Essentials of nucleic acid analysis a robust

approach 1st Edition Jacquie T. Keer download

https://ebookname.com/product/essentials-of-nucleic-acid-
analysis-a-robust-approach-1st-edition-jacquie-t-keer/

Get the full ebook with Bonus Features for a Better Reading Experience on ebookname.com
Instant digital products (PDF, ePub, MOBI) available
Download now and explore formats that suit you...

Principles of Nucleic Acid Structure Stephen Neidle

https://ebookname.com/product/principles-of-nucleic-acid-
structure-stephen-neidle/

Nucleic Acid Testing for Human Disease 1st Edition

Attila Lorincz

https://ebookname.com/product/nucleic-acid-testing-for-human-
disease-1st-edition-attila-lorincz/

Protein Nucleic Acid Interactions Structural Biology

RSC Biomolecular Sciences 1st Edition Phoebe A. Rice

https://ebookname.com/product/protein-nucleic-acid-interactions-
structural-biology-rsc-biomolecular-sciences-1st-edition-phoebe-
a-rice/

Small Animal Dermatology A Color Atlas and Therapeutic

Guide Second Edition Linda Medleau

https://ebookname.com/product/small-animal-dermatology-a-color-
atlas-and-therapeutic-guide-second-edition-linda-medleau/
B K S Iyengar Yoga The Path to Holistic Health 3rd
Edition B.K.S. Iyengar

https://ebookname.com/product/b-k-s-iyengar-yoga-the-path-to-
holistic-health-3rd-edition-b-k-s-iyengar/

Reform Representation and Theology in Nicholas of Cusa

and His Age Variorum Collected Studies 1st Edition H.
Lawrence Bond

https://ebookname.com/product/reform-representation-and-theology-
in-nicholas-of-cusa-and-his-age-variorum-collected-studies-1st-
edition-h-lawrence-bond/

Rethinking Freire Globalization and the Environmental

Crisis 1st Edition Frederique Apffel-Marglin

https://ebookname.com/product/rethinking-freire-globalization-
and-the-environmental-crisis-1st-edition-frederique-apffel-
marglin/

Troilus and Cressida Webster s Chinese Traditional

Thesaurus Edition William Shakespeare

https://ebookname.com/product/troilus-and-cressida-webster-s-
chinese-traditional-thesaurus-edition-william-shakespeare/

Pediatrics 1st Edition Maureen R. Nelson

https://ebookname.com/product/pediatrics-1st-edition-maureen-r-
nelson/
100 Simple Secrets of Great Relationships What
Scientists Have Learned and How You Can Use It 1st
Edition David Niven

https://ebookname.com/product/100-simple-secrets-of-great-
relationships-what-scientists-have-learned-and-how-you-can-use-
it-1st-edition-david-niven/
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

Abbreviations

A adenine
Ax absorbance at x nm
ABRF Association of Biomolecular Research Facilities
AFLP amplified fragment length polymorphism
BBSRC Biotechnology and Biological Sciences Research Council
BIPM International Bureau of Weights and Measures
bp base pair
BSA bovine serum albumin
BSI British Standards Institute
c7dGTP 7-deaza-2 0 -deoxyguanosine triphosphate
C cytosine
cDNA complementary DNA
CGH comparative genome hybridisation
CRM certified reference material
Ct cycle threshold
CTAB cetyltrimethylammonium bromide
CV coefficient of variation
dATP deoxyadenosine triphosphate
dCTP deoxycytidine triphosphate
dGTP deoxyguanosine triphosphate
DEFRA Department of Environment, Food and Rural Affairs
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
DNase deoxyribonuclease
dNTP deoxyribonucleotide triphosphate
DOP-PCR degenerate oligonucleotide primed PCR
dsDNA double-stranded DNA
DTI Department of Trade and Industry
dTTP deoxythymidine triphosphate
dUTP deoxyuridine triphosphate
EBI European Bioinformatics Institute
EC European Community
EDTA ethylenediaminetetraacetic acid

xix
xx Abbreviations
EGTA ethyleneglycol-bis(ß-aminoethyl ether)tetraacetic acid
EQA external quality assessment
ERCC External RNA Control Consortium
ESI electrospray ionisation
EtBr ethidium bromide
FDA Federal Drug Administration
FRET Förster Resonance Energy Transfer
FSA Food Standards Agency
G guanine
GAL gene array list
GITC guanidinium isothiocyanate
GLP Good Laboratory Practice
GLPMA GLP Monitoring Authority
GM(O) genetically modified organism
HSE Health and Safety Executive
HMW high molecular weight
ICP inductively coupled plasma
ICP-OES inductively coupled plasma optical emission spectroscopy
IDMS isotope dilution mass spectrometry
IEC International Electrotechnical Commission
I-PEP improved primer extension pre-amplification
ISO International Organization for Standardization
IUPAC International Union of Pure and Applied Chemistry
kb kilobase
LIMS Laboratory Information Management System
LMP ligation-mediated PCR
LNA locked nucleic acid
LoD limit of detection
LOH loss of heterozygosity
LoQ limit of quantification
MALDI-ToF matrix assisted laser desorption ionisation time of flight
MDA multiple displacement amplification
MGB minor groove binder
MGED Microarray Gene Expression Data Society
MHRA Medicines and Healthcare Products Regulatory Agency
mPCR multiplex PCR
mRNA messenger RNA
MS mass spectrometry
MWM molecular weight marker
n/s nucleotides per second
NCBI National Center for Biotechnology
NIST National Institute of Standards and Technology
NERC Natural Environmental Research Council
NP40 nonidet P40
OD optical density
OECD Organisation for Economic Co-operation and Development
Abbreviations xxi
OES optical emission spectroscopy
PAGE polyacrylamide gel electrophoresis
PBS phosphate buffered saline
PCR polymerase chain reaction
PEG polyethylene glycol
PEP primer extension pre-amplification
PFGE pulsed field gel electrophoresis
PNA peptide nucleic acid
PVP polyvinylpyrrolidone
PT proficiency testing
QA quality assurance
QC quality control
QP quality procedure
qPCR quantitative PCR
qRT-PCR quantitative reverse transcription PCR
RAPD randomly amplified polymorphic DNA
RCA rolling circle amplification
RFLP restriction fragment length polymorphism
RIN RNA integrity number
RM reference material
RNA ribonucleic acid
RNase ribonuclease
rRNA ribosomal RNA
RT-PCR reverse transcriptase PCR
SBE single base extension
SCOMP single cell comparative genomic hybridisation
SDS sodium dodecyl sulfate
SNP single nucleotide polymorphism
SOP standard operating procedure
SSC salt sodium citrate
SSCP single-strand conformation polymorphism
ssDNA single-stranded DNA
STR short tandem repeat
T thymine
Taq Thermus aquaticus
TE tris-EDTA buffer
Tm melting temperature
TLAD T7-based linear amplification of DNA
TMAC tetramethylammonium chloride
Tris tris(hydroxymethyl)aminomethane
U uracil
UKAP United Kingdom Analytical Partnership
UKAS United Kingdom Accreditation Service
UNG uracil-N-glycosylase
UV ultraviolet
VAM Valid Analytical Measurements
xxii Abbreviations
WI working instruction
WGA whole genome amplification

Weights, volumes and concentrations

M molar
mol mole
g gram
L litre
v/v, vol/vol volume per volume
w/v weight per volume

Prefixes
m milli (10
3)
m micro (10
6)
n nano (10
9)
p pico (10
12)
f femto (10
15)
a atto (10
18)
z zepto (10
21)

Time
s second
h hour
min minute
Acknowledgements

We wish to thank all of our colleagues who have contributed to this manual.
Our particular thanks go both to the authors of individual chapters, who are
therefore acknowledged directly, and to Claire English, Shivani Mehta, Carol
Donald, Gavin Nixon and Hernan Valdivia of LGC for their contribution of
additional experimental data in support of the issues raised. Dr Liz Prichard,
Dr Alison Woolford, Dr Carole Foy, Dr Malcolm Burns and Dr David French
of LGC have kindly assisted in preparation of the manuscript by critical
reading and feedback on the material.
The work described was supported under contract with the Department of
Trade and Industry as part of the National Measurement System Measure-
ments for Biotechnology Programme 2004–2007.

Jacquie T. Keer
Lyndsey Birch

xxiii
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 advance-
ment 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, quantita-
tive, 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.

