Taqman allelic discrimination protocol

Based upon information from the recent genetic association studies, two SNPs, that is, rs13266634(Arg276Trp C/T) and rs11558471(A/G) in the SLC30A8 gene, were selected for our study [4 (link)-9 (link)]. The first SNP is non-synonymous, in which the amino acid arginine is changed to tryptophan, while the second one is a synonymous SNP at the 3′-UTR. The SLC30A8 gene structure and location of these two polymorphisms are shown in Figure 1. Genotyping these two SNPs were done using TaqMan SNP genotyping assays purchased from Applied Biosystems (Applied Biosystems, Foster City, USA). Genotyping experiments were performed in ABI 7300 sequence detector with a Taqman allelic discrimination protocol (Applied Biosystems). DNA samples were distributed randomly across plates with cases and controls for genotyping quality control. All PCR reactions were run in 20 μl volumes using 10 to 20 ng genomic DNA. Millipore water was used as negative controls (blanks) on each plate.

INSIG2 (rs7566605) genotyping was performed by the means of TaqMan allelic discrimination protocol (Applied Biosystems, Foster City, CA, USA). Genotype distributions confirmed Hardy-Weinberg equilibrium (HWE, P>0.05) in the non-obese healthy individuals. Genotype calling from real-time PCR data was performed using an algorithm called “best cycle genotyping algorithm”. The quality of the assignment of individual samples to clusters was determined on the basis of silhouette values[36 ].