Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA.
Institute of Forensic Science, Seoul National University College of Medicine, Seoul, South Korea.
Electrophoresis. 2019 Sep;40(18-19):2565-2574. doi: 10.1002/elps.201900118. Epub 2019 Jul 18.
The goal of this study is to develop an epigenetic multiplex for body fluid identification based on tissue specific DNA methylation. A series of genetic loci capable of discerning the origin of DNA as coming from saliva, blood, vaginal epithelia, or semen were used for this application. The markers - BCAS4, CG06379435, VE_8, and ZC3H12D - were amplified together and then sequenced via pyrosequencing. Methylation values for cytosine guanine dinucleotide (CpG) sites at each locus were then measured across the four markers. In total, 124 samples were collected, and bisulfite modified to convert unmethylated DNA to uracil. This converted DNA was then amplified via multiplex PCR with reverse primers containing a biotin molecule. Biotinylated PCR products were then analyzed using pyrosequencing to generate a series of pyrograms containing 18 CpG sites. The percent methylation at each CpG site was determined, and then agglomerative hierarchical cluster analysis was used to create a model to indicate sample origin. Further analysis reduced the number of CpG sites required for optimal determination of body fluid type to five. This study demonstrates an efficient multiplexed body fluid identification process utilizing DNA methylation that can be easily implemented in forensic laboratories.
本研究旨在开发一种基于组织特异性 DNA 甲基化的体液识别表观遗传多重分析方法。为此应用了一系列能够区分 DNA 来源的遗传基因座，如唾液、血液、阴道上皮或精液。标记物 - BCAS4、CG06379435、VE_8 和 ZC3H12D - 一起扩增，然后通过焦磷酸测序进行测序。然后在四个标记物上测量每个基因座的胞嘧啶鸟嘌呤二核苷酸 (CpG) 位点的甲基化值。总共收集了 124 个样本，并进行亚硫酸氢盐修饰，将未甲基化的 DNA 转化为尿嘧啶。然后通过包含生物素分子的反向引物进行多重 PCR 扩增转化后的 DNA。然后使用焦磷酸测序分析生物素化的 PCR 产物，生成包含 18 个 CpG 位点的一系列焦磷酸图。确定每个 CpG 位点的甲基化百分比，然后使用凝聚层次聚类分析创建一个模型来指示样本来源。进一步的分析将确定体液类型所需的 CpG 位点数量减少到五个。本研究展示了一种利用 DNA 甲基化的高效体液识别多重分析方法，可轻松在法医实验室中实施。
