A fluorimetric method for the estimation of G+C mol% content in microorganisms by thermal denaturation temperature.
G+C mol% content in microorganisms is one of the recommended characteristics for the standard description of bacterial species. In this study we present a novel fluorimetric method to estimate the G+C mol% content in microorganisms. Double-stranded DNA was specifically stained with SYBR Green I, and its thermal denaturalization was followed by measuring a decrease in fluorescence using a real-time PCR thermocycler. Unlike most previous determinations of G+C mol%, in this study only DNA from microorganisms with an available completely sequenced genome were used to prepare the calibration curves. Calibration curves showed a linear relationship between G+C mol% content and melting temperature and they were performed both in the absence and presence of 30% formamide. This protocol proves to be a rapid and inexpensive method to estimate DNA base ratios of novel microorganisms.
Gonzalez
J M
JM
Instituto de Recursos Naturales y Agrobiologia, CSIC, Apartado 1052, 41080 Sevilla, Spain. jmgrau@irnase.csic.es
Saiz-Jimenez
C
C
D001482:Base Composition; D002138:Calibration; D004269:DNA, Bacterial / Q000032:analysis*; D005470:Fluorometry; D009691:Nucleic Acid Denaturation; D013696:Temperature
D004269:DNA, Bacterial