DOE Human Genome Program Contractor-Grantee
30. DNA Sequencing by Single Molecule Detection
Peter M. Goodwin, Hong Cai, James H. Werner, James H. Jett, and Richard A. Keller
Bioscience Division, Los Alamos National Laboratory, M888, Los Alamos, NM 87545 USA
We are developing a method, based upon single fluorescent molecule detection, to sequence individual DNA strands. The method consists of: (1) polymerase incorporation of fluorophore-labeled nucleotides into a strand of DNA complementary to the target sequence; (2) anchoring a single fragment of fluorescently-labeled DNA, in flow, upstream of the detection volume of an ultrasensitive fluorescence flow cytometer; (3) exonuclease digestion of the free end of the anchored DNA strand to sequentially release single,fluorophore-labeled nucleotides into the flow stream; and (4) detection and identification of the individual, released fluorophore-labeled nucleotides in the order of exonuclease cleavage. We have made considerable progress towards a demonstration of single molecule DNA sequencing. Up to three of the nucleotide types, labeled with fluorophores, have been incorporated into strands of DNA 2-7 kilobases in length. Multiple strands of fluorescently-labeled DNA have been attached to microspheres and individual microspheres have been anchored in flow upstream of the detection volume. Exonuclease cleavage of fluorescently-labeled DNA on individual microspheres anchored in flow has been observed. We have detected and identified single, tetramethylrhodamine-labeled dUMPs and Rhodamine-6G-labeled dCMPs enzymatically released from DNA strands containing both types of labeled nucleotides. The two fluorescent species were identified by correlated measurements of single molecule fluorescence burst intensity and intra-burst fluorescence lifetime. We present preliminary data demonstrating the detection of single, labeled nucleotides released by the processive exonuclease digestion of a single DNA strand.
This work was supported by the US Department of Energy, Office of Biological and Environmental Research.
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