Abstract
Aberrant CpG island methylation serves as a pivotal biomarker for cancer diagnosis, with accuracy substantially enhanced by analyzing multiple loci. Current techniques, such as bisulfite conversion or restriction enzyme-based methods, often fall short in delivering efficient multiplexed genomic methylation analysis using standard PCR platforms. Herein, we introduce an innovative bisulfite-free, multiplex assay-multiple specific terminal mediated methylation PCR (multi-STEM MePCR). This assay integrates a methylation-dependent restriction endonuclease (MDRE) with a novel multiplex PCR, leveraging innovative stem-loop structured assays for simultaneous detection of multiple CpG sites. As a proof-of-concept, the multi-STEM MePCR platform simultaneously achieved quantification of three methylation model sites down to ten copies per tube, accompanied by a broader linear dynamic range, and attained a sensitivity of 0.1% against a background of 10 000 unmethylated gene copies. Crucially, by markedly minimizing cross-reactivity and reducing competition among targets, this technique adeptly distinguishes between sites exhibiting significant variations in methylation abundance. Additionally, this method effectively detects digestion products of various sizes, demonstrating clinical precision comparable to bisulfite sequencing, yet with simpler operation, less time, and lower cost. This multi-STEM PCR technology pioneers an advanced strategy for multiplexed methylation analysis, which is essential for epigenetic research and clinical DNA methylation diagnostics.