Chunk #12 — Materials and Methods — Genotyping

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Examination of genetic variation in GABRA2 with conduct disorder and alcohol abuse and dependence in a longitudinal study.

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SNP genotyping was performed with TaqMan©® assays for allelic discrimination according to manufacturer's instructions (Applied Biosystems, Foster City, California). Two thousand four hundred and ninety six subjects from CADD and 3,072 subjects from GADD were genotyped. Polymerase Chain Reaction (PCR) reactions were performed with the Biomek® 3000 Laboratory Automation Workstation (Beckman Coulter Inc, Brea, California) and the Dual 384-Well GeneAmp® PCR system 9700 (Applied Biosystems, Foster City, California). To analyze the amplified plates a 7900 Real-Time PCR System (Applied Biosystems, Foster City, California) was used. Based on all the SNPs that have been previously genotyped in these samples from the CADD (33) and GADD (12), DNA samples for subjects with overall call rates <90% were excluded. Three hundred and eighty four samples were genotyped twice for each SNP to determine concordance between replicate reactions; the percent of discordant calls for SNPs in the CADD and GADD, respectively, are shown as follows: rs567926 (0.78%, 0.00%), rs279858 (0.78%, 0.26%), rs279871 (0.52%, N/A), rs279845 (1.30%, 0.00%), rs9291283 (0.26%, 0.78%). Genotype clusters from the amplified 384 well plates were auto-called by the Applied Biosystems