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

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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.