Processing
PrimeTime qPCR Probes
Generate reliable, high-quality gene expression analysis data with double- and single-quenched fluorescent hydrolysis probes.
PrimeTime™ qPCR Probes are 5′ nuclease probes, available with an assortment of reporter-dye combinations that are compatible with common qPCR instruments and that include several license-free combinations.
- Select from a wide range of license-free dyes, including FAM, Yakima Yellow®, SUN™, HEX, Cy® 3, Texas Red®-X, and Cy® 5 dyes
- Reduce background signals, even with longer probes, when using ZEN™ or TAO™ Double-Quenched Probes
- Successfully multiplex with reduced crosstalk using double-quenched probes
- Choose from the convenient 0.5–2.5 nmol packaging size for digital PCR (dPCR) platforms
- Achieve high efficiency of qPCR under fast or standard cycling conditions
- Minimize troubleshooting: PrimeTime qPCR Probes are verified by mass spectrometry* and HPLC-purified
* With the exception of mixed base oligos, which could potentially represent multiple sequences and therefore cannot be accurately evaluated by ESI mass spectrometry.
Yakima Yellow (YAK) is a registered trademark of EliTech Group, Cy is a registered trademark of Cytiva, and Texas Red is a registered trademark of Molecular Probes, Inc.
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Have questions about PrimeTime qPCR Probes?
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Choose from a wide variety of synthesis scales, dyes, and quenchers, including several license-free options. Prices and estimated turnaround times depend on the degree of complexity. For more information on dyes and quenchers, see Product details. Guides for compatible dye and quencher combinations are available in the Resources tab. For multiplex applications, consult the PrimeTime Multiplex Dye Selection tool.
PrimeTime qPCR Probes—standard level
The estimated turnaround time for standard level probes is 3–5 business days. The minimum guarantees are listed for probes that are 15–35 bases in length.
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* ZEN/Iowa Black™ FQ is a double-quenched probe, which provides superior performance compared to traditional single-quenched probes. For more information, download the PrimeTime Custom qPCR Probes Flyer.
PrimeTime qPCR Probes—complex level
The estimated turnaround time for complex level probes is 5–7 business days. The minimum guarantees are listed for probes of 15–35 bases.
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* ZEN/Iowa Black FQ is a double-quenched probe, which provides superior performance compared to traditional single-quenched probes. For more information, download the PrimeTime Custom qPCR Probes Flyer.
TEX™ fluorophore and JOE are trademarsk of ThermoFisher; TYE™ is licensed from ThermoFisher; Texas Red® is a registered trademark of Molecular Probes, Inc.
PrimeTime Mini/Eco qPCR Probes
Available with a 5′ FAM and the option of a 3′ IBFQ quencher alone or in combination with the internal ZEN quencher. Estimated time to shipment is 3–5 days.
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PrimeTime Express qPCR Probes
HPLC purified. Availabile for lengths of 18–35 bases. Shipped dry. Minimum guarantee of 5 nmol.
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* ZEN/Iowa Black™ FQ is a double-quenched probe, which provides superior performance compared to traditional single-quenched probes. For more information, download the PrimeTime Custom qPCR Probes Flyer.
Product details
Double-quenched 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 (Figure 1). This shortened distance, particularly when combined with the traditional 3′ end quencher, leads to a much 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, Double-Quenched Probes also provide consistently reduced Cq values and improved precision when compared to traditional probes. Use of Double-Quenched Probes can allow you to experience both increased sensitivity and precision in their qPCR experiments.
Figure 1. Double-quenched probe showing position of internal ZEN Quencher.
- ZEN Double-Quenched Probes: ZEN Double-Quenched Probes contain a 5′ fluorophore; 3′ Iowa Black™ FQ (IBFQ) quencher; and proprietary, internal ZEN quencher. The most commonly supplied 5′ fluorophores includes FAM, TET, Yakima Yellow, SUN, or HEX.
- TAO Double-Quenched Probes: TAO Double-Quenched Probes are available with a 5′ Cy 5 fluorophore; 3′ Iowa Black RQ (IBRQ) quencher; and proprietary, internal TAO quencher.
For more information download the PrimeTime Custom qPCR Probes Flyer.
