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Progress towards single-molecule DNA sequencing: a one color demonstration
Authors:Werner James H  Cai Hong  Jett James H  Reha-Krantz Linda  Keller Richard A  Goodwin Peter M
Affiliation:Bioscience Division, Los Alamos National Laboratory, Mail Stop J586, Los Alamos, NM 87545, USA. jwerner@lanl.gov
Abstract:Single molecules of fluorescently labeled nucleotides were detected during the cleavage of individual DNA fragments by a processive exonuclease. In these experiments, multiple (10-100) strands of DNA with tetramethyl rhodamine labeled dUMP (TMR-dUMP) incorporated into the sequence were anchored in flow upstream of the detection region of an ultra sensitive flow cytometer. A dilute solution of Exonuclease I passed over the microspheres. When an exonuclease attached to a strand, processive digestion of that strand began. The liberated, labeled bases flowed through the detection region and were detected at high efficiency at the single-molecule level by laser-induced fluorescence. The digestion of a single strand of DNA by a single exonuclease was discernable in these experiments. This result demonstrates the feasibility of single-molecule DNA sequencing. In addition, these experiments point to a new and practical means of arriving at a consensus sequence by individually reading out identical sequences on multiple fragments.
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