Single Cell Single Molecule Digital mRNA Profiling with No PCR Amplification

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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.