Efficient and Sensitive Bisulfite-Free Methylation Detection Approach for Low-Abundance and Highly Fragmented cfDNA

Abstract

Circulating cell-free DNA (cfDNA) has emerged as a promising noninvasive diagnostic tool for liquid biopsy, with abnormal DNA methylation serving as a key biomarker for cancer screening and early diagnosis. However, the low abundance and high fragmentation of cfDNA present significant challenges to the sensitivity and specificity of the current methylation detection methods. We aim to develop a highly sensitive methylation detection method for fragmented cfDNA that does not require bisulfite conversion. In this study, we combined double restriction enzyme digestion with specific terminal-mediated polymerase chain reaction (STEM-PCR) to establish a cfDNA methylation detection method, namely, the dRE-STEM method. The dRE-STEM method could detect methylated cfDNA on the PCR platform without the need for cumbersome bisulfite conversion. A case-control study was conducted to validate the diagnostic value of colorectal cancer (CRC)-related specific methylated locates using the dRE-STEM method. The dRE-STEM method successfully detected methylation ratios as low as 3% using just 4 ng of cfDNA input, and the best diagnostic model achieved 80.2% (95% CI, 70.6–87.4%) sensitivity and 80.9% (95% CI, 71.9–87.7%) specificity for CRC, significantly outperforming conventional protein markers. The dRE-STEM method offers a promising approach for accurate methylation quantification in low-abundance, highly fragmented cfDNA, making it particularly suitable for routine clinical practice.