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目的:建立经济可靠的基于MS-HRM技术的检测DNA甲基化的方法。方法:采用简单的沉淀法纯化回收Bis-DNA(重亚硫酸钠处理后的DNA),首次采用picogreen为MS-HRM分析染料,并用新建立的方法检测结直肠癌患者组织SEPT9启动子甲基化水平。结果:新方法的回收率在63%以上,纯度、得率都显著优于经典方法中的Promega Wizard DNA Clean-up System,Picogreen染料可检测到低至0.5%甲基化。共检测34例结直肠癌组织,其中SEPT9异常甲基化的组织33例,占97%,检出率高于文献报导。结论:本文建立的方法简单快速、灵敏度高、重复性好、经济、可操作性强的优点。
Objective: To establish an economical and reliable method for detecting DNA methylation based on MS-HRM technology. Methods: Bis-DNA was purified and purified by simple precipitation method. For the first time, picogreen was used to analyze the dye of MS-HRM. The methylation level of SEPT9 promoter in tissues of patients with colorectal cancer was detected by a newly established method. Results: The recovery of the new method was above 63%. The purity and yield of the new method were significantly better than the Promega Wizard DNA Clean-up System in the classical method. Picogreen dye could detect as little as 0.5% methylation. A total of 34 cases of colorectal cancer tissues were detected, in which 33 cases of abnormal methylation of SEPT9, accounting for 97%, the detection rate was higher than reported in the literature. Conclusion: The method established in this paper is simple and fast, high sensitivity, good repeatability, economic and practical advantages.