TY - BOOK AU - Brown, T.A. TI - Gene cloning and DNA analysis SN - 9781405181730 U1 - 572.8633 BRO-G PY - 2010/// CY - U.K. PB - Wiley Blackwell KW - Science / Life Sciences / Biology, Science / Life Sciences / Cell Biology, Science / Life Sciences / Microbiology, Science / Life Sciences / Genetics & Genomics, Science / Life Sciences / Molecular Biology N1 - 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 UR - https://www.google.co.in/books/edition/Gene_Cloning_and_DNA_Analysis/yEvt3JdtgTQC?hl=en&gbpv=1&dq=Gene+cloning+and+DNA+analysis+by+brown+T+A&printsec=frontcover ER -