PrimeTime Mini qPCR Probes
The smaller scale and lower price of PrimeTime Mini qPCR probes make them ideal for digital PCR (dPCR) applications, testing a new probe for gene expression or genotyping applications, or for testing the conversion to FAM/IBFQ and FAM/ZEN/IBFQ from other FAM-related quenchers. Mini probes are offered with 5′ FAM, SUN, HEX, and Cy5 fluorophores. Quenchers include 3′ IBFQ alone, double-quenched internal ZEN with 3' IBFQ, or double-quenched internal TAO with 3' IBRQ (Cy5 only).
PrimeTime Eco qPCR Probes
The PrimeTime Eco qPCR Probe is the ideal scale when you need to perform ~500 reactions for gene expression analysis or genotyping. The combination of medium scale and low cost is ideal for initial screening of large sample sets. Eco scales are also amenable to dPCR applications. Eco qPCR Probes are available as double-quenched probes with 5′ FAM, SUN, HEX, and Cy5 fluorophores. Quenchers include 3′ IBFQ alone, double-quenched internal ZEN with 3' IBFQ, or double-quenched internal TAO with 3' IBRQ (Cy5 only).
PrimeTime Express qPCR Probes
Express qPCR Probes are HPLC-purified qPCR probes that are assessed by mass spectrometry for quality control, with traces provided on our website free of charge*. Due to the turnaround time, these probes have limited dye-quencher combinations and synthesis scales.
* With the exception of mixed base oligos, which could potentially represent multiple sequences and therefore cannot be accurately evaluated by ESI mass spectrometry.
Selecting the correct reporter dye and quencher
- Reporter dyes: The correct reporter dye will depend on the type of instrument you are using and the compatibility of the dye with the instrument. Please see the instrument compatibility table for a list of reporter dyes compatible with common instrumentation. For multiplexing applications, we recommend using reporter dyes with the least amount of spectral overlap. For assistance in selecting multiplex dye combinations, use our Multiplexing Dye Selection Tool, which helps you select dyes based on what instrument you have. As an additional reference, for a list of selected IDT dyes and quenchers, please see the dye and quencher wavelength figure and the instrument compatibility table.
- Quenchers: Traditional dark quenchers absorb broadly and do not emit light, which allows for the use of multiple reporter dyes with a given quencher. This characteristic allows for expanded options for multiplex assays. Dark quenchers simplify detection, which makes them compatible with a broad range of image analysis instruments. These double-quenched probes are an improvement over traditional dual-labeled probes and have consistently low background, reduced Cq values, improved precision, and enable the use of longer probes for design in AT-rich regions. Double-quenched probes are available with a variety of fluorophores, including SUN, FAM, HEX, TET, Yakima Yellow, and Cy 5. For more information download the PrimeTime Custom qPCR Probes flyer. We supply commonly used dark quenchers as well as the proprietary dark quenchers, Iowa Black FQ, Iowa Black RQ, and the internal ZEN Quencher. TAMRA is also a quencher option for a FAM reporter dye.
- Summary tables:
Technical overview
Overview of 5′ nuclease assays
Step 1—The primers and probe hybridize in a sequence-dependent manner to the complementary DNA strand. Because the probe is intact, the fluorophore and quencher are in close proximity and the quencher absorbs fluorescence emitted by the fluorophore.
Step 2—The polymerase extends from the primers and begins DNA synthesis.
Step 3—The polymerase reaches the probe and the exonuclease activity of the polymerase cleaves the hybridized probe. As a result of cleavage, the fluorophore is separated from the quencher and fluoresces.
Step 4—The fluorescence is detected by the real time instrument.
These steps are repeated for each PCR cycle and allow detection of specific products. When using intercalating dyes, such as SYBR® Green I (Life Technologies, Inc.), primer-dimers and non-specific products will also contribute to fluorescence. In contrast, the 5′ nuclease assay is specific and fluorescence will only be detected for the DNA sequence to which the probe and primers hybridize.
