TaqMan® probes are dual labeled hydrolysis probes and are a registered trademark of the Roche Molecular Systems, Inc. TaqMan® probes utilize the 5' exonuclease activity of the enzyme
Taq Polymerase for measuring the amount
of target sequences in the samples.
TaqMan® probes consist of a 18-22
bp oligonucleotide probe which is labeled
with a reporter fluorophore at the 5'
end and a quencher fluorophore at the
While carrying out a TaqMan® experiment, a fluorogenic probe, complementary to the target sequence is added to the PCR reaction mixture. This probe is an oligonucleotide with a reporter dye attached to the 5' end and a quencher dye attached to the 3' end. Till the time the probe is not hydrolized, the quencher and the fluorophore remain in proximity to each other, separated only by the length of the probe. This proximity however, does not completely quench the flourescence of the reporter dye and a background flourescence is observed. During PCR, the probe anneals specifically between the forward and reverse primer to an internal region of the PCR product. The polymerase then carries out the extension of the primer and replicates the template to which the TaqMan® is bound. The 5' exonuclease activity of the polymerase cleaves the probe, releasing the reporter molecule away from the close vicinity of the quencher. The fluorescence intensity of the reporter dye, as a result increases. This process repeats in every cycle and does not interfere with the accumulation of PCR product.
In-silico design of TaqMan® probes design with Beacon Designer™ and AlleleID®
PREMIER Biosoft offers AlleleID® and Beacon Designer™ to design TaqMan® probes for your real time PCR assays saving you both time and money. These software products design specific TaqMan® probes and primers for your real time PCR assays that are free of dimers, repeats and runs and ensure signal fidelity.
You can also
design TaqMan® for multiplexing
up to four sequences, avoiding cross
homologies with all probes and primers
preventing competition in multiplex
reactions. Beacon Designer™ and AlleleID® help you evaluate pre-designed TaqMan® probes or design TaqMan® probes for primers.
design parameters used by Beacon Designer™ & AlleleID®
1. Tm Criteria: The primer melting temperature (Tm)
should be around 58-60 oC, and TaqMan® probe
Tm should be 10 oC higher than the Primer
Tm. The Tm of both the primers should
2. Length Criteria: Primers should be 15-30 bases in length.
3. GC Content: The
G+C content should ideally be 30-80%.
If a higher G+C content is unavoidable,
the use of high annealing and melting
temperature co-solvents such as glycerol
would be deemed essential.
4. GC Clamp: The total
number of Gs and Cs in the last five
nucleotides at the 3' end of the primer
should not exceed two. This helps to
introduce relative instability to the
3' end of primers to reduce non-specific
5. Amplicon Length: Maximum amplicon size should not exceed
400 bp (ideally 50-150 bases).
6. Runs and Repeats: The probes should not have runs of identical
nucleotides (especially four or more
consecutive Gs), G+C content should
be 30-80%, there should be more Cs than
Gs, and not a G at the 5' end.
7. Genomic DNA Avoidance: False-positive results are obtained
due to amplification of contaminating
genomic DNA. Thus,In the cDNA preparation,
it is preferable to have primers spanning