Molecular Biology andBiotechnologyFourth Edition

Edited by

John M. Walker and Ralph RapleyUniversity of Hertfordshire, Hatfield, UK

RS'CROYAL SOCIETY OF CHEMISTRY

ContentsChapter 1 Fermentation Technology

Peter F. Stanbury123

IntroductionMicrobial GrowthApplications of Fermentation3.1 Microbial Biomass3.2 Microbial Metabolites3.3 Microbial Enzymes3.4 Transformation Processes3.5 Recombinant ProductsThe Fermentation Process4.1 The Mode of Operation of Fermentation

ProcessesThe Genetic Improvement of Product Formation5.1 Mutation5.2 RecombinationConclusions

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1317172024

Chapter 2 Molecular Analysis and Amplification Techniques 25Ralph Rapley1 Introduction 25

1.1 Enzymes Used in Molecular Biology 252 Extraction and Separation of Nucleic Acids 27

2.1 DNA Extraction Techniques 272.2 RNA Extraction Techniques 29

3 Electrophoresis of Nucleic Acids 304 Restriction Mapping of DNA Fragments 325 Nucleic Acid Blotting and Hybridization 34

5.1 Hybridization and Stringency 346 Production of Gene Probes 37

6.1 DNA Gene Probe Labelling 38

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6.2 Non-radioactive DNA Labelling 396.3 End Labelling of DNA ^ 406.4 Random Primer Labelling of DNA 416.5 Nick Translation labelling of DNA 41

7 The Polymerase Chain Reaction 427.1 Stages and Components of the PCR 457.2 Thermostable DNA Polymerases 487.3 Primer Design in the PCR 487.4 PCR Amplification Templates 507.5 Sensitivity of the PCR 507.6 Modifications of the PCR 527.7 Applications of the PCR 54

8 Alternative Amplification Techniques 559 Nucleotide Sequencing of DNA 57

9.1 Dideoxynucleotide Chain Terminators 589.2 Direct PCR Sequencing 609.3 Cycle Sequencing 619.4 Automated Fluorescent DNA Sequencing 629.5 Maxam and Gilbert Sequencing 63

10 Bioinformatics and the Internet 64

Chapter 3 Recombinant DNA Technology 67Ralph Rapley1 Introduction 672 Constructing Gene Libraries 68

2.1 Digesting Genomic DNA Molecules 682.2 Ligating DNA Molecules 682.3 Considerations in Gene Library Preparation 712.4 Genomic DNA Libraries 722.5 cDNA Libraries 742.6 Linkers and Adaptors 752.7 Enrichment Methods for RNA 762.8 Subtractive Hybridisation 772.9 Cloning PCR Products 77

3 Cloning Vectors 793.1 Plasmid Derived Cloning Vectors 80

3.1.1 Plasmid Selection Systems 813.1.2 pUC Plasmid Cloning Vectors 82

3.2 Virus-based Cloning Vectors 843.2.1 Insertion and Replacement

Cloning Vectors 863.3 M13 and Phagemid-based Cloning Vectors 88

Contents ix

3.3.1 Cloning into Single-stranded PhageVectors 90

3.4 Cosmid-based Cloning Vectors 913.5 Large Insert Capacity Cloning Vectors 933.6 Yeast Artificial Chromosome (YAC) Cloning

Vectors 933.7 Vectors Used in Eukaryotic Cells 94

4 Gene Probes and Hybridisation 954.1 Cloned DNA Probes 954.2 RNA Gene Probes 96

5 Screening Gene Libraries 965.1 Colony and Plaque Hybridisation 965.2 Gene Library Screening by PCR 995.3 Screening Expression cDNA Libraries 995.4 Hybrid Select/Arrest Translation 101

6 Applications of Gene Cloning 1016.1 Sequencing Cloned DNA 1016.2 In vitro Mutagenesis 1026.3 Oligonucleotide-directed Mutagenesis 1036.4 PCR-based Mutagenesis 103

