Molecular Biology and Biotechnology

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ISBN: 9780854046065 | 0854046062
Cover: Paperback
Copyright: 1/1/2001

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As a textbook, Molecular Biology and Biotechnology has always been immensely popular. Now in its fourth edition, it has been completely revised and updated to provide a comprehensive overview of the area, and to reflect all the latest developments. Written by recognised experts, each of the nineteen chapters describes a specific subject area relevant to the subject of biotechnology. The impressive breadth of coverage takes into account both molecular biology and industrial applications and aims to identify the impact that molecular biology has had on the development of biotechnology. Presenting information in an easily assimilated form, Molecular Biology and Biotechnology makes an ideal undergraduate text. It will be of particular interest to students of biology and chemistry, as well as to scientists from outside the field requiring a rapid introduction to the subject.

Fermentation Technology

(25)

Peter F. Stanbury

Introduction

(1)

Microbial Growth

(5)

Applications of Fermentation

(6)

Microbial Biomass

(1)

Microbial Metabolites

(4)

Microbial Enzymes

(1)

Transformation Processes

(1)

Recombinant Products

(1)

The Fermentation Process

(5)

The Mode of Operation of Fermentation Processes

(4)

The Genetic Improvement of Product Formation

(7)

Mutation

(3)

Recombination

(4)

Conclusions

(1)

Molecular Analysis and Amplification Techniques

(42)

Ralph Rapley

Introduction

(2)

Enzymes Used in Molecular Biology

(2)

Extraction and Separation of Nucleic Acids

(3)

DNA Extraction Techniques

(2)

RNA Extraction Techniques

(1)

Electrophoresis of Nucleic Acids

(2)

Restriction Mapping of DNA Fragments

(2)

Nucleic Acid Blotting and Hybridization

(3)

Hybridization and Stringency

(3)

Production of Gene Probes

(5)

DNA Gene Probe Labelling

(1)

Non-radioactive DNA Labelling

(1)

End Labelling of DNA

(1)

Random Primer Labelling of DNA

(1)

Nick Translation labelling of DNA

(1)

The Polymerase Chain Reaction

(13)

Stages and Components of the PCR

(3)

Thermostable DNA Polymerases

(1)

Primer Design in the PCR

(2)

PCR Amplification Templates

(1)

Sensitivity of the PCR

(2)

Modifications of the PCR

(2)

Applications of the PCR

(1)

Alternative Amplification Techniques

(2)

Nucleotide Sequencing of DNA

(7)

Dideoxynucleotide Chain Terminators

(2)

Direct PCR Sequencing

(1)

Cycle Sequencing

(1)

Automated Fluorescent DNA Sequencing

(1)

Maxam and Gilbert Sequencing

(1)

Bioinformatics and the Internet

(3)

Recombinant DNA Technology

(58)

Ralph Rapley

Introduction

(1)

Constructing Gene Libraries

(11)

Digesting Genomic DNA Molecules

(1)

Ligating DNA Molecules

(3)

Considerations in Gene Library Preparation

(1)

Genomic DNA Libraries

(2)

cDNA Libraries

(1)

Linkers and Adaptors

(1)

Enrichment Methods for RNA

(1)

Subtractive Hybridisation

(1)

Cloning PCR Products

(2)

Cloning Vectors

(16)

Plasmid Derived Cloning Vectors

(1)

Plasmid Selection Systems

(1)

pUC Plasmid Cloning Vectors

(2)

Virus-based Cloning Vectors

(2)

Insertion and Replacement Cloning Vectors

(2)

M13 and Phagemid-based Cloning Vectors

(2)

Cloning into Single-stranded Phage Vectors

(1)

Cosmid-based Cloning Vectors

(2)

Large Insert Capacity Cloning Vectors

(1)

Yeast Artificial Chromosome (YAC) Cloning Vectors

(1)

Vectors Used in Eukaryotic Cells

(1)

Gene Probes and Hybridisation

(1)

Cloned DNA Probes

(1)

RNA Gene Probes

(1)

Screening Gene Libraries

(5)

Colony and Plaque Hybridisation

(3)

Gene Library Screening by PCR

(1)

Screening Expression cDNA Libraries

(2)

