Hochparallele Analyse natürlicher mikrobieller Lebensgemeinschaften durch oberflächengebundene Oligonukleotidsonden

Abstract

DNA microarrays represent a high throughput format for the application of nucleic acid probes by reverse hybridization and, therefore, provide a powerful tool for the parallel detection of microorganisms. However, their application in molecular ecology is still hampered by different methodological limitations. In this study, various parameters affecting a surface-mediated hybridization were systematically evaluated with a model system composed of 6 bacterial strains and 20 oligonucleotide probes targeting the 16S rRNA. With adequate hybridization conditions, false-positive signals could be almost completely prevented resulting in clear data interpretation. For microarray analysis of mixed microbial communities, a straightforward protocol for direct chemical labeling of extracted rRNA was established to avoid potential PCR-biases. In samples of different complexity, including an oligotrophic marine system, major bacterial populations (approx. >5%) could specifically be detected by microarray hybridization, as determined by parallel fluorescence in situ hybridization (FISH). However, quantitative FISH data suggested that current microarray analysis provides, at best, semiquantitative information on community composition. Application of phylogenetically redundant and/or nested probe sets is a prerequisite for the reliable hybridization-based detection of microorganisms. A potential reservoir of additional probe target sites is represented by the 23S rRNA. In this study, almost complete 23S rRNA gene sequences were obtained from 11 alpha-proteobacteria isolated from North Sea bacterioplankton. Comparative sequence analysis revealed consistent tree topologies for the alpha-proteobacteria based on their 16S and 23S rRNA genes. The suitability of both markers for the design of specific oligonucleotide probes was systematically evaluated in silico. The overall number of phylogenetic redundant probes available could significantly be increased by also targeting the 23S rRNA.