Use of Subtractive Hybridization To Design Habitat-Based Oligonucleotide Probes for Investigation of Natural Bacterial Communities
AUTOR(ES)
Mau, Margit
FONTE
American Society for Microbiology
RESUMO
We describe a rapid oligonucleotide probe design strategy based on subtractive hybridization which yields probes for 16S rRNA or rRNA genes of individual members of microbial communities that are specific within the context of those communities. This strategy circumvents the need to sequence many similar or identical clones of dominant members of a community. Radioactively labeled subfragments of a cloned 16S rRNA gene sequence for which a probe is required (target) were hybridized with biotinylated total 16S ribosomal DNA (rDNA) amplified from the microbial community, and the hybrids formed were subsequently discarded. The remaining enriched fragments were used to screen a library consisting of cloned subfragments of the target sequence by colony hybridization in order to identify the variable regions of the 16S rRNA gene with the required specificity. The sequencing of random clones in one 16S rDNA library demonstrated that only those clones with 100% sequence identity with the probe fragment were detected by it. Moreover, sequencing of other, randomly selected, probe-positive clones revealed 100% sequence identity with the probe. Probes developed in this way tended to correspond to more variable regions of the 16S rRNA if the target sequences were similar to the sequences of other clones in the library and to less variable regions if the target sequences were phylogenetically isolated within the clone library. Although the absolute specificity of the latter probes, as assessed by comparison with available database sequences, was lower than the absolute specificity of the probes from the more variable regions, they were specific within the context of the environmental samples from which they were derived.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=124691Documentos Relacionados
- Group-specific 16S rRNA hybridization probes to describe natural communities of methanogens.
- Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology.
- Sequence-Based Design of Single-Copy Genomic DNA Probes for Fluorescence In Situ Hybridization
- The use of oligodeoxynucleotide probes in chaotrope-based hybridization solutions.
- Design and synthesis of polyacrylamide-based oligonucleotide supports for use in nucleic acid diagnostics.