Blood genomic dna isolation kit

We selected SNPs based on their functional potential with a minor allele frequency greater than 0.05 in the Asian population and reviewed related literature to identify potential SNPs that could impact EC.
After signing informed consent forms, each subject donated 5 ml of peripheral blood, which was used for genomic DNA extraction. A Blood Genomic DNA Isolation Kit (Axygen Scientific Inc., CA, USA) was used to extract DNA from leukocyte cell pellets according to the manufacturer’s instructions. The DNA purity and concentration were determined by spectrophotometry. All 18 SNPs that were detected at the first stage in the Hangzhou case-control set (discovery set) were also detected using the MassARRAY system (Sequenom Inc., San Diego, California, USA). The genotyping of rs3805322 (ADH4) and rs4822983 (CHEK2) in the Jinan case-control set was performed using TaqMan assays on an ABI 7900 system (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Primers and probes for the two SNPs were supplied by Applied Biosystems. Real-time quantitative polymerase chain reaction (qPCR) was performed under the following conditions: 50°C for 2 minutes, 95°C for 10 minutes, and 45 cycles of 95°C for 30 seconds and 60°C for 1 minute. A random selection of fifteen percent of the samples was reciprocally tested by different persons, and the reproducibility was 99.5%.

Genomic DNAs were extracted from peripheral blood mononuclear cells using the blood genomic DNA isolation kit (Axygen Scientific Inc., 33210 Central Avenue, Union City, California 94587, CA, USA). The reaction system of PCR was as follows: 20 μL of 1X GC‐I buffer (Takara Bio Inc., Otsu, Shiga, Japan), 3.0 mmol/L Mg²⁺, 0.3 mmol/L dNTP, 1 U HotStar Taq polymerase (Qiagen Co., Ltd., Duesseldorf, Germany), 1 μL DNA sample, and 2 μL multiplex PCR primers. The PCR cycle was designed as following: predenaturing at 94°C for 2 min, denaturing at 94°C for 20 sec, annealing at 65°C for 20 sec, extension at 72°C for 30 sec, total 11 cycles; then 94°C for 20 sec, 59°C for 30 sec, 72°C for 90 sec, total 24 cycles; and extension at 72°C for 2 min.