7 Expression of Foreign Genes 1057.1 Production of Fusion Proteins 1067.2 Expression in Mammalian Cells 1097.3 Display of Proteins on Bacteriophage 109

8 Analysing Genes and Gene Expression 1118.1 Identifying and analysing mRNA 1118.2 Reverse Transcriptase PCR (RT-PCR) 1128.3 Analysing Genes in situ 1138.4 Transgenics and Gene Targeting 115

9 Microarrays and DNA Chips 11610 Analysing Whole Genomes 117

10.1 Physical Genome Mapping 11810.2 Gene Discovery and Localisation 12010.3 Human Genome Mapping Project 122

Chapter 4 The Expression of Foreign DNA in Bacteria 125Robert J. Slater and D. Ross Williams1 Introduction 1252 Control of Gene Expression 128

2.1 Prokaryotes 1282.2 Eukaryotes 132

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3 The Expression of Eukaryotic Genes in Bacteria 1343.1 Introns 1353.2 Promoters 1363.3 Ribsome Binding Site 1373.4 Expression of Foreign DNA as Fusion

Proteins 1383.5 Expression of Native Proteins 142

4 Detecting Expression of Foreign Genes 1445 Maximising Expression of Foreign DNA 145

5.1 Optimising Expression in E. coli 1486 Alternative Host Organisms 1507 Future Prospects 1538 Further Reading 153

Chapter 5 Yeast Cloning and Biotechnology J 155Brendan P.G. Curran and Virginia C. Bugeja1 Introduction 1552 Gene Manipulation in S. cerevisiae 155

2.1 Introducing DNA into Yeast 1552.2 Yeast Selectable Markers 1562.3 Vector Systems 156

3 Heterologous Protein Production 1593.1 The Source of Heterologous DNA 1593.2 The Level of Heterologous mRNA Present

in the Cell 1593.3 The Amount of Protein Produced 1603.4 The Nature of the Required Product 162

4 Using Yeast to Analyse Genomes, Genes andProtein-Protein Interactions 1644.1 YAC Technology 1644.2 Gene Knockouts 1694.3 Novel Reporter Systems 172

5 Future Prospects 174

Chapter 6 Cloning Genes in Mammalian Cell-lines 177Edward J. Murray1 Introduction 1772 Methods of DNA Transfection 179

2.1 Calcium Phosphate Co-precipitation 1802.2 DEAE-Dextran 1812.3 Electroporation 1822.4 Protoplast Fusion 182

Contents xi

2.5 Lipofection 1832.6 Polybrene-DMSO Treatment 1842.7 Microinjection 1842.8 Scrapefection 185

3 Requirements for Gene Expression 1854 The DNA Component 189

4.1 Use of Vectors 1894.2 Plasmid-based Vectors 1894.3 Virus-based Vectors 1914.4 Adrenovirus Vectors 1914.5 Retrovirus Vectors 1924.6 Poxviral Vectors 1934.7 Baculovirus Vectors 195

5 Some Considerations in Choice of Cell-line 1966 Transient versus Stable Expression 197

6.1 Selection by Host Cell DefectComplementation 198

6.2 Dominant Selective Techniques 1986.3 Amplifiable Selection Systems 200

Chapter 7 Plant Biotechnology 203Michael G.K. Jones1 Introduction 2032 Applications of Molecular Biology to Speed up

the Processes of Crop Improvement 2032.1 Molecular Maps of Crop Plants 2042.2 Molecular Markers 2052.3 Types of Molecular Markers 2052.4 Marker-assisted Selection 2062.5 Examples of Marker^assisted Selection 2082.6 Molecular Diagnostics 2082.7 DNA Fingerprinting, Variety Identification 2092.8 DNA Microarrays 2102.9 Bioinformatics 210

3 Transgenic Technologies 2113.1 Agrobacterium-mediated Transformation 2113.2 Selectable Marker and Reporter Genes 2113.3 Particle Bombardment 212

