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Gene Cloning and DNA Analysis: An Introduction, 8th Edition contains updated and extended coverage of gene editing strategies like CRISPR/Cas, rewritten chapters on DNA sequencing and genome studies, as well as new material on real-time PCR and typing of human disease mutations. Over 250 full-color illustrations are included to bring to life the comprehensive content. The book also covers topics like:
* The strategies used by researchers and industry practitioners to assemble genome sequences
* Next generation sequencing methods and descriptions of their applications in studying genomes and transcriptomes
* Includes the use and application of gene editing strategies
* Interbreeding between Neanderthals and Homo Sapiens
Gene Cloning and DNA Analysis: An Introduction, 8th Edition is an invaluable introductory text for students in classes like genetics and genomics, molecular biology, biochemistry, immunology, and applied biology. It also belongs on the bookshelves of every professional who desires to improve their understanding of the basics of gene cloning or DNA analysis.
Gene Cloning and DNA Analysis: An Introduction, 8th Edition contains updated and extended coverage of gene editing strategies like CRISPR/Cas, rewritten chapters on DNA sequencing and genome studies, as well as new material on real-time PCR and typing of human disease mutations. Over 250 full-color illustrations are included to bring to life the comprehensive content. The book also covers topics like:
* The strategies used by researchers and industry practitioners to assemble genome sequences
* Next generation sequencing methods and descriptions of their applications in studying genomes and transcriptomes
* Includes the use and application of gene editing strategies
* Interbreeding between Neanderthals and Homo Sapiens
Gene Cloning and DNA Analysis: An Introduction, 8th Edition is an invaluable introductory text for students in classes like genetics and genomics, molecular biology, biochemistry, immunology, and applied biology. It also belongs on the bookshelves of every professional who desires to improve their understanding of the basics of gene cloning or DNA analysis.
T. A. Brown is Emeritus Professor of Biomolecular Archaeology in the Manchester Institute of Biotechnology at the University of Manchester in the United Kingdom. He has published several books on genetics, genomics and biochemistry as well as over 150 research papers.
Preface to the Eighth Edition xv
Part I The Basic Principles of Gene Cloning and DNA Analysis 1
1 Why Gene Cloning and DNA Analysis are Important 3
2 Vectors for Gene Cloning: Plasmids and Bacteriophages 15
3 Purification of DNA from Living Cells 29
4 Manipulation of Purified DNA 53
5 Introduction of DNA into Living Cells 83
6 Cloning Vectors for E. coli 101
7 Cloning Vectors for Eukaryotes 121
8 How to Obtain a Clone of a Specific Gene 145
9 The Polymerase Chain Reaction 169
Part II The Applications of Gene Cloning and DNA Analysis in Research 187
10 Sequencing Genes and Genomes 189
11 Studying Gene Expression and Function 217
12 Studying Genomes 243
13 Studying Transcriptomes and Proteomes 259
Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology 275
14 Production of Protein from Cloned Genes 277
15 Gene Cloning and DNA Analysis in Medicine 301
16 Gene Cloning and DNA Analysis in Agriculture 327
17 Gene Cloning and DNA Analysis in Forensic Science and Archaeology 355
Glossary 377
Index 395
Preface to the Eighth Edition xv
Part I The Basic Principles of Gene Cloning and DNA Analysis 1
1 Why Gene Cloning and DNA Analysis are Important 3
1.1 The early development of genetics 4
1.2 The advent of gene cloning and the polymerase chain reaction 4
1.3 What is gene cloning? 5
1.4 What is PCR? 5
1.5 Why gene cloning and PCR are so important 8
1.5.1 Obtaining a pure sample of a gene by cloning 8
