Primers for amplifying the upstream OPRM1 CpG island using bisulfite-treated DNA were designed using Methyl Primer Express v1.0 (Applied Biosystems, Foster City, CA) and Vector NTI Advance 10 (Invitrogen, Carlsbad, CA). Amplification was performed with 1 μl bisulfite-treated DNA, 1 μM of each primer (primer A: 5′-TTTTTTTTTGTTTTAGTTAGG-3′ and primer B: 5′-CAAATTACCATCTAAATAAA-3′), 250 μM each of dATP, dCTP, dGTP and TTP, 50 mM KCl, 4 mM MgCl2, 0.625 units AmpliTaq Gold® (Applied Biosystems), and 10 mM Tris-HCl (pH 8.3) in 50 μl. Amplification consisted of 5 min at 95°C, 40 cycles of 15 sec at 95°C, 15 sec at 52°C, and 30 sec at 72°C, followed by a final elongation step at 72°C for 7 min. A second round of amplification was performed as above using 1μl of the product of the first amplification using the nested primers C: 5′-TGTAAGAAATAGTAGGAGTTGTGGTAG-3′ and D: 5′-AATAAAACAAATTAACCCAAAAACC-3′. Amplification consisted of 5 min at 95°C, 40 cycles of 15 sec at 95°C, 15 sec at 58°C, and 30 sec at 72°C, followed by a final elongation step at 72°C for 7 min.
