Gene cloning and DNA analysis: an introduction
Saved in:
| Main Author: | |
|---|---|
| Format: | Book |
| Language: | English |
| Published: |
Oxford [u.a.]
Wiley-Blackwell
2010
|
| Edition: | 6. ed. |
| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | XVI, 320 S. Ill., graph. Darst. |
| ISBN: | 9781405181730 9781444334074 |
Staff View
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| 250 | |a 6. ed. | ||
| 264 | 1 | |a Oxford [u.a.] |b Wiley-Blackwell |c 2010 | |
| 300 | |a XVI, 320 S. |b Ill., graph. Darst. | ||
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| 650 | 4 | |a Molecular cloning | |
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| 650 | 4 | |a Cloning, Molecular | |
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| 650 | 4 | |a Sequence Analysis, DNA | |
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Record in the Search Index
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| adam_text | Titel: Gene cloning and DNA analysis
Autor: Brown, Terence A.
Jahr: 2010
Brief Contents
Preface to the Sixth Edition xvi
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 13
3 Purification of DNA from Living Cells 25
4 Manipulation of Purified DNA 45
5 Introduction of DNA into Living Cells 72
6 Cloning Vectors for E. coli 88
7 Cloning Vectors for Eukaryotes 105
8 How to Obtain a Clone of a Specific Gene 126
9 The Polymerase Chain Reaction 147
Part II The Applications of Gene Cloning and
DNA Analysis in Research 163
10 Sequencing Genes and Genomes 165
11 Studying Gene Expression and Function 185
12 Studying Genomes 207
Part III The Applications of Gene Cloning
and DNA Analysis in Biotechnology 223
13 Production of Protein from Cloned Genes 225
14 Gene Cloning and DNA Analysis in Medicine 245
15 Gene Cloning and DNA Analysis in Agriculture 264
16 Gene Cloning and DNA Analysis in Forensic Science and Archaeology 282
Glossary 298
Index 312
Companion website available at www.wiley.com/go/brown/cloning
Contents
Preface to the Sixth Edition xvi
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 3
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? 6
1.5 Why gene cloning and PCR are so important 7
1.5.1 Obtaining a pure sample of a gene by cloning 7
1.5.2 PCR can also be used to purify a gene 9
1.6 How to find your way through this book 11
2 Vectors for Gene Cloning: Plasmids and
Bacteriophages 13
2.1 Plasmids 13
2.1.1 Size and copy number 15
2.1.2 Conjugation and compatibility 16
2.1.3 Plasmid classification 16
2.1.4 Plasmids in organisms other than bacteria 17
2.2 Bacteriophages 17
2.2.1 The phage infection cycle 18
2.2.2 Lysogenic phages 19
Gene organization in the A DNA molecule 19
The linear and circular forms of A DNA 19
M13?a filamentous phage 22
2.2.3 Viruses as cloning vectors for other organisms 24
vii
Contents
3 Purification of DNA from Living Cells 25
3.1 Preparation of total cell DNA 25
3.1.1 Growing and harvesting a bacterial culture 26
3.1.2 Preparation of a cell extract 28
3.1.3 Purification of DNA from a cell extract 29
Removing contaminants by organic extraction and enzyme
digestion 29
Using ion-exchange chromatography to purify DNA from a cell
extract 30
3.1.4 Concentration of DNA samples 30
3.1.5 Measurement of DNA concentration 31
3.1.6 Other methods for the preparation of total celt DNA 32
3.2 Preparation of plasmid DNA 33
3.2.1 Separation on the basis of size 35
3.2.2 Separation on the basis of conformation 36
Alkaline denaturation 36
Ethidium bromide-caesium chloride density gradient
centrifugation 36
3.2.3 Plasmid amplification 39
3.3 Preparation of bacteriophage DNA 39
3.3.1 Growth of cultures to obtain a high A titer 40
3.3.2 Preparation of non-lysogenic A phages 40
