Methylated DNA plays an important role in epigenetic gene regulation, and could therefore serve as a potentially promising biomarker for the diagnosis of cancer and other diseases. Therefore, the development of a simple method for analyzing cytosine methylation in a target gene is required. Here, we report on a new method for the sequence-selective chemical modification of a single cytosine in single-stranded DNA (ssDNA). This method is based on the formation of a DNA three-way junction of the ssDNA and two ssDNA probes, and was successfully applied for a simple polymerase chain reaction (PCR)-based assay for 'pinpoint' methylation analysis.