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PerfectProbe (Orange) Characteristics compared to Taqman (Blue)
- More sensitive detection (earlier CT values)
- Better reproducibility (especially at the limits of detection)
- More fluorogenic than Taqman and Molecular Beacon Probes
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Higher signal to noise ration than Taqman
Custom Real time PCR assays based on the PerfectProbe are available on demand for any human mouse or rat genes
Fig.1 Primers for the human YWHAZ gene were tested with a PerfectProbe (Orange) or Taqman probe (Blue)
What do our customers say?
"....The PerfectProbe technology has greatly improved both replicate reproducibility and the quality of the traces. After working with PerfectProbes I wouldnt go back to using Taqman Probes...."Dr C Boxall. Synairgen Research Plc. Drug discovery company UK .
How do PerfectProbes work?
What are the characteristics of the perfect reporter probe for real-time PCR? Clearly it would have optimal quenching properties and also be very flourogenic. The hairpin structure of Molecular beacons probes leads to very efficient quenching, but this probe is not cleaved and hence the single strengh is low. By contract, the Taqman probe is cleaved but it has a high back ground becuase the dye and quencher are specially far apart at each end of the probe.
We have produce a probe that incorporates the best feature of both systems has is it would have a secondary structure that reduced the spatial separation of quencher and dye leading to a lower background fluorescence. Furthermore it would retain the high fluorogenic potential of a hydrolysis probe (Fig.1). We have designed and validated a probe that is based on this concept. The result is a reporter system with greatly improved signal to noise ratios and detection of amplification at earlier cycle numbers. We have called our probe the PerfectProbeTM.
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Pre amplification Hybridised Post extension |
| TaqmanŽ |
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| Molecular BeaconŽ |
| PerfectProbeTM | *Fluorescence is measured during this stage
Supporting Data
Fig.2a Identical forward and reverse primers for the asthma-associated alternatively spliced gene 1 were used in conjunction with different reporter probes. The TaqmanŽ and PerfectProbe had identical target annealing sequences whilst the Molecular BeaconŽ was shorter but fell within the same target region. Consistent with the diagrams above, the PerfectProbe gave a background level similar to the Molecular BeaconŽ, but had an endpoint fluorescence similar to the TaqmanŽ probe.
Fig.2 Asthma associated gene (AAA1), primary data
Fig.2b The Icycler performs a simple arithmetic baseline subtraction.
Asthma associated gene (AAA1), baseline corrected data, linear plot
Fig.3 Relative quantification using the above assays was performed on a range of hardware platforms. In general the PerfectProbes were between 3 and 5 fold more sensitive. The Rotorgene is atypical in that it derives amplification plots from the signal to noise ratio. Probes with a low background noise are therefore much more sensitive on this system than the TaqmanŽ probe.
Icycler IQ5 (Bio-Rad) Light Cycler (Roche) Rotorgene (Corbett)
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