An Introduction to Genetic Engineering

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1 An Introduction to Genetic Engineering Second edition Desmond S. T. Nicholl Senior Lecturer in Biological Sciences, University of Paisley 7m] CAMBRIDGE = " UNIVERSITY PRESS

2 Contents Preface to the second edition page xi 1 Introduction What is genetic engineering? Laying the foundations First steps What is in stote? 6 Parti: The basis of genetic engineering 9 2 Introducing molecular biology The flow of genetic information The structure of DNA and RNA Gene organisation Gene structure inprokaryotes Gene structure in eukaryotes Gene expression Genes and genomes Genome si%e andcomplexity Genome organisation 24 3 Working with nucleic acids Isolation of DNA and RNA Handling and quantification of nucleic acids Radiolabelling of nucleic acids End labelling ' Nick translation 31

3 vi Contents Labelling by primerextension Nucleic acid hybridisation Gel electrophoresis DNA sequencing Maxam-Gilbert (chemical) sequencing Sanger Coulson (dideoxy or enzymatic) sequencing Electrophoresis and reading of sequences 40 4 The tools of the trade Restriction enzymes cutting DNA Type II restriction endonucleases Use of restriction endonucleases Restriction mapping DNA modifying enzymes Nucleases Polymerases Enzymes that modify the ends of DNA molecules DNA ligase joining DNA molecules 52 Part II: The methodology of gene manipulation 55 5 Host cells and vectors Host cell types Prokaryotic hosts Eukaryotic hosts Plasmid vectors for use in E. coli What areplasmids? Basic cloningplasmids Slightly more exotic plasmid vectors Bacteriophage vectors for use in E. coli Whatare bacteriophages? Vectors based on bacteriophage A Vectors based on bacteriophage M Other vectors Hybridplasmid/phage vectors Vectors for use in eukaryotic cells Artificial chromosomes Getting DNA into cells Transformation and transfection Packagingphage DNA in vitro Alternative DNA delivery methods 83

4 Contents vii 6 Cloning strategies Which approach is best? Cloning from mrna Synthesis of cdna Cloning cdna in plasmid vectors Cloning cdna in bacteriophage vectors Cloning from genomic DNA Genomic libraries Preparation of DNAfragments for cloning legation,packaging and amplification of libraries Advanced cloning strategies Synthesis and cloning of cdna Expression of cloned cdna molecules Cloning large DNAfragments in BACand YACvectors The polymerase chain reaction The (short) history of the PCR The methodology of the PCR The essential features of the PCR The design of primers for PCR DNA polymerases for PCR More exotic PCR techniques PCR using mrna templates Nested PCR Inverse PCR RAPD andseveralotheracronyms Processing of PCR products Applications of the PCR Selection, screening and analysis of recombinants Genetic selection and screening methods The use of chromogenic substrates Insertionalinactivation Complementation of defined mutations Othergenetic selection methods Screening using nucleic acid hybridisation Nucleic acid probes Screening clone banks Immunological screening for expressed genes Analysis of cloned genes Characterisation based on mrna translation in vitro 143

5 viii Contents Restriction mapping Blotting techniques DNA sequencing 148 Part III: Genetic engineering in action Understanding genes and genomes Analysis of gene structure and function A closer look at sequences Finding important regions of genes Investigatinggene expression From genes to genomes Analysinggenomes Mappinggenomes Genome sequencing Sequencing technology Genome projects The human genome project Whose genome, and how many genes does it contain? Genetic and physical maps of the human genome Derivingandassemblingthesequence What next? Genetic engineering and biotechnology Making proteins Native and fusion proteins Yeast expression systems The baculovirus expression system Mammalian cell lines Protein engineering Examples of biotechnological applications of rdna technology Production of enzymes TheBSTstory Therapeuticproducts for use in human health-care Medical and forensic applications of gene manipulation Diagnosis and characterisation of medical conditions Diagnosis of infection Patterns of inheritance Genetically based disease conditions 201

6 Contents ix 11.2 Treatment using rdna technology gene therapy Getting transgenes into patients Gene therapyfor adenosine deaminase deficiency Gene therapy for cysticfibrosis DNAprofiling The history of 'genetic fingerprinting' DNA profiling and the law Mysteries of the past revealed by genetic detectives Transgenic plants and animals Transgenic plants Why transgenicplants? Tiplasmids as vectors for plant cells Making transgenicplants Putting the technology to work Transgenic animals Why transgenic animals? Producing transgenic animals Applications of transgenic animal technology The other sort of cloning Early thoughts and experiments First steps towards cloning Nuclear totipotency Frogs and toads and carrots A famous sheep the breakthrough achieved Beyond Dolly Brave new world or genetic nightmare? Is science ethically and morally neutral? Elements of the ethics debate Does Frankenstein's monster live inside Pandora's box? 262 Suggestions for further reading 263 Using the World Wide Web 266 Glossary 270 Index 287

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