Analytical Molecular Biology -- Contents -- Chapter 1 An Introduction to Analytical Molecular Biology -- 1.1 Introduction -- 1.2 What is Analysis, Why is it Undertaken? -- 1.3 DNA, a Universal Biological Analyte -- 1.4 Sectoral Applications of Analytical Molecular Biology Techniques -- 1.5 Challenges of DNA Analysis -- 1.6 Key Techniques in Analytical Molecular Biology -- 1.7 Future Prospects and Considerations -- 1.8 References and Further Reading -- Chapter 2 Quality in the Analytical Molecular Biology Laboratory -- 2.1 Introduction -- 2.2 VAM Principles -- 2.3 Analytical Context -- 2.4 Method Validation -- 2.4.1 Measures of Validity -- 2.4.2 Valid or Not Valid? -- 2.5 Quality Assurance -- 2.5.1 Calibration -- 2.5.2 Contamination -- 2.5.3 Types of Quality Assurance Standards and Accreditation Schemes -- 2.5.3.1 ISO 9000 Series of Quality System Standards -- 2.5.3.2 Organisation of Economic Co-operation and Development - Good Laboratory Practice -- 2.5.3.3 National Accreditation of Measurement and Sampling -- 2.5.3.4 Clinical Pathology Accreditation Standards -- 2.5.4 Proficiency Testing Schemes -- 2.6 Reference Materials -- 2.6.1 Alternatives to Reference Materials -- 2.7 Summary -- 2.8 Further Reading and Information -- 2.9 References -- Chapter 3 DNA Extraction -- 3.1 Introduction -- 3.2 The Various Steps of DNA Extraction -- 3.3 Choosing the Most Appropriate DNA Extraction Procedure -- 3.4 Validation Issues Arising at the Various Stages of DNA Extraction -- 3.4.1 Sample Storage -- 3.4.2 Sample Preparation -- 3.4.3 Cell and Membrane Lysis -- 3.4.4 Separation of Nucleic Acids from Cell and Matrix Debris -- 3.4.5 Additional Purification of DNA -- 3.4.6 Precipitation and Concentration of DNA -- 3.5 Summary -- 3.6 Genomic DNA Extraction Protocols -- 3.7 References -- Chapter 4 Quantification of Total DNA by Spectroscopy -- 4.1 Introduction.

4.2 Determining DNA Concentration by Ultraviolet Spectroscopy -- 4.2.1 Determining the Extinction Coefficient -- 4.2.2 Practical Aspects of Measuring DNA Concentrations by UV Spectroscopy -- 4.3 Determination of DNA Concentration by Fluorescence Spectroscopy -- 4.3.1 Practical Aspects of Measuring DNA Concentrations by Fluorescence Spectroscopy -- 4.3.2 Preparation of a Calibration Graph -- 4.3.3 Fluorescent Dyes -- 4.3.3.1 Ethidium Bromide -- 4.3.3.2 PicoGreen -- 4.3.3.3 Hoechst 33258 -- 4.4 Summary -- 4.5 References -- Chapter 5 PCR: Factors Affecting Reliability and Validity -- 5.1 Introduction to the Polymerase Chain Reaction -- 5.2 An Illustrative Basic Amplification Protocol -- 5.3 Critical Factors Affecting Amplification and Reproducibility -- 5.3.1 Reaction Components -- 5.3.1.1 Magnesium Chloride -- 5.3.1.2 Thermostable DNA Polymerases -- 5.3.1.3 Deoxynucleotide Triphosphates -- 5.3.1.4 DNA Target -- 5.3.2 Primer Design and Target Selection -- 5.3.3 Thermal Cyclers: Factors Affecting Temperature Control -- 5.3.3.1 Temperature Profile -- 5.3.3.2 Temperature Control -- 5.3.3.3 Ramp Rate -- 5.3.3.4 Alternative Thermal Cyclers -- 5.3.3.5 Summary of Guidelines to Achieve Correct Thermal Profiles -- 5.3.4 Contamination in PCR: Good Housekeeping and Preventative Measures -- 5.3.5 Polymerases: Features Affecting Selection and Use -- 5.3.6 Specificity -- 5.3.6.1 Hot Start PCR -- 5.4 Concluding Comments -- 5.5 References -- Chapter 6 Inhibitors and Enhancers of PCR -- 6.1 Introduction -- 6.2 Factors that Inhibit or Enhance PCR -- 6.2.1 Protocol for Assessing the Effects of PCR Inhibitors and Enhancers -- 6.2.2 A Review of Inhibitors -- 6.2.2.1 Molecular Biological Reagents -- 6.2.2.2 Gel Loading Dyes -- 6.2.2.3 UV-damaged Mineral Oil -- 6.2.2.4 Bodily Fluids -- 6.2.2.5 Faeces -- 6.2.2.6 Food Samples -- 6.2.2.7 Culture Media.