1.5.2 PCR can also be used to purify a gene 10
1.6 How to find your way through this book 11
Further reading 13
2 Vectors for Gene Cloning: Plasmids and Bacteriophages 15
2.1 Plasmids 15
2.1.1 Size and copy number 17
2.1.2 Conjugation and compatibility 18
2.1.3 Plasmid classification 19
2.1.4 Plasmids in organisms other than bacteria 19
2.2 Bacteriophages 19
2.2.1 The phage infection cycle 20
2.2.2 Lysogenic phages 20
2.2.3 Viruses as cloning vectors for other organisms 26
Further reading 27
3 Purification of DNA from Living Cells 29
3.1 Preparation of total cell DNA 30
3.1.1 Growing and harvesting a bacterial culture 30
3.1.2 Preparation of a cell extract 31
3.1.3 Purification of DNA from a cell extract 33
3.1.4 Concentration of DNA samples 37
3.1.5 Measurement of DNA concentration 38
3.1.6 Other methods for the preparation of total cell DNA 39
3.2 Preparation of plasmid DNA 40
3.2.1 Separation on the basis of size 41
3.2.2 Separation on the basis of conformation 42
3.2.3 Plasmid amplification 44
3.3 Preparation of bacteriophage DNA 46
3.3.1 Growth of cultures to obtain a high ¿ titre 47
3.3.2 Preparation of non¿lysogenic ¿ phages 47
3.3.3 Collection of phages from an infected culture 49
3.3.4 Purification of DNA from ¿ phage particles 49
3.3.5 Purification of M13 DNA causes few problems 49
Further reading 51
4 Manipulation of Purified DNA 53
4.1 The range of DNA manipulative enzymes 55
4.1.1 Nucleases 55
4.1.2 Ligases 57
4.1.3 Polymerases 57
4.1.4 DNA modifying enzymes 58
4.2 Enzymes for cutting DNA - restriction endonucleases 59
4.2.1 The discovery and function of restriction endonucleases 60
4.2.2 Type II restriction endonucleases cut DNA at specific nucleotide sequences 61
4.2.3 Blunt ends and sticky ends 62
4.2.4 The frequency of recognition sequences in a DNA molecule 63
4.2.5 Performing a restriction digest in the laboratory 64
4.2.6 Analysing the result of restriction endonuclease cleavage 66
4.2.7 Estimation of the sizes of DNA molecules 68
4.2.8 Mapping the positions of different restriction sites in a DNA molecule 69
4.2.9 Special gel electrophoresis methods for separating larger molecules 70
4.3 Ligation - joining DNA molecules together 72
4.3.1 The mode of action of DNA ligase 72
4.3.2 Sticky ends increase the efficiency of ligation 74
4.3.3 Putting sticky ends onto a blunt¿ended molecule 74
4.3.4 Blunt¿end ligation with a DNA topoisomerase 79
Further reading 81
5 Introduction of DNA into Living Cells 83
5.1 Transformation - the uptake of DNA by bacterial cells 85
5.1.1 Not all species of bacteria are equally efficient at DNA uptake 85
5.1.2 Preparation of competent E. coli cells 86
5.1.3 Selection for transformed cells 86
5.2 Identification of recombinants 88
5.2.1 Recombinant selection with pBR322 - insertional inactivation of an antibiotic resistance gene 89
5.2.2 Insertional inactivation does not always involve antibiotic resistance 90
5.3 Introduction of phage DNA into bacterial cells 92
5.3.1 Transfection 93
5.3.2 In vitro packaging of ¿ cloning vectors 93
5.3.3 Phage infection is visualized as plaques on an agar medium 93
5.3.4 Identification of recombinant phages 95
5.4 Introduction of DNA into non¿bacterial cells 97
5.4.1 Transformation of individual cells 97
5.4.2 Transformation of whole organisms 99
Further reading 99
6 Cloning Vectors for E. coli 101
6.1 Cloning vectors based on E. coli plasmids 102
6.1.1 The nomenclature of plasmid cloning vectors 102
6.1.2 The useful properties of pBR322 102
6.1.3 The pedigree of pBR322 103
6.1.4 More sophisticated E. coli plasmid cloning vectors 104
6.2 Cloning vectors based on ¿ bacteriophage 108
6.2.1 Natural selection was used to isolate modified ¿ that lack certain restriction sites 108
6.2.2 Segments of the ¿ genome can be deleted without impairing viability 108
6.2.3 Insertion and replacement vectors 110
6.2.4 Cloning experiments with ¿ insertion or replacement vectors 112
