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Ionic Current-Based Mapping of Short Sequence Motifs in Single DNA Molecules Using Solid-State Nanopores

机译:使用固态纳米孔的单个DNA分子中短序列基序的离子电流映射

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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.Taql 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 similar to-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.
机译:纳米孔传感器显示出快速,单分子测定DNA序列信息的巨大潜力。这里,我们开发了一种基于离子电流的方法,用于测定具有固态纳米孔的双链DNA分子中的短序列基序的位置。使用DNA-甲基转移酶M.Taql和生物素化的S-腺苷-1-蛋氨酸类咖啡酰基类似物,我们在5'-TCGA-3'序列基序中产生共价连接的生物素标记。然后将单价链霉抗生物素蛋白加入结合生物素化的位点,当DNA通过锥形石渣纳米孔时,产生额外的电流阻断信号。我们确定沿DNA轮廓的易位时间和位置之间的关系,并找到与200bp类似的两个标记站点之间的最小可解析距离。然后,通过确定结合的链霉抗生物素蛋白的位置并表明可以在混合物中同时检测到两个短基因组的位置来表征各种DNA分子。我们的方法提供了一种简单的通用单分子检测平台,其能够以适用于潜在诊断应用的便携式设备的电气格式实现DNA表征。

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