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• More sensitive Detection
• Better reproducibility (especially limits of detection)
• More fluorogenic than Taqman or Molecular Beacon probes
• Better signal to noise ratio than Taqman
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 fluorogenic. 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 because the dye and quencher are specially far apart at each end of the probe.
The probe 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 PerfectProbe™.
The figure below shows Identical forward and reverse primers for the asthma-associated alternatively spliced gene 1 were used in conjunction with different reporter probes. The Taqman® and PerfectProbe had identical target annealing sequences whilst the Molecular Beacon® was shorter but fell within the same target region. Consistent with the diagrams above, the PerfectProbe gave a background level similar to the Molecular Beacon®, but had an endpoint fluorescence similar to the Taqman® probe.
Asthma associated gene (AAA1), primary data
Ashma associated gene (AAA1), baseline corrected data. Linear plot
(The instrument software performs a simple arithmetic baseline subtraction)
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