3.3.3 Collection of phages from an infected culture 42
3.3.4 Purification of DNA from A phage particles 42
3.3.5 Purification of M13 DNA causes few problems 43
A Manipulation of Purified DNA 45
4.1 The range of DNA manipulative enzymes 46
4.1.1 Nucleases 46
4.1.2 Ligases 47
4.1.3 Polymerases 48
4.1.4 DNA modifying enzymes 49
4.2 Enzymes for cutting DNA?restriction endonucleases 50
4.2.1 The discovery and function of restriction endonucleases 51
4.2.2 Type II restriction endonucleases cut DNA at specific nucleotide
sequences 52
4.2.3 Blunt ends and sticky ends 53
4.2.4 The frequency of recognition sequences in a DNA molecule 53
4.2.5 Performing a restriction digest in the laboratory 54
4.2.6 Analysing the result of restriction endonuclease cleavage 56
Separation of molecules by gel electrophoresis 57
Visualizing DNA molecules in an agarose gel 58
4.2.7 Estimation of the sizes of DNA molecules 58
4.2.8 Mapping the positions of different restriction sites in a DNA
molecule 59
4.2.9 Special gel electrophoresis methods for separating larger
molecules 60
Contents
4.3 Ligation?joining DNA molecules together 63
4.3.1 The mode of action of DNA ligase 63
4.3.2 Sticky ends increase the efficiency of ligation 64
4.3.3 Putting sticky ends onto a blunt-ended molecule 64
Linkers 64
Adaptors 65
Producing sticky ends by homopolymer tailing 67
4.3.4 Blunt end ligation with a DNA topoisomerase 69
5 Introduction of DNA into Living Cells 72
5.1 Transformation?the uptake of DNA by bacterial cells 74
5.1.1 Not all species of bacteria are equally efficient at DNA
uptake 74
5.1.2 Preparation of competent E. coli cells 75
5.1.3 Selection for transformed cells 75
5.2 Identification of recombinants 76
5.2.1 Recombinant selection with pBR322?insertional inactivation
of an antibiotic resistance gene 77
5.2.2 Insertional inactivation does not always involve antibiotic
resistance 79
5.3 Introduction of phage DNA into bacterial cells 81
5.3.1 Transfection 81
5.3.2 In vitro packaging of A cloning vectors 81
5.3.3 Phage infection is visualized as plaques on an agar
medium 81
5.4 Identification of recombinant phages 83
5.4.1 Insertional inactivation of a lacZ gene carried by the phage
vector 83
5.4.2 Insertional inactivation of the A cl gene 83
5.4.3 Selection using the Spi phenotype 83
5.4.4 Selection on the basis of A genome size 84
5.5 Introduction of DNA into non-bacterial cells 85
5.5.1 Transformation of individual celts 85
5.5.2 Transformation of whole organisms 85
6 Cloning Vectors for E, coli 88
6.1 Cloning vectors based on £. coli plasmids 89
6.1.1 The nomenclature of plasmid cloning vectors 89
6.1.2 The useful properties of pBR322 89
6.1.3 The pedigree of pBR322 90
6.1.4 More sophisticated E. coli plasmid cloning vectors 90
pUC8?a Lac selection plasmid 92
pGEM3Z?in vitro transcription of cloned DNA 93
6.2 Cloning vectors based on M13 bacteriophage 94
6.2.1 How to construct a phage cloning vector 94
6.2.2 Hybrid plasmid-M 13 vectors 96
Contents
6.3 Cloning vectors based on A bacteriophage 97
6.3.1 Segments of the A genome can be deleted without impairing
viability 98
6.3.2 Natural selection can be used to isolate modified A that lack
certain restriction sites 98
6.3.3 Insertion and replacement vectors 98
Insertion vectors 99
Replacement vectors 100
6.3.4 Cloning experiments with A insertion or replacement
vectors 100
6.3.5 Long DNA fragments can be cloned using a cosmid 101
6.4 A and other high-capacity vectors enable genomic libraries to be
constructed 102
6.5 Vectors for other bacteria 104
7 Cloning Vectors for Eukaryotes 105
7.1 Vectors for yeast and other fungi 105
7.1.1 Selectable markers for the 2 pm plasmid 106
7.1.2 Vectors based on the 2 pm plasmid?yeast episomal
