Department of Bioengineering, ‡Micro and Nanotechnology Laboratory, and §Department of Molecular and Integrative Physiology, University of Illinois at Urbana-Champaign Urbana, Illinois 61801, United States.
ACS Nano. 2015 Jan 27;9(1):290-300. doi: 10.1021/nn5045596. Epub 2015 Jan 15.
DNA methylation is an epigenetic modification of DNA in which methyl groups are added at the 5-carbon position of cytosine. Aberrant DNA methylation, which has been associated with carcinogenesis, can be assessed in various biological fluids and potentially can be used as markers for detection of cancer. Analytically sensitive and specific assays for methylation targeting low-abundance and fragmented DNA are needed for optimal clinical diagnosis and prognosis. We present a nanopore-based direct methylation detection assay that circumvents bisulfite conversion and polymerase chain reaction amplification. Building on our prior work, we used methyl-binding proteins (MBPs), which selectively label the methylated DNA. The nanopore-based assay selectively detects methylated DNA/MBP complexes through a 19 nm nanopore with significantly deeper and prolonged nanopore ionic current blocking, while unmethylated DNA molecules were not detectable due to their smaller diameter. Discrimination of hypermethylated and unmethylated DNA on 90, 60, and 30 bp DNA fragments was demonstrated using sub-10 nm nanopores. Hypermethylated DNA fragments fully bound with MBPs are differentiated from unmethylated DNA at 2.1- to 6.5-fold current blockades and 4.5- to 23.3-fold transport durations. Furthermore, these nanopore assays can detect the CpG dyad in DNA fragments and could someday profile the position of methylated CpG sites on DNA fragments.
DNA 甲基化是 DNA 的一种表观遗传修饰，其中甲基基团添加在胞嘧啶的 5-碳位置。异常的 DNA 甲基化与致癌有关，可以在各种生物流体中进行评估，并可能被用作癌症检测的标志物。需要分析敏感且特异性的针对低丰度和碎片化 DNA 的甲基化靶向检测分析方法，以实现最佳的临床诊断和预后。我们提出了一种基于纳米孔的直接甲基化检测分析方法，该方法绕过了亚硫酸氢盐转化和聚合酶链反应扩增。基于我们之前的工作，我们使用了甲基结合蛋白（MBP），其可以选择性标记甲基化的 DNA。基于纳米孔的检测分析方法通过一个 19nm 的纳米孔选择性地检测甲基化 DNA/MBP 复合物，纳米孔的离子电流阻断更深且持续时间更长，而未甲基化的 DNA 分子由于直径较小而无法检测到。使用亚 10nm 的纳米孔对 90、60 和 30bp 的 DNA 片段进行了超甲基化和未甲基化 DNA 的区分。与未甲基化 DNA 相比，完全与 MBP 结合的超甲基化 DNA 片段的电流阻断程度为 2.1 至 6.5 倍，传输持续时间为 4.5 至 23.3 倍。此外，这些纳米孔检测分析方法可以检测 DNA 片段中的 CpG 二联体，并且有朝一日可以对 DNA 片段上甲基化 CpG 位点的位置进行分析。
