Current status of DNA sequencing by single molecule detection

James H. Werner; Hong Cai; Peter M. Goodwin; Richard A. Keller

doi:10.1117/12.347535

3 May 1999 Current status of DNA sequencing by single molecule detection

James H. Werner, Hong Cai, Peter M. Goodwin, Richard A. Keller

Proceedings Volume 3602, Advances in Fluorescence Sensing Technology IV; (1999) https://doi.org/10.1117/12.347535
Event: BiOS '99 International Biomedical Optics Symposium, 1999, San Jose, CA, United States

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.

Citation Download Citation

James H. Werner, Hong Cai, Peter M. Goodwin, and Richard A. Keller "Current status of DNA sequencing by single molecule detection", Proc. SPIE 3602, Advances in Fluorescence Sensing Technology IV, (3 May 1999); https://doi.org/10.1117/12.347535

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