1
2 Chapter 1
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 valida-
tion 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 assess-
ment 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 manage-
ment 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
Valid Analytical Molecular Biology: The Challenge 3
Valid methodology Trained analysts

• Defined performance • Expert judgement

characteristics • Method choice
• Assess analytical
• Known critical points • Method development problem

• Fit for purpose • Continued training • Define limitations

Quality
Analytical
• Remedial action
Data
• Remedial action • Continued training
• Reference materials • Training records
• Test competencies

• Laboratory design
QA • Calibration of equipment
• Quality procedures/protocols
• Documentation
• Quality consumables/labware

• Reference materials
QC
• Spiked/blank/blind controls
• Proficiency testing/EQA

Quality assurance (QA)/quality control (QC) framework

Figure 1.1 Schematic diagram showing the factors within the laboratory that con-
tribute to the production of reliable data.

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, deter-
mine 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 experi-
ment 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 interpreta-
tion of the analytical data produced and the reporting of results and conclu-
sions. 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
4 Chapter 1
Table 1.1 Stages of the analytical process.
Define the analytical  Define type of data required (qualitative/quantitative)
enquiry  Define use of the data and confidence required in the result
 Define required performance of the method

Assess the sample  Determine nature of the sample

 Define storage, transport and preparation requirements
 Use appropriate sampling procedures
 Ensure trackability through unique sample ID system

Establish constraints  Establish time available for analysis

 Identify equipment and personnel resources available
 Understand any financial limitations
 Determine if there are special safety considerations
 Is the analysis feasible?

Define technical  Identify suitable techniques based on analytical requirement

approach  Match analytical performance to requirements

Select or develop  Select suitable method from published literature, or com-

method mercial kits
 If none suitable, develop in-house method
 Finally, prepare a draft protocol

Validate method  Ensure method performance meets analytical requirements

 Demonstrate the method produces appropriate data
 Document the suitability of the method

Apply the validated  Analyse the samples using the selected, validated approach
method  Use appropriate controls to enable confident interpretation
of results

Interpret and report  Interpretation of the data will depend on the results from QC
the data materials included in the assay as well as test samples them-
selves
 Any limitations of the method should be included in con-
clusions and interpretations of results

reliable results are produced first time. A flawed approach may produce
experimentally valid data that do not directly address the enquiry, or insuffi-
cient 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 do not allow
correct judgement of the actual situation. An overview of the stages to consider
when planning the experimental process is outlined in Table 1.1.

1.3 Principles Underpinning Reliable Measurement

A series of six principles to underpin good experimental practice has been
developed, known as the Valid Analytical Measurement (VAM) principles.
Valid Analytical Molecular Biology: The Challenge 5
Although primarily directed towards chemical analysis, the principles are
generic and fully applicable to biological measurement performed in both
research and more routine laboratory environments. The described approach
requires support and implementation at both a technical and management
level, and the ethos needs to be understood and supported by all staff in the
laboratory.

1.3.1 Understand the Experimental Requirements

Experiments or measurements are generally undertaken to answer a specific
question or to provide a solution to a problem. Understanding the full nature
of the enquiry enables an experimental approach to be developed to produce
sufficient data to fully answer the question.

1.3.2 Use Methods and Equipment which are Fit

for the Intended Purpose
Consistent production of reliable data requires that the methods, instruments,
reagents and software used in an analysis have been tested and shown to
perform as expected. Further information on fulfilling these requirements is
given in Chapters 2 and 3.

1.3.3 Staff Undertaking Analysis Should be Both Qualified and

Competent to Undertake the Task
To ensure that methods and equipment are used correctly, appropriate levels of
staff training and support are required. Formal management schemes address
the continued training and assessment of staff (Chapter 2), and even in
laboratories where no formal system is in place, some level of training is
advisable to avoid time wasted in repeating experimental analyses and the cost
of equipment damage through misuse.

1.3.4 Regular Independent Assessment of Laboratory

Performance
Independent assessment usually takes the form of proficiency testing (PT) or
external quality assessment (EQA) schemes, where samples are distributed to
participating laboratories for analysis. The results are returned and analysed by
the scheme organisers, and a report detailing the performance of the partici-
pants is produced, usually without identifying individual participants. Such
external assessments of performance are useful to confirm that procedures are
producing acceptable in-house results, and results can be compared to those
produced in peer laboratories.
6 Chapter 1
1.3.5 Analytical Consistency
A primary aim of any researcher or analyst is consistently to produce reliable
and valid results. The use of well-defined samples or certified reference mate-
rials (CRMs) can be used on a regular basis to demonstrate the consistent
quality of measurements within a laboratory. In biological analyses few refer-
ence materials are available, but the use of previously characterised samples can
be used to monitor performance over time.

1.3.6 Quality Control and Quality Assurance Framework

Formal management systems (Chapter 2) specify the need for laboratory
management systems, including the use of trained staff, calibrated equipment,
quality protocols and valid methodologies. This is the quality assurance (QA)
framework, which can assist in preventing errors by ensuring the laboratory
and analytical environment is fit for purpose. Quality control (QC) measures
are used in parallel with QA systems, and confirm the quality of data obtained
by the use of control samples and continual monitoring of performance.

1.4 Challenges to Measurement Quality

Despite the establishment of good measurement principles, many technical
challenges remain, arising from a number of factors including the variety of
available methods and platforms, the pace of technological development, lack
of certified reference materials to establish comparability between approaches
and few accessible EQA or PT systems to evaluate comparability between
laboratories. In addition to the practical challenges there is also a number of
administrative and management issues including pressure to publish results
regularly, often high levels of staff turnover and lack of funds and resources for
QC and QA activities.
The analysis of real samples often provides a further challenge, as low
concentrations or inhomogeneous distribution of targets may pose problems.
Difficult samples may originate from a variety of sectoral applications such as
forensic, food or environment, where the DNA analyte may be in association
with an organic matrix, for example, a blood stain on cotton fibre, bacterial
species in milk or genetically modified (GM) soya in processed food products.
A number of common problems that arise in the application of DNA technolo-
gies are considered here.

1.4.1 Low Concentration of Analyte Compared to Matrix

The need to detect, identify and/or quantify very low levels of the target in a
large amount of sample matrix for various applications has led to the develop-
ment of sophisticated DNA extraction and amplification methodologies to
selectively isolate and concentrate the analyte of interest. Examples include
low-level detection of environmental and food pathogens, non-invasive
Valid Analytical Molecular Biology: The Challenge 7
prenatal diagnostic methods and the quantification of DNA contaminants in
biopharmaceuticals.

1.4.2 Complex Matrices

Target analytes present in complex chemical or biological matrices can make
sampling and DNA extraction a difficult undertaking. Challenging matrix
components include naturally occurring secondary compounds which can
interfere with enzyme activity and can cause total inhibition of biological
reactions such as PCR and restriction enzyme digests. Certain components of
biological samples, such as haem and urea, may also affect the analysis. There
may also be difficulties in physically separating the analyte from the matrix, as
clumping and adhesion can make uniform sampling and efficient DNA extrac-
tion difficult.

1.4.3 DNA Degradation

In some instances samples may be subjected to harsh environmental, transport
or storage conditions that can damage the analytes significantly. Poor condi-
tions include industrial processing such as freezing, dyeing, heating, grinding,
tanning, drying and forms of weathering such as those caused by the sun or
rain. Ageing of a sample can also cause physical degradation of the DNA
analyte, and in such instances the use of short DNA targets can enable even
highly degraded materials to yield results. However, it is important to use
calibrators and controls that are in an equivalent physical state to the test
sample, as otherwise the results of the analysis may be affected by any disparate
performance of the intact and degraded materials in the experiment.

1.4.4 Biological Contamination of the Sample

Often the test sample has been contaminated before arrival at the testing
laboratory, so nucleic acids from a variety of sources may be present. The
contamination may be due to environmental insult (for example, bacterial or
fungal contamination), or may inherently be a mixture of target and non-target
material (such as food samples containing a proportion of genetically modified
ingredients). A further problem may arise if the contaminating material contains
chemicals or enzymes that are able to damage DNA within the sample, such as
low pH fruit juices or DNases from contaminating micro-organisms. In this
situation it is not always possible to utilise controls in a similar physical state to
the test samples, and so the likely effect of the contaminant on the results should
be taken into account when interpreting the results of the experiment.

1.4.5 Degradation of Matrix Components

Various components within an analytical matrix can sometimes produce
breakdown products, such as polyphenols, that cause the degradation of
8 Chapter 1
nucleic acids. In such circumstances, some assessment of the level of DNA
degradation is helpful to enable the results of the test to be interpreted
correctly. Tests of the effect of known contaminants may also be performed,
using appropriately spiked samples as controls.

