Entwicklung von Oligonukleotid-Sonden für die Mikroarray-basierte Identifizierung von Plattfisch- und Dorscharten der südlichen Nordsee

Since about ten years DNA microarrays are indispensable tools for genomic and biomedical research. They are used predominantly for expression profiling but are increasingly employed for the analysis of complex environmental samples. This thesis presents the development of a DNA microarray prototype for the identification of gadoid and flatfish species from the southern North Sea. First of all partial sequences of mitochondrial Cytochrome Oxidase I (COI) and 16S rRNA genes were determined for adult gadoid and flatfish specimens. Based on COI and 16S rDNA alignments capture oligonucleotide probes were selected, spotted onto glass slides and the resulting DNA microarrays were tested successfully with fish samples. COI DNA sequences showed sufficient sequence variability for the selection of capture probes even for the discrimination of closely related fish species, e.g. flounder and European plaice, whereas 16S rDNA sequences were too conserved within the families Pleuronectidae and Gadidae to select species specific probes, but were very suitable for the selection of probes for Soleidae and Scophthalmidae species, due to the presence of highly variable regions. The evaluation of 34 COI probes for the identification of Atlantic cod, whiting, flounder, dab, European plaice, American plaice, lemon sole and common sole and 10 16S rDNA probes for turbot, brill, common sole and solenette resulted in 34 species specific, 6 false negative and 4 cross reacting probes. Signal strength varied considerably among all probes and was correlated with the position of the probe binding region and influenced by the concentration of the target DNA and the presence of non target DNA. LNA-modified COI probes showed in some cases improvements concerning the specificity and the sensitivity but altogether increased the proportion of false negative or cross reacting probes. Some probes with HEG2 spacers exhibited increased signal intensities but spacers also resulted in cross hybridizations