4 Applications of Transgenic Technologies 2135 Engineering Crop Resistance to Herbicides 2156 Engineering Resistance to Pests and Diseases 215

6.1 Insect Resistance 215

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6.2 Engineered Resistance to Plant Viruses 2176.3 Resistance to Fungal Pathogens 2196.4 Natural Resistance Genes 2206.5 Engineering Resistance to Fungal Pathogens 2226.6 Resistance to Bacterial Pathogens 2236.7 Resistance to Nematode Pathogens 224

7 Manipulating Male Sterility 2258 Tolerance to Abiotic Stresses 2259 Manipulating Quality 226

9.1 Prolonging Shelf Life 2279.2 Nutritional and Technological Properties 227

9.2.1 Proteins 2279.2.2 Oils 2289.2.3 Manipulation of Starch 2299.2.4 Fructans 230

9.3 Manipulation of Metabolic Partitioning 23010 Production of Plant Polymers and Biodegradable

Plastics 23111 Plants as Bioreactors: Biopharming and

Neutraceuticals 23211.1 Edible Vaccines 23211.2 Production of Antibodies in Plants 23311.3 Plant Neutraceuticals 233

12 Plant Biotechnology in Forestry 23313 Intellectual Property 23414 Public Acceptance 23515 Future Prospects 236

Chapter 8 Molecular, Structural and Chemical Biology inPharmaceutical Research 237Tomi K. Sawyer1 Introduction 2372 Molecular Biology of Disease and in vivo

Transgenic Models 2383 Genomic Protein Targets and Recombinant

Therapeutics 2414 Structural Biology and Rational Drug Design 2475 Chemical Biology and Molecular Diversity 2526 Gene Therapy and DNA/RNA-Targeted

Therapeutics 2577 Future Prospects in Pharmaceutical Research 2598 Conclusions 260

Contents xiii

Chapter 9 Genetically Modified Foods 261Rosa K. Pawsey1 Introduction 2612 Legal Requirements in the Production of Novel

Foods and Processes 2623 Food Crops 2654 Food Animals 2715 Current Trends in Manufactured Foods 2726 Consumer Acceptance and Market Forces 276

Chapter 10 Molecular Diagnosis of Inherited Disease 279Elizabeth Green12

34

IntroductionDirect Detection of Gene Mutations2.1 Detection of Deletions, Duplications and

Insertions2.2 Expansion Mutations2.3 Point Mutations

2.3.1 Allele-specific Oligonucleotides2.3.2 Restriction Enzyme Site Analysis2.3.3 'ARMS'2.3.4 Oligonucleotide Ligation2.3.5 Fluorescently Labelled DNA

SequencingIndirect Diagnosis with Linked Genetic MarkersFuture Prospects

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Chapter 11 DNA in Forensic Science 295Paul Debenham and Peter D. Martin1 Introduction2 MLP and SLP Technology

2.1 MLP/SLP Methods2.1.1 Extraction and Purification of the DNA2.1.2 Quantitation2.1.3 Restriction Endonuclease Digestion of

DNA2.1.4 Electrophoretic Separation2.1.5 Hybridization

2.2 Analysis of Results3 PCR Technology

3.1 The First PCR-based Forensic System

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4 Short Tandem Repeats4.1 Method

4.1.1 Extraction of DNA4.1.2 QuantitationofDNA4.1.3 Amplification of DNA4.1.4 Separation of Products

5 Databases6 Interpretation of the Results7 Mitochondrial DNA8 Y Chromosome Analysis9 The Future

9.1 Capillary Electrophoresis9.2 DNA Chip Technology

Contents

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Chapter 12 Vaccination and Gene Manipulation 317Michael Mackett1 Infectious Disease - The Scale of the Problem 3172 Current Vaccination Strategies 319

