Transtaq hifi buffer

Approximately, 2–3 mL venous blood was taken from each participant. DNA was extracted from leukocytes using the genomic DNA isolation kit (Bioteke, Beijing, China) according to the manufacturer’s directions. The rs2072915 was genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). DNA fragments were amplified using the following primers: 5ʹ-TCTCATGTCCATCAGCTTGG-3ʹ and 5ʹ-CCATGATTTGGGGTGATTTC-3ʹ and digested with the Dpn II restriction enzyme (New England Biolabs, Ipswich, MA). The PCR reaction was performed in 10 μL of the final volume including the following materials: 1 μL template DNA, 10 μM of each primer, 10 X TransTaq HiFi buffer 1 μL, 2.5 mM dNTPs 0.8 μL, 10 X GC enhancer 1 μL and TransTaq HiFi DNA polymerase 0.1 μL (TransGen Biotech, Beijing, China). The annealing temperature of PCR was 58 °C. After amplification, PCR products were digested with Dpn II at 37 °C overnight, yielding a band of 183 bp (T allele) or two bands of 130 bp and 53 bp (A allele). Some samples were randomly selected for replicate genotyping and the results were identical.