ITPA SNP data.xlsx

After obtaining approval from the ethical committee reference number(IRB-UOLFAHS/565) the HCV patients receiving direct-acting antiviral treatment were invited to participate after taking consent. Blood was drawn in aseptic conditions. DNA extraction was performed ITPA gene was amplified by the following conditions: PCR master mix (Thermo scientific by Thermo Fisher) was used with 5-10 ng/µl DNA. The thermal cycle conditions were: Denaturation at 94°C for 10 min then 35 cycles at 95 °C for 1 min, annealing at 55 °C for 30 sec, extension at 72 °C for 30 sec, and final extension at 72 °C for 10 min. PCR products were visualized under ultraviolet light after running gel electrophoresis. PCR product band size was 213bp. Following PCR, ITPA genotypes were investigated using Restriction fragment length polymorphism (RFLP). For this purpose, PCR products were treated overnight with restriction enzyme Xcel to investigate the presence of rs1127354 polymorphisms. In the case of polymorphism rs1127354, 213bp PCR product was digested into fragments 213bp, 135bp, and 78bp for (CC) homozygous dominant allele, (CA) heterozygous allele, and (AA) for homozygous recessive allele respectively. For rs7270101 polymorphism, after overnight treatment of PCR product by Mboll enzyme fragments of 213, 173, 40 bp (AC); 173, 40 bp (CC); and 213 (AA) were obvious.SNPs conformed to the Hardy-Weinberg equilibrium. For further confirmation of RFLP results, Sanger sequencing was conducted.