“… thoughtfully put together …”– “Aslib Book Guide, Vol 64, No 8, Ref 861, August 1999”

“There is value in this book for every laboratory worker to help them improve the quality of their data …”– “The Analyst Web Site, September 1999”

Analytical Molecular Biology

Quality and Validation

By Ginny C. Saunders, Helen C. Parkes

The Royal Society of Chemistry

Copyright © 1999 LGC (Teddington) Ltd
All rights reserved.
ISBN: 978-0-85404-472-6

Contents

Chapter 1 An Introduction to Analytical Molecular Biology Ginny C. Saunders and Helen C. Parkes, 1,
Chapter 2 Quality in the Analytical Molecular Biology Laboratory Ginny C. Saunders, 9,
Chapter 3 DNA Extraction Ginny C. Saunders, 29,
Chapter 4 Quantification of Total DNA by Spectroscopy Paul A. Heaton, 47,
Chapter 5 PCR: Factors Affecting Reliability and Validity David McDowell, 58,
Chapter 6 Inhibitors and Enhancers of PCR Jane Bickley and Daniel Hopkins, 81,
Chapter 7 Random Amplified Polymorphic DNA Analysis Ginny C. Saunders and Daniel Hopkins, 103,
Chapter 8 Development of Multiplex PCR Jo Short and Jim Thomson, 123,
Chapter 9 Membrane Hybridisation Johanne H. Cornett, Jason Sawyer and Della Shanahan, 135,
Chapter 10 Automated Fluorescent DNA Cycle Sequencing N. J. Oldroyd and I. L. Comley, 155,
[Appendix: Glossary of Terms, 173,
Subject Index, 183,

CHAPTER 1

An Introduction to Analytical Molecular Biology

GINNY C. SAUNDERS AND HELEN C. PARKES

1.1 Introduction

DNA technology is having a revolutionary effect on a host of industrial and regulatory sectors. The pace of fundamental innovation in the biosciences shows no signs of abating and continues to reveal new commercial opportunities in both biotechnology and analytical molecular biology. Healthcare, pharmaceutical production, diagnostics, agriculture, animal husbandry, food and forensic analysis are just a few areas where DNA technology is significantly changing the way industry and regulators operate. Clearly, this rapidly developing technology offers tremendous advantages and benefits to bioanalysis with respect to increased scope of application, detection limits, speed, cost and specificity. However, in order to capture and utilise these advantages, there is an urgent need for parallel validation of the analytical techniques employed in DNA-based measurements. The cost of employing invalid or flawed DNA technology would be enormous and highly damaging, both in terms of public perception and financial investment.

Analytical molecular biology has been typically developed in the academic and medical research environments. Here, priorities are understandably concerned with innovation, rather than consideration being given to the more routine applicability, reliability and reproducibility of the methods. Evaluation of these factors and further method validation is therefore an absolute prerequisite for the successful move of techniques from the research laboratory to the analytical laboratory.

Limited discussion at scientific for a has been paid to questioning the validity of DNA-based measurements, despite growing commercial and public activity in these areas. There are possibly three main reasons for the lack of research and debate into the validity of these measurements. First, the excitement of being able to measure where no-one has measured before can lead to an enthusiastic rush of application. Second, regulation of the analysis is generally carried out in-house and not through performance standards set by the larger analytical community. Finally, there is a lack of reference samples such as key analytes contained in complex matrices necessary for the critical comparison of analytical approaches.

This manual aims to introduce and address quality and validation issues that arise in the application of DNA technology and, hopefully, offers a basis for further discussion and debate within the bioanalytical community.

1.2 What is Analysis, Why is it Undertaken?

Analysis is usually initiated, proposed or commissioned by a customer, who can be a private individual or company, public organisation or law enforcement agency such as the police force or trading standard office. Analysis of a material or matrix is undertaken to examine one or more of its constituent parts or analytes. Analytical data are required as an independent source of information in order for the customer to gauge a situation, interpret evidence, decide whether action is required or to ascertain whether certain regulations are being adhered to. The data obtained from analysis are therefore required in a variety of forms:

• Qualitative -confirmation of the presence of an analyte

• Semi-quantitative -provides an estimate of analyte concentration

• Quantitative -provides a well defined value for the amount of analyte

There are also various types of analyses that can be undertaken, each offering different discriminatory powers. These are summarised in Table 1.1.

Analysis should not be viewed as a straightforward exercise or in any sense mundane due to its sometimes routine nature. In reality, analytical methodologies are frequently made up of a complex and evolving mixture of techniques, where specific applications or samples demand appropriate adaptations. A seemingly straightforward implementation of the methodologies and generation of data could arise from either careful and considered planning and validation, showing a dedication to producing quality analytical data, or a complete lack of all the aforementioned qualities. In the second case, implementation appears simple as the task has not been undertaken with due consideration or care. Chapter 2 discusses how to obtain the former scenario and avoid the latter.

