Current DNA sequencing methods rely on amplification of small quantities of sample, followed by a "shot-gun" approach in which 500-base-long sequences from random locations within the genome are established. Tremendous computational effort must then be used to piece together millions of these small sequences into the final genome sequence, which in humans is as large as 3 billion bases. We are developing an alternate strategy in which the action of a single DNA polymerase enzyme is observed as it incorporates individual fluorescently tagged nucleotide analogs to synthesize double stranded DNA. This approach uses the inherent speed, reliability, and processivity of the polymerase/DNA complex to achieve rapid sequencing of hundreds of thousands of bases at a time without the need for expensive amplification and preprocessing of the sample or for complex post-processing of data. Zero-mode waveguides enable the observation of which fluorescent nucleotide analog is being added to the double stranded product in the active site of the enzyme while excluding signal from freely diffusing fluorescent species. The effectiveness of zero-mode waveguides is demonstrated by real-time observation of single molecules of DNA polymerase immobilized inside zero-mode waveguides, using micromolar concentrations of fluorescently labeled nucleotide analogs.
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