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Full phylogenetic study by complete 16S rDNA sequence analysisFull phylogenetic study by complete 16S rDNA sequence analysis

Full phylogenetic study by complete 16S rDNA sequence analysis

Complete 16S rDNA gene sequence analysis including phylogenetic analysis should be part of the description of the type strain of a new species (Stackebrandt et al., 2002).

Required material
A living culture (agar plate or slant) is preferred. Lyophilized material might as well be sent.
An order form (pdf) has to be sent with the samples.

Method
Genomic DNA extraction is performed using the JETFLEX Genomic DNA Purification Kit from Genomed (Löhne, Germany) or the MasterPure™ Gram Positive DNA Purification Kit from Epicentre (Madison, USA).
PCR mediated amplification of the 16S rDNA and purification of the PCR product is carried out as described previously (Rainey et al., 1996). Purified PCR products are sequenced using the BigDye® Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) as directed in the manufacturer's protocol. Sequence reactions are electrophoresed using the Applied Biosystems 3500xl Genetic Analyzer.
The resulting sequence data are put into the alignment editor ae2 (Maidak et al., 1999), aligned manually and compared with representative 16S rRNA gene sequences of most closely related organisms. For comparison 16S rRNA sequences are obtained from the EMBL data base, RDP or our own database (Maidak et al., 1999).
The 16S rRNA gene similarity values are calculated by pairwise comparison of the sequences within the alignment.
For construction of the phylogenetic dendrogram operations of the ARB package (Pruesse et al., 2007) are used: based on the evolutionary distance values the phylogenetic tree is constructed by the neighbor-joining method (Saitou & Nei, 1987) using the correction of Jukes and Cantor (Jukes & Cantor, 1969).

References

  • 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.
  • Rainey, F. A., Ward-Rainey, N., Kroppenstedt, R. M. & Stackebrandt, E. (1996). The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage; proposal of Nocardiopsaceae fam. nov. Int. J. Sys. Bacteriol. 46, 1088-1092.
  • Maidak, B. L., Cole, J.R., Parker, C.T.Jr., Garrity, G.M., Larsen, N., Li, B., Lilburn, T.G., McCaughey, M.J., Olsen, G.J., Overbeek, R., Pramanik, S., Schmidt, T.M., Tiedje, J.M. & Woese, C.R. (1999). A new version of the RDP (Ribosomal Database Project). Nucl. Acids Res. 27, 171-173.
  • Pruesse, E., Quast, C., Knittel, K., Fuchs, B., Ludwig, W., Peplies, J. & Glöckner, F.O. (2007). SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucl. Acids Res. 35, 7188-7196.
  • Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425.
  • Jukes, T. H. & Cantor C. R. (1969). Evolution of protein molecules. In Mammalian protein metabolism, pp. 21-132. Edited by H. N. Munro. New York: Academic press.
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