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SM Cristol, K Rengarajan, M Mehta, JM Nickerson; Quantifying Small DNA Concentrations using Fluorometry . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2470.
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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 R2≷0.96. 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.
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