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5-Methylcytosine is a modified cytosine with a methyl group attached to carbon 5 of its ring. In DNA, it retains the usual pairing relationship with guanine, so DNA methylation can change the chemical information on a sequence without changing its ordinary C/G base-pair identity.

A chemical difference makes the mark experimentally readable. In conventional bisulfite sequencing, treatment converts unmethylated cytosine to a form read as thymine after amplification, while 5-Methylcytosine is relatively protected. Comparing the resulting sequences reveals methylation at individual positions. The method introduced by M. Frommer and colleagues in 1992 turned a modification invisible in a simple four-letter DNA string into a positional readout.^[ref] A methylation measurement must still be interpreted in its genomic and cellular context; the mark does not mean universal shutdown of transcription.

M. Frommer led the 1992 paper establishing a sequence-based display of 5-Methylcytosine in individual DNA strands. The method exploits the different responses of ordinary cytosine and methylated cytosine to bisulfite treatment: ordinary cytosine is preferentially converted to uracil, which is read as thymine after amplification, while methylated cytosine is relatively resistant.^[ref]

This connects chemical modification to an ordinary sequence readout, making bisulfite sequencing a way to map DNA methylation. The untreated reference and conversion controls matter because an observed cytosine/thymine difference could otherwise be mistaken for a sequence variant or incomplete chemical conversion.