Hybrid Select/Arrest Translation

101

(1)

Applications of Gene Cloning

101

(4)

Sequencing Cloned DNA

101

(1)

In vitro Mutagenesis

102

(1)

Oligonucleotide-directed Mutagenesis

103

(1)

PCR-based Mutagenesis

103

(2)

Expression of Foreign Genes

105

(6)

Production of Fusion Proteins

106

(3)

Expression in Mammalian Cells

109

(1)

Display of Proteins on Bacteriophage

109

(2)

Analysing Genes and Gene Expression

111

(5)

Identifying and analysing mRNA

111

(1)

Reverse Transcriptase PCR (RT-PCR)

112

(1)

Analysing Genes in situ

113

(2)

Transgenics and Gene Targeting

115

(1)

Microarrays and DNA Chips

116

(1)

Analysing Whole Genomes

117

(8)

Physical Genome Mapping

118

(2)

Gene Discovery and Localisation

120

(2)

Human Genome Mapping Project

122

(3)

The Expression of Foreign DNA in Bacteria

125

(30)

Robert J. Slater

D. Ross Williams

Introduction

125

(3)

Control of Gene Expression

128

(6)

Prokaryotes

128

(4)

Eukaryotes

132

(2)

The Expression of Eukaryotic Genes in Bacteria

134

(10)

Introns

135

(1)

Promoters

136

(1)

Ribsome Binding Site

137

(1)

Expression of Foreign DNA as Fusion Proteins

138

(4)

Expression of Native Proteins

142

(2)

Detecting Expression of Foreign Genes

144

(1)

Maximising Expression of Foreign DNA

145

(5)

Optimising Expression in E. coli

148

(2)

Alternative Host Organisms

150

(3)

Future Prospects

153

(1)

Further Reading

153

(2)

Yeast Cloning and Biotechnology

155

(22)

Brendan P.G. Curran

Virginia C. Bugeja

Introduction

155

(1)

Gene Manipulation in S. cerevisiae

155

(4)

Introducing DNA into Yeast

155

(1)

Yeast Selectable Markers

156

(1)

Vector Systems

156

(3)

Heterologous Protein Production

159

(5)

The Source of Heterologous DNA

159

(1)

The Level of Heterologous mRNA Present in the Cell

159

(1)

The Amount of Protein Produced

160

(2)

The Nature of the Required Product

162

(2)

Using Yeast to Analyse Genomes, Genes and Protein-Protein Interactions

164

(10)

YAC Technology

164

(5)

Gene Knockouts

169

(3)

Novel Reporter Systems

172

(2)

Future Prospects

174

(3)

Cloning Genes in Mammalian Cell-lines

177

(26)

Edward J. Murray

Introduction

177

(2)

Methods of DNA Transfection

179

(6)

Calcium Phosphate Co-precipitation

180

(1)

DEAE-Dextran

181

(1)

Electroporation

182

(1)

Protoplast Fusion

182

(1)

Lipofection

183

(1)

Polybrene-DMSO Treatment

184

(1)

Microinjection

184

(1)

Scrapefection

185

(1)

Requirements for Gene Expression

185

(4)

The DNA Component

189

(7)

Use of Vectors

189

(1)

Plasmid-based Vectors

189

(2)

Virus-based Vectors

191

(1)

Adrenovirus Vectors

191

(1)

Retrovirus Vectors

192

(1)

Poxviral Vectors

193

(2)

Baculovirus Vectors

195

(1)

Some Considerations in Choice of Cell-line

196

(1)

Transient versus Stable Expression

197

(6)

Selection by Host Cell Defect Complementation

198

(1)

Dominant Selective Techniques

198

(2)

Amplifiable Selection Systems

200

(3)

Plant Biotechnology

203

(34)

Michael G.K. Jones

Introduction

203

(1)

Applications of Molecular Biology to Speed up the Processes of Crop Improvement

203

(8)

Molecular Maps of Crop Plants

204

(1)

Molecular Markers

205

(1)

Types of Molecular Markers

205

(1)

Marker-assisted Selection

206

(2)

Examples of Marker-assisted Selection

208

(1)

Molecular Diagnostics

208

(1)

DNA Fingerprinting, Variety Identification

209

(1)

