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Considerations when adopting published sequences for your own qPCR assay

Increase sensitivity by converting probe sequences to double-quenched ZEN™ or TAO™ probes

Researchers often find probe and primer sequences for a specific qPCR assay in the literature, which they want to use for their own experiments. And why not? These sequences have already been vetted by other scientists and used successfully to generate publishable data.

Check published sequences against current sequence databases

However, before you order assays published in the literature, consider whether they were based on outdated sequence information. With the increasing amount of cataloged sequencing data, there has been a concomitant increase in the number of identified single nucleotide polymorphisms (SNPs) and updated transcript structure and annotation. While the frequency of SNP occurrence may make them unrealistic to avoid when choosing PCR primer and probe sequences (the human genome contains a SNP approximately once every 22 bases), it is important to take their position into consideration. See Considering SNPs when designing PCR and qPCR assays, for data on SNP frequency, and a discussion of the impact SNPs can have on PCR and qPCR assay results. The article also provides recommendations for managing SNP impact on your assay results.

An alternative to published sequences

A quick search of the IDT PrimeTime® Predesigned qPCR Assay database provides assays that cover the human, rat, and mouse transcriptomes that are guaranteed to perform with PCR efficiencies between 90–110% and R2 >0.99. The database design engine uses complete target sequence information that is updated regularly.

Convert literature-derived probe sequences to more sensitive, double-quenched probes

Converting literature-derived probe sequences to double-quenched probes that contain an internal quencher has become popular over the past few years, due to the lower background fluorescence, increased signal, and reduced crosstalk double-quenched probes afford. This adaptation is easily done. IDT provides 2 types of internal quenchers, ZEN™ and TAO™ Internal Quenchers (see sidebar, Double-quenched qPCR probes), that are compatible with a wide range of 5' fluorescent dyes and 3' quenchers.

Double-quenched qPCR probes

While traditional probes have approximately 20–30 bases between the fluorophore and the quencher, the internal ZEN™ or TAO™ quencher decreases that length to only 9 bases. This shortened distance, particularly when combined with the traditional 3' end quencher, leads to more thorough quenching with much lower background and enables the use of much longer probes for designing in AT-rich target regions. In addition to the significantly decreased background, the Double-Quenched Probes also have consistently reduced Cq values and improved precision when compared to traditional probes. Use of the Double-Quenched Probes can allow users to experience both increased sensitivity and precision in their qPCR experiments.

Figure 1. Positioning of internal quencher within a probe sequence.

Evaluate your probe sequence

Adding ZEN or TAO Internal Quenchers into standard hydrolysis probe sequences with 5' fluorescent dyes and 3' quenchers is straightforward and will not appreciably affect probe Tm. However, note that probes from the literature containing MGB (or other Tm boosting modifiers) will have a reduced Tm when ordered without these modifications. Therefore, before ordering, it is a good idea to assess whether the published probe sequence has the ideal Tm to function optimally in your assays.

Use these steps to evaluate the published probe sequence before you order:

  1. Check the Tm of the probe sequence. If the probe Tm is not provided in the literature reference, use our free OligoAnalyzer Tool to calculate it. We recommend that the probe Tm be at least 4°C higher than primer Tms. Note that MGB (or other Tm boosting modified) probes from the literature will have a reduced Tm when ordered without these modifications.
  2. Determine whether you can modify your probe sequence. If the probe vs primer Tms do not meet the above criteria, you may need to either change the position of your assay or modify the probe sequence. Consider a predesigned assay for that exon location as the best primer probe combination is chosen (see the above sidebar, An alternative to published sequences). If the location of your probe cannot be moved, here are a few ways to modify your assay so that probe and primers will work optimally together during PCR.
    • Change the length or position of your probe and/or primers so that primer and probe Tms meet the criteria in step 1 above.
    • Increase the Tm of a short probe by adding bases onto the 5' or 3' end.  Use the OligoAnalyzer Tool to assess Tm, and to rule out dimer formation between probe and primers, as well as secondary structure formation resulting from presence of the added bases.
    • If you do not want to modify your probe sequence, another way to increase probe Tm is to replace 2–4 bases spaced throughout the sequence with LNA bases. LNA (locked nucleic acid) bases are extremely stable and will raise probe Tm. Our Application Support Group can provide guidelines on where in the probe sequence to integrate LNA bases. Contact them at applicationsupport@idtdna.com. Note that LNA probes are not available as double-quenched probes; order them as single quenched probes at LNA PrimeTime Probes.

