Forster resonance energy transfer for studying nucleic acids denaturation: A chemical and biological sciences laboratory experiment

The Förster resonance energy transfer (FRET) melting assay intends to evaluate the unfolding, denaturation process of DNA secondary structures, and its stabilization using compounds known as DNA binders, some of which are highly specific for G-quadruplex DNAs versus duplex DNAs. First, students determined the melting temperature (T_m ) of DNA sequences double labeled with 5'-FAM (fluorescein) and 3'-TAMRA (tetramethylrhodamine) in the absence of DNA binders. Second, they determined the melting temperature of the DNAs in the presence of DNA binders by monitoring fluorescence. After completing this experiment, students understood that this method allows a semiquantitative analysis to test a variety of DNA binders against DNA secondary structures, and it can be used to rapidly identify the most promising drug candidates in the drug development stages at the basic research level.