1.3 DNA, a Universal Biological Analyte

Increasingly high expectations of public health and general quality of life has led to a greater need for the detection and analysis of biological materials. Detection of human, animal, food and environmental pathogens can all inform public health policy. The advent of biological methodologies such as DNA forensics has revolutionised the analysis of scene of crime evidence and provided a valuable tool for law enforcement agencies such as the police, trading standard offices and wildlife protection organisations. Molecular genetic tests have allowed pre-natal detection of genetic diseases and can detect gene mutations which may inform a change of lifestyle.

In spite of the vast variety and complexity of biological materials (matrices and organisms), they share a host of common biomolecules, of which nucleic acids form a major group. Deoxyribonucleic Acid (DNA) is an ideal universal analyte for biological methodologies. It is the genetic material of the majority of forms of life and an identical copy of the genome is contained within nearly every cell of an organism. The DNA of an individual is unique (with the exception of homozygous twins) with respect to the sequential order of the four base constituents, making it an indisputable marker for identification purposes. A genome consists of both highly conserved regions of sequence such as genes and variable, non-conserved regions. Comparable DNA sequences show more similarity between closely related individuals or species and less similarity between distant relatives. Both non-conserved and highly conserved regions of a genome are exploited in analytical molecular biology to detect similarities or differences (known as DNA polymorphisms) of a DNA sequence.

The use of nucleic acids, particularly DNA, as an analyte offers unparalleled sensitivity to biological detection and characterisation techniques. Theoretically, using the polymerase chain reaction (PCR), a single copy of a gene can be detected. In the field of bacterial detection and identification, DNA technology is, in many cases, offering faster analysis times than comparable classical methodologies such as plate culture detections. DNA is also more resistant to degradation than RNA or protein molecules, an important factor when selecting an analyte from highly processed or aged samples.

1.4 Sectoral Applications of Analytical Molecular Biology Techniques

Listed in Table 1.2 is a summary of current applications of analytical molecular biological methodologies. The range is so vast that these techniques could well touch everyone’s life at some time or another and go some way to maintaining the current standard of living expected in the Western world.

1.5 Challenges of DNA Analysis

DNA analysis does, however, have its own challenges. Some major concerns arise from the analysis of ‘real’ samples, as in typical industrial and enforcement situations where non-ideal samples are the norm. Such samples 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, Listeria spp. in milk or Legionella spp. in water.

Some of the challenging situations that exist in the application of DNA technologies are:

• Low concentration of analyte compared to matrix. This has lead to the development 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.

• The varied and complex biological or chemical matrices that are the source of the nucleic acid to be analysed can make DNA extraction a difficult undertaking. Complex chemical or biochemical components of a matrix, such as naturally occurring secondary compounds, can interfere with enzyme activity and can cause total inhibition of biological reactions such as PCR and restriction enzyme digests.

• DNA degradation due to a sample being subjected to harsh conditions. These include industrial processing such as freezing, dying, heating, grinding, tanning, drying and forms of weathering such as those caused by the sun or rain. Such conditions may be in addition to the ageing of a sample, all of which can cause physical degradation of the DNA analyte.

• Biological contamination of the sample can mean that nucleic acids from a variety of sources are present, perhaps due to environmental insult (e.g. bacterial or fungal contamination) or scene of crime samples containing bodily fluid from both the victim and the criminal. Endogenous or exogenous (i.e. from contaminating microorganisms) DNases can cause DNA degradation.

• Degradation of matrix components can sometimes produce breakdown products, such as polyphenols, that cause the degradation of nucleic acids.

• Limited availability of a sample. This may be because the sample represents a unique moment in time or is limited by quantity.

• Lack of suitable controls. There are very few characterised reference samples that can be employed to ensure the accurate calibration of equipment, the correct handling of samples or the applicability of methodologies.

It is partly due to the challenges listed here that there is a wide gap between molecular biological technique development and analytical application, leaving the transition from research to routine somewhat problematic. In order for a technique to become readily accepted as an analytical tool, confidence must be gained in the performance of the technique. An application must appear robust enough to avoid the production of erroneous results and be resistant to small changes in one or more of its parameters.

1.6 Key Techniques in Analytical Molecular Biology

From the wide range of molecular biology techniques available, only a selection is commonly employed in analysis (Table 1.3). Other techniques, such as cloning and transformation, are perhaps more widely employed in biotechnological applications and more state of the art techniques are most likely to be of research interest.

