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. 1995 Dec;61(12):4269–4273. doi: 10.1128/aem.61.12.4269-4273.1995

Rapid differentiation of bacterial species with multiple probes of different lengths in a single slot blot hybridization.

G C Wang 1, Y Wang 1
PMCID: PMC167738  PMID: 8534094

Abstract

We describe a highly efficient method for dot and slot blot hybridizations with multiple oligonucleotide probes for high throughput identification of organisms and studies of microbial community structures. Several probes with distinct specificities were designed to have the same melting temperature but unique lengths by adding different numbers of nonspecific nucleotides to one end. All of the probes were mixed, labelled with 32P, and hybridized to one piece of membrane on which genes coding for 16S rRNAs from different bacterial species had been immobilized. After hybridization, the bound probes were eluted and resolved on a denaturing polyacrylamide gel and the identities of the genes coding for 16S rRNAs were read from an autoradiograph of the gel. The results from the application of this technique to pure actinomycete cultures are reported here.

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Selected References

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  1. Amann R. I., Krumholz L., Stahl D. A. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol. 1990 Feb;172(2):762–770. doi: 10.1128/jb.172.2.762-770.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Amann R. I., Ludwig W., Schleifer K. H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995 Mar;59(1):143–169. doi: 10.1128/mr.59.1.143-169.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burggraf S., Mayer T., Amann R., Schadhauser S., Woese C. R., Stetter K. O. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes. Appl Environ Microbiol. 1994 Sep;60(9):3112–3119. doi: 10.1128/aem.60.9.3112-3119.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. DeLong E. F., Wickham G. S., Pace N. R. Phylogenetic stains: ribosomal RNA-based probes for the identification of single cells. Science. 1989 Mar 10;243(4896):1360–1363. doi: 10.1126/science.2466341. [DOI] [PubMed] [Google Scholar]
  5. Giovannoni S. J., DeLong E. F., Olsen G. J., Pace N. R. Phylogenetic group-specific oligodeoxynucleotide probes for identification of single microbial cells. J Bacteriol. 1988 Feb;170(2):720–726. doi: 10.1128/jb.170.2.720-726.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Kane M. D., Poulsen L. K., Stahl D. A. Monitoring the enrichment and isolation of sulfate-reducing bacteria by using oligonucleotide hybridization probes designed from environmentally derived 16S rRNA sequences. Appl Environ Microbiol. 1993 Mar;59(3):682–686. doi: 10.1128/aem.59.3.682-686.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Larsen N., Olsen G. J., Maidak B. L., McCaughey M. J., Overbeek R., Macke T. J., Marsh T. L., Woese C. R. The ribosomal database project. Nucleic Acids Res. 1993 Jul 1;21(13):3021–3023. doi: 10.1093/nar/21.13.3021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Liesack W., Stackebrandt E. Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J Bacteriol. 1992 Aug;174(15):5072–5078. doi: 10.1128/jb.174.15.5072-5078.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Neefs J. M., Van de Peer Y., De Rijk P., Chapelle S., De Wachter R. Compilation of small ribosomal subunit RNA structures. Nucleic Acids Res. 1993 Jul 1;21(13):3025–3049. doi: 10.1093/nar/21.13.3025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Olsen G. J., Lane D. J., Giovannoni S. J., Pace N. R., Stahl D. A. Microbial ecology and evolution: a ribosomal RNA approach. Annu Rev Microbiol. 1986;40:337–365. doi: 10.1146/annurev.mi.40.100186.002005. [DOI] [PubMed] [Google Scholar]
  11. Olsen G. J., Woese C. R. Ribosomal RNA: a key to phylogeny. FASEB J. 1993 Jan;7(1):113–123. doi: 10.1096/fasebj.7.1.8422957. [DOI] [PubMed] [Google Scholar]
  12. Risatti J. B., Capman W. C., Stahl D. A. Community structure of a microbial mat: the phylogenetic dimension. Proc Natl Acad Sci U S A. 1994 Oct 11;91(21):10173–10177. doi: 10.1073/pnas.91.21.10173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ruff-Roberts A. L., Kuenen J. G., Ward D. M. Distribution of cultivated and uncultivated cyanobacteria and Chloroflexus-like bacteria in hot spring microbial mats. Appl Environ Microbiol. 1994 Feb;60(2):697–704. doi: 10.1128/aem.60.2.697-704.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Stackebrandt E., Witt D., Kemmerling C., Kroppenstedt R., Liesack W. Designation of Streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes. Appl Environ Microbiol. 1991 May;57(5):1468–1477. doi: 10.1128/aem.57.5.1468-1477.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Stahl D. A., Flesher B., Mansfield H. R., Montgomery L. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl Environ Microbiol. 1988 May;54(5):1079–1084. doi: 10.1128/aem.54.5.1079-1084.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Stouthamer R., Breeuwert J. A., Luck R. F., Werren J. H. Molecular identification of microorganisms associated with parthenogenesis. Nature. 1993 Jan 7;361(6407):66–68. doi: 10.1038/361066a0. [DOI] [PubMed] [Google Scholar]
  17. Tsien H. C., Bratina B. J., Tsuji K., Hanson R. S. Use of oligodeoxynucleotide signature probes for identification of physiological groups of methylotrophic bacteria. Appl Environ Microbiol. 1990 Sep;56(9):2858–2865. doi: 10.1128/aem.56.9.2858-2865.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Wagner M., Erhart R., Manz W., Amann R., Lemmer H., Wedi D., Schleifer K. H. Development of an rRNA-targeted oligonucleotide probe specific for the genus Acinetobacter and its application for in situ monitoring in activated sludge. Appl Environ Microbiol. 1994 Mar;60(3):792–800. doi: 10.1128/aem.60.3.792-800.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Woese C. R., Kandler O., Wheelis M. L. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4576–4579. doi: 10.1073/pnas.87.12.4576. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Woese C. R., Stackebrandt E., Macke T. J., Fox G. E. A phylogenetic definition of the major eubacterial taxa. Syst Appl Microbiol. 1985;6:143–151. doi: 10.1016/s0723-2020(85)80047-3. [DOI] [PubMed] [Google Scholar]

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