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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

A catalogue record for this book is available from the British Library

r LGC Limited 2008

All rights reserved

Apart from fair dealing for the purposes of research for non-commercial purposes or for
private study, criticism or review, as permitted under the Copyright, Designs and Patents
Act 1988 and the Copyright and Related Rights Regulations 2003, this publication may not
be reproduced, stored or transmitted, in any form or by any means, without the prior
permission in writing of The Royal Society of Chemistry, or the Copyright owner, or in the
case of reproduction in accordance with the terms of licences issued by the Copyright
Licensing Agency in the UK, or in accordance with the terms of the licences issued by the
appropriate Reproduction Rights Organization outside the UK. Enquiries concerning
reproduction outside the terms stated here should be sent to The Royal Society of
Chemistry at the address printed on this page.

Published by The Royal Society of Chemistry,

Thomas Graham House, Science Park, Milton Road,
Cambridge CB4 0WF, UK

Registered Charity Number 207890

For further information see our website at www.rsc.org

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. Its broad utility has encouraged a rapid and sustained development of
the technology, with a wide range of techniques and products being introduced
each year as well as new technologies emerging from the research base.
Although many of the commercial offerings help the analyst, DNA analysis
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 DNA analysis is a core feature of the book. 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
working at the highest international level, through LGC’s role as the designated
UK National Metrology Institute for chemical and biochemical measurements.
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
x Contents
3.3.8 Working Range and Linearity 51
3.3.9 Measurement Uncertainty 52
3.4 Validation in Practice 53
3.4.1 Outline of the Procedure 53
3.4.1.1 Define the Analytical Requirement 53
3.4.1.2 Write a Draft Protocol 54
3.4.1.3 Investigate the Robustness of the
Technique, and Identify the Critical
Parameters 54
3.4.1.4 Identify Relevant Performance
Parameters, and Determine the Order
of Investigation 54
3.4.1.5 Assess the Performance Characteris-
tics Using Suitable ‘Known’ Materi-
als (RMs, Standards, Spikes) 54
3.4.1.6 Assess Whether the Data Show the
Method is Fit for Purpose 55
3.4.1.7 Define the Limitations of the
Methodology 57
3.4.1.8 Document the Final Protocol and
Method Validation Results 57
Acknowledgements 57
References 57

Chapter 4 DNA Extraction

Ginny C. Saunders and Jennifer M. Rossi

4.1 Introduction 59
4.1.1 Concentration or Amount 60
4.1.2 Purity 60
4.1.3 Integrity 60
4.2 Steps of the DNA Extraction Process 61
4.2.1 Sample Preparation 61
4.2.2 Cell or Membrane Lysis 61
4.2.3 Protection and Stabilisation of Released
DNA 61
4.2.4 Separation of Nucleic Acids from Cell Debris
or Sample Matrix 61
4.2.5 Purification of DNA 61
4.2.6 Concentration of DNA 62
4.3 Choosing an Appropriate DNA Extraction Procedure 62
4.3.1 The History of the Sample 62
4.3.2 The Composition of the Sample 62
4.3.3 Time and Resources Available 63
4.3.4 Standardised Techniques 63
Contents xi
4.3.5 Subsequent Analytical Procedures 63
4.3.6 Potential Impact of Methodology 63
4.4 Validation Issues Arising at the Various Stages
of DNA Extraction 66
4.4.1 Sample Storage 66
4.4.1.1 Incorrect Sample Storage
Temperature 66
4.4.1.2 Incorrect Sample Storage
Environment 66
4.4.2 Sample Preparation 66
4.4.2.1 Homogeneity of Sample 67
4.4.2.2 Surface Area to Lysis Forces Ratio 67
4.4.2.3 Cell or Nucleic Acid Adherence to
Matrix Material 67
4.4.2.4 Contamination 67
4.4.3 Cell and Membrane Lysis 68
4.4.3.1 Inaccessibility of Cells to Lysis Forces 68
4.4.3.2 Type and Amount of Detergent or
Denaturant Used 68
4.4.3.3 Concentration and Activity of Lytic
Enzyme 68
4.4.3.4 Concentration of EDTA in
Extraction Buffer 69
4.4.3.5 Concentration of Salt in Extraction
Buffer 70
4.4.3.6 Extraction Buffer pH 70
4.4.3.7 Excessive Damage of the DNA
Analyte 70
4.4.4 Separation of Nucleic Acids from Cell and
Matrix Debris 71
4.4.4.1 Phenol Quality 71
4.4.4.2 Inefficient Phenol Extraction and
Removal 71
4.4.5 Additional Purification of DNA 71
4.4.5.1 Composition of Extraction Buffer 72
4.4.5.2 Column Cleaning 72
4.4.5.3 RNase Treatment of the Sample 72
4.4.6 Precipitation and Concentration of DNA 74
4.4.6.1 Volume and Temperature of Alcohol
Used and Precipitation Times 74
4.4.6.2 Concentration and Type of Salt 74
4.4.6.3 Degraded DNA 74
4.4.6.4 DNA Concentration 75
4.4.6.5 Pellet Loss 75
4.4.6.6 Pellet Incompletely Re-suspended 75
4.4.6.7 Alcohol Precipitated Inhibitors 76
xii Contents
4.5 Automation of DNA Extraction 76
4.6 DNA Extraction Protocols 77
4.7 Summary 79
References 79

