PCR amplification was carried out for DAT VNTR with primers 5′-TGT GGT GTA GGG AAC GGC CTG AG-3′ and 5′-CTT CCT GGA GGT CAC GGC TCA AGG-3′ (Invitrogen, Carlsbad, CA) in a total volume of 15 μL containing 120 ng of genomic DNA, 1.6 μL of 10× PCR buffer, 0.45 μL of 50 mM MgCl2, 0.25 μL of 5 U/μL Platinum® Taq DNA Polymerase, 1.5 μL each of 10 μM forward and reverse primers, 0.3 μL each of 10 mM dNTPs (all reagents supplied by Invitrogen), and 3.55 μL of nuclease-free water. Amplification was performed via PCR StepOne™ using 48-well plates under the following conditions: 94°C for 3 minutes, followed by 35 cycles of 94°C for 15 seconds and 72°C for 1 minute and 40 seconds, and a final 10-minute incubation at 72°C. Electrophoresis was performed on 15 μL of each sample on 2.0% agarose gels in 1XTAE and visualized with ethidium bromide under UV light. Genotypes were scored by two people independently and rerun when necessary.
