Untersuchungen zur Herstellung und Funktionalität neuartiger Padlock-Sonden für den Nukleinsäurenachweis auf Mikroarrays

Abstract

In this study a new approach for DNA-detection was developed, which combines DNA-microarrays with the principle of padlock probes. For this novel U-shaped capture molecules were developed, which ensure label-free and specific DNA detection on microarrays while simultaneously allowing a novel normalisation strategy of present signal intensities. This approach was implemented for and tested on the sequence detection of various pathogenic microorganisms. The specificity of the system could be demonstrated by differentiating single nucleotide variations of the target sequence. Moreover different variations of the normalisation strategy were tested to further improve the microarray's reproducibility. The reaction conditions of the enzymatic steps involved got optimised. Furthermore the realisability of a new concept for signal amplification was tested for this approach, which is based on in situ concatenation of special padlock probes.