Several imputed variants in an intron of the ALK gene were significantly associated with EEG alpha frequency. We genotyped these variants in 48 DNA samples from individuals in 12 families, including 13 putative carriers of the rare allele in at least one site, 10 of whom had endophenotype data. For each sample, 1 ul DNA at 20 ng/ul was genotyped in a 25 ul reaction along with 12.5 ul TaqMan Universal PCR Master Mix (Life Technologies, Grand Island, NY), 0.625 ul of 40X primer/probe mix, and 10.875 ul water. We used a manufacturer-recommended PCR program: an initial step of 95°C for 10 min, then a 15-s step at 92°, and a 1.5-min step at 60°. Steps 2 and 3 were cycled through 50 times. Each of the four polymorphisms was amplified separately, and the allelic variants for each polymorphism were labeled with VIC and FAM 5′-fluorescent labels. We used an Applied Biosystems PRISM sequence detection 7500 Real Time PCR System to discriminate between alleles, which uses an optical reading of fluorescent markers following a 1-min period of activation at 60°C to assess the amount of probe sequence product.
