Regensburg 2007 – scientific programme

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BP: Fachverband Biologische Physik

BP 10: DNA: supercoils, knots and melting

BP 10.5: Talk

Tuesday, March 27, 2007, 15:15–15:30, H43

DNA melting curves in a snapshot — •Philipp Baaske and Dieter Braun — Ludwig Maximilians Universität München, Center for NanoScience, Amalienstr. 54, 80799 München

We developed a new method for measuring DNA melting curves. It combines fluorescence microscopy with laser based heating of aqueous solutions. The technique allows to measure the stability of DNA in only 150ms.

A IR-laser is focused to a microfluidic chamber (thickness 20 microns) and generates a spatial temperature distribution on the lengthscale of several 100 microns. All temperatures between Tmax (e.g. 90°C) and Tmin (e.g. 20°C) are realized simultaneously. With a CCD camera two images of the fluorescence are taken: one without the laser and one with the laser switched on. From them a melting curve can be derived. The time of 150ms between the images is long enough for equilibration of the temperature and short enough to prevent thermophoresis from affecting the measurement. The state of doublestranded DNA is measured with the use of intercalating dyes and dye-quencher pairs.

In the case of doublestranded DNA the measurements are conducted in nonequlibrium because of the slow hybridization dynamics. For the fast intramolecular DNA hairpin kinetics the new technique compares well with state of the art measurements like uv absorption.

We show the possibility to discriminate single nucleotide polymorphisms (SNPs) in only 150ms, proving the use of an all optical technique for stability analysis of biomolecules.