Gene cloning and DNA analysis

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 λ titre 47

3.3.2 Preparation of non‐lysogenic λ phages 47

3.3.3 Collection of phages from an infected culture 49

3.3.4 Purification of DNA from λ 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 λ 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 λ bacteriophage 108

6.2.1 Natural selection was used to isolate modified λ that lack certain restriction sites 108

6.2.2 Segments of the λ genome can be deleted without impairing viability 108

6.2.3 Insertion and replacement vectors 110

6.2.4 Cloning experiments with λ insertion or replacement vectors 112

6.2.5 Long DNA fragments can be cloned using a cosmid 113

6.2.6 λ 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 μm plasmid 122

7.1.2 Vectors based on the 2 μm 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

10.3 How to sequence a genome 205

10.3.1 Shotgun sequencing of prokaryotic genomes 206

10.3.2 Sequencing of eukaryotic genomes 209

Further reading 215

11 Studying Gene Expression and Function 217

11.1 Studying the RNA transcript of a gene 218

11.1.1 Detecting the presence of a transcript in an RNA sample 219

11.1.2 Transcript mapping by hybridization between gene and RNA 220

11.1.3 Transcript analysis by primer extension 222

11.1.4 Transcript analysis by PCR 223

11.2 Studying the regulation of gene expression 224

11.2.1 Identifying protein binding sites on a DNA molecule 225

11.2.2 Identifying control sequences by deletion analysis 230

11.3 Identifying and studying the translation product of a cloned gene 232

11.3.1 HRT and HART can identify the translation product of a cloned gene 233

11.3.2 Analysis of proteins by in vitro mutagenesis 234

Further reading 240

12 Studying Genomes 243

12.1 Locating the genes in a genome sequence 244

12.1.1 Locating protein‐coding genes by scanning a genome sequence 244

12.1.2 Gene location is aided by homology searching 247

12.1.3 Locating genes for noncoding RNA transcripts 249

12.1.4 Identifying the binding sites for regulatory proteins in a genome sequence 250

12.2 Determining the function of an unknown gene 251

12.2.1 Assigning gene functions by computer analysis 251

12.2.2 Assigning gene function by experimental analysis 252

12.3 Genome browsers 256

Further reading 257

13 Studying Transcriptomes and Proteomes 259

13.1 Studying transcriptomes 259

13.1.1 Studying transcriptomes by microarray or chip analysis 260

13.1.2 Studying transcriptomes by RNA sequencing 261

13.2 Studying proteomes 265

13.2.1 Protein profiling 266

13.2.2 Studying protein–protein interactions 270

Further reading 274

Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology 275

14 Production of Protein from Cloned Genes 277

14.1 Special vectors for expression of foreign genes in E. coli 280

14.1.1 The promoter is the critical component of an expression vector 281

14.1.2 Cassettes and gene fusions 285

14.2 General problems with the production of recombinant protein in E. coli 287

14.2.1 Problems resulting from the sequence of the foreign gene 288

14.2.2 Problems caused by E. coli 289

14.3 Production of recombinant protein by eukaryotic cells 290

14.3.1 Recombinant protein from yeast and filamentous fungi 291

14.3.2 Using animal cells for recombinant protein production 293

14.3.3 Pharming – recombinant protein from live animals and plants 295

Further reading 298

15 Gene Cloning and DNA Analysis in Medicine 301

15.1 Production of recombinant pharmaceuticals 301

15.1.1 Recombinant insulin 302

15.1.2 Synthesis of human growth hormones in E. coli 304

15.1.3 Recombinant factor VIII 305

15.1.4 Synthesis of other recombinant human proteins 308

15.1.5 Recombinant vaccines 308

15.2 Identification of genes responsible for human diseases 314

15.2.1 How to identify a gene for a genetic disease 315

15.2.2 Genetic typing of disease mutations 320

15.3 Gene therapy 321

15.3.1 Gene therapy for inherited diseases 321

15.3.2 Gene therapy and cancer 323

15.3.3 The ethical issues raised by gene therapy 324

Further reading 325

16 Gene Cloning and DNA Analysis in Agriculture 327

16.1 The gene addition approach to plant genetic engineering 328

16.1.1 Plants that make their own insecticides 328

16.1.2 Herbicide‐resistant crops 334

16.1.3 Improving the nutritional quality of plants by gene addition 337

16.1.4 Other gene addition projects 338

16.2 Gene subtraction 339

16.2.1 Antisense RNA and the engineering of fruit ripening in tomato 340

16.2.2 Other examples of the use of antisense RNA in plant genetic engineering 342

16.3 Gene editing with a programmable nuclease 344

16.3.1 Gene editing of phytoene desaturase in rice 344

16.3.2 Editing of multiple genes in a single plant 346

16.3.3 Future developments in gene editing of plants 347

16.4 Are GM plants harmful to human health and the environment? 349

16.4.1 Safety concerns with selectable markers 349

16.4.2 The possibility of harmful effects on the environment 350

Further reading 351

17 Gene Cloning and DNA Analysis in Forensic Science and Archaeology 355

17.1 DNA analysis in the identification of crime suspects 356

17.1.1 Genetic fingerprinting by hybridization probing 356

17.1.2 DNA profiling by PCR of short tandem repeats 357

17.2 Studying kinship by DNA profiling 359

17.2.1 Related individuals have similar DNA profiles 359

17.2.2 DNA profiling and the remains of the Romanovs 360

17.3 Sex identification by DNA analysis 363

17.3.1 PCRs directed at Y chromosome‐specific sequences 363

17.3.2 PCR of the amelogenin gene 364

17.4 Archaeogenetics – using DNA to study human prehistory 365

17.4.1 The origins of modern humans 365

17.4.2 DNA can also be used to study prehistoric human migrations 370