Stability of a surface-bound oligonucleotide duplex inferred from molecular dynamics: a study of single nucleotide defects using DNA microarrays.

Microarray technology uses the sequence dependent hybridization (binding) affinity of surface-bound oligonucleotide strands for the quantification of complex nucleic acid mixtures. In spite of its huge potential in life science and medicine, microarray oligonucleotide hybridization remains far from being understood. Taking advantage of microarray combinatorial possibilities we show that, although surface bound, the hybridization affinities of single-base mismatched oligonucleotides can be derived from first principles using parameters from bulk.

Position dependent mismatch discrimination on DNA microarrays - experiments and model.

Background: The propensity of oligonucleotide strands to form stable duplexes with complementary sequences is fundamental to a variety of biological and biotechnological processes as various as microRNA signalling, microarray hybridization and PCR. Yet our understanding of oligonucleotide hybridization, in particular in presence of surfaces, is rather limited. Here we use oligonucleotide microarrays made in-house by optically controlled DNA synthesis to produce probe sets comprising all possible single base mismatches and base bulges for each of 20 sequence motifs under study.

Impact of point-mutations on the hybridization affinity of surface-bound DNA/DNA and RNA/DNA oligonucleotide-duplexes: comparison of single base mismatches and base bulges.

Background: The high binding specificity of short 10 to 30 mer oligonucleotide probes enables single base mismatch (MM) discrimination and thus provides the basis for genotyping and resequencing microarray applications. Recent experiments indicate that the underlying principles governing DNA microarray hybridization - and in particular MM discrimination - are not completely understood. Microarrays usually address complex mixtures of DNA targets.

Optical study of DNA surface hybridization reveals DNA surface density as a key parameter for microarray hybridization kinetics.

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22 mer strand (bound-DNA) is immobilized via phenylene-diisothiocyanate linker molecule on the substrate, the dye-labeled (Cy3) complementary strand (free-DNA) is in solution in a reaction chamber. We use total internal reflection fluorescence for surface detection of hybridization. As a new feature we perform a simultaneous real-time measurement of the change of free-DNA concentration in bulk parallel to the total internal reflection fluorescence measurement.