of DNA manipulative enzymes
4.2 Enzymes for cutting DNA - restriction endonucleases
Separation of molecules by gel electrophoresis
Visualizing DNA molecules by staining a gel
Visualizing DNA molecules by autoradiography
4.3 Ligation - joining DNA molecules together
Linkers
Adaptors
Chapter 5 Introduction of DNA into Living Cells
5.1 Transformation - the uptake of DNA by bacterial cells
5.2 Identification of recombinants
resistance gene
5.3 Introduction of phage DNA into bacterial cells
5.4 Identification of recombinant phages
5.5 Introduction of DNA into non-bacterial cells
Producing sticky ends by homopolymer tailing
Chapter 6 Cloning Vectors for E. coli
6.1 Cloning vectors based on E. coli plasmids
pUC8 - a Lac selection plasmid
pGEM3Z - in vitro transcription of cloned DNA
6.2 Cloning vectors based on M13 bacteriophage
M13mp7 - symmetrical cloning sites
More complex M13 vectors
6.3 Cloning vectors based on l bacteriophage
sites
Insertion vectors
Replacement vectors
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
The structure and use of a YAC vector
Applications for YAC vectors
7.2 Cloning vectors for higher plants
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
Direct gene transfer into the nucleus
Transfer of genes into the chloroplast genome
Caulimovirus vectors
Geminivirus vectors
7.3 Cloning vectors for animals
P elements as cloning vectors for Drosophila
Cloning vectors based on insect viruses
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
8.2 Direct selection
8.3 Identification of a clone from a gene library
8.4 Methods for clone identification
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
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
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
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
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
Chapter 11 Studying Gene Expression and Function
11.1 Studying the transcript of a cloned gene
Northern hybridization
Reverse transcription-PCR (RT-PCR)
Rapid amplification of cDNA ends (RACE)
RNA sequencing
11.2 Studying the regulation of gene expression
Gel retardation of DNA-protein complexes
Footprinting with DNase I
Modification interference assays
Reporter genes
Carrying out a deletion analysis
11.3 Identifying and studying the translation product of a cloned gene
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
Phage display
The yeast two hybrid system
Chapter 12 Studying Genomes
12.1 Genomics - how to sequence a genome
The H. influenzae genome sequencing project
Problems with shotgun cloning
Clone contig assembly by chromosome walking
Rapid methods for clone contig assembly
Clone contig assembly by sequence tagged site content analysis
Genetic maps
Physical maps
The importance of a map in sequence assembly
12.2 Post-genomics - trying to understand a genome sequence
Searching for open reading frames
Distinguishing real genes from chance ORFs
12.3 Studies of the transcriptome and 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
The promoter must be chosen with care
Examples of promoters used in expression vectors
13.2 General problems with the production of recombinant protein in E. coli
13.3 Production of recombinant protein by eukaryotic cells
Saccharomyces cerevisiae as the host for recombinant protein synthesis
Other yeasts and fungi
Protein production in mammalian cells
Protein production in insect cells
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
Synthesis and expression of artificial insulin genes
Producing vaccines as recombinant proteins
Recombinant vaccines in transgenic plants
Live recombinant virus vaccines
14.2 Identification of genes responsible for human diseases
Locating the approximate position of the gene in the human genome
Identification of candidates for the disease gene
14.3 Gene therapy
Chapter 15 Gene Cloning and DNA Analysis in Agriculture
15.1 The gene addition approach to plant genetic engineering
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
'Roundup Ready' crops
A new generation of glyphosate resistant crops
15.2 Gene subtraction
Using antisense RNA to inactivate the polygalacturonase gene
Using antisense RNA to inactivate ethylene synthesis
15.3 Problems with genetically modified plants
Chapter 16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology
16.1 DNA analysis in the identification of crime suspects
16.2 Studying kinship by DNA profiling
STR analysis of the Romanov bones
The missing children
16.3 Sex identification by DNA analysis
16.4 Archaeogenetics - using DNA to study human evolution
DNA analysis has challenged the multiregional hypothesis
DNA analysis shows that Neanderthals are not the ancestors of modern
Europeans
The spread of agriculture into Europe
Using mitochondrial DNA to study past human migrations into Europe
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
1.6 How to find your way through this book
Chapter 2 Vectors for Gene Cloning: Plasmids and Bacteriophages
2.1 Plasmids
2.2 Bacteriophages
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
Chapter 3 Purification of DNA from Living Cells
3.1 Preparation of total cell DNA
