|Bisulfite conversion||Chemical conversion of unmethylated cytosine to uracil. Methylated cytosines are protected from this conversion allowing to determine DNA methylation at single nucleotide resolution.||
|Methylated DNA enrichment||(Hydroxy-)Methylated DNA is enriched by using specific antibodies (hMeDIP or MeDIP) or proteins (MBD) that specifically bind methylated CpG sites in fragmented genomic DNA.||
|Restriction enzyme-based digestion||Use of (hydroxy)methylation-sensitive or (hydroxy)methylation-dependent restriction enzymes for DNA methylation analysis at specific sites.||
5-methylcytosine (5-mC) has been known for a long time as the only modification of DNA for epigenetic regulation. In 2009, however, Kriaucionis discovered a second methylated cytosine, 5-hydroxymethylcytosine (5-hmC). The so-called 6th base, is generated by enzymatic conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine by the TET family of oxygenases. Early reports suggested that 5-hmC may represent an intermediate of active demethylation in a new pathway which demethylates DNA, converting 5-mC to cytosine. Recent evidence fuel this hypothesis suggesting that further oxidation of the hydroxymethyl group leads to a formyl or carboxyl group followed by either deformylation or decarboxylation. The formyl and carboxyl groups of 5-formylcytosine (5-fC) and 5-carboxylcytosine (5-caC) could be enzymatically removed without excision of the base.