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Use better PCR probes: A second quencher lowers background, increasing signal detection

Figure 1. Internal ZEN™ Quencher Placement. (A) In traditional 5’ nuclease probes, dye and quencher are separated by 20−30 bases. (B) The internal ZEN quencher shortens the distance between 5’ dye and quencher and, in concert with the 3’ quencher, provides greater overall dye quenching, lowering background, and increasing signal detection.

The ZEN quencher is a versatile modification originally developed by IDT as an internal quencher for qPCR 5’-nuclease assay fluorescence-quenched probes (Figure 1). This quencher is placed internally between the 9th and 10th base from the reporter dye on the 5’ end of a probe sequence. A traditional probe is 20−30 bases in length with a terminal dye and quencher. The internal ZEN quencher thus shortens the distance between dye and quencher, and in combina­tion with the terminal 3’ quencher, provides a higher degree of quenching and lowers initial background (see the data at www.idtdna.com/primetime). While traditional probes are limited in length by the proximal quenching power of the quencher modification chosen, by including an internal quencher you can design longer probes, for example, when targeting AT-rich transcripts.

Although the ZEN quencher was developed for qPCR assays, it has several unique properties that make it useful for other applications. The ZEN quencher is a non-base modifier and most modifications of this type have a destabilizing effect when placed internally, between nucleotides in a sequence. However, the ZEN modification actually strengthens duplex formations. It also blocks exonuclease digestion, and is non-toxic in cell and organ culture—it appears to be non-toxic in vivo as well. These properties make the ZEN modification ideal for use in steric blocking antisense oligonucleotides such as anti-miRNA oligonucleotides (AMOs, or “antagomirs”), splice-switching oligonucleotides (SSOs), as well as RNase H active antisense oligos; it is most effective when 2 ZEN modifications are positioned at or near both ends of the oligonucleotide. The ZEN quencher can also be used on the terminal ends of DIG-labeled in situ hybridization (ISH) probes for detection of miRNAs, mRNA, and lncRNAs.

qPCR probes that include the ZEN or TAOTM quencher (dual-quenched probes) can be ordered online at www.idtdna.com. For inquiries on using the ZEN quencher in antisense or DIG/ISH applications, please contact IDT Technical Support at techsupport@idtdna.com.

See what other modifications are available from IDT. You can find a list of standard modifications we make to oligonucleotides on the Modifications page of our online catalog. And if you don’t find what you are looking for, just send us a request. We often synthesize special, non-catalog requests. Contact us at noncat@idtdna.com

Author: Jeremy Pritchard, BA, is a Technical Support Representative at IDT.

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