1.4.6 Limited Availability of the Sample

A sample may be limited because the sample represents a unique moment in
time (for example, a particular stage in disease progression) or is limited by
quantity (such as a scene-of-crime swab or patient biopsy). Samples that are
difficult to replace require extreme care in processing and analysis, as by
definition the analysis cannot easily be repeated.

1.4.7 Lack of Suitable Controls

One of the main challenges in molecular analysis is the lack of certified
reference materials (CRMs), which are a key component in validation and
QA procedures. The majority of physical and chemical measurements are
underpinned by suitable standards but, because of the complexity of the
analytes and the wide range of materials under test, such standards are not
available for most biological applications. In addition, there are very few
characterised reference samples that can be employed to ensure the accurate
calibration of equipment, the correct handling of samples or applicability of
methodologies. Alternatives to the use of CRMs include comparison of the
results from several techniques, or analysis of the same material by several
laboratories, followed by comparison of the results. However, as there are no
materials for which a ‘true value’ is known, objective assessment of method,
equipment and analyst performance is not straightforward.

1.5 Focus on Data Quality

As mentioned already, most QA and QC activity takes place in-house, and may
sometimes be compromised because of resource constraints. An exception is in
analyses where the results may be reported and used in a court of law, such as
short tandem repeat (STR) forensic profiling and the quantitative determina-
tion of GM ingredients in foodstuffs. In such cases, the validity of the analytical
result must be demonstrated absolutely, and is subject to stringent questioning
and challenge by defence lawyers. The presence of an equivalent pressure is not
always evident in other areas of analytical molecular biology. The quality of
data is reliant on the professionalism of the analytical laboratory and the
analysts involved, requiring continual questioning and re-evaluation of the
analytical approach, procedure, staff capabilities and applicability of the test.
Individuals within the laboratory need to look beyond the data that are
produced by the instrument or method to the wider experimental context in
order to correctly interpret results. For example many quantitative PCR
(qPCR) instruments provide quantification results with an apparent accuracy
Valid Analytical Molecular Biology: The Challenge 9
of several decimal places. However, consideration of the likely errors intro-
duced into the process through the various stages including preparation of
standards and reaction set-up indicates that reporting results with this level of
precision may be misleading.
Exercising critical judgement in laboratory set-up, experimental design and
practice and in interpretation of results is central to ensuring consistent
production of reliable data and, most importantly, is in your hands.

Acknowledgements
The author would like to thank Ginny Saunders for information and illustra-
tions, and Lyndsey Birch for many helpful discussions.
CHAPTER 2

Quality in the Analytical

Molecular Biology Laboratory
SALLY L. HOPKINS

LGC, Queens Road, Teddington, TW11 0LY

2.1 Introduction
Definition of terms:
In this chapter the terms traceability and metrological traceability will be used.
It is important for the reader to understand the meanings of these terms as used
here:

Traceability (also known as trackability in the chemical industry) is used to

mean the traceability of an entire experimental procedure, through reagent
batch numbers, sample identifier codes and unique file names;

Metrological traceability is used to mean the ability to trace the value of a
result from experimental processes back through an unbroken chain of
comparison, each with a stated uncertainty, to national or international
standards.

The need for valid practices to produce traceable and robust data of acceptable
quality cannot be disputed in any analytical environment; however the route to
consistently obtaining such analytical data is not necessarily a clear and
straightforward path. This chapter will attempt to demonstrate how imple-
mentation of quality procedures can support the ultimate goal of the analyst,
namely getting the analysis right first time and every time.
Defining quality is difficult and there are many different definitions. One of
the most common themes is that quality is about matching a product or service
with the requirements of the customer.
Most laboratories probably have some form of management system, even if
it is not formally accredited or certified, as without one work may get out of
10
Quality in the Analytical Molecular Biology Laboratory 11
control very quickly. Currently, however, there is increasing emphasis on
formal management systems, certification, compliance and accreditation, as-
sessed by nationally or internationally approved third parties. These formal
systems cost much in time and money to implement, so why should we bother
implementing them at all?
A formal management system is an internationally recognised standard, which
is acknowledged and mutually accepted by customers and other organisations
around the world, identifying that work is of a consistent standard. It provides a
means of structuring the processes and procedures used in an organisation,
making it easier to identify potential risk areas and correct problems. By focusing
on the performance of the organisation and the competency of the staff,
documenting all data and keeping records of processes and procedures, the
volume of errors reduces, and proof of performance is available and defendable
to third parties. This means that the management system could help to improve
the status of a laboratory amongst other companies and customers, as well as
saving money by reducing the number of repeat measurements being carried out.
Both these benefits provide an organisation with a competitive edge.
There is also an increasing drive from customers to find credible laboratories,
which can demonstrate the production of quality data. Customers are now
frequently asking for proof of competence in the form of certification, accredi-
tation or compliance from approved third parties. Customer perceptions were
explored in a UKAP-funded survey, in which private sector customers quoted
appropriate accreditation as the most important attribute for a potential
supplier.1 As well as this, some UK funding bodies are asking for evidence of
best practices and management systems before handing over research grants.2
There is a number of possible consequences in not following a management
system, including potential lack of control over processes and procedures,
inconsistency of analytical approach and lack of measurement and process
traceability, all leading to reduced confidence in the data produced by the
laboratory. The organisation may not be managing its risks effectively and
may, therefore, be open to legal challenge regarding the quality of results or
other work. It is also harder to demonstrate independently the quality of work
carried out, without nationally or internationally recognised third-party con-
firmation of a laboratory’ quality status. Ultimately, if an organisation does not
have independent recognition, in terms of quality, then it risks losing existing
customers and may find it difficult to gain new ones.
The aim of the following sections is to provide a brief guide through some of
the fundamentals of management systems and give some guidance on their
implementation. This may help a laboratory decide which standard best suits
its business needs.

2.2 Management Systems

It is of paramount importance to an analytical laboratory that a management
framework is set up in order to ensure that all analysis is performed with
12 Chapter 2
sufficient assurance of quality. Quality assurance and quality control may be
defined as follows:

Quality assurance (QA)

A planned set of activities designed to ensure that the quality control
programme is effective. It is the overarching system, which plans and
documents the processes involved in ensuring quality output.

Quality control (QC)
A planned system of activities designed to provide a quality product.
These activities are planned in the QA system.

A management system contains policies, procedures and instructions that set

out how things will be done and will also help demonstrate performance of
correct procedure, covering many aspects of administration and laboratory
organisation. Some such aspects are:

Quality policy statement;

General organisation;

Roles and responsibilities of staff;

Document control;

Quality manual.

A range of more specific topics may also be included:

Laboratory environment;

Security;

Facilities and equipment used for testing/calibration;

Employment of suitable staff and their training;

Procedures relating to sample handling and control;

Test methods and procedures;

Policy on subcontracting of work and reporting results;

Use of valid methodologies and QC measures.

An auditing and review policy and procedure must also be in place to ensure
that all documentation and procedures are completed as required. The QA
system is distinct from the QC process. QA is about demonstrating that the
Quality Control process is effective, and how it is maintained under control.
The QC process describes the day-to-day activities which are carried out to
provide a series of checks on the product. This will be described in more detail
in Section 2.4.7.

2.3 Internationally Recognised Assessed Standards

Many organisations operate a highly structured management system similar to
that set out in Figure 2.1.
Quality in the Analytical Molecular Biology Laboratory 13

International Standard

Quality Manual

Quality Procedures

Standard operating Locally held Work Instructions

procedures documents

Records

Figure 2.1 Schematic diagram of a typical management system structure.

