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.
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.
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