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...