Sizing of DNA fragments by flow cytometry

Mitchell E. Johnson; Peter M. Goodwin; W. Patrick Ambrose; John C. Martin; Babetta L. Marrone; James H. Jett; Richard A. Keller

doi:10.1117/12.146730

24 June 1993 Sizing of DNA fragments by flow cytometry

Mitchell E. Johnson, Peter M. Goodwin, W. Patrick Ambrose, John C. Martin, Babetta L. Marrone, James H. Jett, Richard A. Keller

Author Affiliations +

Proceedings Volume 1895, Ultrasensitive Laboratory Diagnostics; (1993) https://doi.org/10.1117/12.146730
Event: OE/LASE'93: Optics, Electro-Optics, and Laser Applications in Scienceand Engineering, 1993, Los Angeles, CA, United States

Abstract

Individual, stained DNA fragments were sized using a modified flow cytometer with high sensitivity fluorescence detection. The fluorescent intercalating dye ethidium homodimer was used to stain stoichiometrically lambda phage DNA and a Kpn I digest of lambda DNA. Stained, individual fragments of DNA were passed through a low average power, focused, mode-locked laser beam, and the fluorescence from each fragment was collected and quantified. Time-gated detection was used to discriminate against Raman scattering from the water solvent. The fluorescence burst from each fragment was related directly to its length, thus providing a means to size small quantities of kilobase lengths of DNA quickly. Improvements of several orders of magnitude in analysis time and sample size over current gel electrophoresis techniques were realized. Fragments of 17.1, 29.9, and 48.5 thousand base pairs were well resolved, and were sized in 164 seconds. Less than one pg of DNA was required for analysis.

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Mitchell E. Johnson, Peter M. Goodwin, W. Patrick Ambrose, John C. Martin, Babetta L. Marrone, James H. Jett, and Richard A. Keller "Sizing of DNA fragments by flow cytometry", Proc. SPIE 1895, Ultrasensitive Laboratory Diagnostics, (24 June 1993); https://doi.org/10.1117/12.146730

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