Cavendish Laboratory, University of Cambridge , JJ Thomson Avenue, Cambridge, CB3 0HE, United Kingdom.
State Key Laboratory of Tribology, Tsinghua University , Beijing 100084, China.
Nano Lett. 2017 Sep 13;17(9):5199-5205. doi: 10.1021/acs.nanolett.7b01009. Epub 2017 Aug 22.
Nanopore sensors show great potential for rapid, single-molecule determination of DNA sequence information. Here, we develop an ionic current-based method for determining the positions of short sequence motifs in double-stranded DNA molecules with solid-state nanopores. Using the DNA-methyltransferase M.TaqI and a biotinylated S-adenosyl-l-methionine cofactor analogue we create covalently attached biotin labels at 5'-TCGA-3' sequence motifs. Monovalent streptavidin is then added to bind to the biotinylated sites giving rise to additional current blockade signals when the DNA passes through a conical quartz nanopore. We determine the relationship between translocation time and position along the DNA contour and find a minimum resolvable distance between two labeled sites of ∼200 bp. We then characterize a variety of DNA molecules by determining the positions of bound streptavidin and show that two short genomes can be simultaneously detected in a mixture. Our method provides a simple, generic single-molecule detection platform enabling DNA characterization in an electrical format suited for portable devices for potential diagnostic applications.
纳米孔传感器在快速、单分子测定 DNA 序列信息方面具有巨大的潜力。在这里，我们开发了一种基于离子电流的方法，用于确定双链 DNA 分子中短序列基序的位置的固态纳米孔。使用 DNA-甲基转移酶 M.TaqI 和生物素化 S-腺苷甲硫氨酸辅因子类似物，我们在 5'-TCGA-3' 序列基序处创建共价连接的生物素标签。然后添加单价链霉亲和素与生物素化位点结合，当 DNA 通过圆锥形石英纳米孔时会产生额外的电流阻断信号。我们确定了跨导时间与 DNA 轮廓上位置之间的关系，并发现两个标记位点之间的最小可分辨距离约为 200bp。然后，我们通过确定结合的链霉亲和素的位置来表征各种 DNA 分子，并表明可以在混合物中同时检测两个短基因组。我们的方法提供了一种简单、通用的单分子检测平台，以电格式对 DNA 进行特征分析，适合用于潜在诊断应用的便携式设备。
