Sodium bisulfite conversion of genomic DNA to differentiate and detect unmethylated versus methylated cytosines is the gold standard for DNA methylation analysis. It provides single nucleotide resolution map of 5-mC of the genome (1). Bisulfite conversion involves the deamination of unmodified cytosines to uracil, leaving the modified bases 5-mC and 5-hmC. This procedure can then be followed either by PCR amplification or massively parallel sequencing methods to reveal the methylation status of every cytosine in gene specific amplification or whole genome amplification.
Treatment of denatured DNA (i.e., single-stranded DNA) with sodium bisulfite leads to deamination of unmethylated cytosine residues to uracil, leaving 5-mC or 5-hmC intact. The uracils are amplified in subsequent PCR reaction as thymines, whereas 5-mC or 5-hmC residues get amplified as cytosines. Comparison of sequence information between the reference genome and bisulfite-treated DNA can provide information about cytosine methylation patterns. Other applications, such as Methylation-Specific PCR (MSP), involve analyzing untreated and bisulfite treated DNA using two different sets of PCR primer pairs, one pair that targets the unaltered, methylated sequence and the other that targets the converted unmethylated sequence. The major drawback to bisulfite conversion is that it does not distinguish between 5-mC and 5-hmC DNA (2).
- Frommer et al. (1992) Proc. Natl. Acad. Sci. 89. 1827-1831. PMID: 1542678
- Huang, Y. et al. (2010) PloS ONE 5, e8888. PMID: 20126651