Certification to a standard is obtained when a specified third party issues a

statement, based on a decision following review, that fulfilment of specified
requirements related to products, processes, systems or persons has been
demonstrated.3

Accreditation is achieved when a third party issues a statement, based on a
decision following review, that competence to carry out a task has been
demonstrated.3

There are four commonly used international standards for laboratories, all of
which complement each other. These are ISO 9001:2000, ISO/IEC 17025:2005,
ISO 15189:2003 and the Principles of Good Laboratory Practice (GLP). ISO
(the International Organization for Standardization) and IEC (the Interna-
tional Electrotechnical Commission) form the specialised system for worldwide
standardisation, while GLP has been developed by the Organisation for Eco-
nomic Co-operation and Development (OECD). The type of analysis the
laboratory carries out will largely govern the standard(s) that it adopts.
As well as the four international standards above, the analytical molecular
biology laboratory may also need to be aware of recommendations highlighted
by funding bodies. Increasingly, funding bodies are asking for assurance of the
quality of data produced. In the UK the Joint Code of Practice for Research2
was issued in 2003 by the Department of Environment, Food and Rural Affairs
(DEFRA), the Food Standards Agency (FSA), the Biotechnology and Biologi-
cal Sciences Research Council (BBSRC) and the Natural Environmental
Research Council (NERC). The code lays out guidelines for the quality of
the research process and the quality of the science carried out, to ensure their
contractors are using ‘best scientific practice’. This code will be discussed along
with the internationally recognised standards, to give laboratories an indication
of what funding bodies will be looking for in the future.
14 Chapter 2
2.3.1 ISO 9001:2000 Quality Management
Systems – Requirements
ISO 9001:20004 is part of a family of standards:

ISO 9000:2000 – Quality Management Systems: Concepts and Vocabu-

lary;

ISO 9001:2000 – Quality Management Systems: Requirements;

ISO 9004:2000 – Quality Management Systems: Guidance for Perform-
ance Improvement.

