Essentials of nucleic acid analysis: a robust approach

273 Pages·2008·76.4 MB·English

by  Jacquie T. Keer

Essentials of Nucleic Acid Analysis
A Robust Approach

Essentials of Nucleic Acid
Analysis
A Robust Approach
Edited by
Jacquie T. Keer and Lyndsey Birch
LGC, Teddington, Middlesex, UK
ISBN:978-0-85404-367-5
AcataloguerecordforthisbookisavailablefromtheBritishLibrary
rLGCLimited2008
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Preface
The last two decades have seen an explosion in the use of DNA analysis, with
key applications encompassing forensic science, pathogen identification, food
authenticity and detection of GMOs, personalised medicine and medical diag-
nostics.Itsbroadutilityhasencouragedarapidandsustaineddevelopmentof
thetechnology,withawiderangeoftechniquesandproductsbeingintroduced
each year as well as new technologies emerging from the research base.
Although manyofthecommercialofferingshelptheanalyst, DNAanalysis
remains a complex multi-step process and achieving a valid result is by no
means a trivial task. This book sets out to guide the analyst through the steps
needed to obtain good quality results. The underlying principles for achieving
this goal were formulated by LGC as the six principles for ensuring valid
analytical measurement, which are detailed in the Introduction. How to apply
these principles to DNAanalysis isa corefeatureof thebook. The authors of
each Chapter are practitioners of the art of DNA analysis in areas where the
quality of the result is critical, be it in forensic applications, food analysis or
workingatthehighestinternationallevel,throughLGC’sroleasthedesignated
UKNationalMetrologyInstituteforchemicalandbiochemicalmeasurements.
Their advice is based on first-hand experience of making high-quality meas-
urements, which takes the reader through the essential elements for making
sound, valid DNA measurements, be they qualitative or quantitative. This
updated volume covers topics such as qPCR and microarray analysis, but the
underlying theme remains one of quality to ensure that the correct result is
achieved first time.
The book is designed to serve as a key component in the DNA
analyst’s toolkit for designing, planning and carrying out high-quality DNA
measurement.
Dr John Marriott
Government Chemist
v

Contents
Abbreviations xix
Acknowledgements xxiii
Chapter 1 Valid Analytical Molecular Biology: The Challenge
Jacquie T. Keer
1.1 Introduction 1
1.2 The Analytical Process 2
1.2.1 Analytical Requirements 2
1.2.2 Stages in the Analytical Process 3
1.3 Principles Underpinning Reliable Measurement 4
1.3.1 Understand the Experimental Requirements 5
1.3.2 Use Methods and Equipment which are Fit
for the Intended Purpose 5
1.3.3 Staff Undertaking Analysis Should be
Both Qualified and Competent to Undertake
the Task 5
1.3.4 Regular Independent Assessment of
Laboratory Performance 5
1.3.5 Analytical Consistency 6
1.3.6 Quality Control and Quality Assurance
Framework 6
1.4 Challenges to Measurement Quality 6
1.4.1 Low Concentration of Analyte Compared
to Matrix 6
1.4.2 Complex Matrices 7
1.4.3 DNA Degradation 7
1.4.4 Biological Contamination of the Sample 7
1.4.5 Degradation of Matrix Components 7
1.4.6 Limited Availability of the Sample 8
1.4.7 Lack of Suitable Controls 8
1.5 Focus on Data Quality 8
Acknowledgements 9
vii
viii Contents
Chapter 2 Quality in the Analytical Molecular Biology Laboratory
Sally L. Hopkins
2.1 Introduction 10
2.2 Management Systems 11
2.3 Internationally Recognised Assessed Standards 12
2.3.1 ISO 9001:2000 Quality Management
Systems – Requirements 14
2.3.2 ISO/IEC 17025:2005 General Requirements
for the Competence of Testing and
Calibration Laboratories 15
2.3.3 ISO 15189:2003 Medical Laboratories –
Particular Requirements for Quality and
Competence 16
2.3.4 Principles of Good Laboratory Practice 1999
(GLP) 16
2.3.5 Joint Code of Practice for Research 16
2.4 Selection and Implementation of a Formal
Management System 17
2.4.1 The Management System 18
2.4.1.1 Quality Manual 19
2.4.1.2 Quality Procedures (QPs) 20
2.4.1.3 Standard Operating Procedures
(SOPs) 20
2.4.1.4 Locally Controlled Documentation 22
2.4.2 Laboratory Environment 22
2.4.2.1 Safety 22
2.4.2.2 Spatial Separation 23
2.4.3 Equipment 24
2.4.3.1 Analytical Requirement 24
2.4.3.2 ‘Ownership’ 24
2.4.3.3 Log Books and Maintenance 24
2.4.3.4 Calibration 25
2.4.4 Reagents 25
2.4.4.1 Reagent Quality 25
2.4.4.2 Storage Conditions 26
2.4.4.3 Reagent Traceability 26
2.4.4.4 Stability/Batch Comparability 26
2.4.5 Analysts 26
2.4.5.1 Culture and Competence 26
2.4.5.2 Training and Development 26
2.4.6 Methods 28
2.4.6.1 Fitness for Purpose 28
2.4.6.2 Documentation 28
2.4.6.3 Metrological Traceability 28
2.4.6.4 Independent Quality Assessment 28
Contents ix
2.4.6.5 Method Validation 29
2.4.6.6 Experimental Design 29
2.4.6.7 Measurement Uncertainty 30
2.4.7 Quality Control 30
2.4.7.1 Reference Materials 31
2.4.7.2 In-house Quality Control Materials 31
2.4.7.3 Performance Control 32
2.4.7.4 Contamination Control 33
2.4.8 Samples 33
2.4.8.1 Chain of Custody 33
2.4.8.2 Sampling and Preparation 34
2.4.8.3 Storage 34
2.4.9 Recording and Reporting 35
2.4.9.1 Electronic Data and Automated
Analysis 35
2.4.9.2 Reporting 36
2.4.10 Archiving 36
2.4.10.1 Electronic Data 37
2.5 Summary 37
Acknowledgements 38
References 38
Chapter 3 An Introduction to Method Validation
Sally L. Hopkins and Vicki Barwick
3.1 Introduction 40
3.1.1 Why and When is Method Validation
Necessary? 41
3.1.1.1 Criticality of the Data 42
3.1.1.2 Uniqueness of the Sample 42
3.1.1.3 Robustness of the Technique 42
3.1.1.4 Expected Level of Utilisation of the
Technique 43
3.2 Planning the Validation Process 43
3.3 Method Performance Parameters 43
3.3.1 Precision 44
3.3.1.1 Repeatability 45
3.3.1.2 Reproducibility 46
3.3.1.3 Intermediate Precision 46
3.3.2 Bias 46
3.3.3 Recovery 47
3.3.4 Accuracy 48
3.3.5 Ruggedness (Robustness) Testing 49
3.3.6 Selectivity 49
3.3.7 Detection Limit (Sensitivity) 50