Using standard extraction methods, DNA was isolated from whole blood, collected from the femoral vein under ketamine anesthesia (15 mg/kg, IM). To genotype for -2232 C>G, a functional SNP that represented one of the major haplotype clades, a portion of the 5′flanking region (−2730 > −2204) was amplified from 25 ng of genomic DNA with flanking oligonucleotide primers (Forward: 5′-GGT TCT CAT TTA AAC CGA GTG ATC-3′; Reverse: 5′-AAG TGG CTC CAA CTA GGG AGT AAG-3′) in 20 μl reactions using AmpliTaq Gold® and 4 mM MgCl2 according to the manufacturers instructions (Invitrogen, Carlsbad, CA, USA). Amplifications were performed on a PerkinElmer thermocycler (9700) with one cycle at 96oC followed by 30 cycles of 94°C/15 sec, 55°C/15 sec, 72°C/30 sec, and a final 3-minute extension at 72°C. Because results of 5′-Exonuclease assay genotyping for -2232C/G were unreliable, we performed restriction digest by EarI (1.5 μl, New England Biolabs, Beverly, MA, USA) using 10 μl of PCR product in a total volume of 20 μl for 4 h at 37°C. Samples were separated by electrophoresis on 10% polyacrylamide gels, and the
