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Quantifying Small DNA Concentrations using Fluorometry

¹ Department of Ophthalmology Emory University Atlanta GA

Commercial Relationships:  S.M. Cristol, None; K. Rengarajan, None; M. Mehta, None; J.M. Nickerson, None.
Grant Identification: FFB, RPB, NIH (P30 EY 6360, RO1 EY09378)

Abstract

Purpose: This study compared the sensitivity of four commercially available fluorophores for rapidly measuring very small concentrations (pg/ml) of DNA by fluorometry. The principle of the assay is to measure enhancement of fluorescence emitted by DNA bound fluorophores. This technique uses inexpensive disposable cuvettes in contrast to A₂₆₀ measurements, which only have a sensitivity on the order of 150 ng/ml.
Methods: Samples ranging from 100 pg/ml to 20 µg/ml of DNA were prepared by a serial dilution of calf thymus DNA and mixed with each dye. Fluorescence measurements were made at room temperature (23-24 ºC) using a photon counting spectrofluorometer. The dyes used to measure fluorescence enhancement were ethidium bromide, Hoechst 33258, PicoGreen, and SYBR Green I. For each dye, the concentration of DNA (over the 0-2 ng/ml range) was modelled as a function of fluorescence intensity using linear regression. Intra-assay repeatability was evaluated using SYBR Green I by making six replicates for each dilution and making a single measurement of each. Inter-assay repeatability was evaluated using SYBR Green I from four lot numbers (of three vendors) measured across two days. The stability of fluorescence was measured over a 24 h interval using SYBR Green I obtained from three vendors.
Results: Within an assay, the coefficient of variation was less than 10% for each fluorophore. No significant difference in fluorescence was observed among the three sources of SYBR Green I. There was no variation between the two lots from a single source. Regression models for ethidium bromide and Hoechst 33258 over the 0-2 ng/ml range were not statistically significant. The models for PicoGreen and SYBR Green I were statistically significant and had values of R₂0.96. From this data, we estimated that the resolution of PicoGreen was about 40% smaller than SYBR Green I.
Conclusion: SYBR Green I and PicoGreen are useful fluorophores for measuring small concentrations of DNA; ethidium bromide and Hoechst 33258 are not. Both SYBR Green I and PicoGreen offer a means of measuring small concentrations of DNA rapidly, inexpensively, and with greater sensitivity than comparable techniques. Because PicoGreen is substantially more expensive than SYBR Green I, we recommend SYBR Green I for assaying small concentrations of DNA.

Keywords: 476 molecular biology • 417 gene/expression

© 2002, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any part of this abstract, contact the ARVO Office at arvo{at}arvo.org.