Institute of Applied Genetics, Department of Forensic and Investigative Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX 76107, USA.
Int J Legal Med. 2013 Mar;127(2):321-33. doi: 10.1007/s00414-012-0770-y. Epub 2012 Sep 18.
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 copurify with the DNA, inhibiting 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, leading 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 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 quantitative PCR and evaluating short tandem repeat typing results. The results support that pressure cycling technology reduces inhibitory effects and thus, in effect, enhances yield of contaminated amplified products of both hematin and humic acid contaminate samples. Based on the results obtained in this study, this method can improve the ability to type challenged or inhibited DNA samples.
在法医人类 DNA 证据分析中，或在任何核酸分析中，一个常见的问题是存在污染物或抑制剂。污染物可能与 DNA 共纯化，抑制下游 PCR，或者即使实际 DNA 量充足，它们也可能有效地将样品表现为包含比 PCR 中存在的模板更少。通常，这些受到挑战的样品表现出等位基因不平衡、等位基因缺失和序列特异性抑制，导致解释困难。减少抑制剂的影响可能会增加受挑战的低模板拷贝样品的有效产量。高压可能会改变一些抑制剂，使其降低 PCR 产物产量的效果降低。为了提高受抑制 DNA 样品的扩增子产量，应用压力循环技术处理暴露于不同浓度血红素（0、1.25、2.5、5 和 7 μM）和腐殖酸（0、1.25、2.5、5 和 7 ng/μL）的 DNA。通过定量 PCR 测量 DNA 量并评估短串联重复序列分型结果来评估高压对抑制剂的影响以及随后对 PCR 过程的影响。结果支持压力循环技术可降低抑制作用，从而有效提高受血红素和腐殖酸污染的扩增产物的产量。基于本研究获得的结果，该方法可以提高对受挑战或抑制 DNA 样品进行分型的能力。
