The detection of DNA methylation with high sensitivity and specificity is important for the early diagnosis of many human diseases, including cancers. Here, we integrated the high specificity of the methylation-dependent restriction endonuclease GlaI for methylation-dependent digestion and the high amplification efficiency of rolling circle amplification (RCA) for the detection of GlaI digestion products. GlaI can only digest a methylated template, leading to the generation of digestion products with specific ends. The specific digestion product can then be ligated to a ligation mediator with a dumbbell structure to generate a complete circular template for further RCA, and the final RCA amplicon can be detected using lateral flow detection (LFD) with the naked eye. The specificity of Gla-RCA not only depends on the specific methylation digestion of GlaI, but only the ligation process of RCA amplification. As a proof of principle, the sensitivity of GlaI-RCA assay was applied to methylated Septin 9 and showed a sensitivity of approximately 1% (50 copies of methylated template per reaction) and no cross-reactivity with 5000 copies of unmethylated DNA used as background. The application of GlaI-RCA was also evaluated with colorectal cancer tissue samples and showed great accordance with standard bisulfite sequencing. A bisulfite-free and LFD-based DNA methylation detection was successfully developed, promising high specificity and rapid visual detection and having a great potential to become a robust tool for DNA methylations analysis.