ISO 9001:2000 is the standard within this family to which organisations will be
assessed and awarded certification. It is this quality management standard that
is commonly used by organisations manufacturing or supplying products or
services in the UK and across the world. This standard replaced the ISO
9001:1994 series of standards, and places more emphasis on customer require-
ments, satisfaction and continual improvement.
This standard is generic in terms of its requirements and can, therefore, be
applied to all types of organisations. It is concerned with controlling processes,
as a way of meeting customer requirements and requires continuous improve-
ment, demonstrating that quality is not a static activity. However, the standard
is not prescriptive in terms of the technical requirements of the organisation or
laboratory.
The certification process involves an organisation registering with an accred-
ited certification body, who will usually discuss the organisation’s needs and
any fees which will be payable. Once the formal application and registration
fees have been received it is usual for a pre-assessment visit to be carried
out to review the organisation’s documentation and further discuss the imple-
mentation of the standard. Once the organisation has put in place all that
was suggested in the pre-assessment, an assessor will visit the organisation
and finally assess their management system against the requirements of
ISO 9001:2000. If the management system complies with these require-
ments, a formal confirmation and a certificate is issued which may then be
used to demonstrate the achieved quality standard to other customers and
organisations.
Once certification has been achieved, the certification body will re-assess the
organisation at regular intervals, to ensure the management system is main-
tained at a satisfactory level. For example, the British Standards Institute (BSI)
will visit at regular intervals (at least every year) to facilitate improvement as
well as checking that the requirements of the standard are still being met.5 The
assessors will report to the organisation any non-conformity against the
standard, as well as suggestions for improving the system, which may not be
classed as non-conformities. Non-conformities must be corrected within a
specified time period and the certifying body will require an action plan to be
provided.
Quality in the Analytical Molecular Biology Laboratory 15
2.3.2 ISO/IEC 17025:2005 General Requirements for the
Competence of Testing and Calibration Laboratories
ISO/IEC 17025:2005 is the current internationally accepted standard for the
accreditation of testing and calibration laboratories.6 This standard replaces the
ISO/IEC 17025:1999 requirements, which in turn replaced ISO Guide 25 and
many national standards. There are two main sections to this standard; one
covering the management requirements and the other covering the technical
requirements. ISO/IEC 17025 was updated in 2005 in order to bring it into
alignment with ISO 9001:2000 (see Section 2.3.1). The main change in the
management section is the specific requirement for continual improvement and
communication with the customer to ensure their requirements are fully met.
Throughout this chapter references to ISO/IEC 17025 refer to the 2005 standard.
ISO/IEC 17025:2005 specifies general requirements for demonstrating
competence to carry out tests or calibrations and covers the use of standard,
non-standard and laboratory-developed methods. This standard contains the
management requirements of ISO 9001:2000, as well as many more technical
specifications, including the laboratory environment, method validation, method
uncertainty, metrological traceability and sampling. The standard is designed to
assess and demonstrate competence for the tests or calibrations being carried
out. If a laboratory is accredited to ISO/IEC 17025:2005 then it is stated that the
management system, for these accredited activities, also meets the principles of
ISO 9001:2000, as stated in the joint ISO-ILAC-IAF communiqué.
The majority of national accreditation bodies across the world accept the
standard, and many of these bodies have mutual recognition agreements to
accept the accreditation made and granted in other countries. In the UK,
the competent body is the United Kingdom Accreditation Service (UKAS).
Accreditation to ISO/IEC 17025:2005 is sought from UKAS for specific tests,
in terms of the scope of a particular method, such as a particular analyte,
matrix and instrument platform. Guidance and information for those seeking
accreditation, including notes to aid implementation, application forms and fee
schedule, are freely available from the UKAS website.7
On receipt of the relevant application forms and the application fee, UKAS
will assign an assessment manager, who will arrange for a pre-assessment to be
carried out. This visit addresses the scope of the accreditation sought. An initial
assessment visit is then carried out to formally assess the applicant against ISO/
IEC 17025:2005. The assessment manager and technical assessors, relevant to
the scope of accreditation being sought, perform the assessment. Any non-
conformities highlighted during the visit are notified to the applicant in writing.
The applicant will be granted accreditation once the non-conformities are
cleared to the satisfaction of UKAS. The maintenance of accreditation is
confirmed by annual surveillance visits, the first of which takes place
six months after the granting of accreditation. A full re-assessment is carried
out every four years. At each of these stages the assessment manager will make
a quotation for the charges due. Where available, participation in relevant
proficiency testing (PT) schemes is also expected of ISO/IEC 17025:2005
Exploring the Variety of Random
Documents with Different Content
 super faciem. meam, et. lacrimans 9» aio. : Heu, heu, heu,
Domine Deus, ergone disperdes omnes reliquias. Israel, effundens.
furorem. tuum. super Hierusalem ὅδ ? Sie, sie nimirum ** puella,
tam 59 gravi tacta *? dolore, post visiones suas consuevit
saepissime, tune. praecipue eum post aliquam Ὁ visionem
magnam"! ad seipsam revertitur, ex intimis cordis alta trahens?
suspiria, graves planctus emittere, tum quia"? ad corpus mortis
huius redire compellitur, tum quia ** de sabbato suae
contemplationis egreditur, tum quia "^ miseris quos in poenis
constitutos viderit ** compatitur. Sicut ** verisimile videtur*5, sic
molestum '? ei, quoties de requie suae contemplationis in
ergastulum sui corporis revertitur, tamquam si quis de mundi huius
amplitudine in aliquem ?? carcerem tenebrosum"! intrare
compelleretur '?, Unde nonnumquam fit ut tam gravi post requiem
suam et tam longa deprimatur infirmitate, et tunc maxime cum
aliquam praecipuam visionem viderit, ut ?? a somno suo excitata, et
cireumstantes videat et sibi colloquentes audiat et prae nimia
debilitate corporis ** verbotenus eis respondere non ** valeat.
Explicit liber secundus **. ANNOTATA. a Fuit Gildoinus secundus
coenobii Fontis Ioannis abbas, qui anno 1172 adhuc in vivis, haud
multo post obiisse credendus est. De eo legesis quae in Gallia christ.,
tom. XII, p. 229. b Abbatia Fontis loannis sita erat in vico hodie
nuncupato Saint-Maurice-sur-Aveyron. Cfr. Patron, Recherches
historiques sur l'Orléanais, tom. 1I, p. 122. Hic autem vicus pertinet
ad praefecuram dictam Loiret, laud procul a Monte-Argiso
(Montargis). De abbatia Fontis Ioannis agitur apud editores Gall.
christ, tom. XII, p. 229, et Ianauschek, Orig. Cistere., tom. I, p. 12. c
Pontiniacum, vicus in. praefectura Yonne, de quo Quantin, Répertoire
archéolog. du départ. de Yonne, p. 47. Abbatia Pontiniacensis est, ut
ait lIanauschek, op. cit., p. 4, secunda Cistercii filia et tertiae lineae
caput ; incepta est anno 1114. d Licet Iuviniacensium comitum
series iam pridem nota sit (1), non paucos tamen hic Vitae locus
vexavit; ita ut nuperrime et ven. vir L. Tridon (2) confessus sit se
aliis meliorem nullam nactum esse fortunam, mec in privato archivo
Castri Fenardi quod adivit, ea. documenta. repperisse quibus ὁ
tenebris eruatur iste comes, qui dicendus est Hainardi IV filius. Hie
vero Rainardus IV uxorem habuit Adelaidem, Nivernensis comitis
filiam (3). e Castrum. Renardi iacet haud longe a. Monte Argiso in
praefectura Loiret. Ex documentis archivi Castri Renardi de quibus
dictum est patet comites Tuviniacenses et dominos de Castro
Renardi arto vinculo fuisse coniunctos. "5 clamans 3.— ὅδ (Et iterum
post visionem — Hier.) om. 9, — 9t pimium 1.— 9 om. 2, 3. — ὅ9
om.2. — 80 tam 3. — *! m, v, 2, 3. — *! trahere 9, 3.— ὅδ (t. q.)
eumque 2.— δὲ (t. q.) eum 9. — 9^ (t. q.) tamen 2.— δὲ viderat 2.
— ** enim «dd. 9, 3, — 98 eur add, 3. — '? est add. 3, 3.— τὸ (in
a.) om. 3.— 51 t e, 2, 3. — 19 compellatur 2, 3. — ^ et 3.— τὸ c. d.
9, 8. — τὸ om, 3. — 18 (e. 1. s.) om. 2. (1) Art de vérifier les dates,
