The chromatin loop between the HERC2 rs12913832 enhancer region and the OCA2 promoter is not caused by allelic differences in the 5′ region of OCA2. (A) Schematic overview of the allele-specific 3C assay. The allele-specific interaction between an ApoI fragment containing HERC2 rs12913832 and an ApoI fragment containing OCA2 rs4778241 is investigated. The presence of the C-allele of rs4778241 generates an additional MfeI restriction site. Primers 1 and 3 are used to detect the 3C product, while primers 2 and 3 are used to normalize the ratio of the A-allele over the C-allele. Ratios are determined by MfeI digestion of PCR products. The additional MfeI site is used to monitor the completeness of digestion. (B) Example of gel images of a representative MfeI digestion of PCR products. In the digested lane, the top band represents the A-allele and the bottom band the C-allele of rs4778241. (C) Quantification of multiple gel images as shown in B. The ratio of the bands generated by primer pair 2 + 3 after MfeI digestion is set to 1. Data are represented as mean ± SEM.
