Assays based on methylation-specific real time PCR allow a very reliable and fast determination of the methylation fingerprint of a certain DNA region. The DNA is amplified by PCR in the presence of a blocker that inhibits the production of either the methylated or the unmethylated DNA species with very high specificity. Due to the high sensitivity of the PCR amplification reaction, minute amounts of methylated DNA in a background of unmethylated seqences and vice versa can be accurately quantified. Therefore this method allows both the characterization of cell types and the assesment of the purity of cellular samples and is preferentially used for established fingerprinting assays.
An additional assay format that can be employed for fingerprinting is a proprietary technology called methylation-specific single base extension (MS-SNuPE). Chemical conversion by bisulfite treatment results in different base pairing preferences of the base cytosine, depending on its previous methylation state. A primer is then annealed adjacent to the cytosine of interest. Chain elongation is allowed to proceed for a single base in the presence of ddNTPs labelled with two different floursecent dyes, one for each potential base pairing preference of the cytosine of interest. Through comparison of the reaction efficiencies the ratio of methylation and demethylation at a certain cytosine position can be accurately determined.