Department of Mechanical Engineering, Johns Hopkins University, Baltimore, MD 21218, United States.
Clin Chim Acta. 2013 Oct 21;425:169-75. doi: 10.1016/j.cca.2013.07.023. Epub 2013 Aug 1.
The use of methylated tumor-specific circulating DNA has shown great promise as a potential cancer biomarker. Nonetheless, the relative scarcity of tumor-specific circulating DNA presents a challenge for traditional DNA extraction and processing techniques. Here we demonstrate a single tube extraction and processing technique dubbed "methylation on beads" that allows for DNA extraction and bisulfite conversion for up to 2 ml of plasma or serum. In comparison to traditional techniques including phenol chloroform and alcohol extraction, methylation on beads yields a 1.5- to 5-fold improvement in extraction efficiency. The technique results in far less carryover of PCR inhibitors yielding analytical sensitivity improvements of over 25-fold. The combination of improved recovery and sensitivity make possible the detection of rare epigenetic events and the development of high sensitivity epigenetic diagnostic assays.
甲基化肿瘤特异性循环 DNA 的应用作为一种有潜力的癌症生物标志物显示出巨大的前景。然而，肿瘤特异性循环 DNA 的相对稀缺性给传统的 DNA 提取和处理技术带来了挑战。在这里，我们展示了一种称为“珠上甲基化”的单管提取和处理技术，该技术可用于提取和 bisulfite 转化多达 2 毫升的血浆或血清。与包括酚氯仿和醇提取在内的传统技术相比，珠上甲基化使提取效率提高了 1.5 至 5 倍。该技术的结果是，PCR 抑制剂的残留量大大减少，分析灵敏度提高了 25 倍以上。回收率和灵敏度的提高使得检测罕见的表观遗传事件和开发高灵敏度的表观遗传诊断检测成为可能。
