Molecular Biology and Biotechnology Fourth Edition This book is dedicated to the memory of Christopher J Dean who died shortly after producing his chapter for this volume Molecular Biology and Biotechnology Fourth Edition Edited by John M Walker and Ralph Rapley University of Hertfordshire, Hatfield, UK RSmC ROYAL SOCIETY OF CHEMISTRY ISBN 0-85404-606-2 A catalogue record for this book is available from the British Library 0The Royal Society of Chemistry 2000 Reprinted 2002 All rights reserved Apart from uny fair dealing for the purposes o j research or private study, or criticism or review us permitted under the terms of the U K Copyright, Designs und Patents Act, 1988, this publication muy not be reproduced, stored or transmitted, in any form or by any means, without the prior permission in writing of The Royul Society of Chemistry, or in the case of reprogruphic reproduction only in accordance with the terms of the licences issued by the Copyright Licensing Agency in the UK, or in accordance with the terms of’the licences issued by the appropriate Reproduction Rights Organization outside the UK Enquiries concerning reproduction outside the terms stated here should be sent to The Royal Society of Chemistry at the address printedon thispuge Published by The Royal Society of Chemistry, Thomas Graham House, Science Park, Milton Road, Cambridge CB4 OWF, U K Registered Charity Number 207890 For further information see our web site at www.rsc.org Typeset in Great Britain Printed in Great Britain by Athenaeum Press Ltd, Gateshead, Tyne and Wear iv Preface One of the exciting aspects of being involved in the field of molecular biology is the ever accelerating rate of progress, both in the development of new methodologies and the practical applications of these methodologies Indeed, such developments led to the idea of the first edition of Molecular Biology and Biotechnology and subsequent editions have reflected the fast moving nature of the area To keep pace with the ever expanding technological changes we have increased the basic molecular biology content of the book from one to two chapters in this latest edition In recent years the development of the World Wide Web has been exponential and now provides an essential source of information and access to databases for the molecular biologist We therefore considered it both appropriate and timely to include a new chapter devoted to the subject of Bioinformatics Other chapter titles remain the same as the previous edition but this should not mask the significant updating of the content of these chapters in response to major developments in each area Indeed, in order to reflect research developments, the majority of these chapters have required a total re-write rather than simple updating PCR (only introduced as stand-alone chapter in the last edition) is firmly established as a day-to-day tool and its revolutionary effect on the field is evidenced by its inclusion in chapters throughout the book Molecular biology continues to profoundly affect progress in areas such as plant biotechnology, food technology (especially the contentious area of genetically modified foods), vaccine development, use and application of monoclonal antibodies, clinical treatment and diagnosis, the production of transgenic plants and animals, and many other areas of research relevant to the pharmaceutical industry All these areas have been fully updated in this edition In addition, we continue to ensure that biotechnology is not just considered at the gene level and full consideration continues to be given to aspects of large-scale production and manufacV vi Preface turing with chapters on fermentation technology, downstream processing and the applications of immobilised biocatalysts Our continued intention is that this book should primarily have a teaching function As such, the book should prove of