Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Journal Article uri icon

Overview

abstract

  • 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.

publication date

  • October 1, 1985

has restriction

  • green

Date in CU Experts

  • May 8, 2014 10:40 AM

Full Author List

  • Lane DJ; Pace B; Olsen GJ; Stahl DA; Sogin ML; Pace NR

author count

  • 6

Other Profiles

International Standard Serial Number (ISSN)

  • 0027-8424

Electronic International Standard Serial Number (EISSN)

  • 1091-6490

Additional Document Info

start page

  • 6955

end page

  • 6959

volume

  • 82

issue

  • 20