plasmids 106
7.1.3 A YEp may insert into yeast chromosomal DNA 107
7.1.4 Other types of yeast cloning vector 108
7.1.5 Artificial chromosomes can be used to clone long pieces of
DNA in yeast 110
The structure and use of a YAC vector 110
Applications for YAC vectors 111
7.1.6 Vectors for other yeasts and fungi 112
7.2 Cloning vectors for higher plants 112
7.2.1 Agrobacterium tumefaciens?nature s smallest genetic
engineer 113
Using the Ti plasmid to introduce new genes into a plant
cell 113
Production of transformed plants with the Ti plasmid 115
The Ri plasmid 117
Limitations of cloning with Agrobacterium plasmids 117
7.2.2 Cloning genes in plants by direct gene transfer 118
Direct gene transfer into the nucleus 118
Transfer of genes into the chloroplast genome 119
7.2.3 Attempts to use plant viruses as cloning vectors 119
Caulimovirus vectors 120
Geminivirus vectors 120
7.3 Cloning vectors for animals 120
7.3.1 Cloning vectors for insects 121
P elements as cloning vectors for Drosophila 111
Cloning vectors based on insect viruses 122
7.3.2 Cloning in mammals 122
Viruses as cloning vectors for mammals 123
Gene cloning without a vector 124
Contents
8 How to Obtain a Clone of a Specific Gene 126
8.1 The problem of selection 126
8.1.1 There are two basic strategies for obtaining the clone you
want 127
8.2 Direct selection 128
8.2.1 Marker rescue extends the scope of direct selection 129
8.2.2 The scope and limitations of marker rescue 130
8.3 Identification of a clone from a gene library 131
8.3.1 Gene libraries 131
8.3.2 Not all genes are expressed at the same time 131
8.3.3 mRNA can be cloned as complementary DNA 133
8.4 Methods for clone identification 133
8.4.1 Complementary nucleic acid strands hybridize to each
other 133
8.4.2 Colony and plaque hybridization probing 133
Labeling with a radioactive marker 136
Non-radioactive labeling 137
8.4.3 Examples of the practical use of hybridization probing 137
Abundancy probing to analyse a cDNA library 137
Oligonucleotide probes for genes whose translation products
have been characterized 138
Heterologous probing allows related genes to be
identified 141
Southern hybridization enables a specific restriction fragment
containing a gene to be identified 142
8.4.4 Identification methods based on detection of the translation
product of the cloned gene 144
Antibodies are required for immunological detection
methods 144
Using a purified antibody to detect protein in recombinant
colonies 145
The problem of gene expression 146
9 The Polymerase Chain Reaction 147
9.1 The polymerase chain reaction in outline 147
9.2 PCR in more detail 149
9.2.1 Designing the oligonucleotide primers for a PCR 149
9.2.2 Working out the correct temperatures to use 152
9.3 After the PCR: studying PCR products 153
9.3.1 Gel electrophoresis of PCR products 154
9.3.2 Cloning PCR products 154
9.3.3 Problems with the error rate of Taq polymerase 157
9.4 Real-time PCR enables the amount of starting material to be
quantified 158
9.4.1 Carrying out a quantitative PCR experiment 159
9.4.2 Real-time PCR can also quantify RNA 160
xjj ¦ Contents
Part II The Applications of Gene Cloning
and DNA Analysis in Research i63
10 Sequencing Genes and Genomes 165
10.1 The methodology for DNA sequencing 165
10.1.1 Chain termination DNA sequencing 166
Chain termination sequencing in outline 166
Not all DNA polymerases can be used for sequencing 168
Chain termination sequencing requires a single-stranded DNA
template 169
The primer determines the region of the template DNA that will
be sequenced 169
10.1.2 Pyrosequencing 171
Pyrosequencing involves detection of pulses of
chemiluminescence 171
Massively parallel pyrosequencing 171
