Optimization of single-molecule fluorescence burst detection of ds-DNA: Application to capillary electrophoresis separations of 100{endash}1000 basepair fragments
- Department of Chemistry, University of California, Berkeley, California (United States) 94720
Methods for optimizing the dye labeling, laser excitation, and data analysis for single-molecule fluorescence burst detection of ds-DNA have been developed and then validated through capillary electrophoresis (CE) separations of 100{endash}1000 basepair (bp) DNA. Confocal microscopy is used to observe fluorescence bursts from individual DNA fragments labeled with the intercalation dye TO6 as they pass through the {approximately}2-{mu}m-diameter focused laser beam. The dye concentration and laser power were optimized by studying fluorescence burst intensities from pBluescript DNA fragments. The optimal TO6 concentration was {le}100nM, and the optimal laser power was {le}1mW. Single-molecule counting was then used to detect CE separations of a 100{endash}1000 hp DNA sizing ladder in 3{percent} linear polyacrylamide. Discrete and baseline-resolved fluorescence bursts were observed in bands as small as 100 bp, and the average burst size within each band increased linearly with fragment size. By counting events using a single optimally chosen discriminator level, we achieve maximum signal-to-noise ratio (S/N) for each fragment size. If the discriminator level is ramped linearly with fragment size to achieve a constant detection efficiency, then the number of events properly reflects the relative fragment concentrations. {copyright} {ital 1997} {ital Society for Applied Spectroscopy}
- DOE Contract Number:
- FG03-91ER61125
- OSTI ID:
- 664575
- Journal Information:
- Applied Spectroscopy, Journal Name: Applied Spectroscopy Journal Issue: 10 Vol. 51; ISSN APSPA4; ISSN 0003-7028
- Country of Publication:
- United States
- Language:
- English
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