Single Cell Single Molecule Digital mRNA Profiling with No PCR Amplification

无需 PCR 扩增的单细胞单分子数字 mRNA 分析

• 批准号: 7936360
• 负责人: XIAOLIANG SUNNEY XIE
• 金额: --
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-22 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7936360
• 关键词: Bacteria; Base Sequence; Biological; Biological Assay; Biological Models; Biomedical Research; Cells; Color; Complementary DNA; Cytolysis; DNA Sequence; DNA-Directed DNA Polymerase; Data; Detection; Diagnosis; Digestion; Exhibits; Fluorescence; Gene Dosage; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Human Genome; Individual; Label; Length; Leukocytes; Malignant Neoplasms; Medical Research; Messenger RNA; Methods; Microfluidics; Monitor; Mycobacterium smegmatis; Nucleotides; Organ; Pathology; Phenotype; Polyphosphates; Preparation; Process; RNA Sequences; RNA-Directed DNA Polymerase; Reaction; Reading; Reporting; Running; Sampling; Surveys; System; Techniques; Time; Tissue Sample; Tissues; Transcriptase; Tuberculosis; Work; Yeasts; charge coupled device camera; cost; digital; fluorescence microscope; fluorophore; inorganic phosphate; laser capture microdissection; lithography; next generation; novel; novel strategies; public health relevance; seal; single molecule; tool

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.

描述（由申请人提供）：通过下一代测序（NGS）方法，可以对许多个体人类基因组进行经济的测序。这些相同的测序方法现在正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，这会引入偏差，扭曲整体 mRNA 分布，并且通常不能应用于单个细胞。利用我们小组最近通过荧光 dNTP 底物合成进行单分子 DNA 测序的工作，我们提出了一种使用储备转录酶对单个 mRNA 分子进行多重测序的新方法，该转录酶在 cDNA 合成过程中采用荧光核苷酸底物直接对 mRNA 进行测序。当储备转录酶掺入非荧光末端磷酸盐标记的核苷酸底物时，会释放荧光多磷酸分子，并进行快速酶消化，产生单个荧光团，其颜色报告了掺入的 dNTP 的身份。为了实现单分子荧光检测，测序反应在密封的亚飞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。我们使用软光刻技术制造了一系列纳米反应器，可以使用荧光显微镜和 CCD 相机同时实时监控数千个独立的测序反应。我们将集成一个微流体系统，该系统可以处理、分离单个裂解细胞中的 mRNA，并将其传递到单分子测序仪。这种新技术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：我们提出了一种新方法，用于对单细胞 mRNA 进行全系统分析，具有单分子敏感性。通过消除 PCR，该方法避免了与低拷贝数基因 PCR 相关的扩增误差和偏差，并提供长读长和轻松的样品制备。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。