Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics

Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. PCR protocols: a guide to methods and applications

We describe a new molecular approach to analyzing the genetic diversity of complex microbial populations. This technique is based on the separation of polymerase chain reaction-amplified fragments of genes coding for 16S rRNA, all the same length, by denaturing gradient gel electrophoresis (DGGE). DGGE analysis of different microbial communities demonstrated the presence of up to 10 distinguishable bands in the separation pattern, which were most likely derived from as many different species constituting these populations, and thereby generated a DGGE profile of the populations. We showed that it is possible to identify constituents which represent only 1% of the total population. With an oligonucleotide probe specific for the V3 region of 16S rRNA of sulfate-reducing bacteria, particular DNA fragments from some of the microbial populations could be identified by hybridization analysis. Analysis of the genomic DNA from a bacterial biofilm grown under aerobic conditions suggests that sulfate-reducing bacteria, despite their anaerobicity, were present in this environment. The results we obtained demonstrate that this technique will contribute to our understanding of the genetic diversity of uncharacterized microbial populations.

Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA

A set of oligonucleotide primers capable of initiating enzymatic amplification (polymerase chain reaction) on a phylogenetically and taxonomically wide range of bacteria is described along with methods for their use and examples. One pair of primers is capable of amplifying nearly full-length 16S ribosomal DNA (rDNA) from many bacterial genera; the additional primers are useful for various exceptional sequences. Methods for purification of amplified material, direct sequencing, cloning, sequencing, and transcription are outlined. An obligate intracellular parasite of bovine erythrocytes, Anaplasma marginale, is used as an example; its 16S rDNA was amplified, cloned, sequenced, and phylogenetically placed. Anaplasmas are related to the genera Rickettsia and Ehrlichia. In addition, 16S rDNAs from several species were readily amplified from material found in lyophilized ampoules from the American Type Culture Collection. By use of this method, the phylogenetic study of extremely fastidious or highly pathogenic bacterial species can be carried out without the need to culture them. In theory, any gene segment for which polymerase chain reaction primer design is possible can be derived from a readily obtainable lyophilized bacterial culture.

16S ribosomal DNA amplification for phylogenetic study.

A 16S rRNA gene database (http://greengenes.lbl.gov) addresses limitations of public repositories by providing chimera screening, standard alignment, and taxonomic classification using multiple published taxonomies. It was found that there is incongruent taxonomic nomenclature among curators even at the phylum level. Putative chimeras were identified in 3% of environmental sequences and in 0.2% of records derived from isolates. Environmental sequences were classified into 100 phylum-level lineages in the Archaea and Bacteria.

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Greengenes, a Chimera-Checked 16S rRNA Gene Database and Workbench Compatible with ARB

Nucleic acid probes capable of specifically hybridizing to rRNA of E coli and Shigella species and not to rRNA of non-E coli/Shigella are described along with methods utilizing such probes for the specific detection of E coli and/or Shigella in food and other samples

Probes for the specific detection of Escherichia coli and Shigella

Comparisons of 5S ribosomal ribonucleic acid (rRNA) nucleotide sequences were used for the phylogenetic characterization of a number of “morphologically conspicuous” sulfide-oxidizing eubacteria and certain of their non-sulfide oxidizing counterparts. Nucleotide sequences were determined for 5S rRNAs isolated from Thiovulum sp., Beggiatoa alba B18LD, Thiothrix nivea JP2, strain 30 (a Thiothrix-like organism), Vitreoscilla beggiatoides B23SS, Vitreoscilla filiformis L1401-7, V.filiformis ATCC 15551, Vitreoscilla stercoraria VT1, and Leptothrix discophora (Stokes). All 5S rRNAs characterized in this study, except that of Thiovulum sp., belong to either the gamma or beta subdivisions of the “purple bacteria,” which form 1 of the 10 major eubacterial divisions so far defined by 16S rRNA oligonucleotide catalog analysis. Although its 5S rRNA is clearly eubacterial in character, Thiovulum sp. appears to have no specific relatives among the eubacteria presently represented in the 5S rRNA data collection.

Phylogenetic Analysis of Certain Sulfide-Oxidizing and Related Morphologically Conspicuous Bacteria by 5S Ribosomal Ribonucleic Acid Sequences

Metazoan phylogeny based on analysis of 18S ribosomal RNA

Nucleic acid probes and methods for detecting Pathogenic Candida Yeasts. Nucleic acid probes are described for detecting yeasts capable of causing Candida infection, specifically yeasts of the genera Candida , Torulopsis , and Yarrowia . The preferred probes are complementary to ribonucleic acid sequences unique to specific Candida species, or groups of such species, and as such can detect the rRNA, rDNA, or polymerase chain reaction amplification products of these genes. Methods for detecting the etiological agent of human Candida fungemia utilizing these probes are also provided.

Nucleic acid probes and methods for detecting pathogenic candida yeasts

Nucleic acid probes and methods for detecting Pathogenic Candida Yeasts. Nucleic acid probes are described for detecting yeasts capable of causing Candida infection, specifically yeasts of the genera Candida, Torulopsis, and Yarrowia. The preferred probes are complementary to ribonucleic acid sequences unique to specific Candida species, or groups of such species, and as such can detect the rRNA, rDNA, or polymerase chain reaction amplification products of these genes. Methods for detecting the etiological agent of human Candida fungemia utilizing these probes are also provided.

David J. Lane

Papers

Probes for the specific detection of Escherichia coli and Shigella

Phylogenetic Analysis of Certain Sulfide-Oxidizing and Related Morphologically Conspicuous Bacteria by 5S Ribosomal Ribonucleic Acid Sequences

Metazoan phylogeny based on analysis of 18S ribosomal RNA

Nucleic acid probes and methods for detecting pathogenic candida yeasts

Nucleic acid probes and methods for detecting pathogenic Candida albicans yeasts