• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of pnasPNASInfo for AuthorsSubscriptionsAboutThis Article
Proc Natl Acad Sci U S A. Oct 1985; 82(20): 6955–6959.
PMCID: PMC391288

Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses.


Although the applicability of small subunit ribosomal RNA (16S rRNA) sequences for bacterial classification is now well accepted, the general use of these molecules has been hindered by the technical difficulty of obtaining their sequences. A protocol is described for rapidly generating large blocks of 16S rRNA sequence data without isolation of the 16S rRNA or cloning of its gene. The 16S rRNA in bulk cellular RNA preparations is selectively targeted for dideoxynucleotide-terminated sequencing by using reverse transcriptase and synthetic oligodeoxynucleotide primers complementary to universally conserved 16S rRNA sequences. Three particularly useful priming sites, which provide access to the three major 16S rRNA structural domains, routinely yield 800-1000 nucleotides of 16S rRNA sequence. The method is evaluated with respect to accuracy, sensitivity to modified nucleotides in the template RNA, and phylogenetic usefulness, by examination of several 16S rRNAs whose gene sequences are known. The relative simplicity of this approach should facilitate a rapid expansion of the 16S rRNA sequence collection available for phylogenetic analyses.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.0M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Zuckerkandl E, Pauling L. Molecules as documents of evolutionary history. J Theor Biol. 1965 Mar;8(2):357–366. [PubMed]
  • Fitch WM, Margoliash E. Construction of phylogenetic trees. Science. 1967 Jan 20;155(3760):279–284. [PubMed]
  • Fox GE, Stackebrandt E, Hespell RB, Gibson J, Maniloff J, Dyer TA, Wolfe RS, Balch WE, Tanner RS, Magrum LJ, et al. The phylogeny of prokaryotes. Science. 1980 Jul 25;209(4455):457–463. [PubMed]
  • Pace B, Matthews EA, Johnson KD, Cantor CR, Pace NR. Conserved 5S rRNA complement to tRNA is not required for protein synthesis. Proc Natl Acad Sci U S A. 1982 Jan;79(1):36–40. [PMC free article] [PubMed]
  • Caruthers MH, Beaucage SL, Becker C, Efcavitch JW, Fisher EF, Galluppi G, Goldman R, deHaseth P, Matteucci M, McBride L, et al. Deoxyoligonucleotide synthesis via the phosphoramidite method. Gene Amplif Anal. 1983;3:1–26. [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Biggin MD, Gibson TJ, Hong GF. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. [PMC free article] [PubMed]
  • Hamlyn PH, Browniee GG, Cheng CC, Gait MJ, Milstein C. Complete sequence of constant and 3' noncoding regions of an immunoglobulin mRNA using the dideoxynucleotide method of RNA sequencing. Cell. 1978 Nov;15(3):1067–1075. [PubMed]
  • Brosius J, Palmer ML, Kennedy PJ, Noller HF. Complete nucleotide sequence of a 16S ribosomal RNA gene from Escherichia coli. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4801–4805. [PMC free article] [PubMed]
  • Gupta R, Lanter JM, Woese CR. Sequence of the 16S Ribosomal RNA from Halobacterium volcanii, an Archaebacterium. Science. 1983 Aug 12;221(4611):656–659. [PubMed]
  • Hagenbüchle O, Santer M, Steitz JA, Mans RJ. Conservation of the primary structure at the 3' end of 18S rRNA from eucaryotic cells. Cell. 1978 Mar;13(3):551–563. [PubMed]
  • Youvan DC, Hearst JE. Reverse transcriptase pauses at N2-methylguanine during in vitro transcription of Escherichia coli 16S ribosomal RNA. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3751–3754. [PMC free article] [PubMed]
  • Youvan DC, Hearst JE. A sequence from Drosophila melanogaster 18S rRNA bearing the conserved hypermodified nucleoside am psi: analysis by reverse transcription and high-performance liquid chromatography. Nucleic Acids Res. 1981 Apr 10;9(7):1723–1741. [PMC free article] [PubMed]
  • Qu HL, Michot B, Bachellerie JP. Improved methods for structure probing in large RNAs: a rapid 'heterologous' sequencing approach is coupled to the direct mapping of nuclease accessible sites. Application to the 5' terminal domain of eukaryotic 28S rRNA. Nucleic Acids Res. 1983 Sep 10;11(17):5903–5920. [PMC free article] [PubMed]
  • Raynal F, Michot B, Bachellerie JP. Complete nucleotide sequence of mouse 18 S rRNA gene: comparison with other available homologs. FEBS Lett. 1984 Feb 27;167(2):263–268. [PubMed]
  • Brownlee GG, Cartwright EM. Rapid gel sequencing of RNA by primed synthesis with reverse transcriptase. J Mol Biol. 1977 Jul;114(1):93–117. [PubMed]
  • Mills DR, Kramer FR. Structure-independent nucleotide sequence analysis. Proc Natl Acad Sci U S A. 1979 May;76(5):2232–2235. [PMC free article] [PubMed]
  • Myers JC, Spiegelman S. Sodium pyrophosphate inhibition of RNA.DNA hybrid degradation by reverse transcriptase. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5329–5333. [PMC free article] [PubMed]
  • Inoue T, Cech TR. Secondary structure of the circular form of the Tetrahymena rRNA intervening sequence: a technique for RNA structure analysis using chemical probes and reverse transcriptase. Proc Natl Acad Sci U S A. 1985 Feb;82(3):648–652. [PMC free article] [PubMed]
  • Woese CR, Gutell R, Gupta R, Noller HF. Detailed analysis of the higher-order structure of 16S-like ribosomal ribonucleic acids. Microbiol Rev. 1983 Dec;47(4):621–669. [PMC free article] [PubMed]
  • Fitch WM, Smith TF. Optimal sequence alignments. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1382–1386. [PMC free article] [PubMed]
  • Kimura M, Ota T. On the stochastic model for estimation of mutational distance between homologous proteins. J Mol Evol. 1972 Dec 29;2(1):87–90. [PubMed]
  • Tomioka N, Sugiura M. The complete nucleotide sequence of a 16S ribosomal RNA gene from a blue-green alga, Anacystis nidulans. Mol Gen Genet. 1983;191(1):46–50. [PubMed]
  • Messing J, Carlson J, Hagen G, Rubenstein I, Oleson A. Cloning and sequencing of the ribosomal RNA genes in maize: the 17S region. DNA. 1984;3(1):31–40. [PubMed]
  • Rubtsov PM, Musakhanov MM, Zakharyev VM, Krayev AS, Skryabin KG, Bayev AA. The structure of the yeast ribosomal RNA genes. I. The complete nucleotide sequence of the 18S ribosomal RNA gene from Saccharomyces cerevisiae. Nucleic Acids Res. 1980 Dec 11;8(23):5779–5794. [PMC free article] [PubMed]
  • Salim M, Maden BE. Nucleotide sequence of Xenopus laevis 18S ribosomal RNA inferred from gene sequence. Nature. 1981 May 21;291(5812):205–208. [PubMed]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


  • Cited in Books
    Cited in Books
    PubMed Central articles cited in books
  • MedGen
    Related information in MedGen
  • PubMed
    PubMed citations for these articles
  • Substance
    PubChem Substance links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...