References >> TaqManTaqMan® Probes
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 3' end.
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.
TaqMan® Probe Applications
1. Quantitative real time PCR.
2. DNA copy number measurements.
3. Bacterial identification assays.
4. SNP genotyping.
5. Verification of microarray results.
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.
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 be equal.
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 priming.
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 exon-exon junctions.