Effects of fluorescent dyes, quenchers, and dangling ends on DNA duplex stability

Single and dual-labeled fluorescent oligodeoxynucleotides are used in many molecular biology applications. We investigated the effects of commonly used fluorescent dyes and quenchers on the thermodynamic stability of a model probe-target DNA duplex. We demonstrate that those effects can be significant. Fluorescent dyes and quenchers were attached to the probe ends. In certain combinations, these groups stabilized the duplex up to 1.8 kcal/mol and increased Tm up to 4.3 °C. None of the groups tested significantly destabilized the duplex. Rank order of potency was, starting with the most stabilizing group: Iowa Black RQ ∼ Black Hole 2 > Cy5 ∼ Cy3 > Black Hole 1 > QSY7 ∼ Iowa Black FQ > Texas Red ∼ TAMRA > FAM ∼ HEX ∼ Dabcyl > TET. Longer linkers decreased stabilizing effects. Hybridizations to targets with various dangling ends were also studied and were found to have only minor effects on thermodynamic stability. Depending on the dye/quencher combination employed, it can be important to include thermodynamic contributions from fluorophore and quencher when designing oligonucleotide probe assays.