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IDT Launches Double-Quenched Probes™ to Increase qPCR Sensitivity
Read time: Less than a minute
Integrated DNA Technologies (IDT) has introduced a new double-quenched probe, which increases the accuracy and reliability of 5’ nuclease qPCR experiments. While traditional probes have 20-30 bases between the dye and quencher, this novel proprietary probe design positions an internal ZEN quencher only 9 bases from the 5’ fluorophore.
This shortened distance, particularly when combined with the standard 3’ quencher, decreases background fluorescence and increases sensitivity. The chemical structure of the ZEN quencher stabilizes duplex formation which allows for its use in previously validated sequences. The improved functionality significantly increases qPCR accuracy and sensitivity when compared to traditional probes.
Due to the incorporation of ZEN at a fixed position 9 bases from the 5’ end, the quencher is always within close proximity of the probe. As such, the initial background fluorescence signal is much lower.
In addition, while traditional probes do not remain well quenched over 30 base pairs, the double-quenched probes maintain a consistently low background even at 40 base pairs or longer. Thus, double-quenched probes result in lower Cq values when compared to traditional probes. This leads to an increase in specificity without any loss in sensitivity or quenching efficiency. This is particularly useful for targets that are AT-rich and require longer probes.
IDT offers a complete range of standard and custom qPCR products including a free online RealTime PCR design tool which optimizes primer and probe sequence for specific user requirements.
This shortened distance, particularly when combined with the standard 3’ quencher, decreases background fluorescence and increases sensitivity. The chemical structure of the ZEN quencher stabilizes duplex formation which allows for its use in previously validated sequences. The improved functionality significantly increases qPCR accuracy and sensitivity when compared to traditional probes.
Due to the incorporation of ZEN at a fixed position 9 bases from the 5’ end, the quencher is always within close proximity of the probe. As such, the initial background fluorescence signal is much lower.
In addition, while traditional probes do not remain well quenched over 30 base pairs, the double-quenched probes maintain a consistently low background even at 40 base pairs or longer. Thus, double-quenched probes result in lower Cq values when compared to traditional probes. This leads to an increase in specificity without any loss in sensitivity or quenching efficiency. This is particularly useful for targets that are AT-rich and require longer probes.
IDT offers a complete range of standard and custom qPCR products including a free online RealTime PCR design tool which optimizes primer and probe sequence for specific user requirements.
