• 2018-07
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  • br Discussion Although DNA has


    Discussion Although DNA has a limited chemical stability it has long been known that DNA in blood stains may remain relative stable for weeks, months, or even years at ambient temperature,1, 2, 3, 4, 5, 6 and samples (e.g., reference samples) that have been transferred to a matrix, e.g., FTA® paper, containing stabilizing agents are stable for at least 4 years. Deposited biological matter is exposed to degradation by endogenic enzymes, microbial exoenzymes and spontaneous chemical reactions.10, 19 In the present work we have addressed the importance of air-humidity since the availability of water is necessary for both microbial activity and for un-catalysed hydrolytic reactions that are known to degrade DNA.11, 12 The influence of the humidity of the surrounding air on the stability of proteins was shown by Sensabaugh, who observed an exponential increase in the inactivation rate of 9-cis-Retinoic Acid phosphatase in semen stains when RH was increased from 40% to 100%. It is therefore noteworthy that we found no indication that RH up to 93% had any significant effect on the stability of DNA in blood stains. The reason may be, that in contrast to the naked molecules of acid phosphatase, DNA in blood stains is most likely still entrapped in the nucleus and tightly bound to histones, which might offer some protection against degradation. At the above mentioned conditions of humidity at room temperature, or 35 °C, DNA was amplifiable for at least seven months. This was supported by the observation that the number of template molecules did not drop significantly over time when RH ≤ 93%. This coincides with the absence of microbial growth on any of the stains incubated at RH ≤ 93%. It should be noted that even at humid locations around the World (Bergen, Brazzaville, Calcutta, Hong Kong, Kuala Lumpur and New Orleans) the average RH does not exceed 90%. Only Kuala Lumpur has a higher morning RH, but it drops considerable during the day, and the average 24-h RH is about 80%. Therefore, even at locations with high humidity ambient conditions are adequate for long time storage of untreated stains. The present results are in agreement with previous observations obtained at ambient temperature.1, 2, 3, 4, 5, 6 In one study, however, EDTA blood was used for the preparation of blood stains, and since EDTA is a biocide and inhibitor of metal-ion catalysed nucleases22, 23, 24, 25 the survival of DNA for 19 month is not directly comparable to the present results on blood and paper without any additives. The reduced DNA stability at 100% RH confirms previous results for incubates at these conditions, although in the present study amplifiable DNA still survived for months. The crucial point appears to be the onset of microbial growth, which happened at room temperature and at 35 °C. In the biologically less favourable temperature range (45–65 °C) microbial growth was not observed, but DNA was evidently less stable since the amplificability of the 1600bp and 273bp fragments dropped to approximately one month, and three months, respectively, at 55 °C and 65 °C. It is, however, noted that even at 45 °C, the 1600bp fragment was amplifiable after eight months. Given the high stability of DNA in dry stains at ambient temperature and humidity, it can be excluded that endogenic enzymes play any significant role in the degradation of DNA. Firstly, the low quantity of water in “dry” stains presumably is too small to allow enzymatic reactions to occur, secondly, most endogenic enzymes become inactivated due to denaturation during the drying process. The present results indicate that degradation of DNA at lower temperatures is mainly exerted by the presence of microbial organisms. At higher temperatures (in the present work 45–65 °C), degradation of DNA is probably the result of spontaneous hydrolytic and oxidative reactions, most notably depurination which results in subsequent breakage of the DNA strand. Such non-enzymatic reactions are often more temperature sensitive than enzymatic processes; for example the rate of depurination increases 6–7 fold for each 10 °C the temperature is raised, as compared to ∼2 fold for enzymatic reactions.26, 27, 28 The result of these non-enzymatic reactions is the accumulation of nucleotide derivatives that affect the ability to PCR amplify the DNA, as well as an exponential increase in shorter and shorter DNA fragments.11, 29, 30