1887

Abstract

Discontinuous DNA complementary to 16S + 23S ribosomal RNA was synthesized by random oligonucleotide priming using reverse transcriptase. cDNA generated from native or denatured rRNA template was labelled by incorporation of either [α-P]dCTP or digoxigenin-labelled dUTP during synthesis, followed by template hydrolysis. The specific activity of the radiolabelled cDNA was 10–10 c.p.m. (g rRNA template) with 60–92% incorporation after 5 h. The length of the reverse transcript was between 20 and 1140 nucleotides and was unaffected by exclusion of primer. The cDNA probe could detect 3 μg rRNA by quantitative slot blot. In the non-radiolabelling digoxigenin system 3 g template gave 0·5–2·0 μg cDNA after 24 h with a length of between 100 and 1225 bases. This probe could detect 50 pg rRNA. Probes were evaluated in the comparison of biotypes by hybridization to Southern blots of restriction-endonuclease-digested total DNA. The digoxigenin-labelled probe was used to identify clinical isolates of to demonstrate its potential use in laboratories requiring high-sensitivity detection without the use of radioisotopes.

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1990-08-01
2024-05-01
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References

  1. Ausubel F.M., Brent R., Kingston R.E., Moore D.D., Smith J.A., Seidman J.G., Struhl K. 1987 Current Protocols in Molecular Biology 1987–1988. New York: John Wiley and Sons;
    [Google Scholar]
  2. Feinberg A.P., Volgelstein B. 1984; Addendum: a technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Analytical Biochemistry 137:266–267
    [Google Scholar]
  3. Grimont F., Grimont P.A.D. 1986; Ribosomal ribonucleic acid gene restriction patterns as potential taxonomic tools. Annales de I’lnstitut Pasteur/Microbiologie 137:165–175
    [Google Scholar]
  4. Hayes L.J., Wright D.J., Archard L.C. 1988; Segmented arrangement of Borreliaduttonii DNA and location of variant surface antigen genes. Journal of General Microbiology 134:1785–1793
    [Google Scholar]
  5. Lior H. 1984; New, extended biotyping scheme for Campylobacter jejuni, Campylobacter coli and “Campylobacter laridis”. . Journal of Clinical Microbiology 20:636–640
    [Google Scholar]
  6. Moureau P., Derclaye I., Gregoire D., Janssen M., Cornelis G.R. 1989; Campylobacter species identification based on polymorphism of DNA encoding rRNA. Journal of Clinical Microbiology 27:1514–1517
    [Google Scholar]
  7. Qu L.H., Michot B., Bachellerie J.-P. 1983; Improved methods for structure probing in large RNAs. Nucleic Acids Research 2:5903–5920
    [Google Scholar]
  8. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, , 2nd edn.. Cold Spring Harbor, NY:: Cold Spring Harbor Laboratory.;
    [Google Scholar]
  9. Tabaqchali S., Silman R., Holland D. 1987; Automation in clinical microbiology: a new approach to identifying microorganisms by automated pattern matching of proteins labelled with 35S-methionjne. Journal of Clinical Pathology 40:1070–1087
    [Google Scholar]
  10. Woese C.R. 1987; Bacterial evolution. Microbiological Reviews 51:221–271
    [Google Scholar]
  11. Youvan D.C., Hearst J.E. 1979; Reverse transcriptase pauses at N2-methylguanine during in vitro transcription of Escherichia coli 16S ribosomal RNA. Biochemistry 76:3751–3754
    [Google Scholar]
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