PDF(Pubmed)
Abstract：
High-resolution melting (HRM) is a cost-efficient tool for targeted DNA methylation analysis. HRM yields the average methylation status across all CpGs in PCR products. Moreover, it provides information on the methylation pattern, e.g., the occurrence of monoallelic methylation. HRM assays have to be calibrated by analyzing DNA methylation standards of known methylation status and mixtures thereof. In general, DNA methylation levels determined by the classical calibration approach, including the whole temperature range in between normalization intervals, are in good agreement with the mean of the DNA methylation status of individual CpGs determined by pyrosequencing (PSQ), the gold standard of targeted DNA methylation analysis. However, the classical calibration approach leads to highly inaccurate results for samples with heterogeneous DNA methylation since they result in more complex melt curves, differing in their shape compared to those of DNA standards and mixtures thereof. Here, we present a novel calibration approach, i.e., temperature-wise calibration. By temperature-wise calibration, methylation profiles over temperature are obtained, which help in finding the optimal calibration range and thus increase the accuracy of HRM data, particularly for heterogeneous DNA methylation. For explaining the principle and demonstrating the potential of the novel calibration approach, we selected the promoter and two enhancers of MGMT, a gene encoding the repair protein MGMT.
摘要：
高分辨率解链(HRM)是一种用于靶向DNA甲基化分析的经济高效的工具。HRM产生PCR产物中所有CpG的平均甲基化状态。此外，它提供了甲基化模式的信息，例如，单等位基因甲基化的发生。HRM测定必须通过分析已知甲基化状态的DNA甲基化标准品及其混合物来校准。总的来说,通过经典校准方法确定的DNA甲基化水平，包括归一化间隔之间的整个温度范围，与焦磷酸测序（PSQ）确定的单个CpG的DNA甲基化状态的平均值非常吻合，靶向DNA甲基化分析的黄金标准。然而,经典的校准方法导致高度不准确的结果样品与异质DNA甲基化，因为它们导致更复杂的熔解曲线，与DNA标准品及其混合物相比，它们的形状不同。这里,我们提出了一种新颖的校准方法，即，温度校准。通过温度校准，获得随温度变化的甲基化曲线，这有助于找到最佳校准范围，从而提高人力资源管理数据的准确性，特别是对于异质DNA甲基化。为了解释原理并展示新颖校准方法的潜力，我们选择了MGMT的启动子和两个增强子，编码修复蛋白MGMT的基因。