10.2 How to sequence a genome 173
10.2.1 The shotgun approach to genome sequencing 174
The Haemophilus influenzae genome sequencing project 174
Problems with shotgun sequencing 176
10.2.2 The clone contig approach 177
Clone contig assembly by chromosome walking 177
Rapid methods for clone contig assembly 178
Clone contig assembly by sequence tagged site content
analysis 179
10.2.3 Using a map to aid sequence assembly 180
Genetic maps 180
Physical maps 181
The importance of a map in sequence assembly 183
11 Studying Gene Expression and Function i85
11.1 Studying the RNA transcript of a gene 186
11.1.1 Detecting the presence of a transcript and determining its
nucleotide sequence 186
11.1.2 Transcript mapping by hybridization between gene and
RNA 188
11.1.3 Transcript analysis by primer extension 190
11.1.4 Transcript analysis by PCR 191
11.2 Studying the regulation of gene expression 192
11.2.1 Identifying protein binding sites on a DNA molecule 193
Gel retardation of DNA-protein complexes 193
Footprinting with DNase I 194
Modification interference assays 194
11.2.2 Identifying control sequences by deletion analysis 197
Reporter genes 197
Carrying out a deletion analysis 198
Contents ? xffi
11.3 Identifying and studying the translation product of a cloned gene 199
11.3.1 HRTand HART can identify the translation product of a cloned
gene 199
11.3.2 Analysis of proteins by in vitro mutagenesis 200
Different types of in vitro mutagenesis techniques 202
Using an oligonucleotide to create a point mutation in a
cloned gene 203
Other methods of creating a point mutation in a cloned
gene 204
The potential of in vitro mutagenesis 205
12 Studying Genomes 207
12.1 Genome annotation 207
12.1.1 Identifying the genes in a genome sequence 208
Searching for open reading frames 208
Simple ORF scans are less effective at locating genes in
eukaryotic genomes 209
Gene location is aided by homology searching 210
Comparing the sequences of related genomes 211
12.1.2 Determining the function of an unknown gene 212
Assigning gene function by experimental analysis requires a
reverse approach to genetics 212
Specific genes can be inactivated by homologous
recombination 213
12.2 Studies of the transcriptome and proteome 214
12.2.1 Studying the transcriptome 215
Studying a transcriptome by sequence analysis 215
Studying transcriptomes by microarray or chip analysis 215
12.2.2 Studying the proteome 217
Separating the proteins in a proteome 217
Identifying the individual proteins after separation 218
12.2.3 Studying protein-protein interactions 220
Phage display 220
The yeast two hybrid system 220
Part III The Applications of Gene Cloning and
DNA Analysis in Biotechnology 223
13 Production of Protein from Cloned Genes 225
13.1 Special vectors for expression of foreign genes in E. coli 227
13.1.1 The promoter is the critical component of an expression
vector 228
The promoter must be chosen with care 228
Examples of promoters used in expression vectors 231
13.1.2 Cassettes and gene fusions 232
xjy Contents
13.2 General problems with the production of recombinant protein in
E. coil 234
13.2.1 Problems resulting from the sequence of the foreign gene 235
13.2.2 Problems caused by E. coli 236
13.3 Production of recombinant protein by eukaryotic cells 237
13.3.1 Recombinant protein from yeast and filamentous fungi 237
Saccharomyces cerevisiae as the host for recombinant protein
synthesis 237
Other yeasts and fungi 238
13.3.2 Using animal cells for recombinant protein production 239
Protein production in mammalian cells 239
Protein production in insect cells 240
13.3.3 Pharming?recombinant protein from live animals and
plants 241
Pharming in animals 241
