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BP: Fachverband Biologische Physik

BP 21: High-Throughput Data and their Analysis

BP 21.2: Vortrag

Mittwoch, 28. März 2007, 18:30–18:45, H43

Optical Study of DNA surface hybridization reveals DNA surface density as a key parameter for interpretation of microarray data — •Wolfgang Michel, Timo Mai, Thomas Naiser, and Albrecht Ott — Experimental Physics 1, University Bayreuth, Germany

We investigate the kinetics of DNA hybridization reactions on glass substrates, where one 22mer 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 (TIRF) 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 TIRF measurement. We observe that the free-DNA concentration decreases considerably during hybridization. We show how the standard Langmuir kinetics needs to be extended to take into account the change in bulk concentration and explain our experimental results. Connecting both measurements we can estimate the surface density of accessible, immobilized bound-DNA. We observe that the fluorescent signal from the surface ceases to be proportional to the number of dye-labeled molecules on the surface for surface-densities of hybridized molecules above 5*10^11 molecules/cm^2. We discuss the implications with respect to DNA microarray detection.