Molecular Biology and Biotechnology, 7/e (HB)

Advances in molecular biology and biotechnology are increasing at a rapid
pace, both in the development of new methodologies and in their practical
applications. This popular textbook has been revised and updated to
provide an overview of this exciting area of bioscience and to reflect a
number of the key developments drivingthis expansion. Chapters on
the basic methods of key technologies such as nucleic acid analysis and
bioinformatics are presented,in addition to genomics and proteomics,
which highlight the impact of molecular biology and biotechnology. New
chapters on important and emergingmethods have been introduced such
as gene editing, next generation sequencing, nanobiotechnology and
molecular modelling.
The first six chapters deal with the core technology used in current
molecular biology and biotechnology. These primarily deal with basic
molecular biology methods such as PCR,cloninggenes and genomes,
protein analysis techniques and recombinant proteinproduction. Later
chapters address major advances in the applications of specialist areas
of molecular biotechnology. Experienced lecturers and researchers
have written each chapter and the information is presented in an easily
assimilated form. This book makes an ideal text for undergraduates
studying these areas and will be of particular interest to students in many
areas of biosciences,biology and chemistry. In addition,it will appeal to
postgraduates and other scientific workers who need a sound introduction
to this ever rapidly advancingand expandingarea.
 

Contents
1 Basic Molecular Biology Techniques
Ralph Rapley
1
1.1 Introduction
1.2 Structure of Nucleic Acids
1.2.1 Primary Structure of Nucleic Acids
1.2.2 Secondary Structure of Nucleic Acids
1.2.3 Denaturation of Double-stranded DNA
1.3 Isolation and Separation of Nucleic Acids
1.3.1 Isolation of DNA
1.3.2 Isolation of RNA
1.3.3 Automated Nucleic Acid Extraction
1.3.4 Enzymes Used in Molecular Biology
1.3.5 Separation of Nucleic Acids
1.3.6 Automated Analysis of Nucleic Acid Fragments
1.3.7 Single-cell DNA Analysis
1.4 Basic Mapping and Analysis of DNA Fragments
1.4.1 DNA Blotting Methods
1.4.2 RNA Blotting
1.5 Gene Probes and Artificial DNA Synthesis
1.5.1 Labelling DNA Gene Probe Molecules
1.5.2 End Labelling of DNA Molecules
1.5.3 Random Primer Labelling
1.5.4 Nick Translation
1.6 The Polymerase Chain Reaction (PCR)
1.6.1 Mechanism and Steps in the PCR
1.6.2 PCR Optimisation Strategies
VIII Contents
1.6.3 PCR Primer Design
1.6.4 Multiplex PCR
1.6.5 Reverse Transcriptase PCR (RT-PCR)
1.7 Quantitative or Real-time PCR (qPCR)
1.7.1 Automation and Refinements in qPCR
1.7.2 Alternative Amplification Methods
1.8 DNA Sequencing
1.8.1 Sanger Chain Termination Sequencing
1.8.2 Dideoxynucleotide Chain Terminators
1.8.3 Sequencing Double-stranded DNA
1.8.4 Automated DNA Sequencing
1.8.5 Next-generation Sequencing (NGS)
1.8.6 Illumina Sequencing Platform
1.8.7 454 Platform and Pyrosequencing
1.8.8 ABI SOLiD Sequencing by Oligonucleotide
Ligation and Detection
1.8.9 Single-molecule Real-time Sequencing
1.9 Microarrays
1.9.1 Mutation and Expression Microarrays
1.9.2 SNP Microarrays
1.10 Conclusion
2 Genes and Genomes
2.1 Introduction
2.1.1 Genomics Background
2.1.2 Genomes and Chromosomes
2.1.3 Genomic Analysis
2.1.4 Gene Identification
2.1.5 Genetic Polymorphism
2.2 Recombinant DNA Technologies
2.2.1 Molecular Cloning Outline
2.2.2 Cloning Vectors
2.2.3 The Cloning Process
2.2.4 DNA Libraries
2.2.5 Alternative Cloning Systems
2.3 Genome Analysis
2.3.1 Mapping Genomes and Genes
2.3.2 Tools for Genetic Mapping
2.3.3 Mutation and Polymorphism Detection
2.4 Genome Projects Background
2.4.1 Mapping and Sequencing Strategies
Contents Contents IX
2.5 Gene Discovery and Localization 78
2.5.1 Laboratory Approaches
2.5.2 Bioinformatics Approaches
2.6 Conclusion
3 Protein Expression and Production
3.1 Introduction 87
3.2 Host-related Issues
3.3 Vectors
3.3.1 Expression Vector Constructs
3.4 Expression Problems
3.5 Fusion Proteins
3.5.1 Solubility-enhancing Tags
3.5.2 Purification-facilitating Tags
3.5.3 Tag Removal
3.6 Cell-free Systems
3.7 High-throughput Approaches
3.8 Other Hosts
3.9 Conclusion
4 Proteins and Proteomics
4.1 Introduction 123
4.1.1 Outline of the Proteomics Workflow
4.2 Protein Chemistry
4.2.1 Protein Structure
4.2.2 Protein Function 128
4.3 Proteomics Workflow: Sample Preparation
4.3.1 Cell Lysis and Protein Extraction
4.3.2 Protein Separation
4.4 Electrophoretic Methods
4.4.1 Zone Electrophoresis
4.4.2 Polyacrylamide Gel Isoelectric Focusing (IEF)
4.4.3 SDS-PAGE
4.5 2D Gel Electrophoresis
4.5.1 The First Dimension: IPG Strip Selection
4.5.3 IPG Strip Rehydration
4.5.4 IPG Strip Equilibration
4.5.5 The Second Dimension: SDS-PAGE
4.5.6 Specific Protein Detection: Western Blotting
4.6 Protein Identification: Mass Spectrometry
4.6.1 Matrix-assisted Laser Desorption/Ionisation
Time-of-Flight Mass Spectrometry(MALDI-TOFMS)
4.6.2 Liquid Chromatography Coupled with Tandem
Mass Spectrometry (LC-MS/MS)
4.7 Protein Identification: Bioinformatics
4.8 Expression Proteomics
4.8.1 Difference Gel Electrophoresis (DIGE)
4.8.2 Isotope-coded Affinity Tags (ICAT)
4.8.3 Protein Microarrays
4.9 Practical Applications
4.10 Conclusion
Bioinformatics Sources Cited in the Text

