2.4 Bisulfite conversion and CpG pyrosequencing analysis
The methylation status of single CpG sites along the studied sequence
was analysed by the Pyrosequencing CpG assay methodology. Bisulfite
conversion reactions for each DNA sample conducted prior to
Pyrosequencing for the complete conversion of the unmethylated Cytosines
(existing as CpG dinucleotides) to Uraciles, with Epitect Bisulfite Kit
(Qiagen).
Sequencing and PCR primers were designed with the PyroMark Assay Design
Software version 2.0 (Qiagen) (Table 2).
TABLE 2: Pyrosequencing primer sets