(Hyper)methylation analysis of the promoter region (-430 to -5 of TSS) of the CCNA1 gene by COBRA and sequence analysis. A: schematic representation of the restriction enzyme sites (B: BstUI and T: TaqI) in the virtual hypermethylated BSP nucleotide sequence after bisulfite treatment. Vertical bars represent CG site, arrow represents TSS (retrieved from Ensembl). B: Result of COBRA analysis of the BSP products of 10 tumour samples (T1-T10), in vitro methylated DNA as a positive control (IV) and leucocyte DNA as a negative (unmethylated) control (L). C. Schematic representation of the sequencing results. From each tumour, the BSP-products were cloned into TOPO-pCR4 (Invitrogen) and sequencing (BaseClear) was performed on M13-PCR products of 7–9 independent clones. Circles represent CG dinucleotides: the darker, the more clones at this site were methylated.