Determination of DNA melting temperatures in diffusion-generated chemical gradients.
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.
Liedl
Tim
T
Department Physik and Center for Nanoscience, LMU Munich, Geschwister-Scholl-Platz 1, 80539 Munich, Germany. tim.liedl@physik.uni-muenchen.de
Simmel
Friedrich C
FC
D004247:DNA / Q000737:chemistry*; D004058:Diffusion; D005559:Formamides / Q000737:chemistry; D009691:Nucleic Acid Denaturation; D013050:Spectrometry, Fluorescence / Q000379:methods*; D044366:Transition Temperature
D005559:Formamides; C031066:formamide; D004247:DNA