DNA isolation and Genotyping
Genomic DNA was extracted from 200 µl of peripheral blood by Macherey-Nagel DNA isolation Kit (Cat No: 740.951.250) following manufacturer instructions.
Polymerase chain reaction (PCR) was used to detect the I/D polymorphism of the ACE gene. A polymerase chain reaction (PCR)-based restriction fragment-length polymorphism (RFLP) method was used to genotype ACE2 rs2106809 and rs2285666 polymorphisms. The PCR was performed in a 25 µl volume for each polymorphism that is containing 10X PCR buffer, 3.0 mM MgCl2, 0.25 mMdNTPs, 1.5 units of Taq polymerase (Promega, Madison, WI), and 0.3 µM each primers (5’ CTG GAG ACC ACT CCC ATC CTT TCT 3’ and 5’ GAT GTG GCC ATC ACA TTC GTC AGA 3’ for ACE I/D polymorphism and F; 5’-GAA AGC CAG ATG CTT TAA CAA G-3’ and R; 5’-TTTTTCCATATCTCTATCTGATCG-3’ for ACE2 rs2106809 polymorphism and F; 5’-CATGTGGTCAAAAGGATATC-3’ R; 5’- AAAGTAAGGTTGGCAGACAT-3’ for ACE2 rs2285666 polymorphism.
For ACE I/D polymorphism the PCR products were run at 2% agarose gel. Allele (I/D) types and genotype for each sample were determined based on the PCR product sizes. The major allele indicates the 490 bp fragment that embraces the 287 bp Alu sequence, known as an insertion, whereas the 190 bp fragment represents the minor allele with the deletion of the Alu sequence. Heterozygosity specifies the combination of both major and minor fragments i.e. 490/190 bp.
For ACE2 rs2106809 polymorphism; The PCR products of 207 base pairs (bp) were digested with restriction enzyme TaqI resulting in two products of 183 bp and 24 bp in the presence of A allele.
For ACE2 rs2285666 polymorphism; The PCR products were incubated with 5 U of AluI restriction enzyme at overnight. After digestion, fragments of 281 and 185 bp identify T allele and a 466 bp band identify C allele.
All digestion products were electrophoresed on 3% agarose gel and visualized by staining with ethidium bromide and evaluated using the gel documentation system (Syngene, Genegenius Bio Imaging System).