Assessment of a Droplet Digital PCR Assay for DNA Methylation-Based Age Prediction from Blood Samples : Pilot Study

DNA methylation is a promising biomarker for forensic age estimation due to its strong correlation with chronological age. This study developed a droplet digital PCR (ddPCR) assay targeting three age-associated markers— ELOVL2, FHL2, and TRIM59—to evaluate its application in forensic cases. The assays were optimized using 100% methylated and unmethylated control DNA to evaluate probe specificity and sensitivity to methylation percentage. The assays exhibited high specificity, successfully distinguishing between methylated and unmethylated targets, and maintained sensitivity even at low methylation levels. Moreover, blood samples from three Korean individuals (aged 25, 42, and 55 years) were analyzed, revealing a clear age-related increase in methylation levels across all markers, with ELOVL2 exhibiting the strongest relation, followed by FHL2 and TRIM59. Reproducibility was evaluated by duplicate testing, with coefficient of variation values below 15% for all markers. These findings highlight the potential application of this ddPCR assay for forensic age estimation.