EvaGreen® (EG) has proved to be the most versatile of the nucleic acid binding dyes. It is a DNA intercalating dye with a novel ‘release-on-demand’ binding mechanism and several characteristics that make it a superior choice for a broad range of applications including qPCR, high resolution DNA melt curve analysis (HRM), routine DNA quantification, digital PCR and capillary gel electrophoresis (CE) (as a gel stain).
In a recent publication, Shoute and Lappnow further explore the binding interactions of EG with dsDNA, calculate the binding constant and propose a model. Their model shows that EG intercalates cooperatively into the dsDNA duplex with a gap of 1 intercalation site between EG binding sites, as expected for a bifunctional molecule. Interestingly, at high [EG]/[base pair] ratios, this intercalation is disrupted, resulting in the formation of anti-parallel stacked chains of EG that bind externally to the duplex DNA.