Ultrasensitive Amplification-Free Quantification of a Methyl CpG-Rich Cancer Biomarker by Single-Molecule Kinetic Fingerprinting

IF 6.7 1区化学 Q1 CHEMISTRY, ANALYTICAL

Analytical Chemistry Pub Date : 2024-10-19 DOI:10.1021/acs.analchem.4c03002

Liuhan Dai, Alexander Johnson-Buck, Peter W. Laird, Muneesh Tewari, Nils G. Walter

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Abstract

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.

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通过单分子动力学指纹技术对富含甲基 CpG 的癌症生物标记物进行超灵敏的免扩增定量分析

人类表观遗传标记中研究得最多的是 DNA 中胞嘧啶的 5-甲基修饰，它作为疾病生物标记的潜力巨大。目前，DNA 甲基化的定量主要依赖于亚硫酸氢盐转化，然后进行 PCR 扩增和 NGS 或芯片分析。PCR 在扩增亚硫酸氢盐转化的甲基化序列和未甲基化序列时可能会出现偏差。在这里，我们将亚硫酸氢盐转化与单分子动力学指纹图谱相结合，开发出一种无需扩增的检测支链氨基酸转氨酶 1（BCAT1）启动子 DNA 甲基化的方法。我们的检测方法可选择性地响应甲基化序列，检测限低于 1 fM，特异性高达 99.9999%。在评估复杂的基因组 DNA 矩阵时，我们能可靠地将全血 DNA 中 BCAT1 启动子处的<5% DNA 甲基化与完全未甲基化的全基因组扩增 DNA 区分开来。总之，这些结果证明了我们的无扩增单分子定量方法的可行性和灵敏度，可以提高甲基化癌症 DNA 生物标记物的早期检测。

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来源期刊

Analytical Chemistry 化学-分析化学

CiteScore

12.10

自引率

12.20%

发文量

1949

审稿时长

1.4 months

期刊介绍： Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.