Current Microbiology

, Volume 27, Issue 3, pp 147–151 | Cite as

Amplification and sequencing of variable regions in bacterial 23S ribosomal RNA genes with conserved primer sequences

  • Guy Van Camp
  • Sabine Chapelle
  • Rupert De Wachter
Article

Abstract

Published bacterial 23S ribosomal RNA sequences were aligned, and universally conserved regions flanking highly variable regions were looked for. In strategically positioned conserved regions, six oligonucleotides suitable for polymerase chain reaction (PCR) and sequencing were designed, allowing fast sequencing of four of the most variable 23S rRNA regions. Two other primers were designed for PCR amplification of nearly complete 23S rRNA genes. All these primers successfully amplified fragments of 23S rRNA genes from seven unrelated bacteria. Four primers were used to determine 938 bp of sequence forCampylobacter jejuni subsp.jejuni. These results indicate that the oligonucleotide sequences presented here are useful for PCR amplification and sequence determination of variable 23S rRNA regions for a broad variety of eubacterial species.

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Literature Cited

  1. 1.
    Betzl D, Ludwig W, Schleifer KH (1990) Identification of lactococci an enterococci by colony hybridization with 23S rRNA-targeted oligonucleotide probes. Appl Env Microbiol 56:2927–2929Google Scholar
  2. 2.
    Casanova JL, Pannetier C, Jaulin C, Kourilsky P (1990) Optimal conditions for directly sequencing double stranded PCR products with Sequenase. Nucleic Acids Res 18:4028PubMedCrossRefGoogle Scholar
  3. 3.
    De Rijk P, Neefs JM, Van de Peer Y, De Wachter R (1992) Compilation os small ribosomal subunit RNA sequences. Nucleic Acids Res 20:2075–2089PubMedCrossRefGoogle Scholar
  4. 4.
    Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853PubMedCrossRefGoogle Scholar
  5. 5.
    Gelfand DH, White TJ (1990) Thermostable DNA polymerases. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. London: Academic Press, pp 129–141Google Scholar
  6. 6.
    Gutell RR, Schnare MN, Gray MW (1992) A compilation of large subunit (23S-and 23S-like) ribosomal RNA structures. Nucleic Acids Res 20:2095–2109PubMedCrossRefGoogle Scholar
  7. 7.
    Höpfl P, Ludwig W, Schleifer KH, Larsen N (1989) The 23S ribosomal RNA higher-order structure ofPseudomonas cepacia and other prokaryotes. Eur J Biochem 185:355–364PubMedCrossRefGoogle Scholar
  8. 8.
    Hultman T, Ståhl S, Hornes E, Uhlén M (1989) Direct solid phase sequencing of genomic and plasmid DNA using magnetic beads as a solid support. Nucleic Acids Res 17: 4937–4946PubMedCrossRefGoogle Scholar
  9. 9.
    Kwok S, Kellogg DE, McKinney N, Spasic D, Goda L, Levenson C, Sninsky JJ (1990) Effects of primer-template mismatches on the polymerase chain reaction: human immunodeficiency virus type 1 model studies. Nucleic Acids Res 18:999–1005PubMedCrossRefGoogle Scholar
  10. 10.
    Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. New York: John Wiley & Sons, pp 115–175Google Scholar
  11. 11.
    Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA 82: 6955–6959PubMedCrossRefGoogle Scholar
  12. 12.
    Leffers H, Kjems J, Ostergaard L, Larsen N, Garrett RA (1987) Evolutionary relationships amongst archaebacteria: a comparative study of 23S ribosomal RNA's of a sulphur dependent extreme thermophile, an extreme halophile and a thermophilic methanogen. J Mol Biol 195:43–61PubMedCrossRefGoogle Scholar
  13. 13.
    Ling LL, Keohavong P, Dias C, Thilly WG (1991) Optimization of the polymerase chain reaction with regard to fidelity: modified T7, Taq, and Vent DNA polymerases. PCR Methods Appl 1:63–69PubMedCrossRefGoogle Scholar
  14. 14.
    Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. New York: Cold Spring Harbor Laboratory PressGoogle Scholar
  15. 15.
    Stackebrandt E, Witt D, Kemmerling C, Kroppenstedt R, Liesack W (1991) Designation of streptomycete 16S and 23S rRNA-based target regions for oligonucleotide probes. Appl Environ Microbiol 57:1468–1477PubMedGoogle Scholar
  16. 16.
    Vandamme P, Falsen E, Rossau R, Hoste B, Segers P, Tytgat R, De Ley J (1991) Revision ofCampylobacter, Helicobacter andWolinella taxonomy: emendation of generic descriptions and proposal ofArcobacter gen. nov. Int J Syst Bacteriol 41:88–103PubMedCrossRefGoogle Scholar
  17. 17.
    Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173:697–703PubMedGoogle Scholar
  18. 18.
    Williams JF (1989) Amplifications 3:19Google Scholar

Copyright information

© Springer-Verlag New York Inc. 1993

Authors and Affiliations

  • Guy Van Camp
    • 1
  • Sabine Chapelle
    • 1
  • Rupert De Wachter
    • 1
  1. 1.Department of BiochemistryUniversity of Antwerp (UIA)Belgium

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