Application And Estabilshment Of Molecular Diagnostic Technique On SNPs Using Unlabled Probes

AIM : To establish the molecular diagnostic technique for genotyping SNPs using unlabled probes.METHODS : The PCR primers and unlabeled probers on the SNP sites of IL-1B gene -511T>C,-31T>C,3954C>T, IL-6 gene -174G>C,-597G>A and IL-6R gene exon 358A > C were designed. The Mg²⁺ concentrations of different dye systems,DNA polymerase, asymmetric PCR primier ratio, template concentration and anneal temperature of PCR reaction system were optimized based on the PCR amplification efficiency and product specificity. The molecular diagnosis methods on these sites were established based on the above optimized PCR-UP system. Besides, the -597G>A and -174G>C sites of IL-6 gene were genetyped in 100 clinical samples using the established PCR-UP systems and validated the detected mutant genotypes with the gold standard of sequencing.RESULTSWe have successfully established the amplification system of asymmetric two-step PCR for genotyping SNPs using fluorescent dye EvaGreen and unlabled probes, which is composed of the suitable concentration of 2.5 mmol/L Mg²⁺, the lowest template concentration of 10ng per 10μl reaction volume, and Realstart Taq DNA polymerase. The primer asymmetry ratios of the IL-1B gene -511T>C,-31T>C,3954C>T sites is 0.02/0.3umol/L, the primer asymmetry ratios of the IL-6 gene-174G>C and -597G>A site is 0.1/0.5 umol/L , the primer asymmetry ratios of of IL-6R gene exon 358A > C site is 0.05/0.5μmol/L.We genotyped on the IL-6 gene-174G>C and -597G>A site for 100 clinical samples using the established asymmetric PCR systems and validated the detected mutant genotypes with the gold standard of sequencing.CONCLUSIONThe molecular diagnostic technique of PCR-UP combined with DNA fluorescent dye EvaGreen is a closed-tube, simple and rapid routine method on genetyping for SNPs...