DNA Microarrays

210

(1)

Bioinformatics

210

(1)

Transgenic Technologies

211

(2)

Agrobacterium-mediated Transformation

211

(1)

Selectable Marker and Reporter Genes

211

(1)

Particle Bombardment

212

(1)

Applications of Transgenic Technologies

213

(2)

Engineering Crop Resistance to Herbicides

215

(1)

Engineering Resistance to Pests and Diseases

215

(10)

Insect Resistance

215

(2)

Engineered Resistance to Plant Viruses

217

(2)

Resistance to Fungal Pathogens

219

(1)

Natural Resistance Genes

220

(2)

Engineering Resistance to Fungal Pathogens

222

(1)

Resistance to Bacterial Pathogens

223

(1)

Resistance to Nematode Pathogens

224

(1)

Manipulating Male Sterility

225

(1)

Tolerance to Abiotic Stresses

225

(1)

Manipulating Quality

226

(5)

Prolonging Shelf Life

227

(1)

Nutritional and Technological Properties

227

(1)

Proteins

227

(1)

Oils

228

(1)

Manipulation of Starch

229

(1)

Fructans

230

(1)

Manipulation of Metabolic Partitioning

230

(1)

Production of Plant Polymers and Biodegradable Plastics

231

(1)

Plants as Bioreactors: Biopharming and Neutraceuticals

232

(1)

Edible Vaccines

232

(1)

Production of Antibodies in Plants

233

(1)

Plant Neutraceuticals

233

(1)

Plant Biotechnology in Forestry

233

(1)

Intellectual Property

234

(1)

Public Acceptance

235

(1)

Future Prospects

236

(1)

Molecular, Structural and Chemical Biology in Pharmaceutical Research

237

(24)

Tomi K. Sawyer

Introduction

237

(1)

Molecular Biology of Disease and in vivo Transgenic Models

238

(3)

Genomic Protein Targets and Recombinant Therapeutics

241

(6)

Structural Biology and Rational Drug Design

247

(5)

Chemical Biology and Molecular Diversity

252

(5)

Gene Therapy and DNA/RNA-Targeted Therapeutics

257

(2)

Future Prospects in Pharmaceutical Research

259

(1)

Conclusions

260

(1)

Genetically Modified Foods

261

(18)

Rosa K. Pawsey

Introduction

261

(1)

Legal Requirements in the Production of Novel Foods and Processes

262

(3)

Food Crops

265

(6)

Food Animals

271

(1)

Current Trends in Manufactured Foods

272

(4)

Consumer Acceptance and Market Forces

276

(3)

Molecular Diagnosis of Inherited Disease

279

(16)

Elizabeth Green

Introduction

279

(1)

Direct Detection of Gene Mutations

280

(11)

Detection of Deletions, Duplications and Insertions

281

(2)

Expansion Mutations

283

(2)

Point Mutations

285

(1)

Allele-specific Oligonucleotides

285

(1)

Restriction Enzyme Site Analysis

286

(1)

`ARMS'

287

(2)

Oligonucleotide Ligation

289

(1)

Fluorescently Labelled DNA Sequencing

290

(1)

Indirect Diagnosis with Linked Genetic Markers

291

(1)

Future Prospects

292

(3)

DNA in Forensic Science

295

(22)

Paul Debenham

Peter D. Martin

Introduction

295

(1)

MLP and SLP Technology

296

(6)

MLP/SLP Methods

299

(1)

Extraction and Purification of the DNA

299

(1)

Quantitation

300

(1)

Restriction Endonuclease Digestion of DNA

300

(1)

Electrophoretic Separation

301

(1)

Hybridization

301

(1)

Analysis of Results

302

(1)

PCR Technology

302

(2)

The First PCR-based Forensic System

304

(1)

Short Tandem Repeats

304

(4)

Method

305

(1)

Extraction of DNA

305

(2)

Quantitation of DNA

307

(1)

Amplification of DNA

307

(1)

Separation of Products

307

(1)

Databases

308

(2)

Interpretation of the Results

310

(1)

Mitochondrial DNA

311

(2)

Y Chromosome Analysis

313

(1)

The Future

313

(4)

Capillary Electrophoresis

313

(1)

