Essentials of Nucleic Acid Analysis: A Robust Approach - Hardcover

Hardcover

ISBN 10: 0854043675 ISBN 13: 9780854043675

Verlag: Royal Society of Chemistry, 2008

Inhaltsangabe

Over the last decade there has been a rapid development of molecular techniques, with an increasing range of instrumentation now available. The development of accompanying reference literature has not kept pace with technological advances and this poses significant challenges to the analyst. 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. The authors of each chapter are practitioners of the art of DNA analysis in areas where the quality of the result is critical. 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. This book provides an indispensable handbook and premier reference for those working in the widely varying areas and specifically in the fields of food analysis and forensic applications.

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Über die Autorin bzw. den Autor

Dr Jacquie T Keer has a background in biochemistry and molecular biology, having done a degree at Oxford and a PhD at St Mary's Hospital Medical School. Dr Jacquie T Keer has a broad academic research background. Her areas of experience include medical microbiology, mammalian molecular genetics, protein analysis and all aspects of DNA and RNA analysis. Dr Jacquie T Keer has subsequently spent several years working in the area of applied molecular biology in support of the National Measurement System at LGC, in its role as the National Measurement Institute for chemical and biological analysis. Dr Jacquie T Keer has been responsible for managing a number of projects including, development of AFLP methods for the detection of Salmonella and VTEC, evaluation of SERRS for highly multiplexed biological assays and detection and quantification of Host Cell protein and DNA in biological products. Areas of expertise include application of real time PCR to the detection and quantification of trace level targets, and development of proficiency testing schemes for the molecular laboratory. Dr Lyndsey Birch has a background in Biochemistry, with both a degree and PhD from Liverpool. Her research work there focussed on cell cycle control and signal transduction pathways, and she has since broadened her experience to include development of novel methods for PCR analysis based on solid-phase capture systems in an in emerging biotechnology context. Dr Lyndsey Birch has subsequently spent several years working in the area of applied molecular biology in support of the National Measurement System at LGC, in its role as the National Measurement Institute for chemical and biological analysis. Areas of expertise include application of real time PCR to the detection and quantification of trace level targets, and development of proficiency testing schemes for the molecular laboratory.

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Over the last decade there has been a rapid development of molecular techniques, with an increasing range of instrumentation now available. The development of accompanying reference literature has not kept pace with technological advances and this poses significant challenges to the analyst. Essentials of Nucleic Acid Analysis A Robust Approach 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. The authors of each chapter are practitioners of the art of DNA analysis in areas where the quality of the result is critical. 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. This book provides an indispensable handbook and premier reference for those working in the widely varying areas and specifically in the fields of food analysis and forensic applications.

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Essentials of Nucleic Acid Analysis

A Robust Approach

By Jacquie T. Keer, Lyndsey Birch

The Royal Society of Chemistry

Copyright © 2008 LGC Limited
All rights reserved.

ISBN: 978-0-85404-367-5

Contents

Abbreviations, xix, 
Acknowledgements, xxiii, 
Chapter 1 Valid Analytical Molecular Biology: The Challenge Jacquie T. Keer, 
Chapter 2 Quality in the Analytical Molecular Biology Laboratory Sally L. Hopkins, 
Chapter 3 An Introduction to Method Validation Sally L. Hopkins and Vicki Barwick, 
Chapter 4 DNA Extraction Ginny C. Saunders and Jennifer M. Rossi, 
Chapter 5 DNA Quantification Paul A. Heaton and Jacquie T. Keer, 
Chapter 6 PCR: Factors Affecting Reliability and Validity Charlotte L. Bailey, Lyndsey Birch and David G. McDowell, 
Chapter 7 Quantitative Real-time PCR Analysis Jacquie T. Keer, 
Chapter 8 Multiplex PCR and Whole Genome Amplification Lyndsey Birch, Charlotte L. Bailey and Morten T. Anderson, 
Chapter 9 Procedures for Quality Control of RNA Samples for Use in Quantitative Reverse Transcription PCR Tania Nolan and Stephen Bustin, 
Chapter 10 Microarrays Sally L. Hopkins and Charlotte L. Bailey, 
Subject Index, 240, 

CHAPTER 1

Valid Analytical Molecular Biology: The Challenge

JACQUIE T. KEER

LGC, Queens Road, Teddington, TW11 0LY

1.1 Introduction

The last decade has seen a rapid increase in the pace of technological advancement and in the uptake of DNA analysis for a range of applications. The increased use of DNA as an analyte reflects its uniform presence in almost all cells of most organisms. In addition the greater stability of DNA, compared to RNA or protein molecules, is ideal for analysis of highly processed or aged samples.

