Epigenomics AG, Berlin, Germany.
Biotechniques. 2009 Sep;47(3):737-44. doi: 10.2144/000113208.
DNA methylation is an important epigenetic mechanism involved in fundamental biological processes such as development, imprinting, and carcino-genesis. For these reasons, DNA methylation represents a valuable source for cancer biomarkers. Methods for the sensitive and specific detection of methylated DNA are a prerequisite for the implementation of DNA biomarkers into clinical routine when early detection based on the analysis of body fluids is desired. Here, a novel technique is presented for the detection of DNA methylation biomarkers, based on real-time PCR of bisulfite-treated template with enzymatic digestion of background DNA during amplification using the heat-stable enzyme Tsp509I. An assay for the lung cancer methylation biomarker BARHL2 was used to show clinical and analytical performance of the method in comparison with methylation-specific PCR technology. Both technologies showed comparable performance when analyzing technical DNA mixtures and bronchial lavage samples from 75 patients suspected of having lung cancer. The results demonstrate that the approach is useful for sensitive and specific detection of a few copies of methylated DNA in samples with a high background of unmethylated DNA, such as in clinical samples from body fluids.
DNA 甲基化是一种重要的表观遗传机制，参与发育、印迹和癌变等基本生物学过程。由于这些原因，DNA 甲基化是癌症生物标志物的一个有价值的来源。用于敏感和特异性检测甲基化 DNA 的方法是将 DNA 生物标志物应用于临床常规的前提，当希望基于体液分析进行早期检测时。在这里，提出了一种基于亚硫酸氢盐处理模板的实时 PCR 检测 DNA 甲基化生物标志物的新方法，该方法在扩增过程中使用耐热酶 Tsp509I 酶解背景 DNA。使用肺癌甲基化生物标志物 BARHL2 的测定来比较该方法与甲基化特异性 PCR 技术的临床和分析性能。当分析来自 75 名疑似患有肺癌的患者的技术 DNA 混合物和支气管灌洗样本时，两种技术的性能相当。结果表明，该方法对于从体液等临床样本中检测高背景未甲基化 DNA 中的几个拷贝甲基化 DNA 非常有用。