DNA Chip Technology

314

(3)

Vaccination and Gene Manipulation

317

(40)

Michael Mackett

Infectious Disease -- The Scale of the Problem

317

(2)

Current Vaccination Strategies

319

(4)

Inactivated Vaccines

319

(2)

Live Attenuated Vaccines

321

(1)

The Relative Merits of Live versus Killed Vaccines

321

(2)

The Role of Genetic Engineering in Vaccine Identification, Analysis and Production

323

(10)

Identification and Cloning of Antigens with Vaccine Potential

323

(1)

DNA/Oligonucleotide Hybridization

324

(1)

Hybrid Selection and Cell-free Translation

324

(1)

Expression Cloning

325

(1)

Genomic Sequencing

326

(1)

Analysis of Vaccine Antigens

327

(2)

B-cell Epitopes

329

(1)

T-cell Epitopes

329

(1)

Generation of Subunit Vaccines

330

(1)

Expression of Potential Vaccine Antigens

330

(3)

Improvement and Generation of New Live Attenuated Vaccines

333

(9)

Improving Current Live Attenuated Vaccines

333

(1)

New Vaccines for Pseudorabies Virus

333

(1)

Improving Attenuation in Vibrio cholerae

334

(2)

Improving Stability - Poliovirus

336

(1)

Recombinant Live Vectors

336

(1)

Vaccinia Virus Recombinants

337

(1)

Recombinant BCG Vaccines

338

(1)

Attenuated Salmonella Strains as Live Bacterial Vaccines

339

(1)

Poliovirus Chimaeras

339

(1)

Cross-species Vaccination, `Live-dead' Vaccines

340

(1)

Other Virus Vectors

341

(1)

Recombinant E. coli Strains

341

(1)

Other Approaches to Vaccines

342

(8)

DNA Vaccines (Genetic Immunisation)

342

(1)

Optimizing Responses

343

(1)

RNA Immunisation

344

(1)

Peptide Vaccines

345

(1)

Anti-idiotypes

346

(1)

Enhancing Immunogenitity and Modifying Immune Responses

347

(1)

Adjuvants, Carriers and Vehicles

347

(1)

Carriers

348

(1)

Mucosal Immunity

349

(1)

Modulation of Cytokine Profile

349

(1)

Modulation by Antigen Targeting

350

(1)

Modulation of Signalling

350

(1)

Summary and Conclusions

350

(3)

Further Reading and Sources of Information

353

(4)

Transgenesis

357

(22)

Linda J. Mullins

John J. Mullins

Introduction

357

(1)

The Production of Transgenic Animals by Microinjection

358

(5)

Transgenic Mice

358

(2)

Transgenic Rats

360

(1)

Choice of Animal

361

(1)

Applications of Micro-injection Techniques to Other Animals

361

(1)

Animal Cloning

362

(1)

Embryo Stem Cell Technology, Homologous Recombination and Transgenesis

363

(3)

General Considerations

366

(2)

The Construct

366

(2)

Aberrant Expression

368

(1)

Design of the Transgenic Experiment

368

(7)

Investigating Gene Expression

368

(1)

Reduction of Gene Function

369

(2)

Cell Ablation

371

(1)

Conditional Gene Alteration

372

(1)

Inducible Gene Targeting Using the Cre-lox System

372

(2)

Tetracycline/Tamoxifen

374

(1)

Commercial Applications

375

(3)

Biopharmaceuticals in Transgenic Animals

375

(1)

Xenografts

376

(1)

Toxicological Applications

377

(1)

Immortomouse

377

(1)

Future Prospects

378

(1)

Protein Engineering

379

(26)

John R. Adair

Introduction

379

(2)

Protein Structures

380

(1)

Tools

381

(11)

Sequence Identification

381

(1)

Sequence Determination and Modelling

381

(1)

Sequence Modification

382

(1)

Site-directed Mutagenesis Methods

383

(1)

Non-PCR Methods

383

(2)

PCR-based Methods

385

(1)

Molecular Evolution

385

(4)

de novo Sequence Design

389

(1)

Expression

389

(2)

Analysis

391

(1)

Applications

392

(12)

Point Mutations

392

(1)

Betaseron/Betaferon (Interferon β-16)

