Detection of single methylated cytosine using junction-forming DNA probes

DNA methylation is an epigenetic mechanism for transcriptional regulation. The methylation process controls cellular differentiation and is defective in many diseases including cancer. Therefore, the development of a simple method for analysing cytosine methylation in a target gene is required. Here we report a conceptually new method for sequence-selective chemical modification of a single cytosine in single-stranded DNA (ssDNA) using two DNA probes to form a DNA three-way junction with the ssDNA. The method was successfully used in a simple quantitative polymerase-chain-reaction-based assay for discrimination of a single methylated cytosine.