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.

/pdf/greengenes-a-chimera-checked-16s-rrna-gene-database-and-4svk1mo01y.pdf

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

Nucleic acid probes capable of specifically hybridizing to rRNA of Campylobacter jejuni, C. coli and C. laridis and not to rRNA or rRNA genes of Pseudomonas aeuroginosa, E. coli or Salmonella typhimunium are described along with methods utilizing such probes for the detection of Campylobacter in clinical, food and other samples.

Detection of campylobacter with nucleic acid probes

Nucleic acid probes capable of specifically hybridizing to rRNA of Campylobacter jejuni. C. coli and C. laridis and not to rRNA or rRNA genes of Pseudomonas aeuroginosa, E. coli or Salmonella typhimunium are described along with methods utilizing such probes for the detection of Campylobacter in clinical, food and other samples.

Detection of campylobacter

Nucleic acid fragments capable of hybridizing to rRNA of a Salmonella species and not capable of hybridizing to rRNA of Escherichia coli.

Oligonucleotide probes for detection of salmonella

The present invention relates to a method of selectively detecting Giardia lamblia in a sample. The method makes use of at least one nucleic acid probe which is a DNA or PNA sequence which hybridizes, under appropriate conditions, to the ribosomal RNA or the ribosomal DNA of Giardia lamblia but does not hybridize to the ribosomal RNA or the ribosomal DNA of other organisms (non-Giardia lamblia organisms) which may be present in a sample.

Nucleic acid probes for the detection of Giardia lamblia

A method for detecting and/or quantitating specific DNA and/or RNA transcripts of human papillomavirus (HPV) is described. Nucleic acid probes specific for the DNA or the transcripts are used to determine the amount of the DNA or transcripts, and to ascertain the degree of amplification of certain high-oncogenic HPV genes. The method provides an accurate and reliable method for prognosticating serious cervical neoplasias and cancers.

David J. Lane

Papers

Detection of campylobacter with nucleic acid probes

Detection of campylobacter

Oligonucleotide probes for detection of salmonella

Nucleic acid probes for the detection of Giardia lamblia

Nucleic acid probe for detection of HPV transcripts.