Center for Genome Technology & Biomolecular Engineering, Department of Chemical Engineering, Columbia University, New York, NY 10027, USA.
Sci Rep. 2012;2:684. doi: 10.1038/srep00684. Epub 2012 Sep 21.
We describe a novel single molecule nanopore-based sequencing by synthesis (Nano-SBS) strategy that can accurately distinguish four bases by detecting 4 different sized tags released from 5'-phosphate-modified nucleotides. The basic principle is as follows. As each nucleotide is incorporated into the growing DNA strand during the polymerase reaction, its tag is released and enters a nanopore in release order. This produces a unique ionic current blockade signature due to the tag's distinct chemical structure, thereby determining DNA sequence electronically at single molecule level with single base resolution. As proof of principle, we attached four different length PEG-coumarin tags to the terminal phosphate of 2'-deoxyguanosine-5'-tetraphosphate. We demonstrate efficient, accurate incorporation of the nucleotide analogs during the polymerase reaction, and excellent discrimination among the four tags based on nanopore ionic currents. This approach coupled with polymerase attached to the nanopores in an array format should yield a single-molecule electronic Nano-SBS platform.
我们描述了一种新颖的基于单分子纳米孔的合成测序（Nano-SBS）策略，该策略通过检测 5'-磷酸修饰核苷酸释放的 4 种不同大小的标签，能够准确地区分 4 种碱基。其基本原理如下。在聚合酶反应过程中，当每个核苷酸被掺入到正在生长的 DNA 链中时，其标签被释放并按释放顺序进入纳米孔。由于标签的独特化学结构，这会产生独特的离子电流阻断特征，从而以单分子水平和单碱基分辨率实现 DNA 序列的电子测定。作为原理验证，我们将四个不同长度的 PEG-香豆素标签连接到 2'-脱氧鸟苷-5'-四磷酸的末端磷酸上。我们证明了核苷酸类似物在聚合酶反应中的有效、准确掺入，以及基于纳米孔离子电流的四个标签的出色区分。这种方法与聚合酶在纳米孔阵列中的结合，应该可以得到一个单分子电子 Nano-SBS 平台。