Order the published probe sequence as a double-quenched probe

Now that you have confirmed your primer and probe sequences are up to date, and determined that the Tm of the probe sequence is optimal for use in your assay, convert a probe of your own design or one from the literature to one containing a ZEN or TAO Internal Quencher. Go to www.idtdna.com/site/order/qpcr/primetimeprobes (Figure 2), and follow these steps to order the double-quenched version of the probe (to enter complete assays of both primer and probe sequences, go to www.idtdna.com/order/qpcrentry.aspx for a similar screen, and follow these same steps):  

Step 1. Provide a name for this sequence entry. (See the Step 1 blue arrow in Figure 2.)

Step 2. Select a synthesis scale. (See the Step 2 blue arrow in Figure 2.)

Step 3. Enter your sequence (e.g., copy and paste), without modifications. (See the Step 3 blue arrow in Figure 2.)

Step 4. Choose your 5' dye + 3' quencher combination from the dropdown list. (See the Step 4 blue arrow in Figure 2.) The dropdown list of modifications is shown in Figure 3. Note that with the 5’ dye choice, a compatible ZEN or TAO internal quencher will be automatically integrated into the appropriate position in your probe sequence.

Note that LNA probes are not available as double-quenched probes; order them as single quenched probes at LNA PrimeTime Probes.

Figure 2. Order probes through the manual entry form. Access this form at: www.idtdna.com/site/order/qpcr/primetimeprobes.

Figure 3. ZEN™ and TAO™ Internal Quenchers are compatible with range of 5' fluorescent dyes.

Checklist for adopting published primer and probe sequences for qPCR assays

  • Check primer and probe sequences against current sequence databases
  • Evaluate Tm of probe
  • Modify probe to adjust Tm, if needed
  • Add ZEN™ or TAO™ Internal Quencher to probe

Further questions about probe design and use of double-quenched probes?

View a list of citations describing research done by your colleagues, who are already successfully using double-quenched probes. IDT also makes available numerous DECODED newsletter articles that give tips for conducting more effective PCR experiments, and provide overviews of the latest PCR techniques and applications. Review other DECODED Online newsletter articles on PCR and qPCR applications.

And you can always contact our Scientific Applications Specialists, who can help you design your probes and assays. Reach them at applicationsupport@idtdna.com.

Product focus: Assays, probes, and tools for qPCR and PCR

PrimeTime® qPCR Assays

  • 5′ nuclease, probe-based assays—the gold standard for quantitative gene expression studies

  • Primer-based assays—designed for intercalating dye experiments

Create custom assays that are designed using our proprietary bioinformatics algorithms for any target and to your specific parameters. Alternatively, select one of our predesigned assays for human, mouse, and rat mRNA targets that are supported by our bioinformatics algorithms and up-to-date sequence information.

Learn more at www.idtdna.com/PrimeTime. For assistance with assay design, contact our scientific application specialists applicationsupport@idtdna.com.

Double-Quenched Probes

ZEN™ and TAO™ Double-Quenched Probes have a 5′ fluorophore, an internal quencher (ZEN or TAO quencher), and Iowa Black FQ as the 3′ quencher. These probes provide consistently earlier Cq values and improved precision, when compared to traditional, single-quenched qPCR probes.

Learn more at www.idtdna.com/qPCRprobes.

Free tools for qPCR and PCR assay design

Explore IDT free, online tools for qPCR primers and probes design and analysis. The design engines for these tools use sophisticated formulas that, for example, take into account nearest neighbor analysis to calculate Tm, and provide the very best qPCR assay designs.

Additional reading

Considering SNPs when designing PCR and qPCR assays—NGS has led to a dramatic increase in identified SNPs. SNPs can pose a problem when they underlie primer or probe sequences used in PCR/qPCR. Learn what effect they can have and how you can minimize their impact on your PCR assays.

Design efficient PCR and qPCR primers and probes using online tools
—Simplify planning of your qPCR experiments using IDT free, online tools for oligonucleotide analysis and PCR primer design. This article provides an overview of our predesigned qPCR assays and the basics of designing customized PCR primers and hydrolysis probes with the PrimerQuest® Tool.

Could your PCR be affected by contamination?—Learn how to prevent false amplification from DNA contamination.

Decrease qPCR background, improve qPCR signal—See data demonstrating that probes containing a second, internal quencher provide increased signal detection and greater assay sensitivity in qPCR assays vs. single-quenched probes, such as probes with BHQ Quenchers. ZEN™ and TAO™ Double-Quenched Probes also make it more feasible to use longer probes due to the resulting lower background fluorescence.

Review other DECODED Online newsletter articles on PCR and qPCR applications.

You can also browse our DECODED Online newsletter for additional application reviews, lab tips, and citation summaries to facilitate your research.

Author: Ellen Prediger, PhD, is a senior scientific writer at IDT.

© 2017 Integrated DNA Technologies. All rights reserved. Trademarks contained herein are the property of Integrated DNA Technologies, Inc. or their respective owners. For specific trademark and licensing information, see www.idtdna.com/trademarks.

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