Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction

TLDR

Small‑subunit rRNA sequences vary phylogenetically and contain conserved segments at species, genus, or kingdom levels. Oligonucleotide primers targeting conserved 5′, 3′, and central 16S rRNA sites across the eubacterial kingdom were used to PCR‑amplify 16S ribosomal DNA from diverse bacterial isolates. Revised, non‑self‑complementary primers produced reproducible amplification, yielding a visible band from as little as 0.4 pg of E.

Abstract

The sequence of small-subunit rRNA varies in an orderly manner across phylogenetic lines and contains segments that are conserved at the species, genus, or kingdom level. By directing oligonucleotide primers at sequences conserved throughout the eubacterial kingdom, we amplified bacterial 16S ribosomal DNA sequences with the polymerase chain reaction. Priming sites were located at the extreme 5' end, the extreme 3' end, and the center of 16S ribosomal DNA. The isolates tested with these primers included members of the genera Staphylococcus, Coxiella, Rickettsia, Clostridium, Neisseria, Mycobacterium, Bilophila, Eubacterium, Fusobacterium, and Lactobacillus and the family Enterobacteriaceae. Initially, the yields from the reactions were erratic because the primers were self-complementary at the 3' ends. Revised primers that were not self-complementary gave more reproducible results. With the latter primers, 0.4 pg of Escherichia coli DNA consistently gave a visible band after amplification. This method should be useful for increasing the amounts of bacterial 16S ribosomal DNA sequences for the purposes of sequencing and probing. It should have a broad range of applications, including the detection and identification of known pathogens that are difficult to culture. This approach may make it possible to identify new, nonculturable bacterial pathogens.