Taqman snp technology

DNA was extracted from the peripheral blood leukocytes using a classical salting out method. The SNPs were genotyped with TaqMan SNP technology from a ready-to-use human assay library (Applied Biosystems, MA, USA) using a high-throughput genotyping system, OpenArray (Life Technologies, CA, USA). A sample without a template was used as a negative control. The negative control was applicable in measuring any false positive signals caused by contamination.

We genotyped 4 common FTO SNPs in rs3751812 (G > T), rs8044769 (C > T), rs8050136 (A > C), and rs9939609 (T > A). DNA was extracted from peripheral blood leukocytes using a classical salting-out method. The SNPs were genotyped with TaqMan SNP technology from a ready-to-use human assay library (Applied Biosystems, MA, USA) using a high-throughput genotyping system, OpenArray (Life Technologies, CA, USA). SNP analysis was performed in duplicate, following the manufacturer’s instructions. We used a sample without template as a negative control to detect possible false positive signals caused by contamination.

We genotyped previously identified MC4R SNPs in: rs17782313, rs12970134, rs633265, and rs1350341. DNA was extracted from peripheral blood leukocytes using a classical salting out method. The SNPs were genotyped with TaqMan SNP technology from ready-to-use human assays library (Applied Biosystems, Foster City, CA, USA) using a high throughput genotyping system (OpenArray, Life Technologies, Waltham, MA, USA). SNP analysis was performed in duplicate according to the manufacturer’s instructions. To detect possible false positive signals caused by contamination, a negative control consisting of a sample without a template was used.

We genotyped for the TCF7L2 SNP in rs7901695. The DNA was extracted from peripheral blood leukocytes, by a salting-out method described previously in detail [19 (link)]. Genotyping for the SNP was conducted using TaqMan SNP technology with a ready-to-use human assay library (Applied Biosystems, Waltham, MA, USA), using an OpenArray System (Life Technologies, Grand Island, NY, USA) as a tool for high-throughput genotyping. The genetic analyses were performed in duplicate in accordance with the manufacturer’s instructions. To detect possible false-positive signals caused by contamination, a negative control consisting of a sample without a template was used.

Based on the available scientific literature^{26 (link)}, we selected and genotyped 4 BDNF polymorphisms that are considered genetic risk factors for obesity: rs6265, rs4923461, rs10501087, and rs10835211. DNA was extracted from the peripheral blood leukocytes by a classical salting-out method. The analyzed SNPs were genotyped with a TaqMan SNP technology from a ready-to-use human assay library (Applied Biosystems, MA, USA) using a high-throughput system (Open Array, Life Technologies, CA, USA). We performed SNP analysis in duplicate, according to the manufacturer’s instructions. As a negative control we used a sample without a template to detect possibly caused by contamination false-positive signals.

DNA was extracted from the peripheral whole blood leukocytes using two extraction methods, the salting-out and the membrane method column separation (QIAamp DNA Blood Mini Kit, Qiagen, Germany). Both were performed in agreement to the manufacturer’s protocols. The purity of DNA was determined based on evaluation of the optical density ratio 260/280 nm.
The single nucleotide polymorphism analysis with was performed using the 7900HT Fast Real-Time PCR System (Applied Biosystems, USA). All SNPs in FOXP3 gene (rs3761549, rs3761548, rs3761547) were genotyped by fluorogenic TaqMan SNP technology from ready to use assays library (Applied Biosystems, Foster City, CA, USA) with the TaqMan Genotyping Master Mix (Applied Biosystems, Foster City, CA, USA) in 20 µl reaction volume. The final concentration of genomic DNA for all samples in the experiment sample was 10 ng/µl. The reactions were carried out under the following conditions: 10 min at 95 °C for starting polymerase activity, 40 cycles of 92 °C for 15 s and 60 °C for 1 min. SNPs analysis was performed in duplicate. As a negative control, a sample without DNA was used. The negative control was used to measure any false positive signal caused by contamination.