Single Cell Single Molecule Digital mRNA Profiling with No PCR Amplification

描述（由申请人提供）：通过下一代测序（NGS）方法，许多个体人类基因组的经济测序已经成为可能。这些相同的测序方法现在正被应用于基因表达谱。然而，下一代方法，当应用于mRNA测序时，依赖于PCR，这引入了偏差，扭曲了mRNA的整体分布，并且通常不能应用于单个细胞。利用我们小组最近通过合成荧光dNTP底物进行单分子DNA测序的工作，我们提出了一种新的方法，使用一种储备转录酶对单个mRNA分子进行多重测序，该转录酶在cDNA合成过程中使用荧光核苷酸底物直接对mRNA进行测序。当一个非荧光的，末端磷酸盐标记的核苷酸底物被储备转录酶掺入后，一个荧光多磷酸盐分子被释放出来，并受到快速的酶消化，产生一个单一的荧光团，其颜色报告了掺入的dNTP的身份。为了允许单分子荧光检测，测序反应在一个密封的亚飞升纳米反应器中连续进行，其中只有一个（或没有）受限制的mRNA分子。利用软光刻技术，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。我们将整合一种微流体系统，该系统处理、分离并将mrna从单个裂解细胞传递到单分子测序仪。这项新技术具有样品制备简单、成本低、信息丰富等特点，将对生物和医学研究产生广泛影响。

DESCRIPTION (provided by applicant): The economical sequencing of a number of individual human genomes has been made possible by next-generation sequencing (NGS) methods. These same sequencing methods are now being applied to gene expression profiling. However, next-generation methods, when applied to mRNA sequencing, rely on PCR, which introduces bias, distorts the overall mRNA distribution, and cannot generally be applied to individual cells. Capitalizing on our group's recent work on single-molecule DNA sequencing by synthesis with fluorogenic dNTP substrates, we propose a novel method for multiplex sequencing of individual mRNA molecules using a reserve transcriptase that employs fluorogenic nucleotide substrates to sequence mRNA directly during the synthesis of cDNA. Upon incorporation of a non-fluorescent, terminal phosphate labeled nucleotide substrate by the reserve transcriptase, a fluorogenic polyphosphate molecule is released, and subjected to fast enzymatic digestion, yielding a single fluorophore, the color of which reports the identity of the incorporated dNTP. To allow single-molecule fluorescence detection, the sequencing reaction takes place continuously in a sealed sub-femtoliter nanoreactor, in which there is only one (or no) confined mRNA molecule. Using soft lithography, we fabricate an array of nanoreactors that allow simultaneous, real-time monitoring of many thousands of isolated sequencing reactions with a fluorescence microscope and CCD camera. We will integrate a microfluidic system that processes, isolates and delivers mRNAs from a single lysed cell to a single-molecule sequencer. The easy sample preparation, low cost, and rich information afforded by this new technique will have a broad impact on biological and medical research. PUBLIC HEALTH RELEVANCE: We propose a new approach for system-wide analyses of mRNAs of a single cell with single-molecule sensitivity. By eliminating PCR, this method circumvents the amplification error and bias associated with PCR for low copy number genes, and offers long read lengths and easy sample preparation. This capability will provide a powerful tool for diagnosis and discovery in biomedical research.