Mboii restriction enzyme

Polymerase chain reaction (PCR) amplification was used to assess the variants of H6PD
(R453Q and D151A) followed by the restriction fragment length polymorphism (RFLP)
analysis. PCR primers for amplification of the region spanning the selected polymorphisms
were designed using NCBI (https://www.ncbi.nlm.nih.gov/) and Oligo 7 software (Table 1).
PCR was performed in a total volume of 25 µl containing 2.5 μL of 10X PCR buffer, 20 pmol
of each forward and reverse primers (SinaClon BioScience Co., Iran), 1.5 mM
MgCl₂ , 0.2 mM dNTPs (SinaClon BioScience Co., Iran), 1 unit of Taq DNA
polymerase (SinaClon BioScience Co., Iran) and about 100 ng of extracted DNA as a
template. The thermal cycler program included an initial denaturation at 95°C for 5
minutes, 35 cycles of denaturation at 95°C for 40 seconds, annealing at 62°C (R453Q)/64°C
(D151A) for 35 seconds, extension at 72°C for 40 seconds, and finally one cycle of
extension at 72°C for 5 minutes. To perform RFLP, 10 µl of PCR products were digested with
2 units of MboII restriction enzyme (Thermo, USA) at 37°C for D151A and PstI restriction
enzyme (Thermo, USA) at 37°C for R453Q, for 15 hours. Products of enzyme digestion were
visualized after 2% agarose gel electrophoresis and staining with GelRed (Kawsarbiotech,
Iran) under ultraviolet light (Figes.1, 2).