Principles and Technical Aspects of PCR Amplification 1st Edition by Elizabeth van Pelt Verkuil, Alex van Belkum, John P Hays ISBN 9781402062414 by Elizabeth Van Pelt-verkuil, Alex Van Belkum, John P. Hays 9789048175796, 9048175798 instant download after payment.

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ISBN 13: 9781402062414
Author: Elizabeth van Pelt Verkuil, Alex van Belkum, John P Hays

Kary Mullis was awarded a Nobel Prize for inventing the PCR technique more than 15 years ago in 1993. Since its "discovery", multiple adaptations and variations of the standard PCR technique have been described, with many of these adaptations and variations currently being used in clinical, diagnostic and academic laboratories across the world. Further, these techniques are being applied at the diagnostic level (e.g. as high throughput testing methodologies to detect minimum residual disease, the presence/absence of specific pathogens etc), as well as to increase our understanding of fundamental disease processes.

Frequently, PCR technicians and specialists limit their understanding of PCR to one particular methodology. However, this approach limits their appreciation of the range of versatile PCR techniques currently available, techniques that may be applicable and indeed more suitable to their own laboratory situation.

This manual aims to provide thereader with a guide to the standard PCR technique and its many available modifications, with particular emphasis on the role of PCR techniques in the diagnostic laboratory (the central theme of this manual). Further, many important technical issues have been addressed, including types of PCR template material, PCR optimization, the analysis of PCR products, quality control and quality assurance, variants and adaptations of the standard PCR protocol, quantitative PCR and in situ PCR. The reader of this manual will be excellently informed about the fundamental principles of PCR and the true potential of PCR within clinical laboratory practice.

Principles and Technical Aspects of PCR Amplification 1st Table of contents:

Chapter 1: The Polymerase Chain Reaction

• An Overview of the PCR Process
• Before PCR and Beyond

Chapter 2: A Brief Comparison Between In Vivo DNA Replication And In Vitro PCR Amplification

• Similarities
• Differences

Chapter 3: The PCR In Practice

• The Basic PCR Reaction
• PCR Thermocycling Parameters (Denaturation, Annealing, Extension)
• PCR Machines (Thermal Cyclers)

Chapter 4: The Different Types And Varieties Of Nucleic Acid Target Molecules

• DNA Targets
• RNA Targets (and Reverse Transcription PCR)
• Sample Preparation and Template Quality

Chapter 5: PCR Primers

• Primer Design Principles
• Primer Properties (Tm, GC content, specificity)
• Primer Concentration and Optimization

Chapter 6: Deoxynucleotide Triphosphates And Buffer Components

• Role of dNTPs
• PCR Buffer Composition (pH, salts, MgCl2)
• Additives for PCR Enhancement

Chapter 7: Taq And Other Thermostable DNA Polymerases

• Characteristics of Taq Polymerase
• Other Thermostable Polymerases and their properties (fidelity, processivity)
• Enzyme Selection and Concentration

Chapter 8: Important Considerations For Typical, Quantitative And Real-Time PCR Protocols

• Optimization Strategies (Annealing temperature, MgCl2, template concentration)
• Hot Start PCR
• Nested PCR
• Multiplex PCR
• Quantitative PCR (qPCR) Principles
• Real-Time PCR Detection Methods

Chapter 9: Analysis Of PCR Amplification Products

• Gel Electrophoresis Methodologies
• Fluorescent or Hapten Labelled Amplimers
• Probe Hybridization Methodologies
• Real-Time Analysis of PCR Amplimers (Intercalating Dyes, FRET, TaqMan probes)
• Nucleic Acid Sequencing of PCR Products

Chapter 10: Ensuring PCR Quality - Laboratory Organisation, PCR Optimisation And Controls

• The Primary Level of Quality Control - Laboratory Organization and the Prevention of PCR Contamination
  • Sources and Routes of Contamination
  • Detecting and Preventing PCR Contamination
• The Secondary Level of PCR Quality Control - PCR Design and Optimization
  • Extrinsic and Intrinsic Factors
  • Developmental Steps to Achieve High Quality PCR Results
  • Use of Positive and Negative Controls
  • Causes and Solutions for False Positive and False Negative PCR Results
• Quality Considerations Specific for RT-PCR Methodologies

Chapter 11: Ensuring PCR Quality - Quality Criteria And Quality Assurance

• Validation of PCR Assays
• Documentation and Record Keeping
• Accreditation and Regulatory Aspects

Chapter 12: Variants And Adaptations Of The Standard PCR Protocol

• Overview of Various PCR Techniques
• Examples of Specialized PCR Applications

Chapter 13: In Situ PCR Amplification (ISA) - Major Considerations, Sample Processing And Applications

• Principles of In Situ PCR
• Sample Preparation for In Situ PC

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Tags: Elizabeth van Pelt Verkuil, Alex van Belkum, John P Hays, Principles, Amplification