6.2.5 Long DNA fragments can be cloned using a cosmid 113
6.2.6 ¿ and other high¿capacity vectors enable genomic libraries to be constructed 114
6.3 Cloning vectors for synthesis of single¿stranded DNA 115
6.3.1 Vectors based on M13 bacteriophage 115
6.3.2 Hybrid plasmid-M13 vectors 117
6.4 Vectors for other bacteria 118
Further reading 119
7 Cloning Vectors for Eukaryotes 121
7.1 Vectors for yeast and other fungi 121
7.1.1 Selectable markers for the 2 ¿m plasmid 122
7.1.2 Vectors based on the 2 ¿m plasmid - yeast episomal plasmids 122
7.1.3 A YEp may insert into yeast chromosomal DNA 124
7.1.4 Other types of yeast cloning vector 124
7.1.5 Artificial chromosomes can be used to clone long pieces of DNA in yeast 126
7.1.6 Vectors for other yeasts and fungi 129
7.2 Cloning vectors for higher plants 129
7.2.1 Agrobacterium tumefaciens - nature's smallest genetic engineer 130
7.2.2 Cloning genes in plants by direct gene transfer 135
7.2.3 Attempts to use plant viruses as cloning vectors 137
7.3 Cloning vectors for animals 138
7.3.1 Cloning vectors for insects 139
7.3.2 Cloning in mammals 141
Further reading 143
8 How to Obtain a Clone of a Specific Gene 145
8.1 The problem of selection 146
8.1.1 There are two basic strategies for obtaining the clone you want 146
8.2 Direct selection 147
8.2.1 Marker rescue extends the scope of direct selection 149
8.2.2 The scope and limitations of marker rescue 150
8.3 Identification of a clone from a gene library 150
8.3.1 Gene libraries 151
8.4 Methods for clone identification 153
8.4.1 Complementary nucleic acid strands hybridize to each other 154
8.4.2 Colony and plaque hybridization probing 154
8.4.3 Examples of the practical use of hybridization probing 157
8.4.4 Identification methods based on detection of the translation product of the cloned gene 164
Further reading 166
9 The Polymerase Chain Reaction 169
9.1 PCR in outline 170
9.2 PCR in more detail 172
9.2.1 Designing the oligonucleotide primers for a PCR 172
9.2.2 Working out the correct temperatures to use 174
9.3 After the PCR: studying PCR products 176
9.3.1 Gel electrophoresis of PCR products 177
9.3.2 Cloning PCR products 178
9.4 Real¿time PCR 180
9.4.1 Carrying out a real¿time PCR experiment 180
9.4.2 Real¿time PCR enables the amount of starting material to be quantified 182
9.4.3 Melting curve analysis enables point mutations to be identified 184
Further reading 185
Part II The Applications of Gene Cloning and DNA Analysis in Research 187
10 Sequencing Genes and Genomes 189
10.1 Chain¿termination DNA sequencing 190
10.1.1 Chain¿termination sequencing in outline 190
10.1.2 Not all DNA polymerases can be used for sequencing 192
10.1.3 Chain¿termination sequencing with Taq polymerase 193
10.1.4 Limitations of chain¿termination sequencing 195
10.2 Next¿generation sequencing 196
10.2.1 Preparing a library for an Illumina sequencing experiment 197
10.2.2 The sequencing phase of an Illumina experiment 199
10.2.3 Ion semiconductor sequencing 201
10.2.4 Third¿generation sequencing 201
10.2.5 Next¿generation sequencing without a DNA polymerase 202
10.2.6 Directing next¿generation sequencing at specific sets of genes 203
...| Erscheinungsjahr: | 2020 |
|---|---|
| Fachbereich: | Biophysik |
| Genre: | Biologie |
| Rubrik: | Naturwissenschaften & Technik |
| Medium: | Taschenbuch |
| Inhalt: |
XIV
416 S. |
| ISBN-13: | 9781119640783 |
| ISBN-10: | 1119640784 |
| Sprache: | Englisch |
| Einband: | Kartoniert / Broschiert |
| Autor: | Brown, T. A. |
| Hersteller: | John Wiley and Sons Ltd |
| Maße: | 240 x 167 x 20 mm |
| Von/Mit: | T. A. Brown |
| Erscheinungsdatum: | 10.12.2020 |
| Gewicht: | 0,832 kg |
T. A. Brown is Emeritus Professor of Biomolecular Archaeology in the Manchester Institute of Biotechnology at the University of Manchester in the United Kingdom. He has published several books on genetics, genomics and biochemistry as well as over 150 research papers.
