Fachet Caitlyn, Quarino Lawrence, Karnas K Joy
Forensic Science Program, Cedar Crest College, 100 College Drive, Allentown, PA, 18104, USA.
Forensic Sci Med Pathol. 2017 Mar;13(1):86-91. doi: 10.1007/s12024-016-9825-6. Epub 2016 Dec 27.
A high resolution melt curve assay to differentiate semen from blood, saliva, urine, and vaginal fluid based on methylation status at the Dapper Isoform 1 (DACT1) gene was developed. Stains made from blood, saliva, urine, semen, and vaginal fluid were obtained from volunteers and DNA was isolated using either organic extraction (saliva, urine, and vaginal fluid) or Chelex 100 extraction (blood and semen). Extracts were then subjected to bisulfite modification in order to convert unmethylated cytosines to uracil, consequently creating sequences whose amplicons have melt curves that vary depending on their initial methylation status. When primers designed to amplify the promoter region of the DACT1 gene were used, DNA from semen samples was distinguishable from other fluids by a having a statistically significant lower melting temperature. The assay was found to be sperm-significant since semen from a vasectomized man produced a melting temperature similar to the non-semen body fluids. Blood and semen stains stored up to 5 months and tested at various intervals showed little variation in melt temperature indicating the methylation status was stable during the course of the study. The assay is a more viable method for forensic science practice than most molecular-based methods for body fluid stain identification since it is time efficient and utilizes instrumentation common to forensic biology laboratories. In addition, the assay is advantageous over traditional presumptive chemical methods for body fluid identification since results are confirmatory and the assay offers the possibility of multiplexing which may test for multiple body fluids simultaneously.
开发了一种高分辨率熔解曲线分析方法，用于根据Dapper异构体1（DACT1）基因的甲基化状态区分精液与血液、唾液、尿液和阴道液。从志愿者处获取血液、唾液、尿液、精液和阴道液制成的污渍，使用有机提取法（唾液、尿液和阴道液）或Chelex 100提取法（血液和精液）分离DNA。然后对提取物进行亚硫酸氢盐修饰，以便将未甲基化的胞嘧啶转化为尿嘧啶，从而产生其扩增子具有根据其初始甲基化状态而变化的熔解曲线的序列。当使用设计用于扩增DACT1基因启动子区域的引物时，精液样本的DNA与其他液体的DNA可通过具有统计学上显著较低的熔解温度来区分。该分析被发现对精子具有特异性，因为输精管切除男性的精液产生的熔解温度与非精液体液相似。储存长达5个月并在不同时间间隔进行测试的血液和精液污渍在熔解温度上几乎没有变化，表明甲基化状态在研究过程中是稳定 的。与大多数基于分子的体液污渍鉴定方法相比，该分析是法医科学实践中更可行的方法，因为它省时且使用法医生物学实验室常用的仪器。此外，该分析优于传统的体液鉴定推定化学方法，因为结果是确定性的，并且该分析提供了多重检测的可能性，即可以同时检测多种体液。
