Flow-based continuous DNA sequencing via single molecule detection of enzymatically cleaved fluorescent nucleotides

Abstract

The development of our rapid, continuous DNA sequencing technology, based on single molecule detection of fluorescently-tagged nucleotides, has proceeded along separate research fronts, each with specific goals: the faithful replication of long sequences of template DNA using one or more fluorescent nucleotide analogues, the incorporation and stable mounting of a single DNA strand into a flow chamber, the enzymatic cleavage of labeled DNA by exonucleases, and the detection of single fluorescent nucleotides in a flow stream by the method of time-gated photon counting. Each individual goal of the sequencing technology has now been realized, and we have begun integrating these efforts in order to demonstrate the feasibility of flow-based sequencing. We are currently detecting photon bursts from TRITC labeled nucleotides which have been cleaved from DNA suspended in our flow cell. The sample size is estimated to be tens of DNA strands.