Antunes Joana, Silva Deborah S B S, Balamurugan Kuppareddi, Duncan George, Alho Clarice S, McCord Bruce
Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA.
Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA; Faculty of Biosciences, Laboratory of Human and Molecular Genetics, PUCRS, 90619-900 Porto Alegre, Brazil.
Anal Biochem. 2016 Feb 1;494:40-5. doi: 10.1016/j.ab.2015.10.002. Epub 2015 Oct 22.
The goal of this study was to develop a method for the detection of semen in biological stains using high-resolution melt (HRM) analysis and DNA methylation. To perform this task, we used an epigenetic locus that targets a tissue-specific differentially methylated region for semen. This specific locus, ZC3H12D, contains methylated CpG sites that are hypomethylated in semen and hypermethylated in blood and saliva. Using this procedure, DNA from forensic stains can be isolated, processed using bisulfite-modified polymerase chain reaction (PCR), and detected by real-time PCR with HRM capability. The method described in this article is robust; we were able to obtain results from samples with as little as 1 ng of genomic DNA. Samples inhibited by humic acid still produced reliable results. Furthermore, the procedure is specific and will not amplify non-bisulfite-modified DNA. Because this process can be performed using real-time PCR and is quantitative, it fits nicely within the workflow of current forensic DNA laboratories. As a result, it should prove to be a useful technique for processing trace evidence samples for serological analysis.
本研究的目的是开发一种利用高分辨率熔解（HRM）分析和DNA甲基化检测生物污渍中精液的方法。为完成这项任务，我们使用了一个靶向精液组织特异性差异甲基化区域的表观遗传位点。这个特定的位点，即ZC3H12D，包含甲基化的CpG位点，这些位点在精液中低甲基化，而在血液和唾液中高甲基化。使用这个程序，可以从法医污渍中分离DNA，通过亚硫酸氢盐修饰的聚合酶链反应（PCR）进行处理，并通过具有HRM功能的实时PCR进行检测。本文所述的方法很可靠；我们能够从仅含1 ng基因组DNA的样本中获得结果。受腐殖酸抑制的样本仍能产生可靠的结果。此外，该程序具有特异性，不会扩增未经过亚硫酸氢盐修饰的DNA。由于这个过程可以使用实时PCR进行，并且是定量的，它非常适合当前法医DNA实验室的工作流程。因此，它应该被证明是一种用于处理微量物证样本进行血清学分析的有用技术。