interest both to undergraduates studying for biological or chemical qualifications and to postgraduate and other scientific workers who need a sound introduction to this ever rapidly expanding area John M Walker Ralph Rapley Contents Chapter Fermentation Technology Peter F Stanbury Introduction Microbial Growth Applications of Fermentation 3.1 Microbial Biomass 3.2 Microbial Metabolites 3.3 Microbial Enzymes 3.4 Transformation Processes 3.5 Recombinant Products The Fermentation Process 4.1 The Mode of Operation of Fermentation Processes The Genetic Improvement of Product Formation 5.1 Mutation 5.2 Recombination Conclusions Chapter Molecular Analysis and Amplification Techniques Ralph Rapley Introduction 1.1 Enzymes Used in Molecular Biology Extraction and Separation of Nucleic Acids 2.1 DNA Extraction Techniques 2.2 RNA Extraction Techniques Electrophoresis of Nucleic Acids Restriction Mapping of DNA Fragments Nucleic Acid Blotting and Hybridization 5.1 Hybridization and Stringency Production of Gene Probes 6.1 DNA Gene Probe Labelling vii 1 6 11 11 12 12 13 17 17 20 24 25 25 25 27 27 29 30 32 34 34 31 38 Contents Vlll 10 6.2 Non-radioactive DNA Labelling 6.3 End Labelling of DNA 6.4 Random Primer Labelling of DNA 6.5 Nick Translation labelling of DNA The Polymerase Chain Reaction 7.1 Stages and Components of the PCR 7.2 Thermostable DNA Polymerases 7.3 Primer Design in the PCR 7.4 PCR Amplification Templates 7.5 Sensitivity of the PCR 7.6 Modifications of the PCR 7.7 Applications of the PCR Alternative Amplification Techniques Nucleotide Sequencing of DNA 9.1 Dideoxynucleotide Chain Terminators 9.2 Direct PCR Sequencing 9.3 Cycle Sequencing 9.4 Automated Fluorescent DNA Sequencing 9.5 Maxam and Gilbert Sequencing Bioinformatics and the Internet Chapter Recombinant DNA Technology Ralph Rapley Introduction Constructing Gene Libraries 2.1 Digesting Genomic DNA Molecules 2.2 Ligating DNA Molecules 2.3 Considerations in Gene Library Preparation 2.4 Genomic DNA Libraries 2.5 cDNA Libraries 2.6 Linkers and Adaptors 2.7 Enrichment Methods for RNA 2.8 Subtractive Hybridisation 2.9 Cloning PCR Products Cloning Vectors 3.1 Plasmid Derived Cloning Vectors 3.1.1 Plasmid Selection Systems 3.1.2 pUC Plasmid Cloning Vectors 3.2 Virus-based Cloning Vectors 3.2.1 Insertion and Replacement Cloning Vectors 3.3 M 13 and Phagemid-based Cloning Vectors 39 40 41 41 42 45 48 48 50 50 52 54 55 57 58 60 61 62 63 64 67 67 68 68 68 71 72 74 75 76 77 77 79 80 81 82 84 86 88 552 Table Chapter I9 Whole cell biosensors Analyte Organism Biosensor Ammonia Biological oxygen demand (BOD) Nitrosomonas sp many Cysteine Glutamate Glutamine Herbicides Proteus morganii Escherichia coli SarcinafIa va Cyanobacteria Nicotinic acid Sulphate Thiamine Lactobacillus arabinosus Desulfovibrio desulfuricans Lactobacillus fermen t i Amperometric (02) Amperometric (02/mediated), polarographic (02) or potentiometric (FWH2) Potentiometric (H2S) Potentiometric (COZ) Potentiometric (NH3) Calorimetric or amperometric (mediated) Potentiometric (H + ) Potentiometric (SO1 ) Amperometric (mediated) cheaper, have longer active lifetimes and are less sensitive to inhibition, pH and temperature variations than the isolated enzymes Against these advantages, such devices usually offer longer response and recovery times, a lower selectivity and they are prone to biocatalytic leakage They are particularly useful where multistep or coenzyme-requiring reactions are necessary The microbial cells may be viable or dead The advantage of using viable cells is that the sensor may possess a self-repair capability but this must be balanced against the gentler conditions necessary for use and problems that might occur due to membrane permeability Different types of whole cell biosensors are shown in Table Biochemical oxygen demand (BOD) biosensors most often use a single selected microbial species