Melting curve analysis in a snapshot

The thermal denaturation of molecules is an essential method in biochemistry and diagnostics, including the measurement of single nucleotide polymorphisms and the binding analysis of proteins. We present a method for the all-optical high speed measurement of melting curves. A thin sheet of water is locally heated with an infrared laser to obtain a spatial temperature distribution between 20 and 100°C. Using a fluorescence microscope a melting curve is recorded within 50ms. This is about 10 000-times faster than state-of-the-art fluorometry and yields the same results for the validation example of a DNA hairpin.

[1]  Michael Zuker,et al.  Mfold web server for nucleic acid folding and hybridization prediction , 2003, Nucleic Acids Res..

[2]  P. Doty,et al.  Determination of the base composition of deoxyribonucleic acid from its thermal denaturation temperature. , 1962, Journal of molecular biology.

[3]  F. Simmel,et al.  Determination of DNA melting temperatures in diffusion-generated chemical gradients. , 2007, Analytical chemistry.

[4]  Dieter Braun,et al.  Extreme accumulation of nucleotides in simulated hydrothermal pore systems , 2007, Proceedings of the National Academy of Sciences.

[5]  C. Wittwer,et al.  Solution-phase DNA mutation scanning and SNP genotyping by nanoliter melting analysis , 2007, Biomedical microdevices.

[6]  K. Voelkerding,et al.  Amplicon DNA melting analysis for mutation scanning and genotyping: cross-platform comparison of instruments and dyes. , 2006, Clinical chemistry.

[7]  Dieter Braun,et al.  Why molecules move along a temperature gradient , 2006, Proceedings of the National Academy of Sciences.

[8]  M. Shriver,et al.  Melting curve analysis of SNPs (McSNP): a gel-free and inexpensive approach for SNP genotyping. , 2001, BioTechniques.

[9]  Dieter Braun,et al.  Thermophoretic depletion follows Boltzmann distribution. , 2006, Physical review letters.

[10]  S. Arduini,et al.  Thermophoresis of DNA determined by microfluidic fluorescence , 2004, The European physical journal. E, Soft matter.

[11]  F. Kramer,et al.  Thermodynamic basis of the enhanced specificity of structured DNA probes. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[12]  Sanjay Tyagi,et al.  Molecular Beacons: Probes that Fluoresce upon Hybridization , 1996, Nature Biotechnology.

[13]  A Libchaber,et al.  Kinetics of conformational fluctuations in DNA hairpin-loops. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[14]  J. Mergny,et al.  Analysis of thermal melting curves. , 2003, Oligonucleotides.