Hairpin competition amplification driven single-CpG-site resolution DNA methylation analysis

DNA methylation is an important epigenetic modification and has been widely used as a biomarker for disease diagnosis, particularly in cancer screening. However, conventional quantitative methylation-specific PCR (qMSP) often lacks single-CpG-site resolution because the methylation states of neighboring CpG sites within primer or probe binding regions can interfere with amplification efficiency. In this work, we report a novel Hairpin Competition qMSP (HC-qMSP) strategy for precise DNA methylation analysis at single-CpG-site resolution. In this method, degenerate bases were introduced at non-target CpG positions within the primer and probe binding regions to eliminate interference from adjacent CpG sites. Meanwhile, a specially designed hairpin primer was introduced so that the extension product forms a hairpin structure that competes with the reverse primer during amplification, enabling selective amplification of methylated templates while suppressing unmethylated sequences. Under optimized conditions, the HC-qMSP assay achieved a discrimination sensitivity down to 0.1%, and showed good linearity across a wide dynamic range. The method was further validated using colorectal cancer tissues and stool samples, successfully distinguishing cancer samples from normal controls. Owing to its simplicity, low cost, and compatibility with conventional real-time PCR platforms, HC-qMSP provides a promising approach for precise DNA methylation analysis and potential clinical diagnostic applications.