Wu Zhongliang, Zhang Ju, Cheng Hong, Liang Ping, Guo Yanhai, Yan Zhen, Zhao Jinrong
Department of Neurology, Xijing Hospital, The Fourth Military Medical University, Xian, China.
Diagn Mol Pathol. 2010 Dec;19(4):232-5. doi: 10.1097/PDM.0b013e3181de4380.
O⁶-Methylguanine-DNA methyltransferase methylation status has a very good predictive value for benefit from alkylating agent therapy. The stratified therapy assignment of patients according to the O⁶-Methylguanine-DNA methyltransferase methylation status requires a standardized diagnostic test. A novel method detecting the promoter methylation status of O⁶-Methylguanine-DNA methyltransferase in tissue samples by a fluorescence polarization assay was developed. A pair of primers was used to amplify a 266 bp fragment in the promoter region of the O⁶-Methylguanine-DNA methyltransferase gene. Two probes specific for either methylated or unmethylated DNA labeled with different fluorophores hybridized with their target amplicons, and the hybridization increased the fluorescence polarization values. The methylation status was determined by the increased fluorescence polarization values. Ninety-seven glioma tumor samples were analyzed in parallel with the new assay and the nested gel-based methylation-specific polymerase chain reaction assay. The results of the methylation status of the fluorescence polarization assay were in good concordance with the results obtained with the nested gel-based methylation-specific polymerase chain reaction assay. The sensitivity and stability of the fluorescence polarization assay have been measured. The coefficient of variation of the reproducibility for the fluorescence polarization assay was <10%. The minimum detection level established with the fluorescence polarization assay was 20 copies/μL. The fluorescence polarization assay allowed the discrimination of the O⁶-Methylguanine-DNA methyltransferase methylation status at individual CpG sites directly in the solution without the 2-step approach with nested primers.
O⁶-甲基鸟嘌呤-DNA甲基转移酶甲基化状态对烷化剂治疗的获益具有很好的预测价值。根据O⁶-甲基鸟嘌呤-DNA甲基转移酶甲基化状态对患者进行分层治疗分配需要标准化的诊断测试。开发了一种通过荧光偏振分析检测组织样本中O⁶-甲基鸟嘌呤-DNA甲基转移酶启动子甲基化状态的新方法。使用一对引物扩增O⁶-甲基鸟嘌呤-DNA甲基转移酶基因启动子区域的一个266 bp片段。两种分别针对甲基化或未甲基化DNA且标记有不同荧光团的探针与其目标扩增子杂交，杂交会增加荧光偏振值。通过荧光偏振值的增加来确定甲基化状态。对97个胶质瘤肿瘤样本同时采用新方法和基于巢式凝胶的甲基化特异性聚合酶链反应法进行分析。荧光偏振分析的甲基化状态结果与基于巢式凝胶的甲基化特异性聚合酶链反应法获得的结果高度一致。已测定荧光偏振分析的灵敏度和稳定性。荧光偏振分析重复性的变异系数<10%。荧光偏振分析确定的最低检测水平为20拷贝/μL。荧光偏振分析无需使用巢式引物的两步法即可直接在溶液中区分单个CpG位点的O⁶-甲基鸟嘌呤-DNA甲基转移酶甲基化状态。
