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Proceedings Paper

Single molecule fluorescence burst detection of DNA separated by capillary electrophoresis
Author(s): Brian B. Haab; Richard A. Mathies
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Paper Abstract

A method has been developed for detecting DNA separated by capillary gel electrophoresis using single molecule photon burst counting. A confocal fluorescence microscope was used to observe the fluorescence bursts from single molecules of DNA multiply labeled with a thiazole orange derivative as they passed through the approximately 2 micrometer diameter focused laser beam. Amplified photoelectron pulses from the photomultiplier are grouped into bins of from 360 - 450 microseconds in duration, and the resulting histogram stored in a computer for analysis. Solutions of M13 DNA were first flowed through the capillary at various concentrations, and the resulting data were used to optimize the parameters for digital filtering using a low-pass Fourier filter, selecting a discriminator level for peak detection, and applying a peak-calling algorithm. The optimized single molecule counting method was then used to detect a separation of pBR 322 DNA from pRL 277 DNA. Clusters of discrete fluorescence bursts were observed at the expected appearance time of each DNA band. These separations were easily detected when only 50 to 100 molecules of DNA per band traveled through the detection region. This new detection technology should lead to the routine analysis of DNA in capillary columns with an on-column sensitivity of approximately 100 DNA molecules per band or better.

Paper Details

Date Published: 25 March 1996
PDF: 8 pages
Proc. SPIE 2705, Fluorescence Detection IV, (25 March 1996); doi: 10.1117/12.236189
Show Author Affiliations
Brian B. Haab, Univ. of California/Berkeley (United States)
Richard A. Mathies, Univ. of California/Berkeley (United States)

Published in SPIE Proceedings Vol. 2705:
Fluorescence Detection IV
E. Roland Menzel; Abraham Katzir, Editor(s)

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