Chapter 5 DNA Quantification

Paul A. Heaton and Jacquie T. Keer

5.1 Introduction 83
5.2 Measurement of DNA Concentration Using
Ultraviolet Spectroscopy 84
5.2.1 Determining the Extinction Coefficient e 84
5.2.2 Practical Aspects of Measuring DNA
Concentrations by UV Spectroscopy 85
5.2.2.1 Calibration of the Spectrophotometer 85
5.2.2.2 Cuvettes 85
5.2.2.3 Sample Preparation 86
5.2.2.4 Reference Blank 86
5.2.2.5 Sample Dilution 86
5.2.2.6 Light Source 86
5.2.2.7 Presence of Contaminants 87
5.3 Determination of DNA Concentration
by Fluorescence Spectroscopy 88
5.3.1 Preparation of a Calibration Graph 88
5.3.2 Practical Aspects of Measuring DNA
Concentrations by Fluorescence Spectroscopy 89
5.3.2.1 Sample Preparation 89
5.3.2.2 Reference Blank 90
5.3.2.3 DNA Standard 90
5.3.2.4 Selecting the Dye 90
5.3.2.5 Dye Concentration 90
5.3.2.6 Microtitre Plates 90
5.3.2.7 Measurement Conditions 90
5.3.3 Fluorescent Dyes 91
5.3.3.1 Ethidium Bromide 91
5.3.3.2 PicoGreens 92
5.3.3.3 SYBR Dyes 92
5.3.3.4 Hoechst 33258 92
5.4 Quantification Using the Polymerase Chain Reaction 93
5.5 Enzymatic Quantification of DNA 93
5.6 Primary Methods of DNA Quantification 94
5.6.1 Gravimetric Analysis 95
5.6.2 Isotope Dilution Mass Spectrometry for
Oligonucleotide Quantification 95
Contents xiii
5.7 DNA Quantification by Constituent Phosphorus
Determination 97
5.8 Comparability of DNA Measurement Methods 98
5.9 Summary 99
References 99

