Epigenetic regulation is important in human health and disease, but the exact mechanisms remain largely enigmatic. DNA methylation represents one epigenetic aspect but is challenging to quantify. In this study, we introduce a digital approach for the quantification of the amount and density of DNA methylation. We designed an experimental setup combining efficient methylation‐sensitive restriction enzymes with digital polymerase chain reaction (PCR) to quantify a targeted density of DNA methylation independent of bisulfite conversion. By using a stable reference and comparing experiments treated and untreated with these enzymes, copy number instability could be properly normalized. In silico simulations demonstrated the mathematical validity of the setup and showed that the measurement precision depends on the amount of input DNA and the fraction methylated alleles. This uncertainty could be successfully estimated by the confidence intervals. Quantification of RASSF1 promoter methylation in a variety of healthy and malignant samples and in a calibration curve confirmed the high accuracy of our approach, even in minute amounts of DNA. Overall, our results indicate the possibility of quantifying DNA methylation with digital PCR, independent of bisulfite conversion. Moreover, as the context‐density of methylation can also be determined, biological mechanisms can now be quantitatively assessed.

中文翻译：

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使用甲基化敏感限制酶和数字 PCR 定量 DNA 甲基化，与亚硫酸氢钠转化无关。

表观遗传调控对人类健康和疾病很重要，但确切的机制在很大程度上仍然是个谜。DNA 甲基化代表了一个表观遗传方面，但难以量化。在这项研究中，我们引入了一种数字方法来量化 DNA 甲基化的数量和密度。我们设计了一种实验装置，将有效的甲基化敏感限制酶与数字聚合酶链反应 (PCR) 相结合，以量化与亚硫酸氢盐转化无关的目标 DNA 甲基化密度。通过使用稳定的参考并比较用这些酶处理和未处理的实验，拷贝数不稳定性可以适当地标准化。计算机模拟证明了该设置的数学有效性，并表明测量精度取决于输入 DNA 的量和甲基化等位基因的分数。这种不确定性可以通过置信区间成功估计。量化各种健康和恶性样本以及校准曲线中的RASSF1启动子甲基化证实了我们方法的高准确性，即使在微量的 DNA 中也是如此。总体而言，我们的结果表明使用数字 PCR 量化 DNA 甲基化的可能性，与亚硫酸氢盐转化无关。此外，由于还可以确定甲基化的背景密度，现在可以定量评估生物学机制。