5 Transgenesis
5.1 Introduction
5.1.1 From Gene to Function
5.2 Transgenesis by DNA Pronuclear Injection
5.2.1 Generation of a Transgenic Mouse
5.2.2 Summary of Advantages and Disadvantages of
Generating Transgenic Mice by Pronuclear
Injection of DNA
5.3 Gene Targeting by Homologous Recombination
in Embryonic Stem Cells
5.3.1 Basic Principles
5.3.2 Generation of a Knockout Mouse
5.3.3 Summary of Advantages and Disadvantages
of Generating Gene Knockout Mice
5.4 Conditional Gene Targeting
5.4.1 Generation of a Conditional Knockout Mouse
Using the Cre/loxP System
5.4.2 Chromosomal Engineering Using the Cre/loxP System
5.4.3 Summary of Advantages and Disadvantages
of Conditional Gene Targeting
5.5 Tetracycline-inducible Gene Switch System
5.5.1 Basic Principles
5.5.2 Generation of a Tetracycline-inducible Transgenic Mouse
5.5.3 Summary of Advantages of Generating Transgenic
XII Contents
7.5 Molecular Engineering of Antibodies
7.5.1 Identification and Analysis of CDRs
7.5.2 Chimaeric Antibodies
7.5.3 Humanised Antibodies
7.5.4 Affinity Maturation of Antibodies
7.5.5 Enhancing Antibody Fc Regions
7.5.6 Engineered Antibody Conjugates
7.6 Surface Display Technology
7.6.1 Phage Display
7.6.2 Phage Display Libraries
7.6.3 Alternative Display Systems
7.7 Production of Antibody Fragments
7.8 Bispecific Antibodies
7.9 Molecular Redirection of Immune Cells
7.10 Single-cell Technology and Transgenics
7.11 Conclusion

8 Human and Animal Cell Culture
8.1 Introduction
8.2 Understanding and Standardising //? Vitro Culture
Systems
8.2.1 The Cells
8.2.2 Culture Conditions
8.2.3 Handling and Maintenance
8.2.4 Cryopreservation
8.3 Quality Assurance
8.3.1 Cell Identity Testing and Other Forms of
Characterisation
8.3.2 Microbial Contamination Testing
8.3.3 Quality Assurance of Reagents and
Other Materials
8.3.4 Quality Assurance of Equipment and Methods
8.4 Documentation
8.5 Safety
8.5.1 Risk Assessment
8.5.2 Potential Pathogens
8.5.3 The Cells Themselves
8.5.4 Pressurised Gases
8.5.5 Liquid Nitrogen
8.5.6 Other Chemicals
8.5.7 Breakage/Malfunction of Equipment
8.6 Legal, Ethical and Regulatory Compliance
Contents Contents XIII
8.7 Education and Training
8.8 Conclusion
9 Genome Editing
9.1 Background
9.2 Targeted Mutagenesis Using ZFNs
9.3 Genome Editing with TALENs
9.4 The CRISPR/Cas System
9.5 Case Study: CRISPR/Cas9 Gene Editing in Crop Plants
9.6 Ethical and Regulatory Issues Related to Genome Editing
10 Genome Sequencing
10.1 Introduction
10.2 A Brief History of DNA Sequencing
10.2.1 Early Steps
10.2.2 Developmental Technology
10.2.3 Current Platforms
10.3 Preparing for Next-generation Sequencing
10.3.1 Experimental Design
10.3.2 Library Construction
10.4 Data Analysis
10.4.1 Quality Control of the Raw Reads
10.4.2 Alignment of the Reads to a Reference Sequence
10.4.3 Generation of the Count Table

