Abstract：
In the context of forensic casework, it is imperative to both establish a DNA profile from biological specimens and accurately identify the specific bodily fluid source. To achieve this, DNA methylation markers have been developed for the differentiation of blood, semen, vaginal epithelial secretions, and saliva samples. Saliva, alternatively referred to as oral fluid, is recognized for its heterogeneous cellular composition, characterized by a mixture of epithelial, leukocytic, and bacterial cells. Consequently, our research has revealed variations in methylation percentages that correlate with the method employed for collecting saliva samples. To investigate these concepts, we scrutinized four CpG markers situated within or in proximity to the BCAS4, SLC12A8, SOX2OT, and FAM43A genes. Subsequently, we designed primers based on bioinformatically transformed reference sequences for these markers and rigorously assessed their quality by examining dimer and hairpin formation, melting temperature, and specificity. These loci were identified as saliva markers based on either buccal swabs or spit collection. Yet, there has been minimal or no research conducted to explore the variations in methylation between different collection methods. For this study, buccal, lip, tongue, spit, and nasal swabs were collected from 20 individuals (N = 100). Mock forensic samples, which include chewing gum (N = 10) and cigarettes (N = 10), were also tested. DNA was extracted, bisulfite converted, then amplified using in-house designed assays, and pyrosequenced. The methylation levels were compared to other body fluids (semen, blood, vaginal epithelia, and menstrual blood [N = 32]). A total of 608 pyrosequencing results demonstrated that sampling location and collection method can greatly influence the level of methylation, highlighting the importance of examining multiple collection/deposition methods for body fluids when developing epigenetic markers.
摘要：
在法医案件工作中，必须从生物标本中建立DNA图谱，并准确识别特定的体液来源。为了实现这一点，DNA甲基化标记已经被开发用于血液的分化，精液,阴道上皮分泌物，还有唾液样本.唾液,或者称为口服液，因其异质的细胞组成而被认可，以上皮混合物为特征，白细胞,和细菌细胞。因此,我们的研究揭示了甲基化百分比的差异,这些差异与收集唾液样本的方法相关.为了研究这些概念，我们仔细检查了位于BCAS4内或附近的四个CpG标记，SLC12A8，SOX2OT，和FAM43A基因。随后，我们设计了基于生物信息转化的参考序列的引物，这些标记，并通过检查二聚体和发夹形成严格评估其质量，熔化温度,和特异性。这些基因座基于口腔拭子或唾液收集被鉴定为唾液标记物。然而，很少或没有进行研究来探索不同收集方法之间的甲基化差异。对于这项研究，颊,唇,舌头,吐口水,从20个人（N=100）收集鼻拭子。模拟法医样本，其中包括口香糖（N=10）和香烟（N=10），也进行了测试。提取DNA,亚硫酸氢盐转化，然后使用内部设计的检测方法进行扩增，并进行了高温测序。甲基化水平与其他体液(精液，血,阴道上皮,和月经血[N=32])。共608次焦磷酸测序结果表明,采样位置和采集方法可以极大地影响甲基化水平,强调在开发表观遗传标记时检查体液的多种收集/沉积方法的重要性。
