Department of Chemistry and Biochemistry, Florida International University, Miami, FL, 33199, USA; General Department of Forensic Science and Criminology, Dubai Police, Dubai, United Arab Emirates.
School of Criminal Justice, Forensic Science and Security, The University of Southern Mississippi, Hattiesburg, MS, 39406, USA.
Anal Biochem. 2020 Dec 15;611:113933. doi: 10.1016/j.ab.2020.113933. Epub 2020 Sep 4.
Discrimination of body fluids can provide important information in the investigation of crime scenes. The goal of this project was to identify new sets of tissue specific differentially methylated regions (tDMRs) and develop assays that can be utilized for forensic discrimination of body fluids, in particular sperm, saliva and blood. In this study, a sample set containing semen with sperm, semen without sperm, buccal swabs, saliva (oral fluids), venous blood, menstrual blood, vaginal secretions, and sweat/skin samples were used to develop four assays. Two methods for the analysis of DNA methylation biomarkers were developed in this paper: pyrosequencing and quantitative PCR/high resolution melt (HRM) analysis. Using an epigenome wide association study, two markers, NMUR2 and UBE2U, were found to be specific for sperm, based on the fact that mean DNA methylation levels for semen (containing sperm cells) were significantly lower than mean DNA methylation levels of other body fluids. In addition, one marker (SA-6) was hypermethylated in saliva when compared to other body fluids. The assays developed for NMUR2, UBE2U and SA-6 markers can be applied in forensic tissue identification using both pyrosequencing and HRM analysis. Additionally, a set of CpG sites in the AHRR locus were hypomethylated in blood when compared to other tissues using pyrosequencing. However, this locus was not amenable to HRM analysis. Overall, this work demonstrates the discovery and application of tDMRs for forensic applications.
体液鉴别可以为犯罪现场调查提供重要信息。本项目的目的是鉴定新的组织特异性差异甲基化区域（tDMR），并开发可用于法医鉴别体液（特别是精子、唾液和血液）的检测方法。在这项研究中，使用了一个包含精液（有精子）、精液（无精子）、口腔拭子、唾液（口腔液）、静脉血、月经血、阴道分泌物和汗液/皮肤样本的样本集，来开发了四种检测方法。本文开发了两种分析 DNA 甲基化生物标志物的方法：焦磷酸测序和定量 PCR/高分辨率熔解（HRM）分析。通过全基因组关联研究，发现了两个标记物 NMUR2 和 UBE2U，它们是精子特异性的，这是基于这样一个事实，即含有精子细胞的精液的平均 DNA 甲基化水平明显低于其他体液的平均 DNA 甲基化水平。此外，与其他体液相比，SA-6 标记物在唾液中呈超甲基化。针对 NMUR2、UBE2U 和 SA-6 标记物开发的检测方法可用于法医组织鉴定，既可以使用焦磷酸测序，也可以使用 HRM 分析。此外，使用焦磷酸测序发现，与其他组织相比，AHRR 基因座中的一组 CpG 位点在血液中呈低甲基化状态。然而，这个基因座不适用于 HRM 分析。总的来说，这项工作展示了 tDMR 用于法医应用的发现和应用。
