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Analysis of the molar G+C of genomic DNA

The mol%G­C content of DNA should be part of the description of the type strain of the type species of a new genus (Stackebrandt et al., 2002).

1-2 g wet biomass (centrifugal pellet) are needed. The biomass can be sent either in frozen state on dry ice or suspended in isopropanol/water (1:1, v/v) at ambient temperature. Lyophilized cells in a corresponding amount can also be sent. However, the use of lyophilized cells may result in a lower DNA yield.

Cells are disrupted by French pressing and DNA is purified on hydroxyapatit according to Cashion et al. (1977). The DNA is hydrolyzed with P1 nuclease and the nucleotides are dephosphorylized with bovine alkaline phosphatase (Mesbah et al., 1989). The resulting deoxyribonucleosides are analyzed by HPLC (Tamaoka & Komagata, 1984). Lambda-DNA and three DNAs with published genome sequences representing a G+C range of 43-72 mol% are used as standards. G+C values are calculated from the ratio of deoxyguanosine and thymidine according to the method of Mesbah et al. (1989).

References


  • Cashion, P., Hodler-Franklin, M. A., McCully, J. & Franklin, M. (1977). A rapid method for base ratio determination of bacterial DNA. Anal Biochem 81, 461-466.
  • Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159-167.
  • Stackebrandt, E., Frederiksen, W., Garrity, G. M., Grimont, P., Kampfer, P., Maiden, M., Nesme, X., Rossello-Mora, R., Swings, J., Truper, H. G., Vauterin, L., Ward, A. C. & Whitman, W. B. (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043-1047.
  • Tamaoka, J. & Komagata, K. (1984). Determination of DNA base composition by reversed-phase high-performance liquid chromatography. FEMS Microbiol Lett 25, 125-128.



Photo of Peter  Schumann
Dr. Schumann, Peter
Phone: +49-531/2616-106

MALDI-TOF mass spectrometry of bacteria, RiboPrinting, peptidoglycan analysis, determination of DNA base composition