During DNA replication (cell division) methyl groups (DNA methylation) are resynthesized on the newly replicated strand by one of the enzymes called DNA Methyl Transferases (DNMT). Those enzymes are not present in standard PCR reaction. Therefore, a PCR product obtained from amplification of a specific locus does not contain information about methylation status of the cytosines and the methylation information is lost.

To analyze the methylation pattern of cytosines within the locus of interest the information about which cytosines are methylated needs to be preserved before PCR amplification is performed. Sodium bisulfite deaminates non-methylated cytosines to uracil and leaves methylated cytosines untouched (in other words methylated cytosines are resistant to modification induced by sodium bisulfite).

Thus, after bisulfite treatment the cytosine content of the template depends on the number of methylated cytosines in the untreated template (protected from change) as shown below in part B and C of the diagram.

PCR products with different number of cytosines can easily be distinguished in post PCR analyses such as Methylation Sensitive High-Resolution Melting (MS-HRM) (see: How does MS-HRM work?).

A: Genomic DNA template (in bold CG sites, which are the dinucleotides undergoing methylation in humans):

Figure 1:

After bisulfite modification:

B: Template with methylated cytosines at CG sites – only Cs not protected by methyl groups are changed by sodium bisulfite:

Figure 2:

C: Template with non-methylated cytosines at CG sites – all the Cs are changed by sodium bisulfite:

Figure 3: