DNA Genotyping via PCR-RFLP Technique

DNA samples were obtained from 400 μL peripheral blood, using Wizard Genomic DNA Purification Commercial Kit (Promega Corporation, Fitchburg, WI, USA). Genotyping was based on polymerase chain reaction (PCR)-restriction fragment length polymorphism technique. For specific DNA amplification, a total amount of 100 ng of genomic purified DNA was amplified in a volume of 25 μL reaction mixture containing 12.5 μL of PCR mastermix, a premixed, ready-to-use solution containing Taq DNA polymerase, deoxynucleotide triphosphates, MgCl₂, and reaction buffers (Promega Corporation); 7.5 μL free nuclease water; 1 μL of bovine serum albumine; 1 μL of each primer; and 1 μL of water suspended DNA. The amplification products were submitted to enzyme digestion (Fermentas; Thermo Fisher Scientific, Waltham, MA, USA) and analyzed by electrophoresis agarose gel (MetaPhor^®; FMC BioProducts, Rockland, ME, USA), allowing detection by ethidium bromide staining of the corresponding genotypes. Specific thermocycling conditions and resulting DNA fragments are presented in Table 1. The genotypic analyses of NOS2A −954G/C (rs2297518) and VEGF +936C/T (rs3025039) were done in accordance with the studies of Kun et al22 (link) and Guan X et al.23 (link)