tom. II, pp. 236 sqq. — (3) La Vie merveilleuse de S. Alpais, p.210,
note.— (3)Cfr. Quantin, DIES TERTIA NOVEMBRIS. 189 VITA D
DIBERVIBERJITUS Incipiunt capitula libri III!. Carvr 1. — De
assumptione beatae Mariae, quam vidit ante Dominum in caelo per
octo dies sollemniter a caelestibus? civibus celebrari. 1.— De
erucifixo quem vidit pendentem in cruce facta ad similitudinem
litterae thau?, et de lampade in thalamo eius divinitus accensa. nr. —
De beato Nicolao, quem vidit in sollemnitate sua candidatorum
turmis pontificum et clericorum cireumdatum, hymnos caelestes
sollemniter decantantium. Iv. — De quodam peccatore qui peccata
sua confessus est presbytero de Cudoth, cuius ipsa peccatum et
confessionem Domino revelante didicit et de praedicto presbytero,
quem dum missam celebraret vidit in spiritu magno lumine
refulgentem. v. — De muliere quae erucem in dextra ferebat*, super
euius brachium dextrum sedebat candidissima columba, et de puero
septenni eam praece- E dente?, qui veniebant ambo ad puellam
ambulantes super aquas. vi. — De candidatis fidelium turmis quas
vidit ascendentes in caelum, et de protoplausto, quem vidit iuxta
magnam arborem * super ripam cuiusdam fontis amoenissimi
stantem. vit. — De regina caelorum et de beata Maria Magdalene et
de saneta Maria Egyptiaca, quas vidit in capella quadam sursum in
excelsis in aere suspensa, et de militia caelestis exercitus, quae
veniebat? ad beatam Mariam. vur. — De Domina nostra, quam vidit
in ecclesia quadam * super altare sedentem, ante quam arbor de !?
terra surgebat variis onerata !' floribus, quos colligebant columbae
descendentes de caelo, et deferebant eos in caelum. ix. — De vidua
grandaeva, euius animam vidit ante beatam Mariam ab angelo suo
secum in caelum duci et inter hymnidicos virginum choros ipsa
iubente collocari. x. — De homine insano quem a duobus spiritibus
immundis vidit in aquam submergi, et animam eius ab immundis
daemonum turmis" ad tormenta detrahi ?*. xi — De multitudine
animarum per pontem ferreum transeuntium, sub quo erant aquae
fetidae, in quibus innumerae torquebantur animae. xii. — De tribus
foeneratoribus quorum animas vidit in poenis flammis ultricibus
cruciari. xu. — De Gisberto * eremita quem vidit capellam suam
thurificantem, et angelum Domini eum praecedentem. xiv. — De
testimonio ^ quod perhibuit diabolus de puella et de duobus
eremitis. xv. — De diabolo, qui venit ad eam in specie canis nigri et
assumpsit formam tauri terribilis '*, et minabatur eam cornibus
impetere. Tit. et Cap. — ! tit. om. 2, num. cap. in [ine ponit 3. — ?
caelestis curiae 2, 3.— ? t. 1. 2, 3.— * gerebat 2, 3.— hp. e. 2,3.—
*t1.2. — a. m.2.— 5 qui veniebant 9,3.— ἢ. 6.4. ν. 3.-- 1.4. 2,3. —
11! honorata 2, 3.— 15 turbis 2, 3. — M distrahi 3, num. X post XI in
g2— Gilleberto ἢ, Gisleberto 3. — 15 (d. t.) testimonium2, 3. — !* t.
tauri f. 2. Cartulaire de l' Yonne, tom. LI, p. 236. XVI. ——
 Videt B. Alpais assumptionem B. M. V.per octiduum in caelo
celebrari. 190 xvi. — Item. de diabolo, qui venil ad eam in specie et
habitu medici multis oneralus 11 phialis, veneniferis potionibus
plenis, admonens eam ut de ipsis biberet. »-xvir. — Item de diabolo,
qui transfiguravit se in angelum lucis, et monebat eam ut se !*
tamquam Deum ?? adoraret. CAPUT PRIMUM De variis visionibus et
revelationibus D. Alpaidis. Incipit liber tertius !. 4. Capvr 1.— In
vigilia 2 ssumptionis beatae Mariae Dei genetricis semperque
virginis? obdormivit in requie sua venerabilis puella; cumque
obdormisset, mulier quaedam super lectum * eius candelam unam
ob devotionem suam obtulit. Duela est igitur in caelum ab angelo
suo, ubi in honorem Virginis matris ante filium Virginis ineffabiliter
gaudebat οἱ congratulabatur omnis curia caelestis, eo quod a filio
suo non solum super humanam?,immo etiam super* angelicam
euriam", mirantibus caelestis curiae ordinibus die illa exaltata sib in
caelis. Gaudebant. omnes in Domino diem festum celebrantes sub
honore beatae Mariae Virginis, pro cuius assumptione cantabant
omnes? canticum novum ante sedem Agni, laudantes et glorificantes
Filium Dei, tenentes cereos ardentes in manibus suis, quos omnes
illuminabat, ut vera? lux illa quae lucere novit eliam his qui in
tenebris sedent οἱ in '? umbra mortis. Genetriei veri luminis hine et
inde assistebant duo candelabra lucentia, scilicet beatus Ioannes
Baptista et sanclus 11 Ioannes evangelista, qui et ipsi gestabant in
manibus suis accensa luminaria. Erat inter eos virgo verecunda "^,
quia ' sola inter eos ἐν cereo carebat; sed sanetus Ioannes
evangelista, eam benigne!? respiciens, cereum unum accensum ei
misit '* per angelum itineris sui praevium. Sollemnitatis illius
beatitudinem ineffabilem melodiae caelestis dulcedinem et?*
suavitatem, beatorum spirituum omniumque caelestium. eivium
iucunditatem et exultationem, tot et tantorum luminum claritatem
15, nullus oculus videre, auris nulla? audire, nullum cor sufficit
excogitare, excedit enim omnem sensum, omnem sermonem??,
omnem humanae mentis intellectum, electorum in illa civitate inclita
gloria, sempiterna sanctorum omnium in tanta sollemnitatis
exultatione felicitas et laetitia, beatorum spirituum Deum et Matrem
Domini collaudantium ordinata militia. In civitate illa inclita erant
omnes sancti fulgentes sicut stellae in perpetuas aeternitates,ubi
unaquaeque?! fulgebat sieut. sol, quorum. omnium lucerna est
agnus filius Virginis ??, euius laudibus nec rosae nec lilia desunt, sed
in eius praeconiis unanimi voce suavissimum melos concinunt flores
rosarum et lilia eonvallium. Dum sic ante filium Virginis et virginem
matrem in utriusque 11 honoratus 3, — !* om, 3. — 19 Dominum 3.
Cap. 1. — ! tit. om, 2. — ? (b. M.) beatissimae 2, 3. — 3 Mariae add.
2, 3. — * lectulum 3. — ^ isnt H — δ omnem add. 3. — * creaturam
2, 3. — 5 cantabant add. 9 — om, 3,-— 1? om, 9, 3. — τι beatus 2.
— 1) veneranda 9, 3. — !! quae 2, 3. — ! (i. e.) om. 2, 3. — !5
benigno 2, — 16 m. e, 2, — '! ut 3, — 15 inaestimabilem add. 9, 3,
— 1? n, a, 2, 8. — 30 ser. o. sen. 9, 3, — 3ι facies add. 3. — 35 v.
9,3, — ?3 vidit 2. — *^ ei add. 9, 3, — ** F. D. c. s. 9,9. — 99 oj, 9
DE BEATA ALPAIDE VIRGINE. honorem coneinerent angelici cives,
venit?? ante D filium suum virgo mater cereumque suum*'
devotissime prima omnium obtulit. Postquam omnes alii secundum
ordines suos cereos suos Filio Dei ?5 obtulerunt, omnium ultima.
cereum suum obtulit ei?* Aupes beatissima "τ. Dumque rediret ab
oblatione, coepit aestuare animo, eo quod de cereis illis caelestibus
nullum habebat, quem secum eum ad corpus reverteretur deferret?
*. In pendulo quippe fuerat animus eius : hine etenim? in tanta
sollemnitate, cum omnes ad oblationem ire conspiceret, offerre sola
tanto regi?" refugere nec volebat nec audebat?!, illinc cereum suum
eum ad se rediret secum afferre ?* votis omnibus desiderabat. Dux
itaque suus, animum eius? intuens, eique compatiens, de uno
cereorum ei minimam? particulam dedit, quam eum ad corpus?
rediis5615 in manu sua tenebat. Cum sic omnes vero lumini,omnem
hominem venientem in hunc mundum illuminanti, luminum. suorum
flammantes radios obtulissent, venit ante filium suum saneta Dei
genetrix cum omni militia caelestium. agminum, adorans et petens
aliquid?! ab eo, fiducialiter ei supplicans in hune modum : Fili, dulcor
unice?5, singulare. gaudium, pia. vota respice tibi supplicantium.
Jesu pie, Jesu bone, [1159 unice qui gro salute humani. generis?
carnis humanae substantiam in utero meo tibi coadunare voluisti,
quiquegenus humanum pretioso sanguine tuo suspensus in eruce
redemisti, respice super hanc familiam tuam in tui nominis hodie
laudibus pro assumplionis meae gaudio congregatam Δ᾽, te
laudantem, te glorificantem in caelis, eo quod. hodie per te sim E
mirabiliter exaltata super choros “ἢ angelorum ad caelestia regna.
Isti vero iam certi, iam de sua incolumitate sunt securi, sed adhuc de
provimorum suorum,qui adhuc peregrinantur in terris,redduntur
salute solliciti. Numquam enim istorum perfecta nec plena** poterit
omnino*?. esse laetitia, donec carorum suorum. parentum, filiorum,
fratrum et sororum **, quae adhuc in illa ** valle lacrimarum*8
detinentur copiosa turba, quam desiderat, quam exspectat, ad.
eorum consortium pariter. et conspectum te duce pervenerit*?. Pro
cunctis itaque fidelibus adhuc in carne manentibus, qui
sollemnitatem assumptionis meae devota mente pro posse suo digne
celebraverunt in terris, tibi supplico, fili dulcissime, postulans, ut
quicumque de mea. assumplione fideliter. gavisi sunt. in. terris?"
remissione peccatorum concessa, per me sociari mereantur angelicis
choris in ista beatitudine, te praestante, qui cum Patre οἱ Spiritu
sancto vivis et gloriaris Deus per omnia saecula saeculorum. Amen.
In hac voce cum omni militia caelestis exercitus flexo genu filium
adoravit regina misericordiae, cui assurgens Dominus precibus eius
misericorditer annuit, eamque in regali cathedra iuxta thronum suum
