International Journal of Systematic and Evolutionary Microbiology, Vol 50, 1463-1469, Copyright © 2000 by Society for General Microbiology

A rapid method for determining the G+C content of bacterial chromosomes by monitoring fluorescence intensity during DNA denaturation in a capillary tube

HX Xu, Y Kawamura, N Li, L Zhao, TM Li, ZY Li, S Shu and T Ezaki
Department of Microbiology, Gifu University, School of Medicine, 40 Tsukasa-machi, 500-8705, Gifu, Japan

A simple and rapid method to determine the G+C content of bacterial chromosomal DNA was developed. It involves determination of T(m) by a Light Cycler and calculation of the G+C content by an empirical formula relating T(m) to G+C content. Instead of a conventional thermal denaturation method, which monitors the increase of absorbance at 260 nm, thermal denaturation was monitored by the decrease of fluorescence intensity in the presence of SYBR Green 1. In this method, the apparent T(m) of DNA was influenced by the concentration of SYBR Green 1, DNA and salt. In addition, when the G+C content was calculated from a linear equation [(mol% G+Cx=mol% G+Cr+1.99(T(m)x-T(m)r), where x is the unknown organism and y is the reference organism], an error value was introduced among strains with extremely low or high G+C content. Based upon five standards (G+C contents in the range 33--66 mol%), a suitable equation was formulated for the capillary method: mol% G+Cx=mol% G+Cr+1.4652(T(m)x-T(m)r)+0.0063(T(m)x(2)-T(m)r(2)). To determine the T(m) of organisms within this range of G+C contents, Escherichia coli ATCC 11775(T) was used as a DNA standard and fixed concentrations of SYBR Green 1, sodium citrate and DNA were used. The data from 37 bacterial strains indicated that this equation behaved well. Because it is rapid and simple, it may prove useful for bacterial identification.

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