Removing contaminants by organic extraction and enzyme digestion
Using ion-exchange chromatography to purify DNA from a cell extract
3.2 Preparation of plasmid DNA
Alkaline denaturation
Ethidium bromide-caesium chloride density gradient centrifugation
3.3 Preparation of bacteriophage DNA
Chapter 4 Manipulation of Purified DNA
4.1 The range of DNA manipulative enzymes
4.2 Enzymes for cutting DNA - restriction endonucleases
Separation of molecules by gel electrophoresis
Visualizing DNA molecules by staining a gel
Visualizing DNA molecules by autoradiography
4.3 Ligation - joining DNA molecules together
Linkers
Adaptors
Chapter 5 Introduction of DNA into Living Cells
5.1 Transformation - the uptake of DNA by bacterial cells
5.2 Identification of recombinants
resistance gene
5.3 Introduction of phage DNA into bacterial cells
5.4 Identification of recombinant phages
5.5 Introduction of DNA into non-bacterial cells
Producing sticky ends by homopolymer tailing
Chapter 6 Cloning Vectors for E. coli
6.1 Cloning vectors based on E. coli plasmids
pUC8 - a Lac selection plasmid
pGEM3Z - in vitro transcription of cloned DNA
6.2 Cloning vectors based on M13 bacteriophage
M13mp7 - symmetrical cloning sites
More complex M13 vectors
6.3 Cloning vectors based on l bacteriophage
sites
Insertion vectors
Replacement vectors
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
The structure and use of a YAC vector
Applications for YAC vectors
7.2 Cloning vectors for higher plants
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
Direct gene transfer into the nucleus
Transfer of genes into the chloroplast genome
Caulimovirus vectors
Geminivirus vectors
7.3 Cloning vectors for animals
P elements as cloning vectors for Drosophila
Cloning vectors based on insect viruses
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
8.2 Direct selection
8.3 Identification of a clone from a gene library
8.4 Methods for clone identification
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
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
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
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
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
Chapter 11 Studying Gene Expression and Function
11.1 Studying the transcript of a cloned gene
Northern hybridization
Reverse transcription-PCR (RT-PCR)
Rapid amplification of cDNA ends (RACE)
RNA sequencing
11.2 Studying the regulation of gene expression
Gel retardation of DNA-protein complexes
Footprinting with DNase I
Modification interference assays
Reporter genes
Carrying out a deletion analysis
11.3 Identifying and studying the translation product of a cloned gene
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
Phage display
The yeast two hybrid system
Chapter 12 Studying Genomes
12.1 Genomics - how to sequence a genome
The H. influenzae genome sequencing project
Problems with shotgun cloning
Clone contig assembly by chromosome walking
Rapid methods for clone contig assembly
Clone contig assembly by sequence tagged site content analysis
Genetic maps
Physical maps
The importance of a map in sequence assembly
12.2 Post-genomics - trying to understand a genome sequence
Searching for open reading frames
Distinguishing real genes from chance ORFs
12.3 Studies of the transcriptome and 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
The promoter must be chosen with care
Examples of promoters used in expression vectors
13.2 General problems with the production of recombinant protein in E. coli
13.3 Production of recombinant protein by eukaryotic cells
Saccharomyces cerevisiae as the host for recombinant protein synthesis
Other yeasts and fungi
Protein production in mammalian cells
Protein production in insect cells
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
Synthesis and expression of artificial insulin genes
Producing vaccines as recombinant proteins
Recombinant vaccines in transgenic plants
Live recombinant virus vaccines
14.2 Identification of genes responsible for human diseases
Locating the approximate position of the gene in the human genome
Identification of candidates for the disease gene
14.3 Gene therapy
Chapter 15 Gene Cloning and DNA Analysis in Agriculture
15.1 The gene addition approach to plant genetic engineering
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
'Roundup Ready' crops
A new generation of glyphosate resistant crops
15.2 Gene subtraction
Using antisense RNA to inactivate the polygalacturonase gene
Using antisense RNA to inactivate ethylene synthesis
15.3 Problems with genetically modified plants
Chapter 16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology
16.1 DNA analysis in the identification of crime suspects
16.2 Studying kinship by DNA profiling
STR analysis of the Romanov bones
The missing children
16.3 Sex identification by DNA analysis
16.4 Archaeogenetics - using DNA to study human evolution
DNA analysis has challenged the multiregional hypothesis
DNA analysis shows that Neanderthals are not the ancestors of modern
Europeans
The spread of agriculture into Europe
Using mitochondrial DNA to study past human migrations into Europe