DNA methylation analysis using High Resolution Melt for body fluid identification in forensic sciences

As submitted by the proposer: For this project we aim to develop biomarkers for body fluid discrimination from crime scene samples. This work is critical since DNA profiles can place a suspect at the crime scene but cannot discriminate innocent from criminal contact. For example, in sexual abuse of minors innocent transfer of DNA from skin would be expected from close relatives, but intimate samples would indicate sexual abuse. Current methods for body fluid identification are based in protein detection, lacking sensitivity and specificity. DNA methylation patterns are stable and regulate tissue-specific gene expression. Our laboratory showed that analysis of DNA methylation in 4 specific genome locations allows discrimination of blood, saliva and semen by pyrosequencing. However, we lack regions capable of discriminating vaginal epithelia. Pyrosequencing allows the quantification of methylation levels but it implies high costs for new equipment purchase and operation. Instead we propose the use of a real time instrument to analyze DNA methylation patterns by High Resolution Melt (HRM). Unmethylated DNA presents a lower melting temperature when compared to methylated DNA. Preliminary results presented in this application show that we can discriminate semen from other body fluids based on melting characteristics of DNA. The aim of this project can be divided in three research goals. First HRM optimization to discriminate blood and saliva (Year 1 January to May) and developmental validation according to SWGDAM guidelines will be performed (Year 1 May to December and Year 2 January to July for vaginal epithelia primers). Second we will use HRM to screen genomic regions capable of discriminating vaginal epithelia (Year 1 May to December) and more regions for blood and saliva discrimination (Year 2 January to July). This will be performed based on the data reported by Park et al. 2014. Third we will develop a multiplex kit that will amplify 4 genomic regions and analyze the melt curve of each region by HRM combined with Plexor technology (Year 2 August to December and all Year 3). We will recruit 40 volunteers from ages 18 and older. From those at least 10 volunteers from each gender since for statistical purposes we need 10 semen and 10 vaginal samples. Semi-annual reports and an official signed copy of the doctoral dissertation will be provided to NIJ. Oral presentation at AAFS and ISHI as well as at TEDxFIU will help disseminate the results obtained for both forensic community and general public. This project contains a research and/or development component, as defined in applicable law. ca/ncf