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

描述(申请人提供)：利用我们团队在单分子酶学方面的经验，我们提出了一种新的方法，使用合成测序的方法对单个核酸分子进行多重测序，该方法使用了荧光核苷酸底物。当聚合酶掺入非荧光的末端磷酸盐标记的核苷酸底物时，荧光聚磷酸分子被释放，并经过快速的酶消化，产生单一的荧光团，其颜色取决于所结合的核苷酸的同一性。为了便于单分子荧光检测，单个核酸分子被限制在密封的亚毫微升纳米反应器中，在其中进行连续的测序反应。使用传统的软光刻，我们制造了一系列纳米反应器，允许使用荧光显微镜和CCD相机同时、实时地监测数千个孤立的测序反应。我们的新方法提供了试剂成本低、读取长度长、样品制备容易以及每分钟几兆基数的高通量。我们还建议将大规模并行的单分子荧光测序仪与处理和传递来自单个细胞的遗传物质的微流控设备相结合。 与公共卫生相关：该项目将在单分子水平上开发新的DNA测序方法，提供一条以不到1000美元的成本进行人类基因组测序的新途径。这种对全基因组进行经济测序的能力将开启个性化医学的新纪元。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: This project will develop new methods of sequencing DNA at the single molecule level, providing a new path towards human genome sequencing for less than $1000. This ability to economically sequence full genomes will usher in a new era of personalized medicine.