Technical innovations include the development of more sensitive, quantitative, high-throughput and massively parallel analyses, all generating new applications and commercial opportunities and covering a wide range of uses. The complete DNA sequence of many genomes has been determined, opening the way for a plethora of new applications, including directed drug discovery and personalised genetic diagnostics and treatment. Forensic analysis, food testing and agriculture are just a few of the many other areas where DNA technology is being adopted, with concomitant changes in regulation and procedures. It is clear that there are significant advantages in using molecular methods, including reduced detection limits, greater speed and scale, lower cost and improved specificity. The potential of novel genetic diagnostic methods, directed drug discovery routes and the increased throughput of massively parallel array-based analyses are strong drivers for even greater uptake of this technology. However, to exploit fully the potential of these developments and remove barriers to wider uptake, there is a need to ensure that molecular analytical methods are reliable, consistent and fit for purpose, in order to avoid the use of biased or flawed techniques and resultant loss of confidence in the techniques.

The majority of technological development occurs in academic or medical research environments, where the main priority is innovation. Consequently little consideration is given to the more routine applicability, reliability and reproducibility of methods, particularly in the early stages of development. Despite evaluation of method performance characteristics and method validation being a prerequisite for the successful move of techniques from the research laboratory to the analytical laboratory, there is resistance to such formal evaluation in some sectors. There are also practical barriers to assessment of method performance, including the lack of reference materials which are necessary for the critical comparison of analytical approaches and the paucity of performance standards in the wider analytical community, as most regulation of analysis is carried out in-house. However, in the light of growing commercial and clinical application, consideration is increasingly being given to the reliability of the technology being used.

Although large volumes of analytical data may be produced from poorly applied methods, generation of dependable results usually requires careful and considered planning and validation. The aim of any experiment is to produce reliable results, and to avoid the need to repeat the analysis because of problems with the reagents, method or equipment used. Consistently 'getting it right first time' depends on a number of factors, including provision of a controlled laboratory environment with calibrated and regularly maintained instruments, use of an effective experimental design and performance of the work by an analyst with sufficient training and experience to correctly perform the method and interpret the result (Figure 1.1). Although it is difficult to estimate the actual cost of poor laboratory practice in wasted time and reagents, the benefits in avoiding repeating work are very clear.

This manual aims to introduce and address quality assurance and validation issues that arise in the application of DNA technology, and to provide a basis for the development of validated methods and experimental good practice. Specifically, Chapters 2 and 3 cover the benefits of formal laboratory management systems and method validation. The remaining chapters in the manual provide information on a range of commonly used techniques, from the initial extraction of DNA from analytical samples and quantification of the amount of DNA present, to a range of downstream processes including various forms of polymerase chain reaction (PCR) amplification and microarray-based analysis.

Analytical laboratories should work to produce quality analytical data, and reading the information presented here should provide a firm foundation for good experimental practice.

1.2 The Analytical Process

1.2.1 Analytical Requirements

Analysis is usually initiated by a 'customer', who can be a private individual or company, public organisation, research funding body or law enforcement agency such as a police force or trading standards office. The results that are produced are usually required for a specific purpose, often as an independent source of information in order to gauge a situation, interpret evidence, determine whether action is required or to ascertain whether certain regulations are being adhered to. Increasingly, some indication of the level of confidence that can be placed in the result is also required, allowing the results of the experiment to be used or interpreted appropriately.

1.2.2 Stages in the Analytical Process

In undertaking an experiment or analysis to address a specific question, a complex procedure is undertaken, beginning with the initial researching of the questions and specific analytical requirements and ending with the interpretation of the analytical data produced and the reporting of results and conclusions. To ensure the process is efficient, careful planning of the work is required. Good experimental design, trained staff and use of suitable methods, equipment, standards and samples can save time in ensuring that sufficient and reliable results are produced first time. A flawed approach may produce experimentally valid data that do not directly address the enquiry, or insufficient data for confident interpretation. Incorrect sample collection or storage could produce erratic results even when a valid method is applied. In addition, use of uncalibrated equipment could generate biased results that...

Details zur Ausgabe

• Verlag: Royal Society of Chemistry
• Erscheinungsdatum: 2008
• Sprache: Englisch
• ISBN 10: 0854043675
• ISBN 13: 9780854043675
• Einband: Gebundene Ausgabe
• Anzahl der Seiten: 272
• Herausgeber: Keer Jacquie T, Birch Lyndsey
• Kontakt zum Hersteller: Nicht verfügbar
• Verantwortliche Person: GPSR Kontakt; gpsr@libri.de; ; Europaallee 1; Bad Hersfeld; 36244; Deutschland