|Genome Sequencing Technologies and Resources
DOE Human Genome Program Contractor-Grantee Workshop
48. DNA Sequencing by Single Molecule Detection
James H. Jett, Peter M. Goodwin,
James H. Werner, Hong Cai, and Richard A. Keller
We are developing a DNA sequencing technique that is based upon single fluorescent molecule detection. The goal is to sequence long strands of DNA approaching 40 kb in length at rates of 50 bases per second. The approach being pursued is to: 1) fluorescently label a strand of DNA by enzymatic incorporation of prelabeled nucleotides; 2) attach a single labeled strand of DNA to a microsphere; 3) suspend the microsphere in the flow stream of a flow cytometer capable of single molecule detection and identification; 4) add an exonuclease with activating cofactors that will cleave sequentially the labeled nucleotides; and 5) identification of the cleaved nucleotides by analysis of laser-induced fluorescence from the label attached to the nucleotides. We have made considerable progress towards demonstrating this approach to DNA sequencing. Briefly, the current status of each of the steps above is as follows. Up to three base types in strands of DNA 2-7 kbp long have been labeled base identifying fluorophors. Multiple strands of labeled DNA have been attached to microspheres and individual microspheres suspended by a laser optical trap in the flow stream of the detection apparatus. Enzymatic activity of several exonucleases on DNA attached to microspheres under flowing conditions has been observed. Fluorescent molecule identification at the single molecule level has been demonstrated by correlated measurements of fluorescent burst intensity and fluorescence lifetime with a single excitation wavelength and a single detection channel. Details of the progress made in each of the steps will be discussed.
This work is supported by the US Department of Energy, Office of Biological and Environmental Research.
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