- DNS-Sonde (1) (remove)
- Characterization of Oligonucleotide Microarray Hybridization: Microarray Fabrication by Light-Directed in situ Synthesis – Development of an Automated DNA Microarray Synthesizer, Characterization of Single Base Mismatch Discrimination and the Position-Dependent Influence of Point Defects on Oligonucleotide Duplex Binding Affinities (2008)
- The present thesis focuses on nucleic acid hybridization between free-floating target sequences and complementary end-tethered oligonucleotide probes on the surface of DNA microarrays. Hybridization experiments were performed on oligonucleotide microarrays (DNA Chips) which were fabricated with an automated synthesis apparatus (developed in the framework of the present thesis). The working principle of the microarray synthesizer is based on a photochemically controlled in situ synthesis process. By means of the combinatorial approach up to 25000 different (arbitrary) probe sequences can be fabricated in parallel - starting from nucleotide building blocks (NPPOC-phosphoramidites) - directly on the surface of the microarray. Great flexibility with regard to the choice of probe sequences is achieved by use of virtual photomasks on the basis of a spatial light modulator (Digital Micromirror Device, DMD, Texas Instruments Inc.). A microscope projection photolithography system is employed to project the virtual masks (i.e. the photomask images shown on the DMD) onto the surface of the microarray substrate. Spatially controlled photodeprotection of photolabile NPPOC protective groups (followed by coupling of a further nucleotide building block) enables massively parallel synthesis of DNA probe sequences. In the automated synthesis process microarrays are routinely fabricated over night. Comparable in situ synthesis systems are currently operated only at very few institutions around the world. We first report the application of phosphorus dendrimer substrates in the in situ synthesis of DNA microarrays. With the phosphorus dendrimer functionalization we obtained superior results in regard to sensitivity, surface homogeneity, signal/background-ratio and reusability of the microarrays. We performed microarray hybridization experiments to investigate the impact of single base defects (deliberately introduced single base mismatches and single base bulges) on the binding affinity of oligonucleotide duplexes. This is particularly interesting with regard to genotyping microarrays which are increasingly employed as a molecular diagnostics tool for the detection of single nucleotide polymorphisms (SNPs). In a number of experiments we investigated the large influence of the single-defect position on duplex binding affinity. The origin of this positional dependence - which is apparently not in agreement with the (two-state) nearest-neighbor model - had not been identified so far. We discovered that the influence of the defect position is not restricted to single base mismatches but can also be observed for single base bulge defects. On the basis of the double-ended zipper model (assuming fluctuating end-domain-opening of the oligonucleotide duplex) we could reproduce the experimentally observed positional influence. Moreover, our theoretical investigations on the zipper model indicate a significant positional influence in regard to the contributions of the individual Watson-Crick nearest-neighbor pairs to the Gibbs free energy of oligonucleotide duplex formation. The present work provides for the first time a theoretical approach for the positional-dependent nearest-neighbor model (PDNN) of Zhang et al.. In the in situ synthesis process of DNA microarrays random point-mutations are introduced into the microarray probe sequences. We have shown - experimentally and by means of a numerical model - that synthesis-related defects significantly affect microarray hybridization characteristics. With regard to single base mismatch discrimination, we discovered significant differences between DNA/DNA- and RNA/DNA hybridization: experimental results indicate an improved discrimination of purine-purine mismatch base pairs in RNA/DNA-duplexes. For the experimentally observed, unexpectedly high stability of Group II single bulges we provide an explanatory approach on the basis of the zipper model. The selection of appropriate (specific and sensitive) probe sequences is of crucial importance for successful application of DNA microarray technology. Our experimental results confirm previous results which show that only a small fraction (in piecewise sections about 20-30%) of a long cRNA target sequence is available for hybridization with the complementary microarray probes. Reduced binding affinities are assumed to originate from the influence of target secondary structure. Using software tools for antisense oligonucleotide design (accounting for target accessibility) we were able to predict efficient microarray probes. We discovered evidence that mechanically stable secondary structures (e.g. double-helical sections) interfere with the microarray surface (sterical hindrance) and thus result in reduced microarray binding affinities.