ISBN-13: 9781119640783 / Angielski / Miękka / 2020 / 432 str.
ISBN-13: 9781119640783 / Angielski / Miękka / 2020 / 432 str.
Preface to the Eighth Edition xvPart I The Basic Principles of Gene Cloning and DNA Analysis 11 Why Gene Cloning and DNA Analysis are Important 32 Vectors for Gene Cloning: Plasmids and Bacteriophages 153 Purification of DNA from Living Cells 294 Manipulation of Purified DNA 535 Introduction of DNA into Living Cells 836 Cloning Vectors for E. coli 1017 Cloning Vectors for Eukaryotes 1218 How to Obtain a Clone of a Specific Gene 1459 The Polymerase Chain Reaction 169Part II The Applications of Gene Cloning and DNA Analysis in Research 18710 Sequencing Genes and Genomes 18911 Studying Gene Expression and Function 21712 Studying Genomes 24313 Studying Transcriptomes and Proteomes 259Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology 27514 Production of Protein from Cloned Genes 27715 Gene Cloning and DNA Analysis in Medicine 30116 Gene Cloning and DNA Analysis in Agriculture 32717 Gene Cloning and DNA Analysis in Forensic Science and Archaeology 355Glossary 377Index 395Preface to the Eighth Edition xvPart I The Basic Principles of Gene Cloning and DNA Analysis 11 Why Gene Cloning and DNA Analysis are Important 31.1 The early development of genetics 41.2 The advent of gene cloning and the polymerase chain reaction 41.3 What is gene cloning? 51.4 What is PCR? 51.5 Why gene cloning and PCR are so important 81.5.1 Obtaining a pure sample of a gene by cloning 81.5.2 PCR can also be used to purify a gene 101.6 How to find your way through this book 11Further reading 132 Vectors for Gene Cloning: Plasmids and Bacteriophages 152.1 Plasmids 152.1.1 Size and copy number 172.1.2 Conjugation and compatibility 182.1.3 Plasmid classification 192.1.4 Plasmids in organisms other than bacteria 192.2 Bacteriophages 192.2.1 The phage infection cycle 202.2.2 Lysogenic phages 202.2.3 Viruses as cloning vectors for other organisms 26Further reading 273 Purification of DNA from Living Cells 293.1 Preparation of total cell DNA 303.1.1 Growing and harvesting a bacterial culture 303.1.2 Preparation of a cell extract 313.1.3 Purification of DNA from a cell extract 333.1.4 Concentration of DNA samples 373.1.5 Measurement of DNA concentration 383.1.6 Other methods for the preparation of total cell DNA 393.2 Preparation of plasmid DNA 403.2.1 Separation on the basis of size 413.2.2 Separation on the basis of conformation 423.2.3 Plasmid amplification 443.3 Preparation of bacteriophage DNA 463.3.1 Growth of cultures to obtain a high lambda titre 473.3.2 Preparation of non-lysogenic lambda phages 473.3.3 Collection of phages from an infected culture 493.3.4 Purification of DNA from lambda phage particles 493.3.5 Purification of M13 DNA causes few problems 49Further reading 514 Manipulation of Purified DNA 534.1 The range of DNA manipulative enzymes 554.1.1 Nucleases 554.1.2 Ligases 574.1.3 Polymerases 574.1.4 DNA modifying enzymes 584.2 Enzymes for cutting DNA - restriction endonucleases 594.2.1 The discovery and function of restriction endonucleases 604.2.2 Type II restriction endonucleases cut DNA at specific nucleotide sequences 614.2.3 Blunt ends and sticky ends 624.2.4 The frequency of recognition sequences in a DNA molecule 634.2.5 Performing a restriction digest in the laboratory 644.2.6 Analysing the result of restriction endonuclease cleavage 664.2.7 Estimation of the sizes of DNA molecules 684.2.8 Mapping the positions of different restriction sites in a DNA molecule 694.2.9 Special gel electrophoresis methods for separating larger molecules 704.3 Ligation - joining DNA molecules together 724.3.1 The mode of action of DNA ligase 724.3.2 Sticky ends increase the efficiency of ligation 744.3.3 Putting sticky ends onto a blunt-ended molecule 744.3.4 Blunt-end ligation with a DNA topoisomerase 79Further reading 815 Introduction of DNA into Living Cells 835.1 Transformation - the uptake of DNA by bacterial cells 855.1.1 Not all species of bacteria are equally efficient at DNA uptake 855.1.2 Preparation of competent E. coli cells 865.1.3 Selection for transformed cells 865.2 Identification of recombinants 885.2.1 Recombinant selection with pBR322 - insertional inactivation of an antibiotic resistance gene 895.2.2 Insertional inactivation does not always involve antibiotic resistance 905.3 Introduction of phage DNA into bacterial cells 925.3.1 Transfection 935.3.2 In vitro