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Mediator Probe PCR

Technique

 

Mediator Probe PCR in an innovative and powerful real-time PCR technique, which allows highly sensitive and selective detection of nucleic acids. Mediator Probe PCR uses label free mediator probes for DNA detection and universal reporter molecules for fluorescence signal generation.

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Figure 1: Reaction mechanism of the Mediator Probe PCR. Here, DNA detection via mediator probes (A – B) and fluorescence signal generation using universal reporter molecules (C – E) are two different steps, independent from each other.1



Advantages

 

Because in Mediator Probe PCR, DNA detection and signal generation are two different processes, both steps can be optimised independently from each other. This leads to several advantages and innovative applications of the technology:

 

1) Optimised fluorogenic universal reporter structures with high signal to noise ratios

Target sequence independent universal reporter molecules are used in Mediator Probe PCR for fluorescence signal generation. Therefore, the fluorescence signal-to-noise ratios of these molecules can be optimised independently from any real-time PCR system, using the mediator extension assay.1 Once optimised, the respective universal reporter will improve the real-time PCR performance of all assays it is used in.

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 Figure 2: Optimised secondary structure of a universal reporter oligonucleotide, simulated with OligoPAD.1

 

 

2) One standard set of universal reporters for different multiplex real-time PCRs

For mulitplex Mediator Probe PCR, one standard set of optimised universal reporters can be used for signal generation. Therefore, each universal reporter type has a specific mediator hybridization site and is labeled with different fluorophores. This enables reporting of mediator probe cleavage in the presence of a specific target DNA-sequence. This standard set can directly be adapted to different target panels by exchanging primer and label free mediator probes.
 
 
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Figure 3: Since fluorogenic universal reporters are target sequence independent, one standard set can be used for the detection of many different target panels.2


 

3) Label free mediator probes with distinct design guidelines

Since universal reporters are used for fluorescence signal generation, mediator probes do not carry any fluorescence labels. Therefore, mediator probe design is not limited by target sequence characteristics, such as GC-quenching or short hybridization sequences. As a result, we were able to derive distinct guidelines for mediator probe sequence design.3 These guidelines are ubiquitous and should be considered in each Mediator Probe PCR, to reach a maximum performance.

 

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Figure 4: The binding energies of a mediator probe significantly influence the assay performance. Therefore, it should be approximately -18 kcal mol-1 between mediator probe and target sequence (left) and -10 kcal mol-1 between mediator probe and universal reporter (right). We recommend using appropriate bioinformatics software tools for binding energy calculation and secondary structure prediction.3

 

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Figure 5: Mediator Probe cleavage. During DNA amplification the Mediator Probe is cleaved by the polymerase. The cleavage site is located at the 3´-end of the mediator after the last base which still binds to the target site.

 

 

Use the Mediator Probe PCR

 

Mediator Probe PCR is protected by the patents/ patent applications based on the patent family published under WO2013079307 (Bifunctional oligonucleotide probe for universal real-time multianalyte detection).

 

 

 

Publications

 

  1. Lehnert M, Kipf E, Schlenker F, Borst N, Zengerle R, von Stetten F. Fluorescence signal-to-noise optimisation for real-time PCR using universal reporter oligonucleotides. Anal. Methods. 2018;10:190. doi: 10.1039/C8AY00812D. 
  2. Wadle S, Lehnert M, Schuler F et al. Simplified development of multiplex real-time PCR through master mix augmented by universal fluorogenic reporters. BioTechniques. 2016;61(3):123–8. doi: 10.2144/000114443.
  3. Wadle S, Lehnert M, Rubenwolf S, Zengerle R, Stetten F von. Real-time PCR probe optimization using design of experiments approach. Biomolecular detection and quantification. 2016;7:1–8. doi: 10.1016/j.bdq.2015.12.002.
  4. Wadle S, Rubenwolf S, Lehnert M et al. Mediator probe PCR: detection of real-time PCR by label-free probes and a universal fluorogenic reporter. Methods in molecular biology (Clifton, N.J.). 2014;1160:55–73. doi: 10.1007/978-1-4939-0733-5_6.
  5. Faltin B, Wadle S, Roth G, Zengerle R, Stetten F von. Mediator probe PCR: a novel approach for detection of real-time PCR based on label-free primary probes and standardized secondary universal fluorogenic reporters. Clinical chemistry. 2012;58(11):1546–56. doi: 10.1373/clinchem.2012.186734

 

 

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