Performance
Dye-quencher combinations
To demonstrate the performance of different dye-quencher combinations, we tested a dilution series and found robustness in PCR efficiency and R2 values across all dye-quencher combinations available (Figure 1).
| Dye-quencher combination | FAM / Iowa Black FQ | FAM / TAMRA | HEX / Iowa Black FQ | TET / Iowa Black FQ | Cy5 /Iowa Black RQ |
|---|---|---|---|---|---|
| Amplification curve |  |  |  |  |  |
| Standard curve |  |  |  |  |  |
| Efficiency | 95.7% | 95.1% | 94.7% | 94.5% | 98.0% |
| Correlation coefficient (R²) | >0.999 | >0.999 | >0.999 | >0.999 | >0.998 |
Figure 1. Demonstrated assay performance with multiple dye–quencher combinations. A plasmid dilution series of the CSK (c-src tyrosine kinase) Assay was used to test different dye-quencher combinations. The data illustrates robustness in PCR efficiency and R2 values close to one across all dye/quenchers available for PrimeTime Assays. All reactions were run with TaqMan® Gene Expression Master Mix (Thermo Fisher) under standard cycling conditions. The first four assays were run on the 7900 Fast Real-Time PCR System (Thermo Fisher) and the final assay (Cy5) was run on the LightCycler® 480 System (Roche).
Figure 2. PrimeTime™ SUN-labeled probes deliver comparable performance to VIC/MGB-NFQ probes. A double-quenched SUN/ZEN/IBFQ probe was compared to a VIC®/MGB-NFQ probe in a qPCR assay for GUSB and PGK1. gBlocks™ Gene Fragments with sequences identical to GUSB and PGK1 were analyzed over 6 sequential 10‑fold dilutions (102–107 copies). Normalized fluorescence values were plotted (A) including and (B) subtracting background fluorescence. Cq values were comparable, and the final R2 value for both standard curves equaled 0.999. Each reaction was run in triplicate using the IDT PrimeTime Gene Expression Master Mix, and data was generated using the QuantStudio™ 7 Flex Real-Time PCR System software (Thermo Fisher) with the following PCR cycling conditions: 10 min 95°C, 40X (15 sec 95°C, 1 min 60°C).
VIC is a registered trademark of Applied Biosystems/LifeTechnologies, Inc.
ZEN Double-Quenched Probes
Achieve maximum quenching for probes as long as 40 bases with ZEN double-quenched probes (Figure 2). Effective quenching means more effective designs, even for AT-rich targets.
Figure 3. Only ZEN Double-Quenched Probes maintain low background signal with increasing probe length. Probes of 2 lengths (20 or 40 bases) with 5 different quenchers were compared in 10 singleplex qPCRs. Six replicate reactions with each probe type were run with 50 ng of cDNA and the TaqMan Gene Expression Master Mix (Thermo Fisher) under standard cycling conditions on the Applied Biosystems 7900HT system. Key: IBFQ = Iowa Black FQ Quencher; BHQ®-1 = Black Hole Quencher®-1 (Biosearch Technologies); Eclipse® = Eclipse quencher (ELITech Group).
TAO Double-Quenched Probes
In qPCR experiments, TAO double-quenched probes exhibit decreased background (Figure 3A) and increased signals (Figure 3B) compared to single-quenched probes.
Figure 4. Increased signal detection and assay sensitivity from TAO Double-Quenched Probes. Three qPCR assays using the same primer and probe sequences were designed to amplify HPRT. The assays differed by the quenchers on the probes—the 5′ Cy 5 fluorophore was combined with either TAO and Iowa Black RQ (IBRQ) quenchers (blue, double-quenched probe), an IBRQ quencher (pink, single-quenched probe), or a Black Hole Quencher-2 (BHQ2) quencher (green, single-quenched probe). A gBlocks™ Gene Fragment (IDT) encoding HPRT sequences was used as template in a 10 µL PCR on a CFX384 instrument (BioRad) using Immolase™ DNA Polymerase (Bioline) and the following cycling conditions: 10 min. 95°C; 45 × (15 sec. 95°C, 1 min. 60°C).
Resources
User guides and protocols
Guides for fluorophore and quencher selection
Instrument compatibility tableDye and quencher wavelength chart
Brochures and flyers
Frequently asked questions
Tools
PrimerQuest™ Design Tool
Design oligos for sequencing, endpoint PCR, and qPCR. Batch design up to 50 sequences at a time. Use our optimized preset design parameters, or customize for your application. Based on the Primer 3 engine.
Multiplex Dye Selection Tool
Select dye combinations compatible with your real-time PCR instrument.
Services
Formulation
We can provide custom formulation for PrimeTime qPCR Probes. Contact Technical and Customer Support for assistance.
GMP and OEM
If you require probes that are approved for use in molecular diagnostic applications, or if you are interested in our OEM services, please click here.
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