Real-time single-molecule nucleic acid sequencing with fluorogenic nucleotides

荧光核苷酸实时单分子核酸测序 项目摘要/摘要 利用我们小组在单分子酶学方面的经验，我们提出了一种新的方法 用于使用合成测序法对单个核酸分子进行多重测序 使用含氟核苷酸底物的方法。在加入非荧光物质后， 聚合酶的末端磷酸盐标记的核苷酸底物，一种荧光聚磷酸盐 分子被释放，并被快速的酶消化，产生一个单一的荧光团，颜色 这取决于被掺入的核苷酸的同一性。为了促进单分子 荧光检测，单个核酸分子被限制在密封的亚飞升中 纳米反应器，在其中连续进行测序反应。使用传统的软件 光刻，我们制造了一系列纳米反应器，允许同时、实时地监测 数以千计的分离测序反应与荧光显微镜和ccd相机。我们的 新的方法具有试剂成本低、读取长度长、样品制备容易、成本高等优点 吞吐量为每分钟几兆基数。我们还建议整合一个大规模并行的 带有微流控装置的单分子荧光测序仪，用于处理和传递基因 来自单个细胞的材料。

Real-time single-molecule nucleic acid sequencing with fluorogenic nucleotides Project Summary/Abstract Capitalizing on our group's experience on single molecule enzymology, we propose a novel method for multiplex sequencing of individual nucleic acid molecules using a sequencing-by-synthesis approach that employs fluorogenic nucleotide substrates. Upon incorporation of a non-fluorescent, terminal phosphate-labeled nucleotide substrate by a polymerase, a fluorogenic polyphosphate molecule is released, and subject to fast enzymatic digestion, yielding a single fluorophore, the color of which is dependent on the identity of the incorporated nucleotide. To facilitate single molecule fluorescence detection, an individual nucleic acid molecule is confined in a sealed sub-femtoliter nanoreactor, in which the sequencing reaction takes place continuously. Using conventional soft lithography, we fabricate an array of nanoreactors that allow simultaneous, real-time monitoring of thousands of isolated sequencing reactions with a fluorescence microscope and CCD camera. Our new approach offers low reagent cost, long read lengths, easy sample preparation, and high throughput at several megabases per minute. We also propose the integration of a massively parallel single molecule fluorogenic sequencer with microfluidic devices that process and deliver genetic material from a single cell.