分子信标熔解曲线法分析SLC11A1基因插入/缺失多态性

Research on the INDEL polymorphism of SLC11A1 gene with molecular beacon melting curve

目的针对溶质转运蛋白家族11成员1蛋白(SLC11A1)基因插入/缺失多态性位点rs17235416,建立基于荧光定量PCR(q PCR)的分子信标熔解曲线法对其进行基因分型。方法针对该位点设计特异性的引物和分子信标探针,联合应用非对称q PCR、分子信标探针和熔解曲线分析等技术建立分子信标熔解曲线法进行基因分型。对210份全血样本提取DNA后,用上述方法和改进的PCR-RFLP法对该位点进行检测和分型,并以测序法验证。结果 210份样本的基因型TGTG+/+、TGTG-/-和TGTG+/-分布频率分别为69.5%、4.8%和25.7%。分子信标熔解曲线法和PCR-RFLP法的分型结果与测序结果完全一致。结论国内外首次成功建立分子信标熔解曲线法检测SLC11A1基因rs17235416位点;该方法在大人群样本检测中具有简便、快速、准确等优点。

Objective To develop a genotyping method to analyze the important INDEL polymorphism （rs17235416） of solute carrier family 11 member 1 protein （SLC11 A1 ） gene by molecular beacon melting curve analysis. Methods Based on the sequence around the polymorphic site, the specific primers and molecular beacon probe were designed, and the asymmetric qPCR, molecular beacon probe and melting curve analysis were used in combination to establish the molecular beacon melting curve analysis method. DNAs extracted from 210 blood samples were detected and genotyped with above method and an improved PCR - RFLP method. Sequencing was performed to confirm the accuracy of these two method＇ s results. Results Three genotypes of SLCllA1 gene, TGTG ＋/＋ , TGTG -/- and TGTG ＋/- , in 210 blood samples were 69.5%, 4.8% and 25.7%, respectively. In comparison with the sequencing results, the present established two methods had 100% consistency. Conclusion A molecular beacon melting curve analysis method was successfully established to genotype the rs17235416 polymorphism for the first time, and it promised the easy, rapid, and reliable application in large samples size study.