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Proceedings Paper

Current status of DNA sequencing by single molecule detection
Author(s): James H. Werner; Hong Cai; Peter M. Goodwin; Richard A. Keller
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Paper Abstract

Our current experiments further the development of a laser- based technique capable of sequencing an individual strand of DNA. We report the detection and identification of fluorescently labeled nucleotides enzymatically cleaved from DNA strands suspended in flow. We used fluorescence lifetime, fluorescence intensity, or a correlated measure of the intensity and lifetime to identify each individual tagged base traversing the detection region with high accuracy. DNA strands containing a single tetramethylrhodamine labeled uracil and/or a single Rhodamine 6G labeled cytosine were attached to polystyrene microspheres. An optical trap was used to capture and hold a single DNA-laden microsphere nominally 20 microns upstream of the detection region of an ultra- sensitive flow cytometer. The addition of an exonuclease cleaved bases from the 3' end of the fluorescently labeled strand. The cleaved, labeled nucleotides were carried by the flow downstream and detected and identified one-at-a-time with high efficiency by laser-induced fluorescence.

Paper Details

Date Published: 3 May 1999
PDF: 12 pages
Proc. SPIE 3602, Advances in Fluorescence Sensing Technology IV, (3 May 1999); doi: 10.1117/12.347535
Show Author Affiliations
James H. Werner, Los Alamos National Lab. (United States)
Hong Cai, Los Alamos National Lab. (United States)
Peter M. Goodwin, Los Alamos National Lab. (United States)
Richard A. Keller, Los Alamos National Lab. (United States)

Published in SPIE Proceedings Vol. 3602:
Advances in Fluorescence Sensing Technology IV
Joseph R. Lakowicz; Steven A. Soper; Richard B. Thompson, Editor(s)

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