2.1 Inactivated Vaccines 3192.2 Live Attenuated Vaccines 3212.3 The Relative Merits of Live versus Killed

Vaccines 3213 The Role of Genetic Engineering in Vaccine

Identification, Analysis and Production 3233.1 Identification and Cloning of Antigens with

Vaccine Potential 3233.1.1 DNA/Oligonucleotide Hybridization 3243.1.2 Hybrid Selection and Cell-free

Translation 3243.1.3 Expression Cloning 3253.1.4 Genomic Sequencing 326

3.2 Analysis of Vaccine Antigens 3273.2.1 B-cell Epitopes 3293.2.2 T-cell Epitopes 329

3.3 Generation of Subunit Vaccines 3303.3.1 Expression of Potential Vaccine

Antigens , 3304 Improvement and Generation of New Live

Attenuated Vaccines 3334.1 Improving Current Live Attenuated Vaccines 333

4.1.1 New Vaccines for Pseudorabies Virus 333

Contents xv

4.1.2 Improving Attenuation in Vibriocholerae 334

4.1.3 Improving Stability - Poliovirus 3364.2 Recombinant Live Vectors 336

4.2.1 Vaccinia Virus Recombinants 3374.2.2 Recombinant BCG Vaccines 3384.2.3 Attenuated Salmonella Strains as Live

Bacterial Vaccines 3394.2.4 Poliovirus Chimaeras 3394.2.5 Cross-species Vaccination, 'Live-dead'

Vaccines 3404.2.6 Other Virus Vectors 3414.2.7 Recombinant E. coli Strains 341

5 Other Approaches to Vaccines 3425.1 DNA Vaccines (Genetic Immunisation) 342

5.1.1 Optimizing Responses 3435.1.2 RNA Immunisation 344

5.2 Pep tide Vaccines 3455.3 Anti-idiotypes 3465.4 Enhancing Immunogenitity and Modifying

Immune Responses 3475.4.1 Adjuvants, Carriers and Vehicles 3475.4.2 Carriers 3485.4.3 Mucosal Immunity 3495.4.4 Modulation of Cytokine Profile 3495.4.5 Modulation by Antigen Targeting 3505.4.6 Modulation of Signalling 350

6 Summary and Conclusions 3507 Further Reading and Sources of Information 353

Chapter 13 Transgenesis 357Linda J. Mullins and John J. Mullins1 Introduction 3572 The Production of Transgenic Animals by Micro-

injection 3582.1 Transgenic Mice 3582.2 Transgenic Rats 3602.3 Choice of Animal 3612.4 Applications of Micro-injection Techniques to

Other Animals 3612.5 Animal Cloning 362

xvi Contents

3 Embryo Stem Cell Technology, HomologousRecombination and Transgenesis

4 General Considerations4.1 The Construct4.2 Aberrant Expression

5 Design of the Transgenic Experiment5.1 Investigating Gene Expression5.2 Reduction of Gene Function5.3 Cell Ablation5.4 Conditional Gene Alteration

