FIG. 2.

Sodium bisulfite genomic sequencing of luciferase regions 1 and 2 from in vivo-methylated pCLH22 DNA. (A) Illustration of the two regions in the luciferase gene of pCLH22 examined by the sodium bisulfite genomic sequencing method after in vivo methylation by Dnmt3a. (B) Luciferase region 1 DNA (approximately 250 bp in length) with 11 CpG sites was PCR amplified, cloned, and sequenced after sodium bisulfite treatment of pCLH22 DNA which had been harvested from transfected 3a-5 cells. (C) Luciferase region 2 DNA (approximately 290 bp in length) with 28 CpG sites from the same pCLH22 DNA described in panel B was analyzed by the sodium bisulfite genomic sequencing method as described above. The filled circles represent methylated CpG sites, and the open circles represent unmethylated CpG sites. Plasmid DNA methylated and sequenced in independent experiments showed similar results. The frequency of methylation at each site was calculated by dividing the number of molecules with methylation at the site by the total number of molecules examined. The overall methylation frequency of each region was calculated by dividing the total number of sites with methylation by the total number of sites examined. The two strands were methylated similarly, and only the top-strand methylation is shown here.