Recombinant proteins from plants 242
Ethical concerns raised by pharming 243
14 Gene Cloning and DNA Analysis in Medicine 245
14.1 Production of recombinant pharmaceuticals 245
14.1.1 Recombinant insulin 246
Synthesis and expression of artificial insulin genes 247
14.1.2 Synthesis of human growth hormones in E. coli 247
14.1.3 Recombinant factor VIII 249
14.1.4 Synthesis of other recombinant human proteins 251
14.1.5 Recombinant vaccines 252
Producing vaccines as recombinant proteins 252
Recombinant vaccines in transgenic plants 253
Live recombinant virus vaccines 253
14.2 Identification of genes responsible for human diseases 255
14.2.1 How to identify a gene for a genetic disease 256
Locating the approximate position of the gene in the human
genome 256
Identification of candidates for the disease gene 258
14.3 Gene therapy 259
14.3.1 Gene therapy for inherited diseases 259
14.3.2 Gene therapy and cancer 260
14.3.3 The ethical issues raised by gene therapy 262
15 Gene Cloning and DNA Analysis in Agriculture 264
15.1 The gene addition approach to plant genetic engineering 265
15.1.1 Plants that make their own insecticides 265
The 5-endotoxins of Bacillus thuringiensis 265
Cloning a 5-endotoxin gene in maize 266
Cloning 6-endotoxin genes in chloroplasts 268
Countering insect resistance to 6-endotoxin crops 269
Contents XV
15.1.2 Herbicide resistant crops 270
Roundup Ready crops 271
A new generation of glyphosate resistant crops 272
15.1.3 Other gene addition projects 273
15.2 Gene subtraction 274
15.2.1 Antisense RNA and the engineering of fruit ripening in
tomato 274
Using antisense RNA to inactivate the polygalacturonase
gene 274
Using antisense RNA to inactivate ethylene synthesis 276
15.2.2 Other examples of the use of antisense RNA in plant genetic
engineering 276
15.3 Problems with genetically modified plants 277
15.3.1 Safety concerns with selectable markers 277
15.3.2 The terminator technology 278
15.3.3 The possibility of harmful effects on the environment 279
16 Gene Cloning and DNA Analysis in Forensic Science
and Archaeology 282
16.1 DNA analysis in the identification of crime suspects 283
16.1.1 Genetic fingerprinting by hybridization probing 283
16.1.2 DNA profiling by PCR of short tandem repeats 283
16.2 Studying kinship by DNA profiling 286
16.2.1 Related individuals have similar DNA profiles 286
16.2.2 DNA profiling and the remains of the Romanovs 286
STR analysis of the Romanov bones 286
Mitochondrial DNA was used to link the Romanov skeletons with
living relatives 287
The missing children 289
16.3 Sex identification by DNA analysis 289
16.3.1 PCRs directed at Y chromosome-specific sequences 289
16.3.2 PCRoftheamelogeningene 290
16.6 Archaeogenetics?using DNA to study human prehistory 291
16.4.1 The origins of modern humans 291
DNA analysis has challenged the multiregional hypothesis 291
DNA analysis shows that Neanderthals are not the ancestors
of modern Europeans 293
16.4.2 DNA can also be used to study prehistoric human
migrations 294
The spread of agriculture into Europe 294
Using mitochondrial DNA to study past human migrations into
Europe 294
Glossary 298
Index 311
Companion website available at www.wiley.com/go/brown/cloning
|
| any_adam_object | 1 |
| author | Brown, Terence A. 1953- |
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| dewey-tens | 570 - Biology |
| discipline | Biologie Chemie |
| edition | 6. ed. |
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| genre | 1\p (DE-588)4151278-9 Einführung gnd-content 2\p (DE-588)4123623-3 Lehrbuch gnd-content |
| genre_facet | Einführung Lehrbuch |
| id | DE-604.BV036425286 |
| illustrated | Illustrated |
| indexdate | 2024-07-09T22:37:48Z |
