应用磁珠法检测并提取尿液游离甲基化DNA

摘要

Objective To establish a simple method to extract the methylated ctDNA in urine using magnetic beads as solid phase adsorption carri⁃er with a specific design reagent system and extraction process,and evaluate its application feasibility for methylated gene detection in urine sample . Methods Fourty cases of methylated ctDNA were extracted in urine using magnetic beads. After methylated modification,the concentration and pu⁃rity of DNA was determined by ultraviolet spectrophotometer. Results The extraction of 50 mL urine can gain 61⁃200 ng/mL methylated ctDNA, and the OD260/280 was 1.8 ± 0.05. Conclusion There are methylated ctDNA exist in the urine which can be extracted by magnetic beads. The estab⁃lished extraction method is simple,effective,and with high purity.%目的：依据血浆中存在游离DNA的理论，采用磁珠作为固相吸附载体并使用特定设计的试剂体系及提取流程，建立一种简便、高效提取尿液样本中游离甲基化DNA的方法，评价其作为用于尿液样本的甲基化基因检测技术的可行性。方法使用磁珠法提取40例成人尿液中游离甲基化DNA，进行甲基化修饰后，紫外分光光度计测定DNA的浓度和纯度。结果结果显示，提取50 mL尿液可得61~200 ng/μL的甲基化DNA，OD260/280为1.8±0.05。使用甲基化阳性对照DNA引物进行PCR及电泳后可见目的条带，说明其纯度可以满足后续甲基化基因检测和PCR等操作要求。结论尿液中确实存在游离甲基化DNA，同时磁珠法提取尿液游离DNA提取过程简单，且提取纯度高，是一种高效的提取方法。