Although rapid, linear and reproducible, they give different results to conventional testing, which involves incubation over days and reflects the varying metabolism of a mixed microbial population Thermophilic organisms have been used in such a biosensor for use in hot wastewater IMMUNOSENSORS Most biosensor configurations may be used as immunosensors33 (Table 8) and some of these have been mentioned earlier Figure 17 shows some of the configurations possible Direct binding of the antigen to immobilized antibody (Figure 17a) or antigen-antibody sandwich (Figure 17b) may be detected using piezoelectric or SPR devices,34 as can antibody release due to free antigen (Figure 17c) E Gizeli and C R Lowe, Curr Opin Biotechnol., 1996,7,66 34C.L Morgan, D J Newman, and C P Price, Clin Chem., 1996,42, 193 33 Biosensors 553 Table A selection of irnmunosensors Analyte Sensing method Biosensor Cocaine Human chorionic gonadotrophin Antibody/Protein A Antibody/catalase An tibody Antibody/peroxidase An ti bod y/ca talase Antibody Antibody/labelled antigen Piezoelectric Amperometric (02) SPR Potentiometric (I-) Amperometric ( ) Evanescent wave Fluorescence Hepatitis B surface antigen Insulin T2 toxin Trinitrotoluene (TNT) Figure 17 Diflerent configurationsfor biosensor immunoassays: ( a ) antigen binding to immobilized antibody, ( b ) immobilized antigen binding antibody which bind free second antigen, ( c ) antibody bound to immobilized antigen partially released by competing free antigen, and ( d ) immobilized antibody binding free antigen and enzyme-labelled antigen in competition Binding of enzyme-linked antigen (Figure 17d) or antibody can form the basis of all types of immunosensors but has proved particularly useful in calorimetric and amperometric devices The amount of enzyme activity bound in these immunosensors is dependent on the relative concentrations of the competing labelled and unlabelled ligands and so it can be used to determine the concentration of unknown antigen concentrations 554 Chapter I9 The main problems involved in developing immunosensors centre on non-specific binding and incomplete reversibility of the antigen-antibody reaction both of which reduce the active area, and hence sensitivity, on repetitive assay Single-use biosensing membranes are a way round this but they necessitate strict quality control during production 10 CONCLUSION Biosensors form an interesting and varied part of biotechnology They have been applied to solve a number of analytical problems and some have achieved notable commercial success They have been slow to evolve from research prototype to the marketplace and have not yet reached their full potential Many more commercial products are expected over the next few years, particularly in medical diagnostics Subject Index Abiotic stress, 225 Abrasives (for cell disruption), 464 Abzymes, 437 Adaptors, 76 Adenovirus, 191 Adjuvants, 347,393 Adsorption (immobilization), 448 Affinity chromatography, 48 I Affinity maturation, 497 Affinity tails (tags), 493 Agarose, 28, see also Electrophoresis Agrobacterium, 17 Allele specific oligonucleotides, 285 Ammonium sulfate precipitation, 47 Amperometric biosensors, 530 Amplification refractory mutation system, 287 Animal cloning, 362 Antibodies chimaeric, 1 Fab, 498 monoclonal, 497-520 plants, from, 233 polyclonal, 497 structure, 498 VH region, 498 VL region, 498 Antigen targeting, 350 Anti-idiotypes, 346 Antisense oligonucleotides, 24 Aqueous two-phase separations, 469 Arabidopsis, 204 ARMS, see Amplification refractory mutation system ARS, see Autonomously replicating sequence Attenuated vaccines, 321,333 Automated fluorescent DNA sequencing, 62 Autonomously replicating sequence, 156 Autoradiography, 39 Avirulence genes, 220 BAC, see Bacterial artificial chromosome Bacillus thuringiensis,2 5-2 17 Bacterial artificial chromosome, 93, 367-368 Bacterial