Direct single-molecule counting of DNA methylation at ultra-low input via a one-step, one-tube methylation-sensitive restriction enzyme (MSRE) workflow

DNA methylation is a key epigenetic mechanism in oncology research, supporting tumor classification, biomarker discovery, and residual disease studies, but measuring it in cell-free DNA (cfDNA) is challenging because cfDNA is scarce and bisulfite conversion causes significant sample loss. Quantification methods add further limits: qPCR depends on relative quantification or standard curves, digital PCR loses sample to dead volume and has limited partitions, and NGS requires complex library prep that raises cost and turnaround time. This poster shows how Countable PCR single-molecule counting, paired with methylation-sensitive restriction enzyme (MSRE) digestion, enables simple, precise, and sensitive absolute quantification of methylated DNA without bisulfite conversion, demonstrated at the hypermethylated MLH1 promoter locus.