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Mediator Probe PCR

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Want a PCR method that gives you high sensitivity and is cost-effective?

Then you might want to try Mediator Probe PCR.  This method gives you sensitivity and limit of detection comparable to that of dual-labeled hydrolysis probes without the high cost. We all know how expensive small batch probe synthesis orders can be, especially when you need multiple fluorescent probes for R&D assay optimization. Mediator Probe PCR cuts those costs by pairing one fluorescent labeled universal reporter with multiple label-free probes. This is a great cost-effective PCR method for academic labs or biotech companies on a budget.

The Method to the Mediator

Mediator Probe PCR is a universal sequence-dependent detection method. It uses a label-free Mediator Probe (MP) and a fluorescent labeled Universal Reporter (UR). The MP contains a DNA target-specific sequence plus a generic “mediator” sequence. The UR contains the same generic “mediator” sequence as the MP plus a fluorophore for detection.  So a single UR can be used to detect multiple label-free mediator probes.

figure1

When a Mediator Probe is added to a reaction containing PCR primers, dsDNA and a UR, the target specific portion of the MP binds to the complementary DNA sequence. The mediator portion of the probe is not complementary to the DNA and remains unbound.

figure2

When amplification reaches the MP, the mediator is cleaved and released into the reaction solution.  The free mediator then binds to its complementary site on the UR.

figure3

Elongation of the mediator sequence results in dequenching of the UR fluorophore by release of the quencher or strand displacement.

figure4

Mediator Probe PCR works with both DNA and RNA targets in real-time PCR assays. In addition there are some real advantages of Mediator Probe/Universal Reporter compared with Dual Labeled Hydrolysis Probes for Real-Time PCR as it is:

  • Cost-effective compared to dual labeled hydrolysis probes (for small batch synthesis, < 40% of the cost)
  • Sensitivity and limit of detection comparable to dual labeled hydrolysis probes
  • Less background variation
  • More efficient quenching due to close proximity of quencher and fluorophore in hairpin structure.

Have you tried mediator probe PCR? We’d love to hear your thoughts on it!

Recommended Reading:

Faltin B, Wadle S, Roth G, Zengerle Rand von Stetten F.  (2012) Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free Primary Probes and Standardized Secondary Universal Fluorogenic Reporters. Clin Chem 58:1546–1556.

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