Pressure Cycling Technology (PCT) Reduces Effects of Inhibitors of the PCR

Pressure Cycling Technology (PCT) Reduces Effects of Inhibitors of the PCR

A common problem in the analysis of forensic human DNA evidence, or for that matter any nucleic acid analysis, is the presence of contaminants or inhibitors. Contaminants may co-purify with the DNA and inhibit downstream PCR, or they may present samples effectively as containing fewer templates than exist in the PCR, even when the actual amount of DNA is adequate. Typically, these challenged samples exhibit allele imbalance, allele dropout and sequence specific inhibition which can lead to interpretational difficulties. Lessening the effects of inhibitors may increase the effective yield of challenged low template copy samples. High pressure may alter some inhibitors and render them less effective at reducing the yield of PCR products. In an attempt to enhance the amplicon yield of inhibited DNA samples, pressure cycling technology (PCT) was applied to DNA exposed to various concentrations of hematin (0, 1.25, 2.5, 5, and 7 µM) and humic acid (0, 1.25, 2.5, 5, and 7 ng/µl). The effect of high pressure on the inhibitors and, subsequently, the PCR process was assessed by measuring DNA quantity by qPCR and evaluating STR typing results. The results support that pressure cycling technology reduces inhibitory effects and, thus, in effect enhances yield of amplified products of both hematin and humic acid contaminated samples. Based on the results obtained in this study, this method can improve the ability to type challenged or inhibited DNA samples.