Golden gate snp genotyping arrays

We collected 10 ml ETDA-anticoagulated venous blood, and separated the plasma and white blood cell layer, and stored the white blood cells in a cryotube at -80° C Celsius refrigerator for DNA extraction. Genomic DNA was extracted using QIAGEN DNA Extraction Kit (QIAGEN Inc.) [37 (link)]. The Illumina Golden Gate SNP Genotyping Arrays was used to genotype 192 SNPs in stage I. The TaqMan platform was taken to genotype 8 SNPs associated with breast cancer prognosis in stage II. We used a 5-μl reaction mixture system with 20 ng of genomic DNA, 2.5 μl of 2×TaqMan Genotyping Master Mix, 0.1 μl of 40×probe and 1.9μl of double distilled water. The PCR reaction conditions were 95° C for 10 minutes, followed by 50 cycles of 92° C for 30 seconds, and 60° C for 1 minutes. We amplified using the 384-well reaction plates and performed genotype analysis using SDS 2.4 software (Applied Biosystems, Foster City, CA, USA). In order to ensure the accuracy and reliability of the experimental results, approximately 5% of the samples were randomly selected for retesting.

In stage I, SNP genotyping was conducted using Illumina Golden Gate SNP Genotyping Arrays according to the manufacturer’s instructions. Only plates with a consistent high call rate in the initial calling were used. If the call rate was <80%, we repeat the experiment. In stage II, genotyping were performed using the TaqMan platform in 384-well plates and read with the Sequence Detection Software on an ABI Prism 7900 instrument according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Primers and probes were supplied by Applied Biosystems, the PCR conditions used were as follows: 50°C for 2 minutes, 95°C for 10 minutes, and 60°C for 1 minute for 40 cycles. After 2 rounds of genotyping, the success rate for genotyping was 99%, and 5% of the samples were selected for replication, the results were 100% concordant.