High resolution melt curve analysis based on methylation status for human semen identification

Area of Science:

• Forensic Science
• Molecular Biology
• Genetics

Background:

• Accurate identification of biological fluids is crucial in forensic investigations.
• Existing methods for body fluid identification have limitations in terms of specificity, time, or confirmatory power.

Purpose of the Study:

• To develop and validate a high-resolution melt curve assay for differentiating semen from other common body fluids.
• To assess the reliability and stability of the assay for forensic casework.

Main Methods:

• DNA was extracted from various body fluid stains (semen, blood, saliva, urine, vaginal fluid).
• Bisulfite modification was performed to convert unmethylated cytosines to uracil.
• A high-resolution melt curve assay targeting the promoter region of the Dapper Isoform 1 (DACT1) gene was employed.

Main Results:

• Semen samples showed a statistically significant lower melting temperature compared to other body fluids.
• The assay demonstrated sperm-specificity, as semen from a vasectomized individual yielded similar results to non-semen fluids.
• Methylation status remained stable in blood and semen stains stored for up to 5 months.

Conclusions:

• The developed DACT1 methylation-based melt curve assay is a reliable and accurate method for semen identification.
• This assay offers advantages over traditional methods, being time-efficient, confirmatory, and potentially multiplexable for forensic applications.