Nanopore-Based Assay for Detection of Methylation in Double-Stranded DNA Fragments
Nanopore-Based Assay for Detection of Methylation in Double-Stranded DNA Fragments
复制标题
DOI：
10.1021/nn5045596
复制
发表时间：
2015-01-01
期刊：
影响因子：
17.1
通讯作者：
Bashir, Rashid
中科院分区：
文献类型：
--
作者：
Shim, Jiwook;Kim, Younghoon;Bashir, Rashid
DNA甲基化是DNA的表观遗传修饰，其中甲基基团被添加在胞嘧啶的5-碳位置。异常的DNA甲基化与癌症发生有关，可以在各种生物液体中进行评估，并可能用作检测癌症的标志物。针对低丰度和片段化DNA的甲基化的分析敏感性和特异性测定是最佳临床诊断和预后所必需的。我们提出了一种基于纳米孔的直接甲基化检测方法，可以避免亚硫酸氢盐转化和聚合酶链反应扩增。在我们先前工作的基础上，我们使用了甲基结合蛋白（MBP），它选择性地标记甲基化的DNA。基于纳米孔的测定通过具有显著更深和延长的纳米孔离子电流阻断的19 nm纳米孔选择性地检测甲基化的DNA/MBP复合物，而未甲基化的DNA分子由于其较小的直径而不可检测。使用亚10 nm纳米孔证明了90、60和30 bp DNA片段上的高甲基化和未甲基化DNA的区分。与MBP完全结合的高甲基化DNA片段在2.1至6.5倍电流阻断和4.5至23.3倍转运持续时间下与未甲基化DNA区分开。此外，这些纳米孔测定可以检测DNA片段中的CpG二联体，并且有朝一日可以分析DNA片段上甲基化CpG位点的位置。
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.