Measuring thermodynamic details of DNA hybridization using fluorescence

Modern real-time PCR systems make it easy to monitor fluorescence while temperature is varied for hundreds of samples in parallel, permitting high-throughput studies. We employed such system to investigate melting transitions of ordered nucleic acid structures into disordered random coils. Fluorescent dye and quencher were attached to oligonucleotides in such a way that changes of fluorescence intensity with temperature indicated progression of denaturation. When fluorescence melting data were compared with traditional ultraviolet optical experiments, commonly used dye/quencher combinations, like fluorescein and tetramethylrhodamine, showed substantial discrepancies. We have therefore screened 22 commercially available fluorophores and quenchers for their ability to reliably report annealing and melting transitions. Dependence of fluorescence on temperature and pH was also investigated. The optimal performance was observed using Texas Red or ROX dyes with Iowa Black RQ or Black Hole quenchers. These labels did not alter two-state nature of duplex melting process and provided accurate melting temperatures, free energies, enthalpies, and entropies. We also suggest a new strategy for determination of DNA duplex thermodynamics where concentration of a dye-labeled strand is kept constant and its complementary strand modified with a quencher is added at increasing excess. These methodological improvements will help build predictive models of nucleic acid hybridization.