eum omni reverentia, sieut matrem suam decebat δ᾽, collocavit. Sie
usque ad octavas assumptionis gloriosae virginis eum. ceteris Agni
sponsis morabatur beata Aupes in caelis, et nono die regresa est?" in
corpore, inventaque?^ est habens in manu sua particulam candelae,
quam ei 0.3. — ?? p, A, 9, — 8 afferret 9, 3. — ?? (h. e.) quia 2— 90
t. p, 5. 3, — 91 aud, n. v. 9, 8. — 33 offerre 2.— 9? e. 8. 9,3. — i m,
ei 8, — 95 suum add. 9, 3. — 9^ rediret 2, — "7 id 3. —! et add. 3,
— *? Dei add. 9, 3, — *? g. h. 2, 3.— “ὁ. Ρ. 8. m. g. 2.— 4? sum 9,
— t$ op, 9. — * oratio add. 3. — '^ 0" 3,— 46 sociorum 1, 9.— 4!
hac 9,— 18 ], v, 2, 3.— !* pervene" rint 2, — 59 f. in t, g. 5. a. 2, 3,
— ^! sublimiter add. 2 3.— 9! om. 3, — 9^ inventa 2, 3. in
 A Crucifizus ei apparet, et lampas in eius thalamo
accenditur. * cod. roie in caelis ab angelo post oblationem acceperat.
Erat autem tam parva, ut eius longitudo latitudinem palmae** non
excederet. De candela ista minimam particulam dedit?? euidam
monacho, de qua postea contigit tale miraculum? fieri. Nam
monachus comitissae Castelli Renardi gravi infirmitate laboranti de
eadem candela minimam particulam misit : quae vino in quo
submersa fuerat potato, pristinae restituta est^" sanitati, Ex illo
tempore quo caelestem? cereum tenuit 5? in manu sua, euius ope
longo caruerat tempore *9, virtute divina recepit ad usum"!
fortitudinis et sanilatis, quod ad usus"? necessarios melius et
expeditius quam antea" eius officio frui potuit. Illud etiam δ dietu
mirabile est *?, quod candelam, quam in vigilia assumptionis beatae
Mariae, sicut antea"* diximus, mulier quaedam" super lectu]um eius,
eum iam obdormisset, obtulerat, nona die reversa ad se**,
advocata"? matre sua. requisivit, admirante simul et obstupescente
matre, quomodo sciret eam oblatam fuisse sibi Ὁ, eo quod ipsa,
quando candela sibi fuit oblata, iam obdormisset. 2. Capvr rt. — In
vigilia beati Michaelis archangeli! replevit thalamum puellae lux et
splendor admirabilis, et claritas Dei cireumfulsit illam ? ;
descenditque iuxta lectulum? eius secus parietem lumine magno
praeeunte, quod totum * illuminabat eius thalamum, imago
quaedam hominis in modum crucifixi eruei affixa clavis *, cuius erux
tria lantum brachia habebat", nam brachio * superiori carebat, quia
in modum figurae thau ὃ facta erat, et ideo similis erat eruci
salvatoris tria tantum brachia habenti *, quae in parte superiori
brachium non habuit 15, sed Pilatus quartum apposuit, quando
titulum salvatoris hebraice, graece οἱ 11 latine 15 in tabula eruci
superposuit. Dumque sic imaginem in cruce pendentem aspiceret,
admirans et stupens, eo quod talem crucem videre non consueverat,
imago illa !? esgrunnivit et caput huc illucque '^ agitavit. Cumque
vocem esgrunnientis audisset et capitis agitationem vidisset, nimio
timore perterrita, caelum ?? aspexit '*, et Spiritu saneto praeventa,
hane ad Dominum non praemeditata romanis verbis effudit
orationem : Je vous aor sanitime roys Qui establistes les deus lois ΕἸ
avoiastes les iii rois Sains esperites soit o moi La toie * meire le
montroit En icelle saintime foi 55 puellae add. 9, 3, — ὅδ d. p. 2,3.
— '* t, m. c. 2. — δ est r. 2, 3. — δ8 illum add.92, 3. — ὅ9 tantum
add. 2, 3.— 9 om. 9, t. c, 3, — 81 (ad u.) om. 3, (τ. ad u.) om. 2. —
** (ad u.) om. 1. — 88 potuisset add. 2, 3. — ** et 3. — *5 om. 3.
— ** ante 9,3. — 9! una 9, 3, — 95 ad se r. 2. — δ ad se add. 2.—
"sro f quuE Cap. 2. — ! om. 2. — ? eam 2, — ὃ lectum 3. — * om. 3.
— 5 er, cl. a. 9, cl. cr. a. 3. — * h. b.3.— * brachii 3. — 5t. f.2, 3, —
? h. b.2, 3. — !? habuerit 2. — !! om. 2. — 19 seriptum add. 2, 3. —
15 pendentis 2, quae in cruce pendebat 3. — 4 coneussit et add. 2,
3. — 15 caelos 2, 8. — 16 respexit 3, — 11 Ge vos aor seintismes
reis Qui estaplutes les elus leis Et envoiastes les tres reis Sanz
esperites seit o moi La toi mere le moitroit En icelle seintisme foi
Que seinte Iglise teint de tei Mame et men cors commant a tei 2. Ge
vos aor seintisme rois DIES TERTIA NOVEMBRIS. 191 Que saincte
eglise tient de toi Marive et mon cors comman a toi V, Cumque 15 in
hune modum seipsam !* Domino suppliciter commendasset, imago,
manibus de cruce ?? disiunctis, caput virgini humiliter? inclinavit, et
ambas manus quasi ad orationem ante se simul coniunxit et ??
inclinato capite, coniunetis manibus, praeeunte luminis sui claritate
??, quae eam in descensu ?* praecesserat ?*, ad caelos ascendit.
Huius caelestis luminis ** claritatem vidit puella quaedam, cognata
venerabilis Aupes??, nomine Maria, cirea lectulum eius clara luce
radiantem, sicut ore proprio mihi dixit, quam tamen cognatus eius,
frater sanctae virginis ?5, quem ad hoc spectaculum advocavit, quia
forte dignus non erat, videre non potuit. Pluries autem accidit ut
lectulum virginis magna subito lux circumfulserit *?, ita quod
thalamum suum totum intus undique repleret, in tantum ut
lampas,quae ante lectulum eius prius inaccensa ?? et ?' sine lumine
manebat ?*, posteaquam magna claritas illa paulatim tota
recessisset, de lumine claritatis illius sine cuiuslibet accendentis
officio remaneret accensa, sicut mihi relatum est a quibusdam. 8.
CarvrT ir. — In sollemnitate beati Nicolai vidit venerabilem virum
pontificalibus indutum infulis, fraglantibus unguentis optimis, virgam
pastoralem habentem * in manu sua. Cuius facies fulgebat sicut sol,
et videbatur ei quod ? ipse beatus Nicolaus erat, cuius nativitas ea
die? in universis sanctorum colebatur * ecclesiis. In eius facie pietas
apparebat δ incomparabilis, in vestimentis eius 5, nive candidioribus,
erat? inaestimabilis odor nimis, cuius dulcedinem et suavitatem si
quis attingeret, omnis mundi huius delectatio ei sorderet. In eius
comitatu erat innumerabilis episcoporum, presbyterorum et
clericorum pompa, vestimentis candidissimis adornata; qui omnes
pariter gaudebant $ in Domino, diem festum celebrantes sollemniter
in honore? tanti pontificis, cuius vita inclita exemplum fuit eis
sanetae conservationis, cuius gloriosis meritis sociari meruerunt
angelieis choris. In honorem beatissimi ' praesulis hymnis canora
caelestibus pontifieum pompa proclamat, sacerdotum turba laeta 11
decantat, clericorum chorus ovans exultat; inter quos gloriosus
pontifex Nicolaus P, elevata voce, Salvatorem suum laudare coepit et
magnificare'?, benedicere et glorificare et in eius laudibus,
cantantibus organis caelestibus, duleisona voeis modulatione
proclamare. Ad cuius angelicum sonum conticuerunt * omnes alii,
mellifluae vocis eis Qui establites los dos leis Et envoiastes les trois
rois Sainz esperites soit o moi La toie mere le motroit En icele sainte
foi Que sainte glise teint de lei , Marine en men cors commant a tei.
Ac super lin. Adoro vos, sanctissime rex, qui fecisti duas leges, qui
misisti tres reges. Sanctus Spiritus sit mecum, tua mater mihi
concedet in illa. sancta fide qua sancta ecclesia tenet de te, animam
meam et corpus commendo tibi 3. 18 cum 3, — ?? om. 1. — Ὁ d. c.
m. 9. — ?! h. v. 2. — 39 sic add, 2. — 38 om. 8. — ?*in d. e. 2. — ?
* claritate add. 8. — 36 gm, 3, — 31 Aupeis 9. — *5 v. s. 9, — 39
circumfulsisset 2, circumfulserat 1. — ?? incensa 9, — 91 on, 9, 3, —
'? pendeD 8. — ! tenentem 2, ὃ. — ? quia 2— ? eratiadi ῷ-4 om. 9,
— ^ et add. Y, 9. — * suis 2. m 7 om. 3. — PODER. debant 3. — ?
honorem 9,3. — ? beati 9. 2" turma 1. 2, t. lecta 3. — !? om. 2. —
13 et add. 2. — '* conticuere 3, 8. , illeeti VITA B. Alpais videt S.
Nicolai in caelis gloriam.
 Cognoscit paenitentis peccatum, 192 illecti duleedine, etin
eius resorfabilem cantilenam devolis intendebant auribus, ultra quam
credibile sit tantae suavitate dulcedinis 15 iucundati. Suspirabat ex
intimis ad vocem pontificis virgo vene rabilis, cuius. cantum explicare
nulla polest ci(hara, cuius sono comparari nulla possunt organa.
Dumque '* militiam caelestis exercitus circa bealum praesulem
conspiceret candidatis ornalam vestibus, vidit ex alia parle venire
quosdam deformes clericos telerrimis vestibus indutos, facie
macilentos, vultu miserabiles, aspectu horribiles ἢ quos eum magna
indignatione, vultu severo ^. respiciens sanclus pontifex, durius
loquebatur ad eos in hunc modum : Quid hic, ministri diaboli,
quaeritis ? aut quid ante me venire praesumitis ing Cur ante faciem
meam ingredi, cur in conspecttt meo apparere non timetis 159
Fugite, fugite hinc cilius ?9, maledicti, non enim est. sors vestra. cum
electis. Domini ?*, nullam. cum iustis participationem habebitis,
nullam in2 me fiduciam h abeatis, quia pro magnis in quibus usque
ad 38 finem vitae vestrae vizistis criminibus, nullum vobis daturus
sum auxilium, nullum a me culpis vestris exigentibus habebitis
subsidium. Nam quando in sollemnitatibus meis. conveniebatis ad.
ecclesias in. honore nominis mei consecratas, ut ibi laudes
dominicas?* decantaretis, meque in aneis operibus, immo Dominum
?* in me operantem, magnificaretis, dominico despecto servitio,
