Epiontis employs a three step approach to discover novel methylation markers. The process starts with the comparative analysis of two cell types, from which DNA is isolated, fragmented, and linked to amplification primers. The subsequent use of methylation-sensitive restriction enzymes allows to set methylation-dependent cuts in the isolated DNA fragments. A PCR reaction will then amplify only previously uncut DNA fragments. Thus, comparative analysis of the two cell types by dot-blot or by DNA chip analysis will reveal differentially methylated regions.
During the crucial validation process bisulfite sequencing is employed to determine specific methylation sites within the identified candidate regions. To thoroughly validate the specificity of the markers, Epiontis built a DNA bank isolated from an extensive range of cell lines and cell types from different tissues. A proprietary assay system (MS-SnuPE, see also under "Assay System") allows the fast development of a protoype assay to be used for many different samples from the DNA bank and thus enables an efficient validation process of the methylation markers.
In a third step, a methylation-sensitive real time PCR assay is developed as a tool for reliable cell type identification in routine applications.