Thiazole Orange as fluorescent marker for the detection and quantification of dsDNA

Thiazole Orange (TO) is a DNA intercalating dye with the potential application for the detection and quantification of double-stranded DNA (dsDNA) during polymerase chain reaction (PCR) amplification. This is because TO emits minimal fluorescence when in aqueous solution but emits nearly 20,000 times more fluorescence when bound to dsDNA. The literature describes high affinity constants for the complexation of TO with dsDNA, as well as studies on the selectivity of TO to evaluate its potential as an intercalator. In general, the affinity of an intercalator, a chemical species of optimal size and nature to intercalate between DNA base pairs, for dsDNA can vary depending on experimental conditions, especially the charge and concentration of the cation present in the solution. In this study, we performed steady-state fluorescence spectroscopy and fluorescence lifetime measurements to study the interaction between TO and dsDNA and analyze the effects of cation concentration and charge on the binding constant. The results obtained show significantly lower affinity constants in the presence of MgCl2 compared to NaCl, as well as an increase in these constants when the ionic strength of the medium decreases. We also studied the possible effect of dsDNA chain size and sequence on the affinity constant, using short hairpin-like chains with predominant AT and GC sequences and longer chains with various sequences of Lambda-DNA. Furthermore, we investigated the application of Thiazole Orange for the detection and quantification of DNA in Loop-mediated isothermal amplification (LAMP) assays, comparing it to a commercial dye.