Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1989 Oct 11;17(19):7843–7853. doi: 10.1093/nar/17.19.7843

Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA.

U Edwards 1, T Rogall 1, H Blöcker 1, M Emde 1, E C Böttger 1
PMCID: PMC334891  PMID: 2798131

Abstract

Using a set of synthetic oligonucleotides homologous to broadly conserved sequences in-vitro amplification via the polymerase chain reaction followed by direct sequencing results in almost complete nucleotide determination of a gene coding for 16S ribosomal RNA. As a model system the nucleotide sequence of the 16S rRNA gene of M.kansasii was determined and found to be 98.7% homologous to that of M.bovis BCG. This is the first report on a contiguous sequence information of an entire amplified gene spanning 1.5 kb without any subcloning procedures.

Full text

PDF
7845

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Dams E., Hendriks L., Van de Peer Y., Neefs J. M., Smits G., Vandenbempt I., De Wachter R. Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res. 1988;16 (Suppl):r87–173. doi: 10.1093/nar/16.suppl.r87. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Dunning A. M., Talmud P., Humphries S. E. Errors in the polymerase chain reaction. Nucleic Acids Res. 1988 Nov 11;16(21):10393–10393. doi: 10.1093/nar/16.21.10393. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. 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]
  5. Gray M. W., Sankoff D., Cedergren R. J. On the evolutionary descent of organisms and organelles: a global phylogeny based on a highly conserved structural core in small subunit ribosomal RNA. Nucleic Acids Res. 1984 Jul 25;12(14):5837–5852. doi: 10.1093/nar/12.14.5837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gyllensten U. B., Erlich H. A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7652–7656. doi: 10.1073/pnas.85.20.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Göbel U., Maas R., Haun G., Vinga-Martins C., Stanbridge E. J. Synthetic oligonucleotide probes complementary to rRNA for group- and species-specific detection of mycoplasmas. Isr J Med Sci. 1987 Jun;23(6):742–746. [PubMed] [Google Scholar]
  8. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Innis M. A., Myambo K. B., Gelfand D. H., Brow M. A. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9436–9440. doi: 10.1073/pnas.85.24.9436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Johnson A. M., Baverstock P. R. Rapid ribosomal RNA sequencing and the phylogenetic analysis of protists. Parasitol Today. 1989 Apr;5(4):102–105. doi: 10.1016/0169-4758(89)90046-x. [DOI] [PubMed] [Google Scholar]
  11. Lane D. J., Pace B., Olsen G. J., Stahl D. A., Sogin M. L., Pace N. R. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6955–6959. doi: 10.1073/pnas.82.20.6955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  13. Nakamaye K. L., Gish G., Eckstein F., Vosberg H. P. Direct sequencing of polymerase chain reaction amplified DNA fragments through the incorporation of deoxynucleoside alpha-thiotriphosphates. Nucleic Acids Res. 1988 Nov 11;16(21):9947–9959. doi: 10.1093/nar/16.21.9947. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Päbo S., Wilson A. C. Polymerase chain reaction reveals cloning artefacts. Nature. 1988 Aug 4;334(6181):387–388. doi: 10.1038/334387b0. [DOI] [PubMed] [Google Scholar]
  15. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  16. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  17. Scharf S. J., Horn G. T., Erlich H. A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. doi: 10.1126/science.3461561. [DOI] [PubMed] [Google Scholar]
  18. Suzuki Y., Nagata A., Ono Y., Yamada T. Complete nucleotide sequence of the 16S rRNA gene of Mycobacterium bovis BCG. J Bacteriol. 1988 Jun;170(6):2886–2889. doi: 10.1128/jb.170.6.2886-2889.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Todd J. A., Bell J. I., McDevitt H. O. HLA-DQ beta gene contributes to susceptibility and resistance to insulin-dependent diabetes mellitus. Nature. 1987 Oct 15;329(6140):599–604. doi: 10.1038/329599a0. [DOI] [PubMed] [Google Scholar]
  20. Wilson K. H., Blitchington R., Hindenach B., Greene R. C. Species-specific oligonucleotide probes for rRNA of Clostridium difficile and related species. J Clin Microbiol. 1988 Dec;26(12):2484–2488. doi: 10.1128/jcm.26.12.2484-2488.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Wong C., Dowling C. E., Saiki R. K., Higuchi R. G., Erlich H. A., Kazazian H. H., Jr Characterization of beta-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. 1987 Nov 26-Dec 2Nature. 330(6146):384–386. doi: 10.1038/330384a0. [DOI] [PubMed] [Google Scholar]
  23. Wrischnik L. A., Higuchi R. G., Stoneking M., Erlich H. A., Arnheim N., Wilson A. C. Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA. Nucleic Acids Res. 1987 Jan 26;15(2):529–542. doi: 10.1093/nar/15.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES