Taqman mgb technology

The whole-genome DNA was isolated and purified from leucocytes of peripheral blood by proteinase K digestion and phenol/chloroform extraction, as described previously [20 (link)]. The two polymorphisms (-765 G > C and 8473 C > T) were genotyped by TaqMan MGB technology (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions. The sequences of primer and probe for the two SNPs are available on request. The reaction mixture of 10 μL contained 20 ng genomic DNA, 3.5 μL of 2 × TaqMan Genotyping Master Mix, 0.25 μL of the primers and probes mix and 6.25 μL of double distilled water. The amplification was performed under the following conditions: 50°C for 2 min, 95°C for 10 min followed by 45 cycles of 95°C for 15 sec, and 60°C for 1 min. Amplifications were performed in the 384-well ABI 7900HT Real Time PCR System (Applied Biosystems), following the manufacturer’s instructions. After the completion of the amplification, the fluorescence intensity in each well of the plate was read and analyzed with SDS 2.4 automated software. Four blank controls were included in each plate to ensure accuracy of the genotyping. About 10% of the samples were randomly selected for repeated assays, and the results were in agreement with the results of the first assays.

Genomic DNA was extracted from peripheral blood leukocytes of 5 mL whole blood of all participants by standard procedures. The 24 htSNPs in the Fusui population were genotyped using the Sequenom MassARRAY system according to the manufacturer’s instructions [27 (link)]. The MassARRAY system is based on multiplexed polymerase chain reaction (PCR) and single-base primer extension technology, and the mass of the extension products is measured with a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry to be correlated with a specific genotype. To ensure genotyping quality, we genotyped 5% randomly selected duplicate samples and 4 blanks in each 384-well plate and the concordance rate was greater than 99%. For primers and probes used for these SNPs, see S3 Table.
The significantly associated SNP (rs3771333) went forward to be genotyped in the Haimen population and cell lines using TaqMan MGB technology (Assay ID: C_25474467_10; Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol. For quality control, we genotyped by direct DNA sequencing a 15% masked, random sample set of the Haimen population and all results were 100% concordance. The experimenters conducted the genotyping in a blind manner and did not know the case-control status.