10.4.4 Differential Gene Expression Analysis
ntroduction to Bioinformatics
Primary and Secondary Databases
          
Contents
11.3 Bioinformatics Data Formats
11.4 NCBI BLAST and PubMed Resources
11.4.1 BLAST
11.4.2 PubMed
11.4.3 OMIM
11.5 Protein Databases
11.5.1 Sequence Alignments and Homology Analysis
11.5.2 Protein Structural Databases
11.6 Specialised Disease-related Databases
11.7 Secondary and Specialised Databases
11.8 Laboratory Bioinformatics
11.8.1 Bioinformatics Resources for PCR
11.8.2 Bioinformatics and NGS Data
11.9 Concluding Remarks

12 Nanotechnology in Medicine
Sara Seriah, Eleni Efthimiadou and Maria Braoudaki
12.1 Introduction
12.2 Nanomaterials
12.2.1 Polymeric Nanoparticles
12.2.2 Dendrimers
12.2.3 Liposomes
12.2.4 Micellar Nanoparticles
12.2.5 Metallic Nanoparticles
12.2.6 Hybrid NPs
12.3 Clinically Approved NPs
12.4 Future Prospects
13 Biosensors
Martin F. Chaplin
13.1 Introduction 357
13.2 The Biological Reaction
13.3 Theory
13.4 Electrochemical Methods
13.4.1 Amperometric Biosensors
13.4.2 Potentiometric Biosensors
13.4.3 Conductimetric Biosensors
Contents Contents XV
13.5 Piezoelectric Biosensors
13.6 Optical Biosensors
13.6.1 Evanescent Wave Biosensors
13.6.2 Surface Plasmon Resonance
13.7 Whole-cell Biosensors
13.8 Receptor-based Sensors
13.9 Conclusion
14 Modelling and Simulation of Proteins
14.1 Introduction
14.2 Prediction of Protein Structure
14.2.1 Comparative Modelling
14.2.2 Threading
14.2.3 Ab Initio Modelling
14.2.4 Contact-based Modelling
14.2.5 Model Assessment and Positive Controls
14.3 Molecular Dynamics Simulation
14.3.1 Computational Representation of Molecules
14.3.2 Simulation Procedures
14.3.3 Limitations of Molecular Dynamics Simulations
14.3.4 Data Analysis of Molecular Dynamics
15 Agricultural Biotechnology
15.1 Introduction
15.2 Today's Agricultural Biotechnologies
15.3 Biotic Stresses
15.3.1 Insect Resistance
15.3.2 Herbicide Tolerance
15.3.3 Other Pathogens
15.4 Tolerance to Abiotic St
15.4.1 Salt Tolerance
15.5 Improved Yield and Nutrition Using Genetic
Modification
15.5.1 Improving Crop Yield Using Genome Editing
Technologies
15.6 Potential to Improve Human Health via the
Biofortification and Nutritional Enhancement of
Food Crops
15.6.1 Golden Rice
15.6.2 Banana21: ‘Golden’ Super Bananas to
Combat Vitamin a Deficiency
15.6.3 Biofortified Maize and Cassava
15.6.4 Nutritionally Enhanced Tomatoes
15.6.5 Designer Oilseed Crops
15.6.6 Plant-made Pharmaceuticals
15.7 Phytoremediation Using Agricultural Biotechnology
15.8 Agricultural Biotechnology and Biofuel Development
15.9 Consumer Acceptance of Plant Biotechnology
15.10 Conclusion
16 Vaccine Design Strategies:
Pathogens to Genomes
16.1 Introduction
16.2 Immunological Considerations in Vaccine Design
16.2.1 Antigen Recognition and Activation of
Innate Immunity
16.2.2 Antigen Processing and the Major
Histocompatibility Complex
16.2.3 Cross-presentation of Antigen by Dendritic Cells
16.2.4 Induction of B and T Cells in Lymph Nodes
16.2.5 Polarisation of T-helper Cells
16.2.6 Protective Immunity Against Acute and Chronic
Infections
16.3 Vaccine Design Strategies
16.3.1 Live Vaccines
16.3.2 Inactivated (Killed) Vaccines
16.3.3 Subunit Vaccines
16.4 Reverse Vaccinology: Genome-based Vaccine Design
16.4.1 Reverse Vaccinology Approach to Vaccine Design.
The Development of the Serogroup B
Meningococcus Vaccine
Contents Contents XVII
 Intellectual Property and
Biotechnology Patents
17.1 Background to Patents
17.2 CRISPR Patents

 17.3 Patent Battles
 17.3.1 Patents at the USPTO
17.3.2 Patents at the EPO
17.4 CRISPR-related Patentscape
17.5 Patent Licensing
Vaccine Design 17.6 It's Not All Over Yet