Table 1.3 identifies eight key techniques which, in combination, represent a powerful collection of methodologies that provide a wide range of analytical approaches. It is therefore obvious that any procedural undertakings that affect the validity of a single analytical technique have the potential to affect a broad range of methodologies. The ‘critical points’ in these key techniques must therefore be well characterised in order to minimise, counteract or, at the very least, understand their effect on the analytical data produced.

1.7 Future Prospects and Considerations

The transfer time of a technique from the research laboratory to the analytical laboratory can vary. This could be dependent upon whether the new analysis is a further application of existing DNA technology, or whether it is an unfamiliar method using novel techniques and equipment. The former may require a shorter time period as reduced training, protocol preparation and validation could be required. In either case, a close working relationship between the researchers and analysts can ease the transition by building a clear understanding of each other’s goals and requirements and working together on common ground.

Plans for the future of analytical applications appear to be progressing towards miniaturisation, parallelisation and automation. In order to achieve this, improvements are required in the areas of sample preparation, assay technology, detection systems and data management. There is also a need to integrate the required steps in an economic way so that a given DNA analysis procedure can be performed substantially quicker and cheaper than existing tests.

Recent advances in the adoption of molecular biology, in particular PCR, 5 as an analytical tool continue to meet a wide demand for ever increasing improvement to levels of detection, accuracy, sensitivity and reliability. Quality should also be at the forefront of demands made on this evolving technology and this subject forms the core theme that runs throughout this book.

The acceptance of DNA profiling as an analytical tool has much to offer us as a lesson to be learnt. This innovative technology, first described by Jeffreys et al., was first used in a court of law in the ‘Pitchfork’ case in Lincolnshire in 1986. Since then, the validity of DNA data submitted as evidence in courts of law has been challenged. The stringent validation and quality processes that are now in place in today’s forensic laboratories have therefore been, to some extent, driven by the pressures of the defence lawyers, continually challenging the analytical process both in this country and abroad. The presence of an equivalent pressure is not always evident in other areas of analytical molecular biology such as environmental or clinical testing. In these cases, the majority of the impetus for ensuring that appropriate data are produced as a matter of course lies with the professionalism of the analytical laboratory and the analysts involved. This is not a task to be undertaken light heartedly. It requires continual questioning and re-evaluation of the analytical approach, procedure, staff capabilities and applicability of the test. Analytical laboratories should, as a priority, work to maintain the confidence of the public and industrial customers by promoting the production of quality analytical data.

CHAPTER 2

Quality in the Analytical Molecular Biology Laboratory

GINNY C. SAUNDERS

2.1 Introduction

The need for valid practices to produce quality data cannot be disputed in any analytical environment; however, the route to consistently obtaining quality analytical data is not necessarily a clear and straightforward path. This chapter will attempt to introduce and highlight the many factors that can and do influence the ultimate goal of the analyst, namely getting the analysis right first time and every time.

There are four key criteria that must be met in order to obtain quality data:

1. A valid methodology

2. A quality assurance system

3. A quality control system

4. Trained and experienced analysts

It is important to note that, throughout the practical chapters of this manual, the assumption is made that the last three of these requirements have been fully met. The first, a valid methodology, is largely the subject of Section 2.4, with additional information presented in each practical chapter as appropriate.

Figure 2.1 demonstrates how these four criteria are inter-linked and work together to yield quality data. In turn, quality results are dependent on all of the criteria being met; a single criterion, even if met, cannot act effectively in isolation from the others. For example, trained and experienced analysts are required both to ensure that the chosen method is fit for its intended purpose and that it is correctly applied. To ensure that this is the case, the analyst will require a full understanding of the scientific principles of the methodology and its limitations. Equally, application of a valid methodology may be invalidated if equipment is not correctly calibrated, or samples are stored incorrectly. These are just two of the many factors that should be managed by a quality system. In addition, methodologies should stand up to external quality control through the use of reference standards or by comparing analysis with those undertaken elsewhere. Quality assurance schemes and experienced analysts should play an important part in determining the cause of any disparity between expected and actual results and defining any remedial or preventative action required.

Adoption of a uniform understanding of terminology commonly used in this area is also required to ensure that these criteria are consistently interpreted and fully met. This chapter sets out to introduce and define various quality and validation terms. Some general terms used throughout the manual are given in Table 2.1.

The Valid Analytical Measurement (VAM) Initiative aims to present a holistic approach to obtaining quality data by presenting six principles for a laboratory to follow. These principles can be applied in a self-regulatory manner and in no way interfere or conflict with the remit of a quality system.

(Continues…)Excerpted from Analytical Molecular Biology by Ginny C. Saunders, Helen C. Parkes. Copyright © 1999 LGC (Teddington) Ltd. Excerpted by permission of The Royal Society of Chemistry.
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