Unlabeled probe genotyping for detec-tion of single nucleotide polymorphism of IL-6 promoter
AIM:To study the practicality of using unlabeled probe genotyping for detecting single nucleotide polymorphism (SNP) of IL-6 gene promoter. METHODS: SNP of IL-6 gene promoter was genotyped with a new fluorescent dye EvaGreen and the latest unlabeled probe technique, and the PCR primers and unlabeled probe were designed on the SNP site - 597G ＞ A of IL-6 gene promoter. The PCR amplification system was optimized, including the concentration of Mg2+, the ratio of asymmetric PCR primer, the lowest concentration of template and the annealing temperature based on the amplification efficiency and production specificity of PCR. One hundred clinical samples from outpatients were genotyped using the optimized PCR system and the detected mutant genotypes were validated with the gold standard of sequencing. RESULTS:We successfully established the amplification system of asymmetric two-step PCR for genotyping on the SNP of IL-6 gene with fluorescent dye EvaGreen and unlabeled probe, which was composed of the suitable concentration of 2.5 mmol/L Mg2+, primer asymmetry ratios of 0.1/0.5 μmol/L, and the lowest template concentration of 10 ng per 10 μL reaction volume. We found seven heterozygous types and one homozygous mutant of the site - 597G ＞ A of IL-6 gene in the detected samples using the optimized asymmetric PCR system, which were confirmed by sequencing. CONCLUSION: The unlabeled probe technique for genotyping on SNP is a simple, economic and routine method that deserves to be spread.
intekleukin-6EvaGreenunlabeled probespolymorphism, single nucleotide
NETL
NSTL
万方数据
维普
