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

描述（由申请人提供）：利用我们小组在单分子酶学方面的经验，我们提出了一种使用合成测序方法对单个核酸分子进行多重测序的新方法，该方法采用荧光核苷酸底物。在通过聚合酶掺入非荧光的末端磷酸盐标记的核苷酸底物后，释放荧光多磷酸盐分子，并进行快速酶消化，产生单个荧光团，其颜色取决于掺入的核苷酸的身份。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纳米反应器中，其中测序反应连续发生。使用传统的软光刻，我们制造了一个阵列的纳米反应器，允许同时，实时监测数以千计的孤立的测序反应与荧光显微镜和CCD相机。我们的新方法具有试剂成本低、读取长度长、样品制备简单以及每分钟数百万次的高通量等优点。我们还提出了一个大规模并行的单分子荧光测序仪与微流控设备，处理和提供遗传物质从一个单一的细胞的整合。 公共卫生相关性：该项目将开发在单分子水平上测序DNA的新方法，为人类基因组测序提供一条新的途径，成本不到1000美元。这种经济地对全基因组进行测序的能力将开创一个个性化医疗的新时代。

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.