Ribosomal RNA-targeted oligonucleotide microarrays (PhyloChips) for analysis of microbial communities in environmental and clinical samples
Fig. 1. Applied multiple probe concept. 16S rRNA-based phylogenetic consensus tree of all recognized sulfate-reducing bacteria of the orders "Desulfobacterales" and "Syntrophobacterales" showing examplarily the hierarchical and parallel specificity of oligonucleotide probes.
|The invention of DNA microarrays provided the basis for a hybridization format that allows greater sample throughput and highly parallel detection of complex microbial communities in the environment. In different kinds of hybridization formats used for microbial community analysis, the ribosomal RNA (gene) is the target molecule of choice. RNA genes consist of highly conserved and variable sequence regions. These sequence regions of different or same degrees of sequence conservation can be exploited for the design of multiple oligonucleotide probes to detect target organisms at different or same phylogenetic levels ("multiple probe concept") (Fig.1). By definition, PhyloChips are DNA microarrays consisting of rRNA-targeted oligonucleotide probes that were designed according to the "multiple probe concept". The great advantage of PhyloChip hybridization is that the reliability of identification is strongly enhanced, because all microorganisms of interest can be detected in a single assay by multiple probes of hierarchical and identical levels of specificity.
Comprehensive PhyloChips were developed for sulfate-reducing prokaryotes (SRP-PhyloChip) (Loy et al. 2002) and bacteria of the provisional betaproteobacterial order "Rhodocyclales" (RHC-PhyloChip) and subsequently evaluated by hybridization with fluorescently labeled 16S rRNA gene PCR amplificates of suitable reference organisms. Furthermore, the applicability of PhyloChips for routine inventorying of prokaryotes in various habitats such as a cyanobacterial mat (Fig.2), periodontal tooth pockets (Fig.3), fen soils, or activated sludge was demonstrated and verified by well-established, microarray-independent, molecular methods.
In essence, PhyloChips are rapid biodiversity screening tools and can be perfectly integrated into the methodological concept of cultivation-independent analysis of complex microbial communities in the environment.
Fig. 2. SRP-PhyloChip diversity survey in the chemocline of a cyanobacterial microbial mat. The microarray results were independently evaluated by amplification, cloning, and comparative sequence analysis of 16S rRNA gene fragments by using group-specific primers.
Fig. 3. SRP-PhyloChip diversity survey in a periodontal tooth pocket. The microarray results were independently evaluated by amplification, cloning, and comparative sequence analysis of 16S rRNA gene fragments by using Desulfomicrobium-specific primers for PCR.
Alexander Loy, Sebastian Luecker, Doris Steger, Manuel Hofer, Ivan Barisic
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