lambda, 85 Bacterial pathogens (plant), 223 Baculovirus, 195 Baker’s yeast, 16 Basic local alignment search tool, 64, 420 Basket centrifuges, 468 Batch centrifuge, 467 B cells, 497 BCG, 338 Becker muscular dystrophy, 28 Betaseron/betaferon, 392 BigBlue Mice, 377 Biocatalysts, 433-444 Biodegradable plastics, 23 1-232 Bioinformatics general, 64,2 10,405-43 NCBI, 410 world wide web in, 408 Biomass (microbial), 555 556 Biopharmaceuticals, 375-376 Biopharming, 232 Bioreactors, plants, 232 Biosensors, 521-524 amperometric, 530 calorimetric, 542 conductimetric, 541 evanescence wave, 548 immunosensors, 552 optical, 545 piezoelectric, 544 potentiometric, 539 whole cell, 551 Biotransformation, 11 BLAST search, see Basic local alignment search tool Blotting, of DNA, 34 Blue/white screening, 83-84 Bovine pappiloma virus, 190 Branched DNA amplification, 56 Calorimetric biosensors, 542 Carriers, 348 Catalytic power, 436 cDNA library, 74-75,99 CalPhos, 181 Cell ablation, 371-372 Cell disruption techniques, 462466 Cell-free translation, 324 Cell fusion, 504 Cellulosome, 440 Centrifuge basket, 468 batch, 467 continuous flow, 467 Chaperones, 150 Chemostat, Chimaeric antibodies, 51 Chloramphenicol acetyl transferase, 369 Chromatography, 472 affinity, 481 expanded bed absorption, 487 gel filtration, 479 high performance, 485 hydrophobic interaction, 484 immobilized metal affinity, 482 ion exchange, 480 membrane, 488 Subject Index Ni-NTA, 107 perfusion, 486 Chymosin, 274 Cloning, 67- 123 Colony hybridization, 96-99 Combinatorial libraries, 252 Conductimetric biosensors, 541 Contigs, 118 Continuous culture, 3-5 Continuous flow centrifuge, 467 Co-precipitation, calcium phosphate in, 180 Cosmids, 91 Covalent binding (immobilization), 450 Cre-lox technology, 372,373 Crop resistance, 215,218 Cross-linking (immobilization), 455 Cryptic signals, 16 Cyanogen bromide, 140 activation, 452 CyberGreen, Cycle sequencing, Cytogenetic map, 121 dA:dT cloning, 78 Data bases enzyme, 414 EST, 41 1-412 forensic science, 308 general, 408-4 16 genome, 413,415 literature, 416 nucleic acid sequence, 10-4 12 protein, 412-414 scanning, 17 second generation multiplex, 309 STS, 41 DEAE-dextran, 181 Death phase, Decay accelerating factor, 376 De novo sequence design, 389,402 Detergents (for cell lysis), 464 Dideoxy sequencing, see Sanger sequencing Dilution rate, Diptheria toxin, 37 DNA adaptors, 76 blotting, 34 Subject Index chips, see DNA microarrays dominant control, 188 electophoresis of, 30-32 extraction of, 27, 305 chelex resin, 306 electroelution, 32 fingerprinting, 34,209 hypervariable, 33 ligase, 26,69 ligation, 68-69 linkers, 75 microarrays, 116,209,240,292 mitochondrial, 1 mutation, point, 285 pollination, 12 polymorphisms, 29 profiling, 296 purification, 28,299 caesium chloride, 28 quantitation, 300, 307 sequencing, 57-63,290 vaccines, 259, 342 vectors, see Vectors Differential display PCR, 114 DNA, biosensors for, 540 DNase, 28 Domain shuffling, 394-397 Double minutes, 200 Downstream processing, 461-496 Drug design, 247 discovery, 248 RNA/DNA, 257,258 Duchenne muscular dystrophy, 28 EBV, see Epstein-Barr virus E coli, expression in, 138-1 50 Electrophoresis agarose gel, 28, 32 capillary, 13 of DNA, 30-32,301 Electroporation, 156, 177, 182, 12 ELISA, 505 2-EMBL, 86 Embryo stem cell, 363 Encapsulation, 453 End labelling of DNA, 40 Entrapment, 453 Entrez, 417 Enzyme databases, 14 557 Enzyme-linked immunosorbent assay, see ELISA Epidermal growth factor, 509 Epstein-Barr virus, 190, 324 EST, 114-1 15,204,240,241,245,411 Ethidium bromide, I Evanescent wave biosensor, 548 Exons, 281 Expanded bed adsorption chromatography, 487 Expansion mutations, 283 Expasy, 14 Exponential phase, Expressed sequence tag, see EST Expression cloning, 325 Expression libraries, 99-101 Expression of proteins, 389 in E coli, 138-1 50 F1 hybrids, 361 Fab, 498 FASTA search, 64,420 Fed-batch