392

(1)

Humalog (Lispro Insulin)

393

(1)

Novel Vaccine Adjuvants

393

(1)

Domain Shuffling (Linking, Swapping and Deleting)

394

(1)

Linking Domains

394

(1)

Domain Fusions for Cell Targeting

394

(1)

Fused Cytokines

395

(1)

Fusions to Stabilize Dimeric Proteins

396

(1)

Swapping Protein Domains

396

(1)

Chimaeric Mouse-Human Antibodies

396

(1)

Polyketide Synthases (PKSs)

397

(1)

Deleting Domains

397

(1)

Whole Protein Shuffling

397

(3)

Protein-Ligand Interactions

400

(1)

Enzyme Modifications

400

(1)

Hormone Agonists

401

(1)

Substitution of Binding Specificities

401

(1)

Towards de novo Design

402

(1)

de novo Design

402

(2)

Conclusions and Future Directions

404

(1)

Bioinformatics

405

(28)

Peter M. Woollard

Introduction

405

(3)

Databases

408

(8)

Sequence Databases

410

(1)

Nucleic Acid Sequence Databases

410

(2)

Protein Sequence Databases

412

(1)

Protein Family and Motif Databases

412

(2)

Genome Databases

414

(1)

Enzyme Databases

414

(2)

Literature Databases

416

(1)

Medline

416

(1)

BIDS Embase

416

(1)

BIDS ISI Citation Indexes and Index to Scientific and Technical Proceedings (ISTP)

416

(1)

Sequence Analysis

416

(11)

Sequence Database Searching

417

(1)

Keyword Searching

417

(1)

Database Scanning

417

(3)

Pairwise and Multiple Sequence Comparisons and Alignments

420

(1)

Pairwise Comparisons

421

(1)

Multiple Sequence Alignments

421

(4)

Improving the Alignment

425

(1)

Profile Searching

426

(1)

Other Nucleic Acid Sequence Analysis

426

(1)

Gene Identification

426

(1)

Restriction Mapping

426

(1)

Single Nucleotide Polymorphisms (SNPs)

426

(1)

Protein Structure

427

(1)

Mapping

428

(1)

Introduction

428

(1)

Linkage Analysis

428

(1)

Physical Mapping

428

(1)

Radiation Hybrids

429

(1)

Primer Design

429

(1)

Bioinformatics Sites and Centres

429

(2)

Local Bioinformatics Services

430

(1)

National EMBnet Nodes

430

(1)

Specialized Sites

430

(1)

Conclusion and Future Prospects

431

(2)

Immobilization of Biocatalysts

433

(28)

Gordon F. Bickerstaff

Introduction

433

(1)

Biocatalysts

433

(11)

Enzymes

434

(1)

Specificity

435

(1)

Catalytic Power

436

(1)

Ribozymes

437

(1)

Abzymes

437

(2)

Multienzyme Complexes

439

(1)

PDC

439

(1)

Proteosome

439

(1)

Cellulosome

440

(1)

Multienzyme Complexes and Immobilization Technology

441

(1)

Cells

442

(1)

Animal Cells

442

(1)

Plant Cells

443

(1)

Microorganisms (Bacteria, Yeast and Filamentous Fungi)

443

(1)

Biocatalyst Selection

444

(1)

Immobilization

444

(12)

Choice of Support Material

446

(2)

Next Generation of Support Material

448

(1)

Choice of Immobilization Procedure

448

(1)

Adsorption

448

(2)

Covalent Binding

450

(3)

Entrapment

453

(1)

Encapsulation

454

(1)

Cross-linking

455

(1)

Properties of Immobilized Biocatalysts

456

(4)

Stability

456

(1)

Catalytic Activity

457

(3)

Applications

460

(1)

Downstream Processing: Protein Extraction and Purification

461

(36)

Mike D. Scawen

P.M. Hammond

Introduction

461

(1)

Cell Disruption

462

(4)

Enzymic Methods of Cell Disruption

463

(1)

Chemical Methods of Cell Lysis

463

(1)

Alkali

463

(1)

Detergents

464

(1)

Physical Methods of Cell Lysis

464

(1)

Osmotic Shock

464

(1)

Grinding with Abrasives

464

(1)

