High resolution melt curve analysis based on methylation status for human semen identification
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- 作者：Caitlyn Fachet ; Lawrence Quarino…
- 关键词：DNA methylation status ; High resolution melt assay ; Semen identification ; DACT1
- 刊名：Forensic Science, Medicine, and Pathology
- 出版年：2017
- 出版时间：March 2017
- 年：2017
- 卷：13
- 期：1
- 页码：86-91
- 全文大小：
- 刊物主题：Pathology; Forensic Medicine; Criminology and Criminal Justice, general;
- 出版者：Springer US
- ISSN：1556-2891
- 卷排序：13
文摘
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.