Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease
Fig 4
Allele-specific bisulfite pyrosequencing.
To analyze the allele-specific DNA methylation, we individually measured the DNA methylation of each allele and compared methylation between the two alleles. Thus, the allele-specific polymerase chain reaction (PCR) was performed and the methylation levels of each of the two alleles, given allele-specific amplicons, were measured separately using pyrosequencing. The allele-specific PCR primers were designed such that each primer of 3′ end was a pair of base of heterozygous SNP. (a) The reference sequence of rs36221701 before and after bisulfite conversion. Rs36221701 contained two CpG sites denoted as CpG1 and CpG2 in 5′ to 3′ direction. By bisulfite conversion, unmethylated C is converted to U (U is converted to T by PCR). Rs362210701 (C/T) is converted to (T/T); it was impossible to distinguish the converted T and original T. Thus, we were unable to detect the origin of the allele for this SNP by PCR primers. (b) Allele-specific PCR. We designed PCR primer for rs13239907 (A/G), which is located 297 bp downstream of rs36221701 and was in complete linkage disequilibrium with rs36221701 (A→C, G→T: r2 = 1.0) in JPT data from 1,000 genome project. The forward primer was common to both alleles; reverse primers were designed such that each primer of 3′ end was a pair of bases of genotypes (A or G: complementary strand). (c) Allele-specific pyrosequencing. The two given allele-specific amplicons were separately analyzed using pyrosequencing. Sequence primer 1 targeted rs13239907 and was used to check the accuracy of allele-specific PCR, whereas sequence primer 2 targeted CpG1 and CpG2 was used to analyze the ratio of methylation around rs36221701. (d) Primer details. The 3′ end of PCR primer R was a pair of base of heterozygous SNP. 5′ end of PCR primer R was biotinylated.