Solid Shear

465

(1)

Liquid Shear

465

(1)

Initial Purification

466

(3)

Debris Removal

466

(1)

Batch Centrifuges

467

(1)

Continuous-flow Centrifugation

467

(1)

Basket Centrifuges

468

(1)

Membrane Filtration

468

(1)

Aqueous Two-phase Separation

469

(2)

Precipitation

471

(1)

Ammonium Sulfate

471

(1)

Organic Solvents

471

(1)

High Molecular Weight Polymers

472

(1)

Heat Precipitation

472

(1)

Chromatography

472

(19)

Scale-up and Quality Management

472

(3)

Method Selection

475

(1)

Selection of Matrix

475

(4)

Gel Filtration

479

(1)

Ion Exchange Chromatography

480

(1)

Affinity Chromatography

481

(3)

Hydrophobic Interaction Chromatography

484

(1)

High Performance Chromatographic Techniques

485

(1)

Perfusion Chromatography

486

(1)

Expanded Bed Adsorption

487

(1)

Membrane Chromatography

488

(1)

Maintenance of Column Packing Materials

488

(1)

Equipment for Large-scale Chromatography

489

(2)

Control and Automation

491

(1)

Ultrafiltration

491

(1)

Design of Proteins for Purification

492

(4)

Inclusion Bodies

492

(1)

Affinity Tails

492

(4)

Future Trends

496

(1)

Monoclonal Antibodies

497

(24)

Christopher J. Dean

Introduction

497

(1)

Antibody Structure

498

(1)

Preparation of Hybridomas by Somatic Cell Fusion

499

(9)

Principle of the Technology

499

(1)

Choice of Myeloma Cell-line

500

(1)

Choice of Host for Production of Immune B-cells

501

(1)

Immunogen and Route of Immunization

502

(1)

Preparation of Myeloma Cell-line and Host Immune Lymphocytes for Fusion

503

(1)

Hybridoma Formation by Somatic Cell Fusion

504

(1)

Screening Hybridoma Culture Supernatants

504

(2)

Cloning Hybridomas

506

(1)

Bulk Production, Isolation and Purification of Monoclonal Antibodies

506

(1)

Bulk Production

506

(1)

Isolation and Purification

507

(1)

Examples of the Preparation of Rat Monoclonal Antibodies Which Have Been Used to Investigate the Structural and Functional Properties of Macromolecules

508

(4)

HIV I gp 120

508

(1)

mAbs to Growth Factor Receptors

509

(2)

Monoclonal Antibodies for Clinical Application

511

(1)

Generation of Monclonal Antibodies Using Recombinant Gene Technology

512

(5)

Isolation of Immunoglobulin Variable Region Genes and Expression on the Surface of Bacteriophage

512

(1)

Isolation of mRNA for VH and VL and Generation of cDNA

513

(1)

PCR Amplification of cDNAs for Antibody VH and VL

514

(1)

Linking of VH and VL to Give scFv

515

(1)

Insertion of scFv into Phagemid Vector

515

(1)

Expression of scFv on the Surface of Bacteriophage

516

(1)

Screening Phage Display Libraries of Immunoglobulin Genes

516

(1)

Preparation of Soluble scFv

517

(1)

Screening Supernatants Containing Soluble scFv

517

(1)

Monoclonal Antibodies in Biomedical Research

517

(2)

Monoclonal Antibodies in the Diagnosis and Treatment of Disease

519

(2)

Biosensors

521

(34)

Martin F. Chaplin

Introduction

521

(5)

The Biological Reaction

526

(1)

Theory

527

(3)

Electrochemical Methods

530

(12)

Amperometric Biosensors

530

(9)

Potentiometric Biosensors

539

(2)

Conductimetric Biosensors

541

(1)

Calorimetric Biosensors

542

(2)

Piezoelectric Biosensors

544

(1)

Optical Biosensors

545

(6)

Evanescent Wave Biosensors

548

(2)

Surface Plasmon Resonance

550

(1)

Whole Cell Biosensors

551

(1)

Immunosensors

552

(2)

Conclusion

554

(1)

Subject Index

555

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Molecular Biology and Biotechnology

Summary

Table of Contents

Supplemental Materials