Multi-STEM MePCR: a bisulfite-free, multiplex, high-sensitive and high-specific assay to measure DNA methylation

Aberrant CpG island methylation serves as a pivotal biomarker for cancer diagnostics, 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. Here, 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. For proof of concept, the multi-STEM MePCR platform achieves quantification of three methylation model sites simultaneously down to 10 copies per tube, accompanied by a broader linear dynamic range, and attaining 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 abundances. Additionally, this method effectively detects digestion products of various sizes, demonstrating clinical precision comparable to bisulfite sequencing, yet with simpler operation, shorter time, and lower cost. This multi-STEM PCR technology pioneers an advanced strategy for multiplexed methylation analysis, essential for epigenetic research and clinical DNA methylation diagnostics.