SR3335 br Materials and methods br

Materials and methods
Results
Discussion
Acknowledgements This research was funded by the South African Medical Research Council, National Research Foundation and the University of KwaZulu-Natal Research Incentive fund.
Introduction Most forensic laboratories have experienced the inability to detect or amplify DNA from moist biological stains or reference samples on cotton buds, which had been stored and/or shipped at soaking wet conditions. It has been shown that the inactivation rate of a protein (acid phosphatase) in semen stains is exponentially related to the relative humidity, the rate being 1,000,000-fold higher at 100% humidity than at 40% [1]. The degradation of DNA often parallels the degradation of proteins, and many of the processes that degrade DNA depend on the presence of water [2], [3]. Therefore, we expected that DNA in biological stains and reference samples would show a rapid decay at high relative humidity. To see if this was true, stains of whole blood or buccal SR3335 were incubated at various conditions of relative humidity and temperature. The quality and quantity of the remaining DNA was assessed by PCR.
Methods and materials Whole blood (5 μl) or buccal cells were spotted onto pieces (4×4 mm) of Whatman filter paper No. 3 and air-dried overnight. The stains were incubated in closed boxes at 0%, 50%, 80% and 100% relative humidity at room temperature, 35, 45, 55 and 65 °C. Constant humidity was maintained by the inclusion of H2O, saturated solutions of (NH4)2SO4 (80% humidity), NaHSO4, H2O (50% humidity) or silica gel. One series of bloodstains on microscopic slides was transferred to soaking wet (Milli-Q grade water) cotton buds and incubated at 100% humidity at room temperature. DNA was extracted from the stains using QIAamp® DNA Mini Kit (Qiagen, Germany). PCR of a 1600 bp segment was performed using primers targeting the human ACP1 locus from intron 4 to exon 5 and a 273 bp segment was amplified using primers targeting exon 4 at the human HFE-locus. Real time PCR was performed using ABI-Prism-7000 SDS (Applied Biosystems) and primers and probe targeting a 147 bp segment in exon 4 of the human HFE-gene.
Results
Discussion The results showed a surprising stability of DNA in stains even at extreme environmental conditions (35–65 °C, 100% relative humidity), provided the stains had been dried prior to incubation. We had expected a rapid decay of DNA at high relative humidity but observed, on the contrary, that even long DNA fragments were amplifiable after month of incubation. The appearance of fungal/microbial growth on some of the stains at 100% humidity may explain the decrease in amplifiable DNA observed for these stains. However, the average environmental humidity is well below 100% even during wet seasons, e.g. the average value is 90% for the winter month in Denmark [4] and it does not exceed 82% at any time of the year in tropical Darwin, Australia [5]. Therefore, even in humid climates simple air-drying seems to be adequate for the preservation for month of DNA in stains and reference samples to be analysed for STRs and SNPs. This may be especially attractive to developing countries that want to implement modern DNA-profiling techniques but find the purchasing of expensive sampling kits prohibitive.