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，我们提出了一种利用荧光核苷酸底物的合成测序方法对单个核酸分子进行多重测序的新方法。当聚合酶与非荧光的末端磷酸盐标记的核苷酸底物结合时，一个荧光多磷酸盐分子被释放出来，并受到快速酶切，产生单个荧光团，其颜色取决于所结合核苷酸的身份。为了方便单分子荧光检测，单个核酸分子被限制在一个密封的亚飞升纳米反应器中，测序反应在其中连续进行。利用传统的软光刻技术，我们制造了一组纳米反应器，可以用荧光显微镜和CCD相机同时实时监测数千个分离的测序反应。我们的新方法具有试剂成本低，读取长度长，样品制备简单，以及每分钟几兆碱基的高通量。我们还建议将大规模平行的单分子荧光测序仪与处理和传递单细胞遗传物质的微流控装置集成在一起。

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.