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，对许多单个人类基因组进行经济测序成为可能。这些同样的测序方法现在正被应用于基因表达谱分析。然而，当下一代方法应用于信使核糖核酸测序时，依赖于聚合酶链式反应，这引入了偏差，扭曲了整个信使核糖核酸的分布，并且一般不能应用于单个细胞。在本课题组最近利用荧光dNTP底物合成单分子DNA测序的基础上，我们提出了一种新的方法，该方法使用一种储备转录酶来对单个mRNA分子进行多重测序，该转录酶在合成cDNA的过程中使用荧光核苷酸底物直接对mRNA进行测序。当储备转录酶将非荧光的末端磷酸盐标记的核苷酸底物掺入时，荧光聚磷酸分子被释放，并经过快速的酶消化，产生单个荧光团，其颜色报告被结合的dNTP的身份。为了实现单分子荧光检测，测序反应在密封的亚毫微升纳米反应器中连续进行，其中只有一个(或没有)受限的mRNA分子。使用软光刻，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。我们将集成一个微流控系统，该系统处理、分离并将单个裂解细胞的mRNAs传递到单分子测序仪。这项新技术提供的样品制备简单、成本低、信息量丰富，将对生物和医学研究产生广泛的影响。 公共卫生相关性：我们提出了一种新的方法，用于对具有单分子敏感性的单个细胞的mRNAs进行全系统分析。通过取消PCR，该方法避免了低拷贝数基因的扩增误差和偏差，并提供了较长的读取长度和容易的样品制备。这一能力将为生物医学研究中的诊断和发现提供强大的工具。

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.