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Basic tools and techniques in molecular biology

Ralph Rapley and David Whitehouse

2.1 Introduction

The purpose of this chapter is to provide a perspective on basic molecular biology techniques that are of special relevance to forensic genetics. Genetic polymorphisms are the most valuable tools for human identification and for determining genetic relationships and have consequently become a mainstay for forensic science. Throughout the history of forensic genetics, genetic polymorphisms have been studied at various levels from the cellular and serological, such as the determination of blood groups and HLA types, gene product analysis, such as the red cell isozyme and serum protein polymorphisms, to the direct examination of nuclear and mitochondrial DNA. The last has afforded a variety of polymorphic systems, notably fragment length polymorphisms, due to variable number tandem repeats (VNTRs) and single nucleotide polymorphisms (SNPs). DNA analysis offers exquisite resolution compared with the former cellular and gene product analysis approaches to characterizing genetic variation; for the first time it has enabled a direct view of the entire genome. It is the techniques and procedures for working with and analysing segments of DNA that are the subject matter of this chapter.

2.2 Isolation and separation of nucleic acids Isolation of DNA

The use of DNA for forensic analysis or manipulation usually requires that it is isolated and purified to a certain extent (see Chapter 3). It should be noted

Molecular Forensics. Edited by Ralph Rapley and David Whitehouse Copyright 2007 by John Wiley & Sons, Ltd.

that the level of purity of template DNA to be amplified by the polymerase chain reaction is frequently far less critical than the knowledge that the sample to be analysed is uncontaminated and exclusively contains only material from the stated source. Figure 2.1 illustrates a general scheme for DNA extraction.

DNA is recovered from cells by the gentlest possible method of cell rupture to prevent the DNA from fragmenting by mechanical shearing. This is usually in the presence of EDTA, which chelates the Mg2+ ions needed for enzymes that degrade DNA, termed DNases. Ideally the cell membrane should be solubilized using detergent, and cell walls, if present, should be digested enzymatically (e.g. lysozyme treatment of bacteria). If physical disruption is necessary, it should be kept to a minimum, and should involve cutting or squashing of cells rather than the use of shear forces. Cell disruption should be performed at 4°C, using disposable plastics where possible; all glassware and solutions are autoclaved to destroy DNase activity (Cseke et al., 2004). Techniques such as laser microdissection are being investigated as exciting new tools for the recovery of genetic material from crime scenes (see Chapter 10). After release of nucleic acids from the cells, RNA can be removed by treatment with ribonuclease (RNase), which

Figure 2.1 General steps involved in extracting DNA

from cells or tissues is usually heat treated to inactivate any DNase contaminants (Cseke et al., 2004). The other major contaminant, protein, is removed by shaking the solution gently with water-saturated phenol, or with a phenol/chloroform mixture, either of which will denature proteins but not nucleic acids. Centrifugation of the emulsion formed by this mixing produces a lower, organic phase, separated from the upper, aqueous phase by an interface of denatured protein. The aqueous solution is recovered and deproteinized repeatedly, until no more material is seen at the interface. Finally, the deproteinized DNA preparation is mixed with sodium acetate and absolute ethanol, and the DNA is allowed to precipitate out of solution in a freezer. After centrifugation, the DNA pellet, which can be washed in 70% ethanol to remove excess salt, is redissolved in a buffer containing EDTA to inactivate any DNases present. This solution can be stored at 4°C for at least a month.

DNA solutions can be stored frozen for prolonged periods, although repeated freezing and thawing tends to damage long DNA molecules. The procedure described above is suitable for total cellular DNA, mitochondrial DNA and DNA from microorganisms and viruses. However, in the case of the extraction of DNA from difficult sources such as hair shafts, a more vigorous preparatory step is required, such as disrupting the starting material in a tissue grinder. The integrity of the DNA can be checked by agarose gel electrophoresis and the concentration of the DNA can be determined spectrophotometrically.

Contaminants may also be identified by scanning UV spectrophotometry from 200 nm to 300 nm. A ratio of 260 : 280 nm of approximately 1.8 indicates that the sample is free of protein contamination, which absorbs strongly at 280 nm.

Automated and kit-based extraction of nucleic acids

Automation and kit-based manipulations in molecular biology are steadily increasing, and the extraction of nucleic acids by these means for forensic analysis is no exception (see Chapter 3). There are many commercially available kits for nucleic acid extraction. Although many rely on the methods described here, their advantage lies in the fact that the reagents are standardized and quality-control-tested, providing a high degree of reliability. Essentially the same reagents for nucleic acid extraction may be used in a format that allows reliable and automated extraction. This is of particular use where a large number of DNA extractions are required (Montpetit et al., 2005).

