The polymerase chain reactionis a revolutionary technique for enabling the detection and analysis of minute amounts of nucleic acids. Here, Eva Harris makes this elegantly simple technique accessible to researchers, physicians, and laboratory workers throughout the world.

1 Introduction 2 PCR Technology 2.1. Description of the Technique 2.1.1. Historical Overview 2.1.2. The Molecular Basis of PCR 2.1.3. Advantages and Disadvantages 2.2. Technical Details 2.2.1. Components of the PCR Mixture 2.2.2. Thermal Cycling Parameters 2.2.3. Optimizing 2.2.4. Potential Problems 2.2.5. Detection of Products 2.3. Frequently-Used PCR-Based Techniques 3 Principles of Sustainable Technology Transfer 3.1. A Low-Cost Methodology 3.1.1. Appropriate Technology 3.1.2. Alternative Techniques 3.1.3. Simplification of Protocols 3.1.4. In-House Preparation of Reagents 3.1.5. Recycling 3.1.6. Donated Materials 3.2. Knowledge-Based Participatory Transfer Process 3.3. Appropriate Application 3.3.1. General Considerations 3.3.2. An Evaluation Framework 3.3.3. Assessment Criteria for PCR 3.3.4. Case-by-Case Evaluation of PCR Applied to Infectious Diseases 3.4. Intra-Reginal Cooperation 4 Case Study: The AMB/ATT Program 4.1. Introduction 4.2. Program Description 4.2.1. Objectives 4.2.2. Format 4.3. Program Structure 4.3.1. Phase I 4.3.2. Phase II 4.3.3. Phase III 4.4. Program Development 4.5. Examples of Projects 4.6. International Courses II Selected Protocols 5.1. PCR Protocols 5.1.1. Overview 5.1.2. Equipment, Materials, Control, and Procedures Common to All PCR Protocols 5.1.3. Dengue Virus 5.1.4. New World Leishmania 5.1.5. Mycobactrium tuberculosis 5.1.6. Plasmodium falciparum and Plasmodium vivax 5.1.7. Vibrio cholerae 5.1.8. Diarrheagenic E. coli and Shigella 5.1.9. Chlamydia trachomatis and Neisseria gonorrhoeae 5.1.10. Leptospira 5.1.11. Trypanosoma cruzi 5.2. Nonradioactive DNA Probes: V. cholerae Colony Blot 5.2.1. Labeling the Probe Using PCR 5.2.2. Preparation of the Colony Blot 5.2.3. Hybridization 5.2.4. Visualization 6 Rapid Cloning of PCR Products 6.1. Primer Design 6.2. Preparation of PCR Products for Cloning 6.2.1. Digestion of Vector and PCR Products 6.2.2. Preparative Agarose Gel 6.2.3. Purification of the Excised DNA Fragments Using Silica Particles 6.3. Ligation 6.3.1. Checking DNA Fragment Concentration by Agarose Gel Electrophoresis 6.3.2. Ligation Reaction 6.4. Preparation of Competent Cells and Transformation 6.4.1. Preparation of Competent Cells 6.4.2. Transformation 6.5. Checking Clones by PCR 6.6. Plasmid Purification 6.7. Analysis of Clones by Restriction Enzyme Digestion 6.8. Agarose Gel Electrophoresis III Appendix A. Construction of Laboratory Equipment B. In-House Preparation of Reagents B.1 Useful Formulas B.2 Solutions B.3 Preparation of Selected Reagents B.4 DNA Size Markers C. Inventory for a PCR Laboratory D. Good Laboratory Practice D.1 General Tips D.2 Calibration of Adjustable Pipettors E. Prevention of Cross-Contamination F. PCR Troubleshooting Guide and Flow-Chart G. Workshop Organization and Teaching Tips H. Sample Charts and Worksheets I. Useful World Wide Web Sites

Eva HarrisFaculty, School of Public Health, University of California, Berkeley

"Makes the polymerase chain reaction (PCR) technique more accessible to researchers, physicians, and laboratory workers throughout the world. Describes the theoretical basis of the technique, practical details, and the philosophy behind the technology transfer program that the author has developed over the past 10 years. A low-cost approach is outlined for use in the study of infectious diseases, which can be applied to other technologies and applications. This approach is especially useful for laboratories in developing countries, and in high school undergraduate and continuing education programs in the US. Appendices give information on construction of equipment, and contain sample charts and worksheets. Includes b&w photos of equipment, and of men and women performing lab procedures. The author is on the faculty of the School of Public Health at UC-Berkeley."--SciTech Book News |k No