摘要
目的:探讨无标记探针技术检测IL-6基因启动子单核苷酸多态性(SNP)的实用性.方法:采用新型荧光染料EvaGreen和无标记探针技术对IL-6基因启动子SNP进行基因分型.以其-597G>A位点为例设计PCR扩增引物和无标记探针,按PCR扩增效率和产物特异性进行Mg2+浓度、不对称PCR引物比例以及模板浓度等条件的优化.并用此优化体系基因分型100例临床标本,杂合型与突变型以测序验证.结果:结果显示EvaGreen荧光PCR检测体系是最适Mg2+浓度为2.5mmol/L,不对称PCR引物比例为0.1/0.5μmol/L,模板浓度为10ng/10μL的两步法不对称PCR.该体系对100例样本进行IL-6基因-597G>A位点基因分型,发现7例杂合型和1例突变型,经测序与检测结果一致.结论:无标记探针技术检测基因SNP是一种值得推广的简便易行、低成本、常规化的基因分型方法.
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 Mg^2＋ , 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 Mg^2＋ , primer asymmetry ratios of 0.1/0.5 umol/L, and the lowest template concentration of 10 ng per 10uL 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.
出处
《第四军医大学学报》
北大核心
2009年第16期1484-1487,共4页
Journal of the Fourth Military Medical University
