The efficiency of quenching via methods such as FRET is extremely sensitive to the distance between the fluorophore and the quencher (RFQ). By increasing the effectiveness of the quenching, background fluorescence is reduced and sensitivity is improved.
Duplex stability and melting temperature (Tm) are also important factors when considering probe design, since each denaturing phase requires that high temperatures are reached (95–96°C) to ensure sufficient separation of the DNA duplex. In subsequent annealing steps, the temperature must be lowered below the Tm to enable target nucleic acids and primers to re-bind. At this point, temperatures must remain high enough to avoid nonspecific hybridization.
The quencher and fluorophore are typically separated by between 20 and 30 bases, which limits quencher efficiency. In order to increase the effectiveness of the probe and decrease the RFQ, shorter oligonucleotides can be synthesized. However, these sequences will only hybridize strongly to complementary DNA at lower temperatures, which are unfavorable for qPCR assays.
Even with the use of Tm-enhancing modifications, such as the inclusion of LNA bases or minor groove binders, probe length is still usually between 14 and 18 bases. Any method that permits the placement of the fluorophore and quencher within closer proximity of one another will improve quencher efficiency and subsequently enhance the probe performance.
For this reason, Integrated DNA Technologies (IDT) has developed a double-quenched probe that is stable at higher temperatures and minimizes the occurrence of background fluorescence, enabling researchers to gain highly precise and sensitive data.