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被应用于基因表达谱分析。然而，下一代方法在应用于mRNA测序时依赖于PCR，这引入了偏差，扭曲了整体mRNA分布，并且通常不能应用于单个细胞。利用我们小组最近的单分子DNA测序与荧光dNTP底物的合成工作，我们提出了一种新的方法，使用储备转录酶，采用荧光核苷酸底物直接在cDNA的合成过程中测序mRNA的单个mRNA分子的多重测序。在通过储备转录酶掺入非荧光的末端磷酸标记的核苷酸底物后，释放荧光多磷酸分子，并进行快速酶消化，产生单个荧光团，其颜色报告掺入的dNTP的身份。为了允许单分子荧光检测，测序反应在密封的亚飞升纳米反应器中连续发生，其中仅存在一个（或没有）受限的mRNA分子。使用软光刻，我们制造了一个阵列的纳米反应器，允许同时，实时监测成千上万的孤立的测序反应与荧光显微镜和CCD相机。我们将整合一个微流控系统，处理，分离和提供mRNA从一个单一的裂解细胞到一个单分子测序仪。这种新技术所提供的简单样品制备、低成本和丰富的信息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，用于单分子灵敏度的单细胞mRNA的全系统分析。通过消除PCR，该方法避免了与低拷贝数基因的PCR相关的扩增误差和偏倚，并且提供了长读取长度和容易的样品制备。这种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.