COMPARATIVE STUDY OF TWO DNA EXTRACTION METHODS IN DIFFERENT TISSUES AND CONDITIONS OF DEGRADATION

Abstract

The aim of this study is to compare two methods of extraction of DNA from different tissues (spleen, liver, lungs, kidney, heart, muscles, and pancreas) with Ph-CL and QIAAamp® DNA Mini kit and to confirm which of these two methods provide better results. Degradation of the tissues was in 3 controlled conditions in a period of 6 months: room temperature, in a refrigerator at +4 and outside temperature in the period 2.3-17.8.2010. In total, 210 analyses were performed and 20-30 mg of tissue was taken. Quantification was performed with Quantifiler kit on 7500 RealTime PCR ABI and analysed with 7500 System SDS version 1, 2, 3 software. The temperature was continuously measured with digital TESTO thermometers on each 2 hours. T-test was used to compare the two extraction protocols from tissues. Comparation of different tissues which were degradated in 3 controlled conditions and extracted in 5 periods was performed with ANOVA. This study shows that bigger yield of DNA from the tissues can be isolated with PhCl extraction method, but, also, with QIAAamp® DNA Mini kit, even though less amount of isolated DNA is obtained, it can be used for further PCR reaction. PhCl method is slow, cancerogenous, more expensive and the large amount of DNA can make a problem in the amplification process. Comparative results from different tissues show that there is no big difference in the extracted yield of DNA. We proved that in putrefied tissues non toxic and fast method can be used for DNA extraction. Our study shows that the isolated DNA does not decrease with the increase of PMI and ambiental temperature, most probably due to the short decay period of 6 months that we were analysing. However, in forensic DNA analysis we should take into account that the quality and quantity of extracted DNA depends on the ambiental conditions, PMI and the type of tissue. The fastest degradation happens in tissues which have more proteolotyc enzymes.