Automated DNA extraction techniques for forensic analysis
Many different methods are used to extract DNA from the wide range of specimens commonly found at crime scenes. Techniques range from simple alkaline lysis followed by neutralization (Klintschar and Neuhuber, 2000), to the well-known salting-out method (Miller et al., 1988) that is used in cases of higher cell concentrations, to a simple closed-tube method utilizing a thermostable proteinase (Moss et al., 2003). Only a few of these methods are suitable for automation, and many of the steps involved are associated with a high risk of contamination. Many methods also require centrifugation and solvent extraction steps (Sambrook et al., 1989; Walsh et al., 1991) and thus are also not easily adapted for automation. Most of the methods used for DNA extraction can meet only one of the several standards for an optimal DNA extraction process: high DNA yield, rapidity of the method (McHale et al., 1991), high throughput and high DNA quality (Akane et al., 1994; Klintschar and Neuhuber, 2000). Thus, for a long time the method of choice for many forensic samples was the traditional but hazardous use of phenol-chloroform extraction, which had to be performed under stringent safety measures (Butler, 2005). The end-product of the extraction sometimes was not sufficiently pure and still needed to undergo further purification steps using membranes or columns, so this method is not ideal in several respects. In order to assess the utility of other methodologies for DNA extraction, we first examine the principal steps in isolating DNA from biological material.
Molecular Forensics. Edited by Ralph Rapley and David Whitehouse Copyright 2007 by John Wiley & Sons, Ltd.
The first step in any DNA extraction method is to break the cells open in order to access the DNA within. Although DNA may be isolated by 'boiling' cells (Starnbach et al., 1989), this rather crude means of disrupting the cell does not produce DNA that is always of sufficient quality and purity to be used in downstream analytical techniques such as polymerase chain reaction (PCR) amplification. DNA isolated by simple boiling generally fails as a substrate for further analysis because it has not been sufficiently separated from structural elements and DNA-binding proteins, and these impurities compromise downstream procedures. In order for DNA to be released cleanly, the phospholipid cell membranes and nuclear membranes have to be disrupted in a process called lysis, which uses a detergent solution (lysis buffer), often containing the detergent sodium dodecyl sulphate (SDS), which disrupts lipids and thus disrupts membrane integrity. Lysis buffer also contains a pH-buffering agent to maintain the pH of the solution so that the DNA stays stable: DNA is negatively charged due to the phosphate groups on its structural backbone, and its solubility is charge-dependent and thus pH-dependent. Proteinases, which are enzymes that digest proteins, are generally added to lysis buffer in order to remove proteins bound to the DNA and to destroy cellular enzymes that would otherwise digest DNA upon cell lysis. The lysis procedure sometimes calls for the use of heat and agitation in order to speed up the enzymatic reactions and the lipid solubilization.
DNA extraction: purification and efficient removal of PCR inhibitors
Cell or tissue samples may contain elements that inhibit the DNA extraction process at any of the various steps involved in DNA isolation. These inhibitors may interfere at any step of the process, but are generally problematic in three areas:
1. Interference with the cell lysis, the first step in DNA preparation.
2. Interference by degrading nucleic acids or by otherwise preventing their isolation after lysis is complete.
3. Inhibition of polymerase activity during the PCR amplification of target DNA after successful purification.
We focus here on the third type of inhibition: interference with PCR. After the initial isolation of DNA, it must be separated from the other cellular compo nents that remain after the lysis procedure. This is often followed by further washing steps, which function to remove any remaining substances that could inhibit amplification of the DNA by PCR and its subsequent analysis. A wide range of PCR inhibitors have been reported (see Wilson, 1997, for review). Common inhibitors include various body fluid components (e.g. haemoglobin, melanin, urea) as well as chemical reagents that are frequently used in clinical and forensic science laboratories (e.g. heparin, formalin, Ca2+). Inhibitors also include microorganism populations, which are frequently an overrepresenta-tion of bacterial cells or food constituents found at the scene. Similarly, environmental compounds at the crime scene or in the forensic laboratory can also act as PCR inhibitors. These include organic and phenolic compounds, glycogen, polysaccharides, humic acids, fats and laboratory items such as pollen, glove powder and plasticware residue. Carry-over of compounds such as those used in cell lysis (e.g. proteolytic enzymes or denaturants) and phenolic compounds from DNA purification procedures can also be problematic. Many of these PCR-inhibitory compounds, such as polysaccharides, urea, humic acids, haemoglobin, melanin in hair samples or indigo dyes from denim, exhibit a solubility similar to that of DNA, therefore they are not completely removed during classical extraction protocols such as detergent and phenol-chloroform extraction, and persist as contaminants in the final DNA preparation. Several methods have been developed to remove these contaminants, including glass bead extraction, size-exclusion chromatography, spin column separation, agarose-embedded DNA preparation or immunomagnetic separation (Wilson, 1997; Moreira, 1998).
There are, in general, three primary techniques used in forensic DNA laboratories: the phenol-chloroform extraction method, Chelex extraction and magnetic affinity solid-phase extraction. The principles of all three methods are shown in Figure 3.1 and are described in the following sections.
The standard phenol-chloroform extraction protocol in use today (Figure 3.1a) is described in Sambrook et al. (1989). Samples are incubated with enzymatic lysis buffer (e.g. 10 mM Tris-HCl pH 7.4, 400 mM NaCl, 2 mM Na2EDTA pH 8.1, 1% SDS and 667 |g/ml proteinase K) overnight at 37°C or for 2 hours at 56°C to partially digest cellular proteins. The resultant liquid-phase cell lysate is treated with equilibrated phenol, and the aqueous and organic phases are mixed thoroughly and then separated by centrifugation. The DNA remains in the aqueous phase while the cellular proteins are extracted into the organic
Extraction: bloodstain on textile a: PHENOL/CHLOROFORM
P—-1 Addition of Lysis Lysis LJ Solution (with \/ Proteinase K)
Incubation (37 or 56°C)
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