人类研究最深入的表观遗传标记是 DNA 中胞嘧啶的 5-甲基修饰，它作为疾病生物标记具有巨大的潜力。目前，DNA 甲基化的定量很大程度上依赖于亚硫酸氢盐转化，然后进行 PCR 扩增和 NGS 或微阵列分析。 PCR 在亚硫酸氢盐转化的甲基化序列与非甲基化序列的差异扩增中存在潜在偏差。在这里，我们将亚硫酸氢盐转化与单分子动力学指纹分析相结合，开发了一种无需扩增的支链氨基酸转氨酶 1 (BCAT1) 启动子 DNA 甲基化检测方法。我们的检测选择性响应甲基化序列，检测限低于 1 fM，特异性为 99.9999%。通过评估复杂的基因组 DNA 矩阵，我们可靠地区分全血 DNA 中 BCAT1 启动子处 <5% 的 DNA 甲基化与完全未甲基化的全基因组扩增 DNA。总而言之，这些结果证明了我们的无扩增单分子定量方法在改善甲基化癌症 DNA 生物标志物的早期检测方面的可行性和敏感性。

The most well-studied epigenetic marker in humans is the 5-methyl modification of cytosine in DNA, which has great potential as a disease biomarker. Currently, quantification of DNA methylation relies heavily on bisulfite conversion followed by PCR amplification and NGS or microarray analysis. PCR is subject to potential bias in differential amplification of bisulfite-converted methylated versus unmethylated sequences. Here, we combine bisulfite conversion with single-molecule kinetic fingerprinting to develop an amplification-free assay for DNA methylation at the branched-chain amino acid transaminase 1 (BCAT1) promoter. Our assay selectively responds to methylated sequences with a limit of detection below 1 fM and a specificity of 99.9999%. Evaluating complex genomic DNA matrices, we reliably distinguish <5% DNA methylation at the BCAT1 promoter in whole blood DNA from completely unmethylated whole-genome amplified DNA. Taken together, these results demonstrate the feasibility and sensitivity of our amplification-free, single-molecule quantification approach to improve the early detection of methylated cancer DNA biomarkers.