Figure 2.

Example of a pyrogram (eg, sample 3). The percentage of CpG methylation is shown above and was directly taken from pyrogram evaluation. The sequencing direction is reverse (5′ → 3′) because of working with a reverse sequencing primer, whereas the forward strand was labeled with biotin. The sequence analyzed is presented below: dispensation order (DO) is the order of nucleotides pipetted by the pyrosequencer and red nucleotides served as control. CpGs analyzed are given in blue. sequence to analyze (STA) represents the analyzed DNA sequence from −101 nt to –68 nt. RS (mod) is the reverse sequence after bisulfite treatment. FS (mod) stands for forward sequence after bisulfite treatment. The yellow box marks the localization of the forward PCR primer used for methylation-specific PCR (MSP) analysis. The left white box marks the localization of the pyrosequencing primer (reverse). The right white box marks the site of the forward PCR primer for pyrosequencing. Major and minor transcription start points (TSP) as well as transcription factor-binding sites are given according to Iwahashi et al.²⁹