6.2.2.8 Wooden Toothpicks -- 6.2.2.9 Plant Polysaccharides -- 6.2.2.10 Polyphosphates in Fungi -- 6.2.2.11 Humic Acid -- 6.2.2.12 Pollen -- 6.2.3 A Review of Approaches for Overcoming Inhibition and Enhancing PCR -- 6.2.3.1 Tetramethylammonium Chloride -- 6.2.3.2 Betaine -- 6.2.3.3 Glycerol -- 6.2.3.4 DMSO -- 6.2.3.5 Formamide -- 6.2.3.6 Non-ionic Detergents -- 6.2.3.7 Protein Additives: Bovine Serum Albumin and gp32 -- 6.2.3.8 Spermine/Spermidine -- 6.2.3.9 Nucleotide Analogues -- 6.3 Concluding Comments -- 6.4 References -- Chapter 7 Random Amplified Polymorphic DNA Analysis -- 7.1 Introduction -- 7.1.1 RAPD Reproducibility and Repeatability -- 7.2 Applications of RAPD Analysis -- 7.3 RAPD Methodology -- 7.3.1 Extraction of DNA -- 7.3.2 Estimation of DNA Concentration -- 7.3.3 Optimisation of PCR-RAPD Cycling Conditions -- 7.3.4 Trouble Shooting During Optimisation -- 7.3.5 Visualisation of RAPD Profiles -- 7.3.6 Analysis of RAPD Data -- 7.4 Validation Issues of RAPD Analysis -- 7.4.1 DNA Quality and Quantity -- 7.4.2 Biological Contamination -- 7.4.3 Taq Polymerase Enzyme -- 7.4.4 Thermal Cyclers -- 7.4.5 Primer Concentration and Design -- 7.4.6 Denaturation Time -- 7.4.7 Annealing Temperature -- 7.4.8 Ramp Rate -- 7.4.9 Extension Time -- 7.4.10 Intensities of Amplified Products -- 7.4.11 The Addition of PCR Enhancers -- 7.4.12 Interpretation of RAPD Profile Data -- 7.5 Related Technologies -- 7.6 Concluding Comments -- 7.7 References -- Chapter 8 Development of Multiplex PCR -- 8.1 Introduction -- 8.2 Design of a Multiplex PCR -- 8.2.1 Choosing Targets -- 8.2.2 Position of Primers -- 8.2.3 Primer Design -- 8.2.4 Initial PCR Development -- 8.2.5 Reaction Components -- 8.2.6 Cycling Parameters -- 8.2.7 Untemplated Nucleotide Addition -- 8.2.8 Overcoming Mis-priming Events -- 8.3 Detection Strategies -- 8.3.1 Agarose Gel Electrophoresis.

8.3.2 Polyacrylamide Gel Electrophoresis -- 8.3.3 Solid Phase Capture -- 8.4 Summary -- 8.5 References -- Chapter 9 Membrane Hybridisation -- 9.1 Introduction -- 9.1.1 Hybridisation Theory -- 9.1.2 Kinetics of Hybridisation -- 9.1.2.1 Hybridisation with a Single Membrane-bound Sequence -- 9.2 The Various Stages of Membrane Hybridisation -- 9.2.1 DNA Extraction -- 9.2.2 Nucleic Acid Gel Electrophoresis -- 9.2.3 Immobilisation of DNA onto the Membrane -- 9.2.4 Preparation and Labelling of Nucleic Acid Probes -- 9.2.5 Hybridisation -- 9.2.6 Detection -- 9.3 Validation of Membrane Hybridisation -- 9.3.1 Introduction -- 9.3.2 DNA Extraction -- 9.3.3 Immobilisation of Nucleic Acid to Membrane -- 9.3.3.1 Membrane Type -- 9.3.3.2 Transfer of Nucleic Acid to Membrane -- 9.3.3.3 Method of Immobilisation -- 9.3.3.4 Size and Nature of the Nucleic Acid -- 9.3.4 Nucleic Acid Probes -- 9.3.4.1 Probe Production and Labelling -- 9.3.4.2 Probe Concentration -- 9.3.5 Hybridisation and Washing -- 9.3.5.1 Temperature and Ionic Strength: Effects on Hybridisation Rate -- 9.3.5.2 Temperature and Ionic Strength: Effects on Hybridisation Stringency -- 9.3.5.3 Temperature Control -- 9.3.5.4 Other Components of Hybridisation Solutions -- 9.3.5.5 Time Period of Hybridisation -- 9.3.5.6 Re-use of Probe and Membranes -- 9.3.6 Detection -- 9.4 Summary -- 9.5 References -- Chapter 10 Automated Fluorescent DNA Cycle Sequencing -- 10.1 Introduction -- 10.2 Development of a Fluorescent DNA Sequencing Protocol -- 10.2.1 Template Preparation -- 10.2.1.1 Plasmid DNA as a Sequencing Template -- 10.2.1.2 PCR Products as Sequencing Templates -- 10.2.2 The Fluorescent Cycle Sequencing Reaction -- 10.2.3 Template Sequence Composition -- 10.2.4 Post-reaction Purification -- 10.2.5 Gel Preparation -- 10.2.6 Data Analysis and Interpretation -- 10.3 Summary -- 10.4 References.

Appendix: Glossary of Terms -- Subject Index.