1. © Copyright 2009 by the American Association for Clinical Chemistry Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free Primary Probes and Standardized Secondary Universal Fluorogenic Reporters B. Faltin, S. Wadle, G. Roth, R. Zengerle, and F. von Stetten November 2012 www.clinchem.org/content/58/11/1546.full © Copyright 2012 by the American Association for Clinical Chemistry

2. © Copyright 2009 by the American Association for Clinical Chemistry Background  Polymerase chain reaction (PCR) in clinical diagnostics  Amplification of DNA  Various applications, e.g. Genotyping (e.g. single nucleotide polymorphisms) Quantification (e.g. pathogen load) Expression profiling (e.g. cancer screening) Figure 1. Schematic representation of the PCR principle. Denaturation of target DNA Annealing of primers Primer elongation

3. © Copyright 2009 by the American Association for Clinical Chemistry Background (continued)  Real-time PCR (e.g. hydrolysis probe PCR)  Advantages Specificity, sensitivity Low time-to-result Multiplex analyses  Disadvantages Cost-intensive (individual probe for each target) Uneven background signal of different probes Primer elongation and cleavage of hydrolysis probe Figure 2. Hydrolysis probe: structure (top) and cleavage during primer elongation (bottom). Annealing of primers and hydrolysis probe

4. © Copyright 2009 by the American Association for Clinical Chemistry Question  Why is hydrolysis probe PCR cost-intensive during assay development or when applied to numerous different targets?

5. © Copyright 2009 by the American Association for Clinical Chemistry Methods  Mediator probe PCR  Novel approach for real time amplification  Mediator probe (MP) Target-specific 3’ region (probe) Generic 5’ region (mediator) Label free  Universal reporter (UR) Fluorophore and quencher Hairpin conformation Mediator hybridization site Figure 3. Structure of mediator probe (top) and universal reporter (bottom). * * Quencher and fluorophore should be in close proximity *

6. © Copyright 2009 by the American Association for Clinical Chemistry Methods (continued) Figure 4. Mediator probe PCR: (A) Target DNA, (B) Denaturation, (C) Annealing of MP and primers, (D) Primer elongation; cleavage of MP; release of mediator, (E) Annealing of mediator to the UR, (F) Elongation of the mediator, (G) Degradation of the 5’ terminus of the UR. The quencher is released from the UR, or (H) Displacement of the 5’ terminus; unfolding of the hairpin and dequenching. Denaturation of target DNA Target DNA Mediator probePolymerase Annealing of primers and mediator probe Primer elongation and cleavage of mediator probe; release of mediator Degradation of 5’ terminus Displacement of 5’ terminus Mediator elongation Mediator annealing to universal reporter Primer Mediator

7. © Copyright 2009 by the American Association for Clinical Chemistry Question  Which requirements must be fulfilled for the sequence design of the mediator probe and the universal reporter?

8. © Copyright 2009 by the American Association for Clinical Chemistry Figure 5. Intraassay imprecision betweeen MP PCR and Hydrolysis probe PCR. Back-calculated copy numbers of the MP PCR (abscissa) are plotted against results of the hydrolysis probe PCR (ordinate). Calculation for 5 different DNA concentrations with 8 replicates each. Results

9. © Copyright 2009 by the American Association for Clinical Chemistry Figure 6. Duplex amplification of various HPV18 DNA concentrations and 300 copies of H. sapiens ACTB. The calculated copy numbers of HPV18 are plotted for the MP PCR (abscissa) and the hydrolysis probe PCR (ordinate). Results(continued)

10. © Copyright 2009 by the American Association for Clinical Chemistry Figure 7. Amplification of a DNA dilution series of HPV18 (a) and E. coli (b). Back-calculated copy values for MP PCR (abscissa) were plotted against values for hydrolysis probe (HP) PCR (ordinate). Results (continued)

11. © Copyright 2009 by the American Association for Clinical Chemistry Results (continued) Figure 8. Limit of detection. MP PCR (black), hydrolysis probe PCR (gray). 95 % Hydrolysis probe PCR: 85 copies / rxn Mediator probe PCR: 78 copies / rxn

12. © Copyright 2009 by the American Association for Clinical Chemistry Figure 9. Efficiency of fluorescence quenching. Specific hydrolysis probes (left panel) and universal reporters (right panel). Results(continued)

13. © Copyright 2009 by the American Association for Clinical Chemistry Results (continued) Table 1. Costs savings for MP PCR compared to hydrolysis probe PCR. Above the break even point of 4 oligonucleotides MP PCR is cheaper than hydrolysis probe PCR. Calculated are oligonucleotide synthesis costs for a different number of targets (0.05 nmol synthesis scale). Costs for oligo synthesis ($) Number of targets1410 Hydrolysis probe PCR2459802450 MP PCR 1 6558951500 UR600675 950 MP55220 550 1 Cost of MP PCR = Cost of UR + Cost of MP

14. © Copyright 2009 by the American Association for Clinical Chemistry Question  What is the advantage of the MP PCR over hydrolysis probe PCR?

15. © Copyright 2009 by the American Association for Clinical Chemistry Conclusion  MP PCR is an alternative real-time PCR technique  LOD, inter- and intraassay imprecision, duplex capability of MP PCR are comparable to hydrolysis probe PCR  Low cost synthesis of target specific, label free MPs  Only one universal fluorogenic reporter (UR) is required to monitor the amplification of different samples  Cost savings in UR synthesis due to economy of scales  UR has target independent, high efficiency of quenching

16. © Copyright 2009 by the American Association for Clinical Chemistry Thank you for participating in this month’s Clinical Chemistry Journal Club. Additional Journal Clubs are available at www.clinchem.org Follow us