Taqman snp genotyping assay c 11592758 10

Amplified samples were then diluted to a working concentration and genotyped for 2 in the BDNF gene rs6265 (C_000011.10:g.27658369C>T) and rs4923461 (NC_000011.10:g.27635363A>G) using TaqMan SNP genotyping assay C_11592758_10 and C____50562_10 (Applied Biosystems, Inc) respectively. Individual allele determinations were made using TaqMan Genotyping Master Mix (Life Technologies, Catalog 4371357) with amplification on a GeneAmp 9700 (Applied Biosystems) and analyzing the endpoint fluorescence using a Tecan M200 and JMP 10.0 (SAS, Inc.). Human DNA from cell lines was purchased from Coriell Cell Repositories for all representative genotypes in duplicate using previously reported genotyped from NCBI. These and no template controls were run alongside study samples representing 9% of the total data output. Any samples that were not able to be genotyped to a 95% or greater confidence were repeated under the same conditions.

Saliva derived Human DNA samples were diluted to a working concentration and genotyped for the rs6265 (C_000011. 10:g.27658369C.T) SNP in the BDNF gene using TaqMan SNP genotyping assay C_115 92758_10 (Applied Biosystems). Individual allele determinations were made using TaqMan Genotyping Master Mix (Life Technologies, Catalog 4371357) with amplification on a GeneAmp 9700 (Applied Biosystems) and analyzing the endpoint fluorescence using a Tecan M200 and JMP 10.0 (SAS, Inc.). Call rate for the BDNF Val66Met genotype was 100% (206/206). The majority of adolescents had the Val/Val genotype (N=152), followed by the Val/Met genotype (N=48), and the Met/Met genotype (N=6). Consistent with other investigations of the BDNF Val66Met polymorphism (e.g., Gunnar et al., 2012 (link); Hariri et al., 2003 (link)) and functional differences across genotypes (Soltész et al., 2014 ), the Met/Met and Val/Met genotypes were collapsed into an any Met allele group. The genotype distribution for the BDNF Val66Met polymorphism was in Hardy-Weinberg equilibrium (χ²(1)=.83, ns). There were no differences in race/ethnicity by genotype χ²(6)=10.88, ns).

Saliva samples for DNA analyses were collected under researcher
observation using Oragene saliva collection kits (DNA Genotek, Kanata, Canada).
Genotyping was performed at the UCLA Genotyping and Sequencing Core. The
Val66Met polymorphism (rs6265) was genotyped using a 5′ nuclease assay to
discriminate between the two alleles (i.e., Val vs. Met; Taqman SNP Genotyping
Assay C_11592758_10, Applied Biosystems, Grand Island, NY). Polymerase chain
reactions were performed using 5-μL reaction volumes in 384-well plates
with 5 ng of DNA and Taqman genotyping master mix from Applied Biosystems. The
standard protocol provided with the kit was followed. End point reads of
fluorescence levels were obtained with an ABI 7900HT Sequence Detection System.
The genotype frequencies at Val66Met in the present sample were Val/Val = 73,
Val/Met = 28, and Met/Met = 3, and did not deviate from Hardy–Weinberg
equilibrium, χ² (1, 104) = 0.03, p = .99.