Chapter 6 PCR: Factors Affecting Reliability and Validity

Charlotte L. Bailey, Lyndsey Birch and
David G. McDowell

6.1 Introduction 101

6.1.1 Real-time PCR 103
6.2 The Amplification Protocol 103
6.3 DNA Template 105
6.3.1 Integrity 105
6.3.2 Concentration 105
6.4 Reaction Components and Conditions Affecting
Amplification and Reliability 106
6.4.1 Reaction Buffer 106
6.4.2 Magnesium Chloride 107
6.4.3 Deoxynucleotide Triphosphates 107
6.4.4 Water 107
6.4.5 Primer Design and Target Selection 107
6.4.5.1 Primer Design 107
6.4.5.2 Target Selection 109
6.4.6 Thermostable DNA Polymerases 109
6.4.6.1 Factors Affecting Choice
of Polymerase 110
6.4.7 Hot-start Mechanisms 110
6.4.7.1 Wax 112
6.4.7.2 Antibody 112
6.4.8 PCR Optimisation 112
6.4.9 Inhibitors and Enhancers 113
6.5 Thermal Cycling 119
6.5.1 Cycle set-up 119
6.5.1.1 Denaturation 119
6.5.1.2 Annealing 119
6.5.1.3 Extension 119
6.5.1.4 Cycle Number 120
6.5.2 Thermal Cycler 120
6.5.3 Temperature Control 120
6.5.3.1 Block Control 121
6.5.3.2 Reaction Control 121
6.5.4 Ramp Rate 121
6.5.5 Alternative Thermal Cyclers 122
xiv Contents
6.6 Contamination Control 122
6.6.1 Physical Laboratory Separation and
Dedicated Equipment 123
6.6.2 Pipettes 124
6.6.3 Methods of Decontamination 124
6.6.3.1 Uracil-N-glycosylase and
dUTP 124
6.6.3.2 Ultraviolet Light 128
6.6.3.3 Chemical Decontamination 128
6.7 Post-PCR Analysis 128
6.8 Conclusions 128
References 129

Chapter 7 Quantitative Real-time PCR Analysis

Jacquie T. Keer

7.1 Introduction 132

7.2 Approaches to Product Detection 133
7.2.1 The 5 0 Nuclease Assay 134
7.2.2 Molecular Beaconst 135
7.2.3 Hybridisation Probes 136
7.2.4 Scorpiont Primers 138
7.2.5 Plexort Primer Technology 138
7.2.6 Melting Curve Analysis 139
7.2.7 Choice of Fluorophores 140
7.3 Range of Instruments 141
7.4 Practical Aspects of qPCR Analysis 144
7.4.1 Assay Design 144
7.4.1.1 Target Sequence 145
7.4.1.2 Probe and Primer Design 145
7.4.2 PCR Master Mix 146
7.4.2.1 Magnesium Chloride 146
7.4.2.2 DNA Polymerase 147
7.4.3 Cycling Conditions 147
7.4.4 Primer and Probe Optimisation 149
7.4.5 Target Level 150
7.4.6 Contamination Control 152
7.4.7 Experimental Design 153
7.4.7.1 Use of Controls 153
7.4.7.2 Level of Replication 153
7.4.7.3 Randomisation 154
7.4.8 Data Analysis 154
7.4.8.1 Basic Mathematics of PCR
Amplification 155
7.4.8.2 Data Normalisation 155
Contents xv
7.4.8.3 Routes to Determining Amplification
Efficiency 156
7.4.8.4 Outlier Identification 156
7.4.9 Validation 156
7.5 Quantification of Low Levels of Target Analyte 157
7.5.1 Level of Variability 158
7.5.1.1 Sample Handling 158
7.5.1.2 Amplification Cycles 159
7.5.1.3 Replication Level 159
7.5.1.4 Data Handling 159
7.6 Standards and Comparability 161
7.6.1 Quantitative Standards 161
7.6.1.1 Instrument Calibration 162
7.6.1.2 Comparability 162
7.6.1.3 Measurement Uncertainty 163
7.7 Summary 163
References 164