culture, Fermentation processes, 12-1 Fingerprinting, 34,209 FISH, see Fluorescent in situ hybridization FLAVRSAVR tomato, 227,263 Fluorescent in situ hybridization, 114-115 Food animals, 271 Food enzymes, 275 Foot and mouth disease, 328 Forensic science, 295 Forestry, biotechnology in, 233-234 Fragile X syndrome, 284 French press, 465 Fructans, 230 Functional genomics, 240 Fungal pathogens, resistance to, 19220,222 Fusion proteins, 106-109, 138-141 G proteins, 173 Gancyclovir, 37 I Gel filtration chromatography, 479 GelStar, 31 Gene avirulance, 220 dosage, 149 558 Gene (cont.) expression in cell lines, 109, 197 control of, 128 detection of, 144 in E coli, 106, 138-1 50 maximising, I45 in transgenes, 369 knockouts, 169,240,370 linkage analysis, 428 mapping of, 120,426,428 mutation, 280 natural resistance, 220,22 identification, 122,426 probes, 37-38 labelling 38-43 multi locus, 296-299 PCR, 54 silencing, 18 splicing, 133 therapy, 257 transient, 179 Genetic immunization, 342 Genetically modified considerations, 235 Genetically modified crops, 235 Genetically modified foods, 261-278 Genetically modified organisms, 262 Genome databases, 413,415 Genome size, 244 Genome survey sequences, 41 Genomic library, 72-73 Genomic sequencing, 326 GM foods, see Genetically modified foods GMOs, see Genetically modified organisms GRAS, 265 Green fluorescent proteins, 367 Growth rate (microbial), GSS, see Genome survey sequences HAMA, see Human anti-mouse antibody HAT medium, 504 HBV, see Hepatitis B virus Hepatitis B virus, 331 Herpes simplex virus, 371 High performance liquid chromatography, 292,485 Subject Index High throughput screening, 242 HIV, 508 HLA-DQ locus, 304 Homogeneously staining regions (HSR), 200 Homologous recombination, 363366 Hormone agonists, 40 HPLC, see High performance liquid chromatography HSR, see Homogeneously staining regions HSV, see Herpes simplex virus HTF islands, 121 Humalog, 393 Human anti-mouse antibody, 1 Human genome project, 117, 122123,279 Human growth hormone, 142-144 Hybridization, 34-37 colony, 96-99 in situ, 113 oligonucleotide, 324 plaque, 96-99 Hybrid arrest/select translation, 101, 324 Hybridomas, 499 Hybrid selection, 324 Hydrophobic interaction chromatography, 484 Idiophase, IgG, see Antibodies Immobilization (of enzymes and cells), 433-460 Immobilized metal affinity chromatography, 482 Immortomouse, 377-378 Immunoglobulin G, see Antibodies Immunosensors, 552 Inactivated vaccines, 19 Inclusion bodies, 492 Insect resistance, 15 A-Insertion vectors, 86 ‘In silico’ research, 66,406 In vitro mutagenesis, 102-103, 385 Introns, 135-1 36 Ion exchange chromatography, 480 Ion-selective field effect transistor, 540 Subject Index ISFET, see Ion-selective field effect transistor Isoschizomers, 27 karyoplasts, 362 Killed vaccines, 32 Knockout, see Gene knockouts Labelling, of DNA 3’ end labelling, 40-41 5’ end labelling, 40-41 labels, 39 nick translation, 41-43 non-radioactive, 39 random hexamer, 41 Lac operon, 130 Lambda EMBL, 86 Lambda insertion vectors, 86 Lambda phage, 85 Lambda replacement vectors, 86 Lambda ZP, 84-87, 126 LCR, see Ligase chain reaction Leader peptide, 131 Libraries, gene, 71 Ligase chain reaction, 55 Ligation, see DNA Linked gene markers, 29 Linkers, see DNA Lipofection, 183 Liquid shear, 465 Lispro insulin, 393 Live-dead vaccines, 340 Live vaccines, 32 1, 333 Log phase, Lysine synthesis, 18 M 13,89-90 life cycle, 89 MALDI-TOF, 209,315 Mammalian cell line cloning, 177 Manton-Gaulin homogenizer, 465 Mapping, of crop plants, 204 Marker assisted selection, 208, 21 Maxam and Gilbert sequencing, 57, 63 Maximum specific growth rate, Membrane chromatography, 488 Membrane filtration, 468 Messenger RNA, 30,72, 13 Metabolic partitioning, 230 59 Microarrays, see DNA, microarrays Microbial biomass, Microbial growth, 1-6 Microinjection, 184, 358-363 Microsatellite DNA, 206 Molecular diagnostics, 