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TaqMan qPCR is real-time PCR that reads out through a hydrolysis probe: a short oligonucleotide carrying a reporter dye at one end and a quencher at the other, which the polymerase cuts during amplification so that fluorescence rises as product is made. Because the signal depends on the probe binding its target, it differs from a dye that reports any double-stranded DNA. This page explains how the probe works, which TaqMan assay format answers which question, and what a lab should record when it orders or reports a probe assay.
Definition and scope
“TaqMan” is the name of a family of probe-based real-time PCR chemistries, and “probe qPCR” is the general term for the same hydrolysis-probe approach. Each probe has a reporter dye on its 5′ end and a quencher on its 3′ end. While the probe is intact, the quencher suppresses the reporter (Thermo Fisher, How TaqMan assays work). The physics behind this is distance dependence: quenching in this kind of dual-labelled probe depends on the sixth power of the distance between the two fluorophores (Patterson et al., 1996, Nucleic Acids Research 24(18):3656-3658).
This page covers the concept and the record-keeping around it. It is not a bench protocol. For setting up a reaction, follow the instructions for your master mix and instrument; the general reaction set-up is in PCR protocol basics, and the wider real-time PCR context is in the qPCR and RT-qPCR guide.
How a TaqMan assay works in practice
A standard gene expression assay uses a pair of unlabelled primers plus one probe, labelled with FAM or VIC at the 5′ end and carrying an MGB group and a non-fluorescent quencher (NFQ) at the 3′ end (Thermo Fisher, How TaqMan assays work). Across a cycle:
- Denaturation: the probe is free in solution and its signal stays quenched.
- Annealing: the primers and the probe bind their complementary target sequence.
- Extension: the Taq polymerase 5′ nuclease activity cleaves the bound probe, separating the dye from the quencher. Fluorescence increases in proportion to the amount of amplicon synthesised.
The two modifications on the 3′ end do different jobs. The minor groove binder (MGB) increases the melting temperature without increasing probe length, which allows shorter probes and widens the melting-temperature difference between a matched and a mismatched probe. The non-fluorescent quencher improves spectral resolution when several dyes are used together (Thermo Fisher, How TaqMan assays work).
“qPCR protocol TaqMan”: where the numbers come from
Cycling temperatures, times, primer and probe concentrations are set by the master-mix and instrument manufacturers, so they are deliberately not listed here. Take them from the instructions supplied with your reagents and confirm them against your own validation data, such as a standard curve. Hydrolysis probes also produce no melt curve, so melt-based checks do not apply; melt curve analysis explains why.
TaqMan assay formats and what each one answers
The same probe chemistry is packaged for different questions. The table below is limited to what the manufacturer’s explanation states.
| Question | What the assay contains | How the result is read |
|---|---|---|
| Gene expression | Two unlabelled primers and one FAM- or VIC-labelled MGB/NFQ probe | Fluorescence rises as the target amplicon is synthesised |
| Genotyping (SNP) | Two probes, one FAM and one VIC, each with MGB and NFQ | Only the probe matching the target allele is cleaved, so the dye that increases indicates the allele |
| Copy number | A Copy Number Assay and a Reference Assay run together in the same reaction | Relative quantitation against a reference sequence present in two copies per diploid genome |
| microRNA | Advanced miRNA assays, which need cDNA synthesis first | Quantified by qPCR after the cDNA step |
| Rare mutation detection (castPCR) | Allele-specific TaqMan qPCR plus an MGB blocker | Mutation level estimated from the delta Ct |
If your question is absolute copy number without a standard curve, a different method may fit better; see digital PCR vs qPCR.
What to record when ordering and reporting a probe assay
Probe assays are often bought as pre-designed products whose sequences the vendor may not release. The MIQE 2.0 update (Clinical Chemistry 71(6):634-651) sets out the reporting expectations that matter for a lab ordering and documenting them. The original MIQE guidelines (Bustin et al., 2009, Clinical Chemistry 55(4):611-622) state that full disclosure of all reagents, sequences and analysis methods is necessary.
- Probe design: reporter dye, quencher and any MGB modification, as listed on the assay documentation.
- Sequences: disclosure of primer and probe sequences is “highly desirable and strongly encouraged” in MIQE 2.0. Where a commercial pre-designed assay vendor does not release them, the assay context sequence must be submitted.
- Supplier details: manufacturer, catalogue number and batch number.
- Revalidation triggers: MIQE 2.0 states that optimisation and validation of assays must be repeated when changing kits or suppliers, and that a change of reagent batch should be verified.
- Performance figures: limit of detection and lower limit of quantification, dynamic range, and prediction intervals.
A fuller reporting checklist is in the MIQE reporting checklist, and normalisation choices are covered in reference gene selection and validation. The instrument side is described in what is a thermal cycler.
Common misunderstandings
“I can run a melt curve to check my TaqMan assay.”
No. The signal comes from probe cleavage, not from a double-stranded DNA dye, so there is no melt curve to read for a hydrolysis probe assay.
“Ct and Cq are different things.”
MIQE 2.0 defines Cq as the quantification cycle, formerly known as Ct or Cp. It also says Cq values should be converted into efficiency-corrected target quantities, and that statistics should not be run on raw Cq values.
“A shorter probe must bind more weakly.”
Not for MGB probes. The MGB group increases the melting temperature without increasing probe length, which is what allows shorter probes (Thermo Fisher, How TaqMan assays work).
“The same assay from another supplier is interchangeable.”
MIQE 2.0 says assays must be re-optimised and re-validated when kits or suppliers change.
FAQ
What is a TaqMan qPCR probe?
A hydrolysis probe with a reporter dye on the 5′ end and a quencher on the 3′ end. Polymerase 5′ nuclease activity cleaves it when it is bound to the target, releasing fluorescence.
What is the difference between probe qPCR and dye-based qPCR?
Probe qPCR needs a labelled probe to bind and be cleaved before a signal is produced, which also means no melt curve. The chemistry comparison is in the qPCR guide.
Which dyes do TaqMan gene expression probes use?
FAM or VIC at the 5′ end, with MGB and a non-fluorescent quencher at the 3′ end (Thermo Fisher, How TaqMan assays work).
Do I have to publish probe sequences?
MIQE 2.0 strongly encourages it; if a vendor will not release them, the assay context sequence must be submitted, together with manufacturer, catalogue and batch numbers.
Sources
- Thermo Fisher Scientific, How TaqMan assays work, checked 2026-10-29.
- MIQE 2.0, Clinical Chemistry 71(6):634-651, doi:10.1093/clinchem/hvaf043, checked 2026-10-29.
- Bustin SA et al., The MIQE guidelines, Clinical Chemistry 2009;55(4):611-622, doi:10.1373/clinchem.2008.112797, checked 2026-10-29.
- Patterson GH et al., Nucleic Acids Research 1996;24(18):3656-3658, doi:10.1093/nar/24.18.3656, checked 2026-10-29.
