Intron 6 of MUTYH
(Major allele sequence (IVS6+35G);
minor allele sequence (IVS6+35A);
was inserted into the multicloning site of the pGL4.10 vector (Promega) and designated pIVS6+35G and pIVS6+35A.
Fragments of the MUTYH promoter (-2000 to +200 and −1000 to +200) were prepared by PCR.
The oligonucleotide5′-AACTCGAGGGGAATTTACCGATGCCCAGAACG-3′ (position +177 to +200 with XhoI linker) was used as the downstream primer for all reactions.
Each of the following oligonucleotides (with KpnI linker) was used as an upstream primer to amplify specific deletion fragments: 5′-GAGGTACCATGTTCACCCTGGGAGAAAGAGAAGTC-3′ (-2000 to −1974) for p-2KB+IVS6+35G and p-2KB+IVS6+35A, and 5′-GAGGTACCCCCACAAGCCTTTGTAACCCACGTGTT-3′ (-1000 to −974) for p-1KB+IVS6+35G and p-1KB+IVS6+35A.
The amplified fragments were then subcloned into XhoI/KpnI-digested pIVS6+35G and pIVS6+35A.
Promoter activities were measured by a Dual Luciferase Reporter Assay System (Promega) and an LD 400 luminometer (Beckman Coulter, Brea, CA).
Each experiment was independently repeated six times and each sample was run in triplicate.