Ribosome-based Single Molecule Method to Acquire Sequence Data from Genomes

描述(申请人提供)：提出了一种从单个核酸分子中获取序列数据的方法。该方法涉及基于参与蛋白质生物合成的分子的荧光共振能量转移分析(FRET)。使用几种配置的荧光相关光谱仪从单分子中获取荧光信号，包括在溶液中、表面上和纳米空间(零模波导)中的测量。该项目的具体目标是：(I)使用荧光染料和猝灭剂进行定点标记；(Ii)优化FRET分析；(Iii)构建合成模板并在该模板上展示系统的性能；(Iv)研究能够增强荧光的纳米结构；(V)研究系统中单分子的行为；以及(Vi)展示系统获得大量序列数据的能力。我们还将探索将纳米颗粒用作荧光标签和猝灭剂。一旦完全开发，这种方法将允许对包括全基因组在内的多种类型的核酸进行快速分析，并将根据美国国立卫生研究院1000美元的基因组计划，实现超低成本的基因组测序。该方法可用于体外核酸诊断和药物开发。

DESCRIPTION (provided by applicant): A method is presented for acquiring sequence data from single nucleic acid molecules. The approach involves a fluorescence resonance energy transfer assay (FRET) based on molecules involved in protein biosynthesis. The fluorescence signal is acquired from single molecules using a fluorescence correlation spectroscope in several configurations, including measurements in solution, on surfaces and in nanocavities (zero-mode waveguides). The project's specific aims are to: (i) perform site-directed labeling with a fluorescent dye and quencher; (ii) optimize the FRET assay; (iii) construct a synthetic template and demonstrate the performance of the system on this template; (iv) investigate nanostructures capable of enhancing fluorescence, (v) study the behavior of single molecules in the system; and (vi) demonstrate the capability of the system to acquire high volumes of sequence data. We will also explore the use of nanoparticles as fluorescent tags and quenchers. The method, once fully developed, will allow fast analyses of many types of nucleic acids, including whole genomes, and will lead to ultra-low cost genome sequencing, in accordance with the NIH's $1,000 genome program. The method can be used for nucleic acid diagnostics in vitro and in drug discovery.