Essentials of Nucleic Acid Analysis sets out to guide the analyst through the steps needed to obtain good quality results in DNA analysis.
The underlying principles for achieving this goal were formulated by LGC (formerly the Laboratory of the Government Chemist) as the six principles for ensuring valid analytical measurement, which are detailed in the introduction.
The reader is also provided with guidelines for method validation and quality control of established and emerging DNA measurement techniques.
Technical details and examples of application of key techniques in nucleic acid analysis are provided while highlighting best practice, available standards and practical advice on improving measurement quality.
Contents
1. Valid Analytical Molecular Biology: The Challenge
- The Analytical Process
- Principles Underpinning Reliable Measurement
- Challenges to Measurement Quality
2. Quality in the Analytical Molecular Biology Laboratory
- Management Systems
- Internationally Recognised Assessed Standards
- 1 ISO 9001:2000 Quality Management Systems – Requirements
- Selection and Implementation of a Formal Management System
3. An Introduction to Method Validation
- Planning the Validation Process
- Method Performance Parameters
- Validation in Practice
4. DNA Extraction
- Steps of the DNA Extraction Process
- Choosing an Appropriate DNA Extraction Procedure
- Validation Issues Arising at the Various Stages of DNA Extraction
- Automation of DNA Extraction
- DNA Extraction Protocols
5. DNA Quantification
- Measurement of DNA Concentration Using Ultraviolet Spectroscopy
- Determination of DNA Concentration by Fluorescence Spectroscopy
- Quantification Using the Polymerase Chain Reaction
- Enzymatic Quantification of DNA
- Primary Methods of DNA Quantification
- DNA Quantification by Constituent Phosphorus Determination
- Comparability of DNA Measurement Methods
6. PCR: Factors Affecting Reliability and Validity
- The Amplification Protocol
- DNA Template
- Reaction Components and Conditions Affecting
- Hot-start Mechanisms
- Contamination Control
- Post-PCR Analysis
7. Quantitative Real-time PCR Analysis
- Approaches to Product Detection
- Range of Instruments
- Practical Aspects of qPCR Analysis
- Quantification of Low Levels of Target Analyte
- Standards and Comparability
8. Multiplex PCR and Whole Genome Amplification
- Introduction to Multiplex PCR
- Design and Optimisation of mPCR
- Detection Strategies
- Applications of mPCR
- Advantages and Disadvantages of mPCR
- Introduction to WGA
- WGA Methodologies
- WGA Applications and Characteristics
9. Procedures for Quality Control of RNA Samples for Use in Quantitative Reverse Transcription PCR
- RNA Extraction Approaches
- RNA Quality
- RNA Quantification
- Effect of RT Experimental Design on qPCR Data
10. Microarrays
Index
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