208 Molecular evolution, 355 Monoclonal antibodies, 49 7-520 Motifs, in proteins, 412 mRNA,see Messenger RNA Mucosal immunity, 349 Multienzyme complexes, 439 Multi locus probes, 296-299 Multiple cloning site, 86 Muscular dystrophy, see Becker muscular dystrophy Mutagenesis, 102-103, 385 Mutamouse, 377 Mutation, 17-24 Mycoprotein, 262 NASBA, 57 Natural resistance genes, 220-222 NCBI, 241,412 Nematode pathogens, resistance to, 224-225 Neomycin phosphotransferase, 1 Nested PCR, 52 Neutraceuticals, 232-233 Nicin controlled expression (NICE), 152 Nick translation, 41-43 Northern blotting, 35, 111 Nucleic acid sequence based amplification, see NASBA Occlusion bodies, 195 Oligo dT, 30 Oligonucleotide directed mutagenesis, 103-104,383 Oligonucleotide ligation assay, 289 Open reading frames, 125, 169 Optical biosensors, 545 Organic solvent precipitation, 47 Osmotic shock, 464 P artifical chromosome, 367 Particle bombardement, 12 pBR322, 80, 141, 147, 190, see also Vectors 560 PCR, see Polymerase chain reaction Penicillin, production of, 16 Peptide nucleic acids, 259 Peptide vaccines, 345 Perfusion chromatography, 486 Periplasmic secretion, 150 PFGE, see Pulsed field gel electrophoresis &Phage, 85 Phage display libraries, 16 Phagemids, 88-9 , 515 Pharmocogenomics, 259 PicoGreen, 300 Piezoelectric biosensors, 544 Plant biotechnology, 204-236 oils, 228 polymers, 23 shelf life, 227 viruses, 17 Plasmids, 80-84, 126, 189 PNA, see Peptide nucleic acids Point mutations, 285, 392 Polybrene, 184 Polymerase chain reaction, 42-55 alternatives to, 55 annealing, 46 applications of, 53 cloning, 77-79 differential display, 14 expression, 107 general, 42-54,302 library screening, 99 multiplex, 28 1, 282 mutagenesis, 103, 385 nested, 52 pre-screening, 284 primers, 44, 54, 222, 302,429 design of, 48 fluorescent, 283 megaprimers, 105 quantitative ,283 RT-PCR, 50, 112 sensitivity, 50 Taq DNA polymerase, 46 templates, 50 thermostable polymerases, 48, 54 touchdown, 49 Poliovirus, 336, 339 Polybrene-DMSO, 184 Subject Index Polyclonal antibodies, 497 Polyketides, 397 Polyketide synthase, 397 Polymorphism, 206 PolyA tail, 30, 74, 133 Poly dC, 74 Post-translational modification, 163 Potentiometric biosensors, 539 Pox virus, 193 Primers, 75, see also Polymerase chain reaction, primers Probes, see Gene probes Productivity (chemostat), 13-14 Promoters CAT, 186 enhancers, 132, 187 general, 106, 136-137, 146, 185, 186 TATA, 185 Proteasome, 439 Protein databases, 12-414 de novo design, 389,402 domain shuffling, 394 expression, 389 immobilization of, 433 inclusion bodies, 492 ligand interactions, 400 modelling, 38 1, motifs, 412 purification, 461-496 shuffling, 397 structure, 380 Protein engineering, 379-404 Protein-ligand interactions, 400 Protein resource foundation (PRF), 412 Proteomics, 66,260 Protoplast fusion, 182 Pruteen, 22 Pseudorabies virus, 333 pUC, 82-84, see also Vectors Pulsed field gel electrophoresis, 30 Pyruvate dehydrogenase, 439 Q-b replicase, 56 Quantitative PCR, 283 Quantitative trait loci (QTL), 207 Quorn, 7,262 Subject Index Rabbit reticulocyte lysate, 106 Radiation hybrids, 429 Random hexamer labelling, Random primer labelling, RAPDS (random amplified polymorphic DNA), 205-206 Recombinant DNA technology, 67123 Recombinant proteins, 12 Recombination, 20 A-Replacement vectors, 86 Resistance to bacterial pathogens, 223 to fungal pathogens, 219 insects, 214 to nematode pathogens, 224 pathogen derived, 21 to pests, 21 to plant viruses, 217 Restriction endonuclease analysis, 32, 286 digestion of DNA, 25, 301 Restriction enzymes, 27 Restriction fragment length polymorphism, 32-34,205 Restriction mapping, 32 Retinoblastoma, 365 Retrovirus, 192 Reverse transcriptase, 74 Reverse transcriptase-PCR, 50, 112113 RFLP, see Restriction fragment length polymorphism Ribonuclease protection assay, 12 Riboprobes, 87 Ribosome binding site, 130, 135 