5.4.1 Inducible Gene Targeting Using theCre-lox System

5.4.2 Tetracycline/Tamoxifen6 Commercial Applications

6.1 Biopharmaceuticals in Transgenic Animals6.2 Xenografts6.3 Toxicological Applications6.4 Immortomouse

7 Future Prospects

Chapter 14 Protein EngineeringJohn R. Adair1 Introduction

1.1 Protein Structures2 Tools

2.1 Sequence Identification2.2 Sequence Determination and Modelling2.3 Sequence Modification

2.3.1 Site-directed Mutagenesis Methods2.3.1.1 Non-PCR Methods2.3.1.2 PCR-based Methods

2.4 Molecular Evolution2.5 de novo Sequence Design2.6 Expression2.7 Analysis

3 Applications3.1 Point Mutations

3.1.1 Betaseron/Betaferon (Interferon /?-16)3.1.2 Humalog (Lispro Insulin)3.1.3 Novel Vaccine Adjuvants

3.2 Domain Shuffling (Linking, Swapping andDeleting)

Contents xvii

3.2.1 Linking Domains 3943.2.1.1 Domain Fusions for Cell

Targeting 3943.2.1.2 Fused Cytokines 3953.2.1.3 Fusions to Stabilize Dimeric

Proteins 3963.2.2 Swapping Protein Domains 396

3.2.2.1 Chimaeric Mouse-HumanAntibodies 396

3.2.2.2 Polyketide Synthases (PKSs) 3973.2.3 Deleting Domains 397

3.3 Whole Protein Shuffling 3973.4 Protein-Ligand Interactions 400

3.4.1 Enzyme Modifications 4003.4.2 Hormone Agonists 4013.4.3 Substitution of Binding Specificities 401

3.5 Towards de novo Design 4023.5.1 de novo Design 402

4 Conclusions and Future Directions 404

Chapter 15 Bioinformatics 405Peter M. Woollard1 Introduction 4052 Databases 408

2.1 Sequence Databases 4102.1.1 Nucleic Acid Sequence Databases 4102.1.2 Protein Sequence Databases 4122.1.3 Protein Family and Motif Databases 412

2.2 Genome Databases 4142.3 Enzyme Databases 4142.4 Literature Databases 416

2.4.1 Medline 4162.4.2 BIDSEmbase 4162.4.3 BIDS ISI Citation Indexes and Index to

Scientific and Technical Proceedings(ISTP) 416

3 Sequence Analysis 4163.1 Sequence Database Searching 417

3.1.1 Keyword Searching 4173.1.2 Database Scanning 417

3.2 Pairwise and Multiple Sequence Comparisonsand Alignments 420

xviii Contents

3.2.1 Pairwise Comparisons3.2.2 Multiple Sequence Alignments3.2.3 Improving the Alignment3.2.4 Profile Searching

3.3 Other Nucleic Acid Sequence Analysis3.3.1 Gene Identification3.3.2 Restriction Mapping3.3.3 Single Nucleotide Polymorphisms

(SNPs)4 Protein Structure5 Mapping

5.1 Introduction5.2 Linkage Analysis5.3 Physical Mapping5.4 Radiation Hybrids5.5 Primer Design

6 Bioinformatics Sites and Centres6.1 Local Bioinformatics Services6.2 National EMBnet Nodes6.3 Specialized Sites

7 Conclusion and Future Prospects

Immobilization of BiocatalystsGordon F. Bickerstaff1 Introduction2 Biocatalysts

2.1 Enzymes2.1.1 Specificity2.1.2 Catalytic Power

2.2 Ribozymes2.3 Abzymes2.4 Multienzyme Complexes

2.4.1 PDC2.4.2 Proteosome2.4.3 Cellulosome2.4.4 Multienzyme Complexes and

Immobilization Technology2.5 Cells

2.5.1 Animal Cells2.5.2 Plant Cells2.5.3 Microorganisms (Bacteria, Yeast and

Filamentous Fungi)

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Contents xix

2.6 Biocatalyst SelectionImmobilization3.1 Choice of Support Material

3.1.1 Next Generation of SupportMaterial

3.2 Choice of Immobilization Procedure3.2.1 Adsorption3.2.2 Covalent Binding3.2.3 Entrapment3.2.4 Encapsulation3.2.5 Cross-linking

Properties of Immobilized Biocatalysts4.1 Stability4.2 Catalytic ActivityApplications

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Chapter 17 Downstream Processing: Protein Extraction andPurification 461Mike D. Scawen and P.M. Hammond12

3

45

IntroductionCell Disruption2.1 Enzymic Methods of Cell Disruption2.2 Chemical Methods of Cell Lysis

