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Aquatic Microbial Ecology

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AME 36:99-105 (2004)  -  doi:10.3354/ame036099

Use of the SYBR Green I fluorescent dye and a centrifugal filter device for rapid determination of dissolved DNA concentration in fresh water

Kazuaki Matsui1,*, Nobuyoshi Ishii2, Mie Honjo3, Zen¹ichiro Kawabata3

1Laboratory of Environmental Biotechnology, Faculty of Engineering, Tohoku Gakuin University, 1-13-1 Chuo, Tagajo, Miyagi 985-8537, Japan
2Environmental and Toxicological Science Research Group, National Institute of Radiological Sciences, 9-1 Anagawa-4-chome, Inage-ku, Chiba-shi 263-8555, Japan
3Center for Ecological Research, Kyoto University, Kamitanakami Hirano-cho 509-3, Otsu 520-2113, Japan

ABSTRACT: A rapid fluorometric assay using the fluorescent dye SYBR Green I was established to determine the concentration of dissolved nucleic acids in fresh water. The sensitivity of SYBR Green I to double stranded DNA (dsDNA) ( λHind III digest) was as low as 50 pg, 200 times more sensitive than Hoechst 33258. SYBR Green I bound to dsDNA emitted 10 times stronger fluorescence than when bound to single stranded DNA or RNA, indicating its selectivity for dsDNA measurement. The dissolved DNA (dDNA) concentration in fresh water determined using SYBR Green I was almost the same as that obtained using Hoechst 33258. This suggests that the dDNA measured by SYBR Green I is comparable to that determined by Hoechst 33258 in previous studies. To reduce preparation time, the dDNA in lake water filtrate was precipitated by ethanol and purified using a centrifugal filter device. The overall preparation process takes only a few hours and requires only 10 ml of water. The process described here may, therefore, facilitate Œsame day¹ measurement of dDNA dynamics in freshwater environments.

KEY WORDS: Dissolved DNA · SYBR Green I · Ethanol precipitation · Centrifugal filter device · Extracellular DNA · Fresh water

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