Ribozymes, 240,370,437 RNA extraction of, 29 ribosomal, 30 RNA vaccines, 342 RNase,28 RT-PCR, see reverse transcriptasePCR Runaway replication, 149 Sanger sequencing, 57 ScFv, see Single chain antibodies SCP, see Single cell protein Scrapefection, 177, 185 56 Screening, gene libraries, 96-101 Secondary metabolites (microbial), 7-10 Selection, marker assisted, 208 Sephadex, 480 Sequence retrieval system, 417 Sequence alignments, 421 clustering, 424 databases, 10-412 identification, of genes, 426 minisatellites, 296 profile search, 426 signal, 161 SNP analysis, 426 Sequence tagged sites, 19,4 11,429 Sequencing (DNA), 57-63 chain terminators in, 58 cycle sequencing, DNA, 57,290 M13,57 Maxam and Gilbert, 57,63 PCR products, 60 Sanger, 57 Shear, see Solid shear and Liquid shear Shelf life, see Plants Shine-Dalgarno sequence, see Ribosome binding site Short tandem repeats (STR), 304, 31 Signal transduction, 173 Simple sequence repeats (SSR), 205 Single cell protein, Single chain antibodies, 15,516 Single locus probes, 296,298 Single nucleotide polymorphisms, 315,426 Site-directed mutagenesis, 383 Small nuclear ribonuclear proteins, 133 SNPs, see Single nucleotide pol ymorphisms Solid shear, 465 Southern blotting, 34,285 Soya (GM), 276 Specific growth rate (p),4 Splicing, 133 SRS, see Sequence retrieval system Starch, 229 562 Sterility, manipulation of, 225 Stocky ends, 26 Stringency, 34-35, see also Hybridization Structure motifs (protein), 380 STS, see Sequence tagged sites Subtilisin, 127 Subtractive hybridization, 77 Subunit vaccines, 330 Support materials (for immobilization), 446 Surface plasmon resonance, 35, 550 SV40, 191,369,378 SWISPROT 412 T cells, 497 Tamoxifen, 374 Taq DNA polymerase, see PCR TaqMan assay, 55,292 Ti plasmid, 95 Tissue plasminogen activator (tPA), 397 tPA, see Tissue plasminogen activator Transgenes animal models, 239 biopharmaceuticals, 375 commercialization, 14 mice, 358-360 rats, 360-361 technologies, 15, 1-2 13, 238 Transfect ion methods of, 179 polycation-assisted, 177 Transformation, 156,2 1 Trophophase, Ultrafiltration, 49 Uracil-N-glycosylase, Vaccines, 247, 17-353 adjuvants, 347,393 anti-idiotype, 346 attenuated, 321, 333 DNA, 342 edible, 232 inactivated, 19 ligand interaction, 400 live, attentuated, 321, 333 peptide, 345 Subject Index RNA, 344 subunit, 330 Vaccinia virus, 193-195, 337, 340 Variable number tandem repeats, 33, 297 Vectors dT vectors, 78 phagemids, 88 plasmid general, 80-84, 126, 189 pBR322,80, 141, 147, 190 PUC, 82-84 selection systems, viral based adenovirus, 191 baculovirus, 195 cassette, 147 cosmids, 91 REMBL, 86 expresssion, 141 M 13,89-9 poxvirus, 193 retrovirus, 192 vaccinia virus, 194 AZAP, 8647,126 VH, 498 Vibrio cholerae, 334 VL, 498 VNTR, see Variable number tandem repeats Western blotting, 145 Wheat germ extract, 106 Whole-cell biosensors, 551 X chromosome, 28 X-gal, 84 Xenografts, 376 YAC, see Yeast, artificial chromosome Y chromosome, 296,3 13 Yeast artificial chromosome (YAC), 9394, 164-1 69 centromeric plasmid (YCp), 157 cloning, 155-1 75 episomal plasmids (Yep), 94, 156, 157 genome analysis, 164 563 Subject Index selectable markers, 156-1 58 two-hybrid system, 157 vector systems, 156 Yep, see Yeast, episomal plasmid Yield factor, Zoo blotting, 35 A-ZP, 86-87, 126 ... Molecular Biology and Biotechnology Fourth Edition This book is dedicated to the memory of Christopher J Dean who died shortly after producing his chapter for this volume Molecular Biology and. .. Chapter Molecular, Structural and Chemical Biology in Pharmaceutical Research Tomi K Sawyer Introduction Molecular Biology of Disease and in vivo Transgenic Models Genomic Protein Targets and Recombinant... Targets and Recombinant Therapeutics Structural Biology and Rational Drug Design Chemical Biology and Molecular Diversity Gene Therapy and DNA/RNA-Targeted Therapeutics Future Prospects in Pharmaceutical