packaging of lambda cloning vectors 935.3.3 Phage infection is visualized as plaques on an agar medium 935.3.4 Identification of recombinant phages 955.4 Introduction of DNA into non-bacterial cells 975.4.1 Transformation of individual cells 975.4.2 Transformation of whole organisms 99Further reading 996 Cloning Vectors for E. coli 1016.1 Cloning vectors based on E. coli plasmids 1026.1.1 The nomenclature of plasmid cloning vectors 1026.1.2 The useful properties of pBR322 1026.1.3 The pedigree of pBR322 1036.1.4 More sophisticated E. coli plasmid cloning vectors 1046.2 Cloning vectors based on lambda bacteriophage 1086.2.1 Natural selection was used to isolate modified lambda that lack certain restriction sites 1086.2.2 Segments of the lambda genome can be deleted without impairing viability 1086.2.3 Insertion and replacement vectors 1106.2.4 Cloning experiments with lambda insertion or replacement vectors 1126.2.5 Long DNA fragments can be cloned using a cosmid 1136.2.6 lambda and other high-capacity vectors enable genomic libraries to be constructed 1146.3 Cloning vectors for synthesis of single-stranded DNA 1156.3.1 Vectors based on M13 bacteriophage 1156.3.2 Hybrid plasmid-M13 vectors 1176.4 Vectors for other bacteria 118Further reading 1197 Cloning Vectors for Eukaryotes 1217.1 Vectors for yeast and other fungi 1217.1.1 Selectable markers for the 2 mum plasmid 1227.1.2 Vectors based on the 2 mum plasmid - yeast episomal plasmids 1227.1.3 A YEp may insert into yeast chromosomal DNA 1247.1.4 Other types of yeast cloning vector 1247.1.5 Artificial chromosomes can be used to clone long pieces of DNA in yeast 1267.1.6 Vectors for other yeasts and fungi 1297.2 Cloning vectors for higher plants 1297.2.1 Agrobacterium tumefaciens - nature's smallest genetic engineer 1307.2.2 Cloning genes in plants by direct gene transfer 1357.2.3 Attempts to use plant viruses as cloning vectors 1377.3 Cloning vectors for animals 1387.3.1 Cloning vectors for insects 1397.3.2 Cloning in mammals 141Further reading 1438 How to Obtain a Clone of a Specific Gene 1458.1 The problem of selection 1468.1.1 There are two basic strategies for obtaining the clone you want 1468.2 Direct selection 1478.2.1 Marker rescue extends the scope of direct selection 1498.2.2 The scope and limitations of marker rescue 1508.3 Identification of a clone from a gene library 1508.3.1 Gene libraries 1518.4 Methods for clone identification 1538.4.1 Complementary nucleic acid strands hybridize to each other 1548.4.2 Colony and plaque hybridization probing 1548.4.3 Examples of the practical use of hybridization probing 1578.4.4 Identification methods based on detection of the translation product of the cloned gene 164Further reading 1669 The Polymerase Chain Reaction 1699.1 PCR in outline 1709.2 PCR in more detail 1729.2.1 Designing the oligonucleotide primers for a PCR 1729.2.2 Working out the correct temperatures to use 1749.3 After the PCR: studying PCR products 1769.3.1 Gel electrophoresis of PCR products 1779.3.2 Cloning PCR products 1789.4 Real-time PCR 1809.4.1 Carrying out a real-time PCR experiment 1809.4.2 Real-time PCR enables the amount of starting material to be quantified 1829.4.3 Melting curve analysis enables point mutations to be identified 184Further reading 185Part II The Applications of Gene Cloning and DNA Analysis in Research 18710 Sequencing Genes and Genomes 18910.1 Chain-termination DNA sequencing 19010.1.1 Chain-termination sequencing in outline 19010.1.2 Not all DNA polymerases can be used for sequencing 19210.1.3 Chain-termination sequencing with Taq polymerase 19310.1.4 Limitations of chain-termination sequencing 19510.2 Next-generation sequencing 19610.2.1 Preparing a library for an Illumina sequencing experiment 19710.2.2 The sequencing phase of an Illumina experiment 19910.2.3 Ion semiconductor sequencing 20110.2.4 Third-generation sequencing 20110.2.5 Next-generation sequencing without a DNA polymerase 20210.2.6 Directing next-generation sequencing at specific sets of genes 20310.3 How to sequence a genome 20510.3.1 Shotgun sequencing of prokaryotic genomes 20610.3.2 Sequencing of eukaryotic genomes 209Further reading 21511 Studying Gene Expression and Function 21711.1 Studying the RNA transcript of a gene 21811.1.1 Detecting the presence of a transcript in an RNA sample 21911.1.2 Transcript mapping by hybridization between gene and RNA 22011.1.3 Transcript analysis by primer extension 22211.1.4 