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DNA - DNA hybridizationDNA - DNA hybridization

DNA - DNA hybridization

DNA-DNA hybridization is necessary for the description of a new species within a taxon when strains share more than 97% 16S rRNA gene sequence similarity (Stackebrandt & Goebel, 1994, Tindall et al., 2010). This limit is controversially discussed (Stackebrandt & Ebers, 2006).

Required material
3g of wet biomass from all strains included in the analysis are needed. It must be suspended in iso-propanol / water (1:1, v/v) (first suspend biomass in water, mix well and then mix 1:1 with isopropanol) which can be sent in tightly sealed screw cap tubes (e.g. 50 ml Falcon tubes) at ambient temperature. Alternatively centrifuged biomass might be sent on dry ice (best option).
Lyophilized cells in corresponding amount can also be sent. However, the use of lyophilized cells may result in a lower DNA yield.

An order form from our homepage http://www.dsmz.de/identification/files/idform.pdf
has to be sent with the samples.

Please specify pairings in the table like:
Strain A Strain B
Strain B X -
Strain C X X

Method
Cells are disrupted by using a Constant Systems TS 0.75 KW (IUL Instruments, Germany) and the DNA in the crude lysate is purified by chromatography on hydroxyapatite as described by Cashion et al. (1977).
DNA-DNA hybridization is carried out as described by De Ley et al. (1970) under consideration of the modifications described by Huss et al. (1983) using a model Cary 100 Bio UV/VIS-spectrophotometer equipped with a Peltier-thermostatted 6x6 multicell changer and a temperature controller with in-situ temperature probe (Varian).

References

  • Stackebrandt, E. & Goebel, B.M. (1994). Taxonomic Note: A Place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bact 44, 846-849.
  • Tindall, B.J., Rosselló-Móra, R., Busse, H.-J., Ludwig, W. & Kämpfer, P. (2010). Notes on the characterization of prokaryote strains for taxonomic purposes. Int J Syst Evol Microbiol 60, 249-266.
  • Stackebrandt, E. & Ebers, J. (2006). Taxonomic parameters revisited: tarnished gold standards. Microbiology Today 33, 152–155.
  • 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.
  • De Ley, J., Cattoir, H. & Reynaerts, A. (1970). The quantitative measurement of DNA hybridization from renaturation rates. Eur J Biochem 12, 133-142.
  • Huss, V. A. R., Festl, H. & Schleifer, K. H. (1983). Studies on the spectrophotometric determination of DNA hybridization from renaturation rates. Syst Appl Microbiol 4, 184-192.



Photo of Cathrin  Spröer
Dr. Spröer, Cathrin
Phone: +49-531/2616-101

Partial and complete 16S rRNA gene sequence analysis, phylogenetic analysis, spectroscopic DNA-DNA hybridization