There are three major technical issues involved during extraction of nucleic acids from low-DNA, forensic or environmental samples: (1) extraction efficiency; (2) how well the extracted nucleic acids actually represent those in the sample; and (3) chemical and nucleic acid contamination. If sample size and quality is sufficient, the efficiency of extraction may not be a serious issue, as many analyses only require small amounts of nucleic acid template. However, given the increased risk of artifacts when working with low concentrations of extracted nucleic acids (see Section 2.1), efficient nucleic acid extraction is generally desirable. Obviously, as molecular target numbers approach limits of reliable detection, such as in very small or dilute samples, extraction efficiency becomes an important issue. For highly dilute samples, ultracentrifugation devices may facilitate the concentration of large volume extractions. Also, although many samples may contain sufficient nucleic acids for proper analyses, these nucleic acids can be bound to the sample substrate or contain intermolecular cross-links that inhibit downstream analyses. Addition of PTB (N-phenacylthiazolium bromide) appears to break intermolecular cross-links caused by Maillard reactions, releasing more PCR amplifiable DNA during the extraction process (Poinar et al, 1998). In addition, many cells, cell walls or organism structures can be highly impervious to lysis. Numerous detergents, lytic enzymes and physical disruption methods have been introduced to facilitate liberation of nucleic acids from recalcitrant samples (Griffiths et al., 2000; Hurt et al., 2001). However, it must be kept in mind that there is a general tradeoff between the rigorousness of extraction methods and the quality of nucleic acids extracted, and decreased nucleic acid quality again increases risks of downstream artifacts. Although manufactured kits have recently become available for the routine and potentially standardized extraction of nucleic acids from diverse sample types, it must be remembered that such kits have not been rigorously validated across a full range of organisms and sample types.
If research goals include comparisons of target frequencies it is essential that extracted nucleic acid targets are in the same ratio as present in the original sample. Unfortunately, nucleic acids from different organisms, tissues or substrates are not liberated and recovered equally, and this potential for bias must be recognized when interpreting results. This issue is less serious if one is comparing like samples, as any extraction biases can be assumed to remain constant across all samples. The relative representation levels of extracted nucleic acids may be a moot point if single targets are being analyzed, but is a serious issue for complex samples and analyses.
Contamination during extraction procedures can be of two types: co-extraction of substances that may inhibit downstream analyses and the introduction of contaminant sequences during extraction procedures that can lead to the recovery of erroneous results. The former contamination type is generally the result of failure to eliminate compounds from the sample that inhibit downstream analyses, whereas the latter introduces false sequences into the sample. Co-extraction of contaminating DNA and enzymatic inhibitors is a serious issue, potentially leading to false negative results or compounding artifact risks. The range of starting material and preservation conditions, in the case of ancient DNA studies, is extremely broad including soils, sediments, rocks, vegetation, water, ice, archival museum and pathology specimens (dried ethanol and/or formalin fixed skins, hair, feather, bones and tissues), archaeological material (e.g. bone, clothing, pottery, stone tools, food residues), faeces, naturally or artificially mummified remains, coprolites (subfossil faeces) and forensic specimens (e.g. body fluids, hair, skin, stool). This diversity of material and preservation creates a range of challenges for DNA extraction and subsequent enzymatic manipulations, and often necessitates customized extraction and purification procedures. Compounds that are co-extracted with nucleic acids, such as phenolic compounds and humic acids, often require additional purification steps or extract dilution prior to downstream analyses, both of which can decrease target density and therefore increase contamination risk (e.g. by necessitating use of additional PCR cycles; see Section 3).
The concerns associated with the introduction of contaminant sequences during extraction procedures are very similar to those encountered in sample collection and storage. Again, concerns are greatest when dealing with samples containing low-target numbers and/or nucleic acid quality. Preparations of sample material prior to actual extraction procedures must be carried out in an otherwise DNA-free laboratory environment. Also, all reagents and equipment used for the extraction must be free of nucleic acid contamination. It is generally assumed that commercial kits and reagents are free of nucleic acids, but this is often not the case, and precautionary UV treatment of all materials is therefore recommended.
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