2.3 Automated analysis of nucleic acid fragments

Gel electrophoresis remains the established method for the separation and analysis of nucleic acids. Indeed a number of automated systems using precast gels are available that are gaining popularity. This is especially useful in situations where a large number of samples or high-throughput analysis is required. In addition, new technologies such as Agilents' Lab-on-a-chip have been developed that obviate the need to prepare electrophoretic gels. These systems employ microfluidic circuits where a small cassette unit that contains interconnected micro-reservoirs is used. The sample is applied in one area and driven through microchannels under computer-controlled electrophoresis. The channels lead to reservoirs allowing, for example, incubation with other reagents such as dyes for a specified time. Electrophoretic separation is thus carried out in a micro-scale format. The small sample size minimizes sample and reagent consumption, and as such is useful for DNA and RNA sample analysis. In addition the units, being computer controlled, allow data to be captured within a very short time-scale (see He et al., 2001). Alternative methods of analysis, including denaturing high-performance liquid chromatography-based approaches, have gained in popularity, especially for mutation analysis (Underhill et al., 2001). Mass spectrometry is also becoming increasingly used for nucleic acid analysis (Oberacher et al., 2006).

2.4 Molecular biology and bioinformatics

Databases and basic bioinformatics

Bioinformatics has become a vital resource for applied forensic molecular biology and is a key component of the routine detection and identification of short tandem repeat (STR) profiles in forensic casework (see Chapter 11). The National DNA Database (NDNAD) established in 1995 was the first forensic science database. It contains STR profiles from subjects in the UK. Samples are normally taken from mouth swabs, though less frequently blood samples are taken. The NDNAD also contains information on samples from volunteers and crime scenes (Parliamentary Office of Science and Technology, 2006). Many countries now maintain their own forensic DNA databases. For example, in the USA the FBI has developed the Combined DNA Index System (CODIS).

The emergence of nucleic acid and the accompanying bioinformatics tools has been driven principally by the Human Genome Project with its need to store, analyse and manipulate vast numbers of DNA sequences. There are now a huge number of sequences stored in genetic databases from a variety of other organisms. The largest of the sequence databases include GenBank at the National Institutes of Health (NIH) in the USA, EMBL at the European Bioinformatics Institute (EBI) at Cambridge, UK and the DNA database of Japan (DDBJ) at Mishima in Japan. All the genome databases are accessible to the public via the Internet.

2.5 The polymerase chain reaction (PCR)

Basic concept of the PCR

The polymerase chain reaction or PCR is currently the mainstay of forensic molecular biology. One of the reasons for the wide adoption of the PCR globally is the elegant simplicity of the reaction and relative ease of the practical manipulation steps. Indeed, combined with the relevant bioinformatics resources for its design and for determination of the required experimental conditions, it provides a rapid means for DNA identification and analysis. It has opened up the investigation of cellular and molecular processes to those outside the field of molecular biology (Altshuler, 2006).

The PCR is used to amplify a precise fragment of DNA from a complex mixture of starting material, usually termed the template DNA, and in many cases requires little DNA purification. It does require the knowledge of some DNA sequence information, which flanks the fragment of DNA to be amplified (target DNA). From this information two oligonucleotide primers may be chemically synthesized, each complementary to a stretch of DNA to the 3' side of the target DNA, one oligonucleotide for each of the two DNA strands (Figure 2.2). It may be thought of as a technique analogous to the DNA replication process that takes place in cells since the outcome is the same: the generation of new complementary DNA stretches based upon the existing ones. It is also a technique that has replaced, in many cases, the traditional DNA cloning methods since it fulfils the same function - the production of large amounts of DNA from limited starting material - however this is achieved in a fraction of the time needed to clone a DNA fragment. Although not without its drawbacks, the PCR is a remarkable development that is changing the approach of many scientists to the analysis of nucleic acids and continues to have a profound impact on core biosciences and biotechnology.

Stages in the PCR

The PCR consists of three defined sets of times and temperatures, termed steps: (i) denaturation, (ii) annealing and (iii) extension. Each of these steps is repeated 30-40 times, termed cycles (Figure 2.3). In the first cycle the double-stranded template DNA is (i) denatured by heating the reaction to above 90°C. Within the complex DNA the region to be specifically amplified (target) is made accessible. The temperature is then cooled to between 40 and 60°C. The precise temperature is critical and each PCR system has to be defined and optimized. One useful technique for optimization is Touchdown PCR where a programmable cycler is used to incrementally decrease the annealing temperature until the optimum is derived. Reactions that are not optimized may give rise to other

Complex genomic 'template' DNA

Complex genomic 'template' DNA

PCR primers designed to each DNA strand that flanks region to be amplified

Primer!

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