Chapter 8 Multiplex PCR and Whole Genome Amplification

Lyndsey Birch, Charlotte L. Bailey and
Morten T. Anderson

8.1 Introduction to Multiplex PCR 167

8.1.1 Number of Targets Amplified During
Multiplex PCR 168
8.2 Design and Optimisation of mPCR 168
8.2.1 Design Strategy 168
8.2.2 Amplification Target 169
8.2.3 Primer Positioning 169
8.2.4 Primer Design 170
8.2.5 Standardisation of Oligonucleotide Tm 171
8.2.5.1 Base Analogues 171
8.2.5.2 Peptide Nucleic Acid (PNA) 171
8.2.5.3 Locked Nucleic Acid (LNA) 171
8.2.6 Optimisation 174
8.2.6.1 Initial Assay Development 174
8.2.6.2 Reaction Components 174
8.2.6.3 Cycling Parameters 175
8.2.7 Overcoming Mis-Priming Events 176
8.2.8 Specificity 176
8.2.9 Untemplated Nucleotide Addition 177
8.3 Detection Strategies 177
8.4 Applications of mPCR 177
8.5 Advantages and Disadvantages of mPCR 177
8.6 Introduction to WGA 179
xvi Contents
8.7 WGA Methodologies 179
8.7.1 Degenerate Oligonucleotide Primed PCR 179
8.7.2 Primer Extension Pre-amplification 180
8.7.3 Improved Primer Extension Pre-amplification 180
8.7.4 Multiple Displacement Amplification 180
8.7.5 Ligation-mediated PCR 181
8.7.6 T7-based Linear Amplification of DNA 181
8.8 WGA Applications and Characteristics 182
Acknowledgements 184
References 184

Chapter 9 Procedures for Quality Control of RNA Samples for Use in

Quantitative Reverse Transcription PCR
Tania Nolan and Stephen Bustin

9.1 Introduction 189

9.2 RNA Extraction Approaches 189
9.2.1 Freezing 189
9.2.2 Sulfate 190
9.2.3 Guanidinium Isothiocyanate 190
9.2.4 Phenol 190
9.2.5 Additional Purification 190
9.2.6 Extraction from Archival Tissue Samples 191
9.3 RNA Quality 192
9.3.1 RNA Integrity Number 193
9.3.2 Spectrophotometric Measurement 194
9.3.3 Presence of Inhibitors 194
9.4 RNA Quantification 199
9.4.1 Significance of Quantification 199
9.4.2 Methods of Quantification 201
9.5 Effect of RT Experimental Design on qPCR Data 201
9.6 Conclusion 204
References 205

Chapter 10 Microarrays
Sally L. Hopkins and Charlotte L. Bailey

10.1 Introduction 208

10.1.1 What are Microarrays? 208
10.1.2 A Note about Nomenclature 210
10.1.3 Types of Microarrays 210
10.1.3.1 Applications of DNA Microarrays 212
10.1.3.2 Impact of Applications 213
10.2 Technology Status 214
10.2.1 Current Problems 214
10.2.2 Controlling Experimental Uncertainties 216
Contents xvii
10.2.2.1 Experimental Design 217
10.2.2.2 Microarray Layout and Content 219
10.2.2.3 Target Quality 220
10.2.2.4 Array Handling and Hybridisation 221
10.2.2.5 Gene List Files 222
10.2.2.6 Image Acquisition and Processing 223
10.2.2.7 Normalisation 224
10.2.2.8 Critical Data Assessment 224
10.2.2.9 Drawing Biological Conclusions 225
10.2.2.10 Data Management 225
10.2.3 Technology Solutions 226
10.3 Current Commercial Microarray Quality Controls 226
10.3.1 Printing Controls 227
10.3.2 Universal Reference RNA 228
10.3.3 Spike-in Controls 229
10.4 Microarray Standardisation Initiatives 229
10.4.1 Microarray Gene Expression Data Society
(MGED) 230
10.4.2 External RNA Control Consortium (ERCC) 230
10.4.3 Microarray Quality Control (MAQC) Project 231
10.4.4 Association of Biomolecular Research
Facilities (ABRF) Microarray Research
Group (MARG) 231
10.4.5 Measurements for Biotechnology (MfB)
Programme 232
10.4.5.1 Specificity Standards and
Performance Indicators 232
10.4.5.2 Comparability of Gene
Measurements 234
10.4.5.3 Quality Metrics/Increasing
Confidence in Toxicogenomic
Measurement 234
10.4.5.4 Standard Units to Measure Gene
Expression 235
10.5 Summary 235
References 235

Subject Index 240

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