title: Determination of DNA melting temperatures in diffusion-generated chemical gradients.
abstract: For fast and reliable determination of DNA melting temperatures with single-nucleotide resolution in a microfluidic setup, stable gradients of the denaturing agent formamide were generated by means of diffusion. Formamide lowers the melting temperature of DNA, and a given formamide concentration can be mapped to a corresponding virtual temperature along the formamide gradient. We applied this concept to determine the melting temperatures of five sets of dye- and quencher-labeled oligonucleotides of different lengths. Differences in the length of complementary sequences of only one nucleotide as well as a single nucleotide mismatch can be detected with this method. Comparison with conventional melting temperature measurements based on temperature scans yields very good agreement.
authors.affiliation: Department Physik and Center for Nanoscience, LMU Munich, Geschwister-Scholl-Platz 1, 80539 Munich, Germany. tim.liedl@physik.uni-muenchen.de
authors.forename: Tim
authors.initials: T
authors.lastname: Liedl
authors.forename: Friedrich C
authors.initials: FC
authors.lastname: Simmel
mesh_terms: D004247:DNA / Q000737:chemistry*; D004058:Diffusion; D005559:Formamides / Q000737:chemistry; D009691:Nucleic Acid Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D044366:Transition Temperature
chemical_list: D005559:Formamides; C031066:formamide; D004247:DNA