Preface to the Eighth Edition xv
Part I The Basic Principles of Gene Cloning and DNA Analysis 1
1 Why Gene Cloning and DNA Analysis are Important 3
2 Vectors for Gene Cloning: Plasmids and Bacteriophages 15
3 Purification of DNA from Living Cells 29
4 Manipulation of Purified DNA 53
5 Introduction of DNA into Living Cells 83
6 Cloning Vectors for E. coli 101
7 Cloning Vectors for Eukaryotes 121
8 How to Obtain a Clone of a Specific Gene 145
9 The Polymerase Chain Reaction 169
Part II The Applications of Gene Cloning and DNA Analysis in Research 187
10 Sequencing Genes and Genomes 189
11 Studying Gene Expression and Function 217
12 Studying Genomes 243
13 Studying Transcriptomes and Proteomes 259
Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology 275
14 Production of Protein from Cloned Genes 277
15 Gene Cloning and DNA Analysis in Medicine 301
16 Gene Cloning and DNA Analysis in Agriculture 327
17 Gene Cloning and DNA Analysis in Forensic Science and Archaeology 355
Glossary 377
Index 395
Preface to the Eighth Edition xv
Part I The Basic Principles of Gene Cloning and DNA Analysis 1
1 Why Gene Cloning and DNA Analysis are Important 3
1.1 The early development of genetics 4
1.2 The advent of gene cloning and the polymerase chain reaction 4
1.3 What is gene cloning? 5
1.4 What is PCR? 5
1.5 Why gene cloning and PCR are so important 8
1.5.1 Obtaining a pure sample of a gene by cloning 8
1.5.2 PCR can also be used to purify a gene 10
1.6 How to find your way through this book 11
Further reading 13
2 Vectors for Gene Cloning: Plasmids and Bacteriophages 15
2.1 Plasmids 15
2.1.1 Size and copy number 17
2.1.2 Conjugation and compatibility 18
2.1.3 Plasmid classification 19
2.1.4 Plasmids in organisms other than bacteria 19
2.2 Bacteriophages 19
2.2.1 The phage infection cycle 20
2.2.2 Lysogenic phages 20
2.2.3 Viruses as cloning vectors for other organisms 26
Further reading 27
3 Purification of DNA from Living Cells 29
3.1 Preparation of total cell DNA 30
3.1.1 Growing and harvesting a bacterial culture 30
3.1.2 Preparation of a cell extract 31
3.1.3 Purification of DNA from a cell extract 33
3.1.4 Concentration of DNA samples 37
3.1.5 Measurement of DNA concentration 38
3.1.6 Other methods for the preparation of total cell DNA 39
3.2 Preparation of plasmid DNA 40
3.2.1 Separation on the basis of size 41
3.2.2 Separation on the basis of conformation 42
3.2.3 Plasmid amplification 44
3.3 Preparation of bacteriophage DNA 46
3.3.1 Growth of cultures to obtain a high ¿ titre 47
3.3.2 Preparation of non¿lysogenic ¿ phages 47
3.3.3 Collection of phages from an infected culture 49
3.3.4 Purification of DNA from ¿ phage particles 49
3.3.5 Purification of M13 DNA causes few problems 49
Further reading 51
4 Manipulation of Purified DNA 53
4.1 The range of DNA manipulative enzymes 55
4.1.1 Nucleases 55
4.1.2 Ligases 57
4.1.3 Polymerases 57
4.1.4 DNA modifying enzymes 58
4.2 Enzymes for cutting DNA - restriction endonucleases 59
4.2.1 The discovery and function of restriction endonucleases 60
