PCR and qPCR
Support and Educational Content

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

Quick facts:

Availability: DNA or RNA

Location: Internal

Scales: 100 nmol-Large Scale

Purification: HPLC required for dual-labeled probes, not for other applications

IDT Ordering Symbol: /ZEN/, placed between 2 nucleotides

ZEN Internal Quencher

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

Product focus—Oligos, oligo modifications, dsDNA fragments

Custom oligonucleotides and primers

You can order up to 1 µmol desalted, custom synthesized DNA oligonucleotides and they will be shipped to you the next business day (larger scales are shipped within 5 business days). You can also specify whether to receive them dried down or hydrated, and whether you want them already annealed. Every IDT oligonucleotide you order is deprotected and desalted to remove small molecule impurities. Your oligos are quantified twice by UV spectrophotometry to provide an accurate measure of yield. Standard oligos are also assessed by mass spectrometry for quality you can count on.

Learn more or order now.

Oligo modifications

Review a list of the common modifications IDT can add to oligonucleotides here. Not finding a modification you need on the IDT website? IDT will consider any modification you need. Just send your request to noncat@idtdna.com.

Custom dsDNA fragments

Rather than annealing oligonucleotides to obtain dsDNA fragments, when your fragment size is 125 bp or longer, it might make more sense to order gBlocks® Gene Fragments. gBlocks Gene Fragments are double-stranded, sequence-verified, DNA genomic blocks, 125–2000 bp in length, that can be shipped in 2–5 working days for affordable and easy gene construction or modification. These dsDNA fragments have been used in a wide range of applications including CRISPR-mediated genome editing, antibody research, codon optimization, mutagenesis, and aptamer expression. They can also be used for generating qPCR standards.

Learn more about gBlocks Gene Fragments at www.idtdna.com/gblocks.

Additional reading

DNA oligonucleotide resuspension and storage—You just received your newly synthesized oligonucleotides. Now what? Here are some guidelines and recommendations on how to resuspend and store your oligos.

Oligonucleotide modifications: Choosing the right mod for your needs—Learn about our broad family of oligonucleotide modifications, and get suggestions for selecting modifications that can help you in your research.

Modifications that block nuclease degradation—Are you working with your oligos in cells culture or in vivo? Find out which modifications can be added to an oligo to limit nuclease degradation.

Need a non-standard modification?—Need a modification you don’t find on our website? IDT offers 89 modifications that are not listed in our online catalog. A few of the more popular ones are described along with information on how to order them. IDT will consider any modification you have in mind. Just make a request at noncat@idtdna.com.

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

© 2013, 2016 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. MGB Eclipse is a registered trademark of Elitech Group. For specific trademark and licensing information, see www.idtdna.com/trademarks.

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