Transcript analysis by PCR 22311.2 Studying the regulation of gene expression 22411.2.1 Identifying protein binding sites on a DNA molecule 22511.2.2 Identifying control sequences by deletion analysis 23011.3 Identifying and studying the translation product of a cloned gene 23211.3.1 HRT and HART can identify the translation product of a cloned gene 23311.3.2 Analysis of proteins by in vitro mutagenesis 234Further reading 24012 Studying Genomes 24312.1 Locating the genes in a genome sequence 24412.1.1 Locating protein-coding genes by scanning a genome sequence 24412.1.2 Gene location is aided by homology searching 24712.1.3 Locating genes for noncoding RNA transcripts 24912.1.4 Identifying the binding sites for regulatory proteins in a genome sequence 25012.2 Determining the function of an unknown gene 25112.2.1 Assigning gene functions by computer analysis 25112.2.2 Assigning gene function by experimental analysis 25212.3 Genome browsers 256Further reading 25713 Studying Transcriptomes and Proteomes 25913.1 Studying transcriptomes 25913.1.1 Studying transcriptomes by microarray or chip analysis 26013.1.2 Studying transcriptomes by RNA sequencing 26113.2 Studying proteomes 26513.2.1 Protein profiling 26613.2.2 Studying protein-protein interactions 270Further reading 274Part III The Applications of Gene Cloning and DNA Analysis in Biotechnology 27514 Production of Protein from Cloned Genes 27714.1 Special vectors for expression of foreign genes in E. coli 28014.1.1 The promoter is the critical component of an expression vector 28114.1.2 Cassettes and gene fusions 28514.2 General problems with the production of recombinant protein in E. coli 28714.2.1 Problems resulting from the sequence of the foreign gene 28814.2.2 Problems caused by E. coli 28914.3 Production of recombinant protein by eukaryotic cells 29014.3.1 Recombinant protein from yeast and filamentous fungi 29114.3.2 Using animal cells for recombinant protein production 29314.3.3 Pharming - recombinant protein from live animals and plants 295Further reading 29815 Gene Cloning and DNA Analysis in Medicine 30115.1 Production of recombinant pharmaceuticals 30115.1.1 Recombinant insulin 30215.1.2 Synthesis of human growth hormones in E. coli 30415.1.3 Recombinant factor VIII 30515.1.4 Synthesis of other recombinant human proteins 30815.1.5 Recombinant vaccines 30815.2 Identification of genes responsible for human diseases 31415.2.1 How to identify a gene for a genetic disease 31515.2.2 Genetic typing of disease mutations 32015.3 Gene therapy 32115.3.1 Gene therapy for inherited diseases 32115.3.2 Gene therapy and cancer 32315.3.3 The ethical issues raised by gene therapy 324Further reading 32516 Gene Cloning and DNA Analysis in Agriculture 32716.1 The gene addition approach to plant genetic engineering 32816.1.1 Plants that make their own insecticides 32816.1.2 Herbicide-resistant crops 33416.1.3 Improving the nutritional quality of plants by gene addition 33716.1.4 Other gene addition projects 33816.2 Gene subtraction 33916.2.1 Antisense RNA and the engineering of fruit ripening in tomato 34016.2.2 Other examples of the use of antisense RNA in plant genetic engineering 34216.3 Gene editing with a programmable nuclease 34416.3.1 Gene editing of phytoene desaturase in rice 34416.3.2 Editing of multiple genes in a single plant 34616.3.3 Future developments in gene editing of plants 34716.4 Are GM plants harmful to human health and the environment? 34916.4.1 Safety concerns with selectable markers 34916.4.2 The possibility of harmful effects on the environment 350Further reading 35117 Gene Cloning and DNA Analysis in Forensic Science and Archaeology 35517.1 DNA analysis in the identification of crime suspects 35617.1.1 Genetic fingerprinting by hybridization probing 35617.1.2 DNA profiling by PCR of short tandem repeats 35717.2 Studying kinship by DNA profiling 35917.2.1 Related individuals have similar DNA profiles 35917.2.2 DNA profiling and the remains of the Romanovs 36017.3 Sex identification by DNA analysis 36317.3.1 PCRs directed at Y chromosome-specific sequences 36317.3.2 PCR of the amelogenin gene 36417.4 Archaeogenetics - using DNA to study human prehistory 36517.4.1 The origins of modern humans 36517.4.2 DNA can also be used to study prehistoric human migrations 370Further reading 374Glossary 377Index 395
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.
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