4.2.2 Type II restriction endonucleases cut DNA at specific nucleotide sequences 61
4.2.3 Blunt ends and sticky ends 62
4.2.4 The frequency of recognition sequences in a DNA molecule 63
4.2.5 Performing a restriction digest in the laboratory 64
4.2.6 Analysing the result of restriction endonuclease cleavage 66
4.2.7 Estimation of the sizes of DNA molecules 68
4.2.8 Mapping the positions of different restriction sites in a DNA molecule 69
4.2.9 Special gel electrophoresis methods for separating larger molecules 70
4.3 Ligation - joining DNA molecules together 72
4.3.1 The mode of action of DNA ligase 72
4.3.2 Sticky ends increase the efficiency of ligation 74
4.3.3 Putting sticky ends onto a blunt¿ended molecule 74
4.3.4 Blunt¿end ligation with a DNA topoisomerase 79
Further reading 81
5 Introduction of DNA into Living Cells 83
5.1 Transformation - the uptake of DNA by bacterial cells 85
5.1.1 Not all species of bacteria are equally efficient at DNA uptake 85
5.1.2 Preparation of competent E. coli cells 86
5.1.3 Selection for transformed cells 86
5.2 Identification of recombinants 88
5.2.1 Recombinant selection with pBR322 - insertional inactivation of an antibiotic resistance gene 89
5.2.2 Insertional inactivation does not always involve antibiotic resistance 90
5.3 Introduction of phage DNA into bacterial cells 92
5.3.1 Transfection 93
5.3.2 In vitro packaging of ¿ cloning vectors 93
5.3.3 Phage infection is visualized as plaques on an agar medium 93
5.3.4 Identification of recombinant phages 95
5.4 Introduction of DNA into non¿bacterial cells 97
5.4.1 Transformation of individual cells 97
5.4.2 Transformation of whole organisms 99
Further reading 99
6 Cloning Vectors for E. coli 101
6.1 Cloning vectors based on E. coli plasmids 102
6.1.1 The nomenclature of plasmid cloning vectors 102
6.1.2 The useful properties of pBR322 102
6.1.3 The pedigree of pBR322 103
6.1.4 More sophisticated E. coli plasmid cloning vectors 104
6.2 Cloning vectors based on ¿ bacteriophage 108
6.2.1 Natural selection was used to isolate modified ¿ that lack certain restriction sites 108
6.2.2 Segments of the ¿ genome can be deleted without impairing viability 108
6.2.3 Insertion and replacement vectors 110
6.2.4 Cloning experiments with ¿ insertion or replacement vectors 112
6.2.5 Long DNA fragments can be cloned using a cosmid 113
6.2.6 ¿ and other high¿capacity vectors enable genomic libraries to be constructed 114
6.3 Cloning vectors for synthesis of single¿stranded DNA 115
6.3.1 Vectors based on M13 bacteriophage 115
6.3.2 Hybrid plasmid-M13 vectors 117
6.4 Vectors for other bacteria 118
Further reading 119
7 Cloning Vectors for Eukaryotes 121
7.1 Vectors for yeast and other fungi 121
7.1.1 Selectable markers for the 2 ¿m plasmid 122
7.1.2 Vectors based on the 2 ¿m plasmid - yeast episomal plasmids 122
7.1.3 A YEp may insert into yeast chromosomal DNA 124
7.1.4 Other types of yeast cloning vector 124
7.1.5 Artificial chromosomes can be used to clone long pieces of DNA in yeast 126
