Figure 4. 3C-SNP analysis.

(A–E) show SNP sequencing results with electropherograms of the sequence and allele ratios by pyrosequencing (PSQ) analysis. A diagram indicating the 3C ligation product with the orientation of primers and SNPs is given below each sequence result. (A) 3C ligation product of restriction sites a and m which represents DMR0 and the enhancer showing preferentially monoallelic interaction at a SNP within fragment a-b1. The a-b1 fragment is close to the P0 and P2 promoters in the flanking BamHI fragments. (Promoters can extend up to 5 Kb upstream of the transcription start site and the resolution of the 3C technique does not enable distinction between different promoters when they are in close proximity). (B) SNP analysis of 3C ligation product of restriction sites c2 and m representing an interaction between the enhancer and IGF2 promoters near restriction site c2. Both SNPs in this region show monoallelic interactions. (C) Allelic analysis of 3C ligation product of restriction sites a and h, using a SNP near restriction site h in the CCD shows that associations between CTCF AD/DMR0 and CCD are biallelic. This SNP also showed that CTCF bound to both alleles at the CCD in Figure 2. (D) SNP analysis of 3C ligation product of restriction sites k and q indicative of an interaction between the ICR and the downstream CTCF DS showing a monoallelic interaction. (E) SNP analysis of 3C ligation products between ICR and the CTCF AD/DMR0 (restriction sites a–k). Both sides of the hybrid product show biallelic associations.