Figure 2

Restriction Fragment Length Polymorphism Analysis: The C/T polymorphism alters a Sma-I site in the SNP (rs1001179), therefore an antisense primer 5'-gagcctcgccccgccggcccg-3' that incorporates a mutation in order to abolish an exisiting Sma-I site was used together with sense primer 5'-attccgtctgcaaaactggc-3' which amplifies a 126 bp fragment. Hot Start PCR reactions were performed using 10 ng genomic DNA, without knowledge of sample status, using the following conditions; initial denaturation at 95°C for 15 min followed by 35 cycles of 94°C for 30 s, 60°C for 45 s, and 72°C for 30 s, with a final extension at 72°C for 10 min. The entire reaction was then digested with 2 U of Sma-I and analysed on 2% agarose gels after staining with ethidium bromide and visualization on a FluorChem UV imaging system. The T allele eliminates a SmaI restriction site. 99 bp and 27 bp fragments are visualized in the wild-type C allele (see lanes 6, 7, 8, 10, 11, 12, 13, and 14) while the full length PCR product are seen in lanes 2, 3, 4, 5 and 9.