7.1.6 Vectors for other yeasts and fungi 129
7.2 Cloning vectors for higher plants 129
7.2.1 Agrobacterium tumefaciens - nature's smallest genetic engineer 130
7.2.2 Cloning genes in plants by direct gene transfer 135
7.2.3 Attempts to use plant viruses as cloning vectors 137
7.3 Cloning vectors for animals 138
7.3.1 Cloning vectors for insects 139
7.3.2 Cloning in mammals 141
Further reading 143
8 How to Obtain a Clone of a Specific Gene 145
8.1 The problem of selection 146
8.1.1 There are two basic strategies for obtaining the clone you want 146
8.2 Direct selection 147
8.2.1 Marker rescue extends the scope of direct selection 149
8.2.2 The scope and limitations of marker rescue 150
8.3 Identification of a clone from a gene library 150
8.3.1 Gene libraries 151
8.4 Methods for clone identification 153
8.4.1 Complementary nucleic acid strands hybridize to each other 154
8.4.2 Colony and plaque hybridization probing 154
8.4.3 Examples of the practical use of hybridization probing 157
8.4.4 Identification methods based on detection of the translation product of the cloned gene 164
Further reading 166
9 The Polymerase Chain Reaction 169
9.1 PCR in outline 170
9.2 PCR in more detail 172
9.2.1 Designing the oligonucleotide primers for a PCR 172
9.2.2 Working out the correct temperatures to use 174
9.3 After the PCR: studying PCR products 176
9.3.1 Gel electrophoresis of PCR products 177
9.3.2 Cloning PCR products 178
9.4 Real¿time PCR 180
9.4.1 Carrying out a real¿time PCR experiment 180
9.4.2 Real¿time PCR enables the amount of starting material to be quantified 182
9.4.3 Melting curve analysis enables point mutations to be identified 184
Further reading 185
Part II The Applications of Gene Cloning and DNA Analysis in Research 187
10 Sequencing Genes and Genomes 189
10.1 Chain¿termination DNA sequencing 190
10.1.1 Chain¿termination sequencing in outline 190
10.1.2 Not all DNA polymerases can be used for sequencing 192
10.1.3 Chain¿termination sequencing with Taq polymerase 193
10.1.4 Limitations of chain¿termination sequencing 195
10.2 Next¿generation sequencing 196
10.2.1 Preparing a library for an Illumina sequencing experiment 197
10.2.2 The sequencing phase of an Illumina experiment 199
10.2.3 Ion semiconductor sequencing 201
10.2.4 Third¿generation sequencing 201
10.2.5 Next¿generation sequencing without a DNA polymerase 202
10.2.6 Directing next¿generation sequencing at specific sets of genes 203
...| Erscheinungsjahr: | 2020 |
|---|---|
| Fachbereich: | Biophysik |
| Genre: | Biologie |
| Rubrik: | Naturwissenschaften & Technik |
| Medium: | Taschenbuch |
| Inhalt: |
XIV
416 S. |
| ISBN-13: | 9781119640783 |
| ISBN-10: | 1119640784 |
| Sprache: | Englisch |
| Einband: | Kartoniert / Broschiert |
| Autor: | Brown, T. A. |
| Hersteller: | John Wiley and Sons Ltd |
| Maße: | 240 x 167 x 20 mm |
| Von/Mit: | T. A. Brown |
| Erscheinungsdatum: | 10.12.2020 |
| Gewicht: | 0,832 kg |
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