A Low-Cost Approach to PCR: Appropriate Transfer of Biomolecular Techniques

Paperback | January 1, 1999

byEva Harris

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The polymerase chain reaction (PCR) is a technique used to replicate specific pieces of DNA millions of times, which permits the detection and analysis of minute amounts of nucleic acids. Since its introduction in the late 1980s, this technique has been applied not only in molecular biologyresearch but also in fields as diverse as anthropology, phylogeny, and forensics. However, despite the large impact of PCR, many of its applications remain within the confines of research and the academic environment. Now, in A Low-Cost Approach to PCR: Appropriate Transfer of BiomolecularTechniques, Dr. Eva Harris makes this elegantly simple technique more accessible to researchers, physicians, and laboratory workers throughout the world. She provides a description of the theoretical basis of the technique, the practical details of the method, and the philosophy behind thetechnology transfer program that she developed over the last ten years. The book serves as a guide for potential users in developing countries and for scientists in developed countries who may wish to work abroad. In addition, the low-cost approach outlined in this book can be useful for highschool, undergraduate, or continuing education programs in the United States. While the specific applications of PCR outlined in the book are immediately useful to the study of infectious diseases, the approach presented can be generalized to a number of other technologies and situations. The bookwill help laboratories in many areas of the world generate information on site for use by physicians, epidemiologists, public health workers, and health policy professionals to develop new strategies for disease control.

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The polymerase chain reaction (PCR) is a technique used to replicate specific pieces of DNA millions of times, which permits the detection and analysis of minute amounts of nucleic acids. Since its introduction in the late 1980s, this technique has been applied not only in molecular biologyresearch but also in fields as diverse as anth...

Eva Harris is at University of California, Berkeley.

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Format:PaperbackDimensions:328 pages, 9.21 × 6.1 × 0.79 inPublished:January 1, 1999Publisher:Oxford University Press

The following ISBNs are associated with this title:

ISBN - 10:0195119266

ISBN - 13:9780195119268

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Table of Contents

1 Introduction2 PCR Technology2.1. Description of the Technique2.1.1. Historical Overview2.1.2. The Molecular Basis of PCR2.1.3. Advantages and Disadvantages2.2. Technical Details2.2.1. Components of the PCR Mixture2.2.2. Thermal Cycling Parameters2.2.3. Optimizing2.2.4. Potential Problems2.2.5. Detection of Products2.3. Frequently-Used PCR-Based Techniques3 Principles of Sustainable Technology Transfer3.1. A Low-Cost Methodology3.1.1. Appropriate Technology3.1.2. Alternative Techniques3.1.3. Simplification of Protocols3.1.4. In-House Preparation of Reagents3.1.5. Recycling3.1.6. Donated Materials3.2. Knowledge-Based Participatory Transfer Process3.3. Appropriate Application3.3.1. General Considerations3.3.2. An Evaluation Framework3.3.3. Assessment Criteria for PCR3.3.4. Case-by-Case Evaluation of PCR Applied to Infectious Diseases3.4. Intra-Reginal Cooperation4 Case Study: The AMB/ATT Program4.1. Introduction4.2. Program Description4.2.1. Objectives4.2.2. Format4.3. Program Structure4.3.1. Phase I4.3.2. Phase II4.3.3. Phase III4.4. Program Development4.5. Examples of Projects4.6. International CoursesII Selected Protocols5.1. PCR Protocols5.1.1. Overview5.1.2. Equipment, Materials, Control, and Procedures Common to All PCR Protocols5.1.3. Dengue Virus5.1.4. New World Leishmania5.1.5. Mycobactrium tuberculosis5.1.6. Plasmodium falciparum and Plasmodium vivax5.1.7. Vibrio cholerae5.1.8. Diarrheagenic E. coli and Shigella5.1.9. Chlamydia trachomatis and Neisseria gonorrhoeae5.1.10. Leptospira5.1.11. Trypanosoma cruzi5.2. Nonradioactive DNA Probes: V. cholerae Colony Blot5.2.1. Labeling the Probe Using PCR5.2.2. Preparation of the Colony Blot5.2.3. Hybridization5.2.4. Visualization6 Rapid Cloning of PCR Products6.1. Primer Design6.2. Preparation of PCR Products for Cloning6.2.1. Digestion of Vector and PCR Products6.2.2. Preparative Agarose Gel6.2.3. Purification of the Excised DNA Fragments Using Silica Particles6.3. Ligation6.3.1. Checking DNA Fragment Concentration by Agarose Gel Electrophoresis6.3.2. Ligation Reaction6.4. Preparation of Competent Cells and Transformation6.4.1. Preparation of Competent Cells6.4.2. Transformation6.5. Checking Clones by PCR6.6. Plasmid Purification6.7. Analysis of Clones by Restriction Enzyme Digestion6.8. Agarose Gel ElectrophoresisIII AppendixA. Construction of Laboratory EquipmentB. In-House Preparation of ReagentsB.1 Useful FormulasB.2 SolutionsB.3 Preparation of Selected ReagentsB.4 DNA Size MarkersC. Inventory for a PCR LaboratoryD. Good Laboratory PracticeD.1 General TipsD.2 Calibration of Adjustable PipettorsE. Prevention of Cross-ContaminationF. PCR Troubleshooting Guide and Flow-ChartG. Workshop Organization and Teaching TipsH. Sample Charts and WorksheetsI. Useful World Wide Web Sites

Editorial Reviews

"The polymerase chain reaction (PCR) is a technique used to replicate specific pieces of DNA millions of times, which permits the detection and analysis of minute amounts of nucleic acids. . . . [T]his technique has been applied not only in molecular biology research but also in fields asdiverse as anthropology, phylogeny and forensics. However, despite the large impact of PCR, many of its applications remain within the confines of research and the academic environment. This book attempts to make the PCR technique more accessible to researchers, physicians and laboratory workersthroughout the world by providing a description of the theoretical basis of the technique, practical details of the method, and the philosophy behind the technology transfer programme that the author has developed over the last 10 years. . . . The book should help laboratories in many areas of theworld generate information on site for use by physicians, epidemiologists, public health workers . . ."--CAB Abstracts