Abstract
Determination of whole-genome DNA–DNA similarity is today a standard technique for species delineation in microbial taxonomy. However, these studies demand hard-to-perform and time-consuming experiments. Herein, we present an easy and rapid fluorimetric method to estimate DNA–DNA relatedness between microbial strains from differences of the thermal denaturation temperatures of hybrid and homologous genomic DNA. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence. A quantitative, real-time PCR thermocycler was used to perform the experiment and obtain fluorescence determinations at increasing temperatures. The proposed method was validated by comparing species of the hyperthermophilic genera Pyrococcus and Thermococcus. The method proves to be an easy, rapid, and inexpensive alternative to estimate DNA–DNA relatedness between closely related species.
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Acknowledgements
The authors thank Dr. Luz Candenas de Lujan and Dr. Francisco Pinto for the use of an iQ iCycler and their valuable assistance and comments, and Dr. R. Rosello-Mora for his useful comments on preliminary stages of this study. The authors acknowledge funding from the Spanish Ministry of Education and Sciences, projects REN2002-00041/GLO and BTE2002-04492-C02-01.
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Gonzalez, J.M., Saiz-Jimenez, C. A simple fluorimetric method for the estimation of DNA–DNA relatedness between closely related microorganisms by thermal denaturation temperatures. Extremophiles 9, 75–79 (2005). https://doi.org/10.1007/s00792-004-0417-0
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DOI: https://doi.org/10.1007/s00792-004-0417-0