| institution | BVB |
| isbn | 9781405181730 9781444334074 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-020222052 |
| oclc_num | 437306611 |
| open_access_boolean | |
| owner | DE-355 DE-BY-UBR DE-20 DE-11 DE-M49 DE-BY-TUM DE-703 DE-19 DE-BY-UBM DE-188 |
| owner_facet | DE-355 DE-BY-UBR DE-20 DE-11 DE-M49 DE-BY-TUM DE-703 DE-19 DE-BY-UBM DE-188 |
| physical | XVI, 320 S. Ill., graph. Darst. |
| publishDate | 2010 |
| publishDateSearch | 2010 |
| publishDateSort | 2010 |
| publisher | Wiley-Blackwell |
| record_format | marc |
| spelling | Brown, Terence A. 1953- Verfasser (DE-588)113251661 aut Gene cloning and DNA analysis an introduction T. A. Brown 6. ed. Oxford [u.a.] Wiley-Blackwell 2010 XVI, 320 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier genes cabt DNA cloning cabt DNA sequencing cabt nucleotide sequences cabt Molecular cloning Nucleotide sequence DNA Analysis Cloning, Molecular DNA, Recombinant analysis Sequence Analysis, DNA Gentechnologie (DE-588)4071722-7 gnd rswk-swf Klonierung (DE-588)4192480-0 gnd rswk-swf Sequenzanalyse Chemie (DE-588)4132277-0 gnd rswk-swf DNS (DE-588)4070512-2 gnd rswk-swf Genklonierung (DE-588)4123275-6 gnd rswk-swf 1\p (DE-588)4151278-9 Einführung gnd-content 2\p (DE-588)4123623-3 Lehrbuch gnd-content Genklonierung (DE-588)4123275-6 s DE-604 DNS (DE-588)4070512-2 s Sequenzanalyse Chemie (DE-588)4132277-0 s Gentechnologie (DE-588)4071722-7 s 3\p DE-604 Klonierung (DE-588)4192480-0 s 4\p DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020222052&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 3\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 4\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Brown, Terence A. 1953- Gene cloning and DNA analysis an introduction genes cabt DNA cloning cabt DNA sequencing cabt nucleotide sequences cabt Molecular cloning Nucleotide sequence DNA Analysis Cloning, Molecular DNA, Recombinant analysis Sequence Analysis, DNA Gentechnologie (DE-588)4071722-7 gnd Klonierung (DE-588)4192480-0 gnd Sequenzanalyse Chemie (DE-588)4132277-0 gnd DNS (DE-588)4070512-2 gnd Genklonierung (DE-588)4123275-6 gnd |
| subject_GND | (DE-588)4071722-7 (DE-588)4192480-0 (DE-588)4132277-0 (DE-588)4070512-2 (DE-588)4123275-6 (DE-588)4151278-9 (DE-588)4123623-3 |
| title | Gene cloning and DNA analysis an introduction |
| title_auth | Gene cloning and DNA analysis an introduction |
| title_exact_search | Gene cloning and DNA analysis an introduction |
| title_full | Gene cloning and DNA analysis an introduction T. A. Brown |
| title_fullStr | Gene cloning and DNA analysis an introduction T. A. Brown |
| title_full_unstemmed | Gene cloning and DNA analysis an introduction T. A. Brown |
| title_short | Gene cloning and DNA analysis |
| title_sort | gene cloning and dna analysis an introduction |
| title_sub | an introduction |
| topic | genes cabt DNA cloning cabt DNA sequencing cabt nucleotide sequences cabt Molecular cloning Nucleotide sequence DNA Analysis Cloning, Molecular DNA, Recombinant analysis Sequence Analysis, DNA Gentechnologie (DE-588)4071722-7 gnd Klonierung (DE-588)4192480-0 gnd Sequenzanalyse Chemie (DE-588)4132277-0 gnd DNS (DE-588)4070512-2 gnd Genklonierung (DE-588)4123275-6 gnd |
| topic_facet | genes DNA cloning DNA sequencing nucleotide sequences Molecular cloning Nucleotide sequence DNA Analysis Cloning, Molecular DNA, Recombinant analysis Sequence Analysis, DNA Gentechnologie Klonierung Sequenzanalyse Chemie DNS Genklonierung Einführung Lehrbuch |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=020222052&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT brownterencea genecloninganddnaanalysisanintroduction |