(550d) Real Time Detection of DNA Hybridization and Melting On Spotted Oligonucleotide Arrays by Total Internal Reflection Fluorescence

Complementary base pair interactions between DNA strands form the basis for many methods of DNA detection and identification. DNA microarrays allow the parallel investigation of the ability of a particular sequence to bind to libraries of differing DNA sequences. In most cases, such measurements of DNA-DNA interactions focus on single time results, typically at one temperature, that are performed ex situ. As a result, kinetic and thermodynamic information on the factors that influence DNA binding at surfaces are limited. Our laboratory uses an in situ method for detecting nucleic acid hybridization and melting events in real time at arrayed oligonucleotide surfaces. These measurements are made at the solid-liquid interface using total internal reflection fluorescence (TIRF) imaging. The arrays are constructed by covalently end-immobilizing single-stranded DNA molecules of chosen sequences onto microscopic glass slide surfaces. We have investigated the influences of target DNA concentrations, nucleotide mismatches, and ionic strength on the hybridization kinetics, and used a mathematical model that includes the effects of diffusion and solution-phase depletion in order to obtain estimates for the hybridization rate constants on the surface from our experimental data. The stability of and interactions between DNA duplexes are investigated by measurements of melting curves for hybridized structures formed to the microarrayed surface. TIRF imaging of the microarray through programmed temperature profiles allows direct, concurrent measurements for DNA-DNA interactions. The resulting melting curves of the surface oligos are compared with those predicted from solution phase melting models, showing that single-base mutations are detectable by this in situ method. The talk will examine the abilities of this approach in providing enhanced detection abilities of single- and multiple nucleotide mutations from known gene sequences.