高分辨率熔解曲线技术在大豆基因分型中的应用

Application of High Resolution Melting Curve Technology in Soybean Genotyping

高分辨熔解曲线(high resolution melting, HRM)分析技术是一种高效,快速,便捷的核苷酸检测方法。本实验从染料用量、模板浓度、产物片段长度3个方面探索了适宜HRM分析的最佳条件,并利用HRM技术对单核苷酸(single nucleotide polymorphism, SNP)及插入/缺失(insertion-deletion, InDel)检测及基因分型等方面进行了分析。结果表明:10μL反应体系中,20×Eva Green饱和染料加入0.1~0.3μL,模板浓度加入10~200 ng不影响HRM分辨率;扩增产物长度在70~190 bp范围内,插入/缺失片段长度3~13 bp范围内,高分辨熔解曲线均有较好的分辨效果。利用HRM分析技术检测分析F4个体基因型,结果与PAGE电泳分析完全一致。本实验结果说明,HRM技术与传统凝胶电泳基因分型方法相比,具有操作简单、分辨率高、准确快速、成本低等优点,是基因型鉴定的最佳选择。

High resolution melting（HRM） technology is a kind of efficient, rapid and convenient nucleotide detection method. We explored the optimum condition for HRM from 3 aspects which are dye amount, template concentration and length of PCR production. We also used HRM to analyze a single nucleotide polymorphism and insertion-deletion as well as genotyping. In the 10 μL reaction system, 20×Eva Green saturated dye was added to0.1-0.3 L, and template concentration added 10-200 ng did not affect HRM resolution. High resolution melting appeared a good result when the amplicon length wihtin the range of 70-190 bp and insertion/deletion length within 3-13 bp. HRM analysis technique was used to detect and analyze the genotype of F4 individuals. The results were in good agreement with page electrophoresis analysis. Comparing with traditional gel electrophoresis, HRM was a good choice for genotyping with advantages of high resolution, simple operation, rapid, high accuracy and low cost.