ACE Gene D/I Polymorphism Identification and Classification

Detailed description of the procedure of ACE gene D/I genotype identification has been described previously (Comey et al., 1994; Sun et al., 2018; He et al., 2019). DNA samples were extracted from buccal mucosal cells by cotton swabs. ACE gene D/I polymorphism was determined using polymerase chain reaction (PCR). DNA samples were extracted using 5% chelex-100 (165 μl) and proteinase K (3 μl, 20 mg/μl) and amplified using the forward primer of 5’-CTG GAG ACC ACT CCC ATC CTT TCT-3’ and the reverse primer 5’-GAT GTG GCC ATC ACA TTC GTC AGA T-3’ (Lian et al., 2012). The PCR reaction system (15 μl) consisted of ddH₂O (10.8 μl), dNTP (1.2 μl), 10 × buffer (1.5 μl), TAKARA HS Taq polymerase (0.1 μl), each primer (0.2 μl), and template (1.0 μl). The DNA was amplified by 35 cycles using PCR Thermal (iCycler, Bio-Rad, United States). Every cycle was started with pre-denaturation at 94°C for 5 min, followed by 30 s of denaturation at 94, 55, and 72°C. The amplification was ended with a final elongation of 10 min at 72°C and kept at 15°C. To identify the ACE D/I genotype, 2 μl PCR amplicon was electrophoresed using a 2.5% agarose gel with the presence of a 190 bp fragment for D allele and a 470 bp fragment for I allele.