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

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.

Biogeography of bacterial communities in hot springs: a focus on the actinobacteria

“…Bacterial 16S rRNA gene amplification was performed using primer pair 341F (5'-CCTACGGGAGGCAGCAI-3') [21], and 908R (5'-CCGTCAATTCMTTTGAGTTI-3') [25]. For actinobacterial-specific amplifications, primers 226-243-F (5'-GGATGAGCCCGCGGCCTA-3') [17] and (A3R) 1414-1430-R (5-CCAGCCCCACCTTCGAC-3) [36] were used.…”

Characterization of a marine-isolated mercury-resistant Pseudomonas putida strain SP1 and its potential application in marine mercury reduction

“…Genomic DNA from SP1 was extracted according to the method described previously (Syn and Swarup 2000). The 16S rRNA gene was PCR amplified using primers 8F and 1492R according to the method described by Lane et al (1985). When the TA cloning strategy was employed, the purified PCR products were directly ligated into pMD18-T. Sequencing was carried out by Nuosai Sequencing Company (Beijing, China)…”

Microbiology: The new germ theory

“…The research community, intent on studying natural ecosystems in their entirety, made efforts to work out what all the unculturables are. In 1985, a team of microbiologists published a technique that could census bacteria and archaea in a sample by sequencing the 16S ribosomal RNA gene, which is different for every species 1 . These types of genetic survey quickly became routine in the environmental microbiology field.…”