2.2.1 Alkali2.2.2 Detergents

2.3 Physical Methods of Cell Lysis2.3.1 Osmotic Shock2.3.2 Grinding with Abrasives2.3.3 Solid Shear2.3.4 Liquid Shear

Initial Purification3.1 Debris Removal3.2 Batch Centrifuges3.3 Continuous-flow Centrifugation3.4 Basket Centrifuges3.5 Membrane FiltrationAqueous Two-phase SeparationPrecipitation5.1 Ammonium Sulfate5.2 Organic Solvents5.3 High Molecular Weight Polymers5.4 Heat Precipitation

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6 Chromatography 4726.1 Scale-up and Quality Management 4726.2 Method Selection 4756.3 Selection of Matrix 4756.4 Gel Filtration 4796.5 Ion Exchange Chromatography 4806.6 Affinity Chromatography 4816.7 Hydrophobic Interaction Chromatography 4846.8 High Performance Chromatographic

Techniques 4856.9 Perfusion Chromatography 4866.10 Expanded Bed Adsorption 4876.11 Membrane Chromatography 4886.12 Maintenance of Column Packing Materials 4886.13 Equipment for Large-scale Chromatography 4896.14 Control and Automation 491

7 Ultrafiltration 4918 Design of Proteins for Purification 492

8.1 Inclusion Bodies 4928.2 Affinity Tails 492

9 Future Trends 496

Chapter 18 Monoclonal Antibodies 497Christopher J. Dean1 Introduction 4972 Antibody Structure 4983 Preparation of Hybridomas by Somatic Cell Fusion 499

3.1 Principle of the Technology 4993.2 Choice of Myeloma Cell-line 5003.3 Choice ofHostfor Production of Immune

B-cells 5013.4 Immunogen and Route of Immunization 5023.5 Preparation of Myeloma Cell-line and Host

Immune Lymphocytes for Fusion 5033.6 Hybridoma Formation by Somatic Cell

Fusion 5043.7 Screening Hybridoma Culture Supernatants 5043.8 Cloning Hybridomas 5063.9 Bulk Production, Isolation and Purification

of Monoclonal Antibodies 5063.9.1 Bulk Production 5063.9.2 Isolation and Purification 507

Contents xxi

4 Examples of the Preparation of Rat MonoclonalAntibodies Which Have Been Used to Investigatethe Structural and Functional Properties ofMacromolecules 5084.1 HIV I gp 120 5084:2 mAbs to Growth Factor Receptors 5094.3 Monoclonal Antibodies for Clinical

Application 5115 Generation of Monclonal Antibodies Using

Recombinant Gene Technology 5125.1 Isolation of Immunoglobulin Variable Region

Genes and Expression on the Surface ofBacteriophage 5125.1.1 Isolation of mRNA for VH and VL and

Generation of cDNA 5135.1.2 PCR Amplification of cDNAs for

Antibody VH and VL 5145.1.3 Linking of VH and VL to Give scFv 5155.1.4 Insertion of scFv into Phagemid Vector 5155.1.5 Expression of scFv on the Surface of

Bacteriophage 5165.1.6 Screening Phage Display Libraries of

Immunoglobulin Genes 5165.1.7 Preparation of Soluble scFv 5175.1.8 Screening Supernatants Containing

Soluble scFv 5176 Monoclonal Antibodies in Biomedical Research 5177 Monoclonal Antibodies in the Diagnosis and

Treatment of Disease 519

Chapter 19 Biosensors 521Martin F. Chaplin1 Introduction 5212 The Biological Reaction 5263 Theory 5274 Electrochemical Methods 530

4.1 Amperometric Biosensors 5304.2 Potentiometric Biosensors 5394.3 Conductimetric Biosensors 541

5 Calorimetric Biosensors 5426 Piezoelectric Biosensors 544

xxii Contents

7 Optical Biosensors 5457.1 Evanescent Wave Biosensors 5487.2 Surface Plasmon Resonance 550

8 Whole Cell Biosensors 5519 Immunosensors 55210 Conclusion 554

Subject Index 555