propriis voluptatibus. carnis et gulae. illecebris serviebatis. Nam haec
erat. causa adventus vestri ad. sollemnitatem, ut commessationibus
et ebrielatibus et luzuriis vestris in. unum congregati liberius
vacarelis. Coronabalis vos rosis antequam marcescerent,nullum erat
pratum quod ** non pertransiret. luxuria. vestra. In carne vestra
seminastis, nunc seminum vestrorum fructum metelis. corruptionem.
Ignem. luxuriae desiderastis, ignem semper amastis, ignem.
invenistis, in igne semper ardebitis. Recedite igilur hinc, maledicti, ite
in ignem aeternum, qui praeparatus ?* est diabolo, cui servistis, et.
ministris. eius?9, cum. quibus in aeternum ardebitis . Adiuvet nos
beatus 30 Nicolaus, ut meritis suis et precibus huius sanetae
virginis,cui seipsum ostendere dignatus est, aeternam damnationem
evadere valeamus et ad vitam pervenire sempiternam. Amen. 4.
CaPtT Iv.— Accessit ad sacerdotem de Cudoth quidam parochianus
eius ἢ, paenitentiam. volens agere? de peccalis suis, et omnia
peccata sua, quorum memor esse poterat, per humilem
confessionem ei patefecit. Inter quae criminale quoddam ?, quod
ipse solus noverat, pro quo laieus* in conscientia sua conquerebatur,
confiteri ? non erubuit, el paenitentiam quam ei presbyter * iniunxit,
humiliter suscepit. Postea presbyter venit ad virginem,ut ante eam,
sieut consueverat, priusquam capella sua facta esset, officium de
missa, epistulam et evangelium legeret. Quo perleeto, quaesivit ab
eo puella, cur eo die solito tardius advenisset. Cumque se * vellet
apud eam exeusare, ait illa: Scio, domine mi, scio causam morae
vestrae : quia hodie venit ad vos quidam. parochia35 ἃ. s, 9, 3.— 1δ
sic add, 2, 3, — 17 s, v. 2, 3. — !5 (aut quidpraes.) om, 2. — 1" ad
quid ante me venire praesumilis add. 9, — 30 ocius 3. —?! Dei 2, —
33 3. — 38 in 2, — ** d. 1. 9, d. 1. sollemniter 3, — 35 Deum 3. —
?* quo 2, 3.— ὅτ paratus 3, — 35 (m. e.) om. 1. — 39 (Recedite-
ard.) om. 2.— 9? beatissimus 2. Cap. 4. — ! suus 2, 3. —? a. v, 2. —
? peccatum add. 9, 3. — * acrius 3. — " (c. conf.) torquebatur 2, 3.
— * p. e. ds τ requisivit 3.— δ om. 3. — s. v. 2, 3. — 1 om, 2.— 11
1ῃ vinculis suis add, 3. — 13 conversationis 2, — !? gj, 2, 3, — 4 d,
DE BEATA ALPAIDE VIRGINE. nus vester, qui omnia peccata Sua,
vobis salubri * confessione patefecit. Illud quoque. crimen.
pessimum, quod ipse commiserat, pro quo diabolus eum 19 artius *
ligatum tenebat, ex toto corde paenitens ore proprio vobis
confessus. est, salubri profecto accepto consilio, quia per condignae
paenitentiae fructum el sanctae confessionis sacramentum a se
diaboli iugum excussit. Et '? tunc presbytero dixit ^ peccatum, quod
ὁ ille commiserat. Obstupuit. presbyter, admirans quomodo id sciret.
vel quis ei peceatum hominis illius revelasset ; praesertim eum
tantum ille qui peccatum fecerat, et presbyter, cui illud confessus
fuerat, criminis illius conscii fuissent '*. 5. Altera quoque vice, cum
ante eam officium missae dixisset, ait illi : Cur hodie solito tardius
venistis ? Qui presbyter se occupatum fuisse respondit. Tunc illa :
Scio, inquit, scio occupationem vestram. Nam per domum praepositi
venistis et ante uxorem eius, quae pro puero, quem muper peperit,
infirma iacet in lectulo, officium missae 5, epistulam. et evangelium
legistis. Quod ita. fuisse verum ?? presbyter ?^ mihi dixit, admirans
quomodo id ei fuisset revelatum *!. 6. Altera die, cum missam in
ecclesia sua celebrasset, et ante puellam cantata missa venisset, tale
ab ea responsum accepit : Non est modo facies vestra. tantae
claritatis mec tantae pulchritudinis splendore plena, quantae. erat
hodie cum ante saerum 9? altare sacris indutus vestibus sacramentis
spiritualibus astaretis. Cui presbyter : Quomodo scis qualis tum
fuerim, cum me in ecclesia non videres 99, Ad quem illa : Zmmo vidi
vos astantem sanclo. allari magno. circumdatum. lumine, quando
sacrosanctum. Domini nostri. Iesu. Christi corpus inter manus
vestras super calicem elevastis **, in tres partes divisistis ; quarum
unam in calicem in sanguinem ** misistis. Hoc est enim verum ?*
corpus,quod Salvator nosler in utero 31 piyginis Mariae pro nobis
assumpsit, quod. pro nobis in cruce fuit suspensum, clavis affizwm,
lancea. vulneratum, ad salutem. omnium. credentium. Hic est
gloriosus eliam 35 ille sanguis, quem in. calice tenebatis, qui de
pretioso corpore Salvatoris in. cruce fuit ejf usus in remissionem
peccatorum nostrorum. Hoc corde credo, hoc ore. confiteor, in. hac
fide debent. tanta sacramenta. accipere? fideles christiani, ut ab eo
salvari mereantur, a quo. sunt ?? redempti , qui cum. Patre et sancto
Spiritu ? vivit et regnat Deus per omnia saecula saeculorum. Amen.
*. Capvr v. — Una sabbatorum in adventu Domini, aspiciens a longe
vidit quandam maximam? aquam et mulierem speciosissimam, vene
rando ὃ habitu decoratam, super aquas siccis vestigiis ambulantem :
quae in manu sua dextera tenebat unam pulcherrimam * erucem,
super cuius brachium dextrum stabat candidissima quaedam
columba oculis in. caelum intenta. Praecedebat mulierem puer
septennis, speciosus forma prae filiis hominum, cuius aspectu
desiderabili non poterat virgo saliari. Veniebant ambo pariter ad
puellam. mirantem et obstupescentem, eo quod p. 2, 3. — homo
add.9,3. — 15 Caput v add. 3, item de visione add. 9; — 11 venisti
2. — 18. et add. 2. —* v.f. 2,3. — ? ipse add. 2,3.— ?* cap. v add.3,
item de altera visione add. 9, — 3 sanctum 3. — "8 videris 9, 3. —
** elevatum 2, 3.— 38 sanguine 3,.— 5 illud add. 9, 3.— ?"
beatissimae add. 9, 3. — 38. e, g. 2, 3. — 39 suscipere 9, 3. — *? et
add. 9, 3.— *18.$.9, 3. Cap. 5. — ! n 3. —? m.q. 2, 3. — ?
reverendo 2, 3.— * p. u. 2, ὃ. siccis D et parochum de Cudot videt
orantem. super aegros, E et in missa. luce splendentem. Sancta
mulier cum puero septenni d columba € apparet.
 Visio de fidelibus Adamo. B euntibus in caelum et de quasi?
super terram. ambulabant 9. Occurrit. eis obviam * puella volens
osculari erucem, quam in manu sua femina portabat ; sed in corde
suo nimis pavebat ut, si crucem tangeret, columba quae desuper *
sedebat, inde territa avolaret "ἃ et ne columbam terreret, osculata
est crucem in inferiori parte sub manu tenentis illam '^ ; et in osculo
sensit tantam suavitatem odoris ?' de ligno crucis egredientem, ut
omnium aromatum vinceret suavitatem. Osculata cruce, dedit ei
benedictionem puer elevata manu, signum crucis faciens super eam
; postea mulier, signo salutiferae crucis elevato, de ipsa eruce eam
"^ signavit, et sic columba desuper crucem ?? avolante 1" et ad
caelos gratulabundo volatu ascendente, puer post columbam et
mulier crucem tenens post puerum in caelum pariter ascenderunt.
CAPUT SECUNDUM De visionibus quas in caelo vidit B. Alpais. 8. Carr
σι". — Alio tempore ducta est ad quendam?fluvium maximum,
volebatque trans torrentem transire in quaedam, quae ultra ripam
fluminis erant, amoenissima prata vernantibus herbis, et floribus
odoriferis? virentia. Nullus ibi pons, nulla, qua. transire posset, erat
navicula ; unde nimis erat ambigua, quid agere. quo ire deberet
ignorans, eum subito prospiciens videt elegantem forma * iuvenem,
quasi in aetate quindecim annorum super aquas fluminis a pratis ad
se venientem. Cumque iam prope esset, dextram ei porrigens nutu
eam advocavit. Vocata super amnem ascendit, et sequebatur eum 5
admirans quod siecis vestigiis super aquas ^ pariter ambularent.
Transacto ἴ flumine, intraverunt in prata delectabilia virentibus
herbis, purpureis rosis, vernantibus violis et omnimodis floribus
suaviter redolentia.Super flores in pratis ita leviter* gradiebantur,
quod eos pedibus suis non conculcabant. Sub ? pedibus
ambulantium inconcussae stabant rosae. Post terga transeuntium
stabat ' herba stans recta !!, sicut. prius stelerat, ac si pedes eorum
eam minime tetigissent. Cum omnia illa prata B pertransissent,
intraverunt in pomerium quoddam magnum et delectabile, generibus
eunetis '? arborum copiose repletum, in quo innumerabiles ὁἢ
animae sanctorum, ineffabili laetitia et exultalione simul 15
gaudentium '*, quarum aliae purpureis aliae candidatis erant
Welcome to our website – the ideal destination for book lovers and
knowledge seekers. With a mission to inspire endlessly, we offer a
vast collection of books, ranging from classic literary works to
specialized publications, self-development books, and children's
literature. Each book is a new journey of discovery, expanding
knowledge and enriching the soul of the reade

Our website is not just a platform for buying books, but a bridge
connecting readers to the timeless values of culture and wisdom. With
an elegant, user-friendly interface and an intelligent search system,
we are committed to providing a quick and convenient shopping
experience. Additionally, our special promotions and home delivery
services ensure that you save time and fully enjoy the joy of reading.

Let us accompany you on the journey of exploring knowledge and

personal growth!

ebookname.com