For researchers performing qPCR using 5’ nuclease assays, balancing signal-to-noise and probe specificity can be a challenge. Designing probes in AT-rich regions requires longer probes to be specific, but choosing probe length can pose a dilemma. Longer probes provide higher Tms, allowing for the use of higher melting temperatures, which can prevent nonspecific probe:target hybridization. However, as probe length increases, the distance between the fluorophore reporter and quencher at the probe end increases, resulting in reduced quenching and thus, poor signal to background discrimination.
Until recently, Tm enhancers (such as Locked Nucleic Acid [LNA] bases and Minor Groove Binder [MGB]), or internal quenchers attached to thymidine bases have been used to design properly quenched, short probes for qPCR. When incorporated into the probe, Locked Nucleic Acid (LNA) bases and Minor Groove Binder (MGB) modifications alter DNA structure and increase probe stability in duplex formation, increasing probe melting temperature. Thus, shorter probes with high melting temperatures and good quenching can be designed. However, these types of modifications are not only more costly, but also extremely challenging to design as melting temperature prediction algorithms for such modifications are not widely known. Also, internal quenchers attached to thymidine bases require the presence of thymidine residues within the probe sequence.
ZEN Double-Quenched Probes obviate the need for LNAs, MGB, or internal quenchers attached to thymidine bases. ZEN quencher is an internal quencher that is placed directly between DNA bases and is used in addition to a 3’ quencher. It is incorporated at a fixed posistion, 9 bases from the 5’ end, which ensures that this quencher is always in close proximity to the fluorophore. This shortened distance, combined with the presence of the standard 3’ quencher, allows the design of longer probes with sufficient melting temperature for qPCR, without diminishing quencher efficacy. In fact, while traditional probes do not remain well quenched over 30 bp, ZEN Double-Quenched Probes maintain a consistently low background even when longer than 40 bp.
ZEN Double-Quenched Probes can be coupled with several different fluorescent dyes. See the PrimeTime Custom qPCR Probes flyer for details.