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Mediator Probe PCR: A Novel Approach for Detection of Real-Time PCR Based on Label-Free 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 © Copyright 2009 by the American Association for Clinical Chemistry

Background Ø Polymerase chain reaction (PCR) in clinical diagnostics § Amplification of DNA § Background Ø 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) Denaturation of target DNA Annealing of primers Primer elongation Figure 1. Schematic representation of the PCR principle. © Copyright 2009 by the American Association for Clinical Chemistry

Background (continued) Ø Real-time PCR (e. g. hydrolysis probe PCR) § Advantages • Specificity, Background (continued) Ø 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 Annealing of primers and hydrolysis probe Primer elongation and cleavage of hydrolysis probe Figure 2. Hydrolysis probe: structure (top) and cleavage during primer elongation © Copyright 2009 (bottom). by the American Association for Clinical Chemistry

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

Methods Ø Mediator probe PCR § Novel approach for real time amplification § Mediator Methods Ø 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). * by the American Association for Clinical proximity © Copyright 2009 Quencher and fluorophore should be in close. Chemistry

Methods (continued) Target DNA Mediator annealing to universal reporter Denaturation of target DNA Polymerase Methods (continued) Target DNA Mediator annealing to universal reporter Denaturation of target DNA Polymerase Mediator probe Mediator elongation Primer Annealing of primers and mediator probe Degradation of 5’ terminus Displacement of 5’ terminus Mediator Primer elongation and cleavage of mediator probe; release of mediator 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. © Copyright 2009 by the American Association for Clinical Chemistry

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

Results Figure 5. Intraassay imprecision betweeen MP PCR and Hydrolysis probe PCR. Back-calculated copy Results 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. © Copyright 2009 by the American Association for Clinical Chemistry

Results (continued) Figure 6. Duplex amplification of various HPV 18 DNA concentrations and 300 Results (continued) Figure 6. Duplex amplification of various HPV 18 DNA concentrations and 300 copies of H. sapiens ACTB. The calculated copy numbers of HPV 18 are plotted for the MP PCR (abscissa) and the hydrolysis probe PCR (ordinate). © Copyright 2009 by the American Association for Clinical Chemistry

Results (continued) Figure 7. Amplification of a DNA dilution series of HPV 18 (a) Results (continued) Figure 7. Amplification of a DNA dilution series of HPV 18 (a) and E. coli (b). Back-calculated copy values for MP PCR (abscissa) were plotted against values for hydrolysis probe (HP) PCR (ordinate). © Copyright 2009 by the American Association for Clinical Chemistry

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

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

Results (continued) Costs for oligo synthesis ($) Number of targets 1 4 10 Hydrolysis Results (continued) Costs for oligo synthesis ($) Number of targets 1 4 10 Hydrolysis probe PCR 245 980 2450 MP PCR 1 655 895 1500 UR 600 675 950 MP 55 220 550 1 Cost of MP PCR = Cost of UR + Cost of MP 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). © Copyright 2009 by the American Association for Clinical Chemistry

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

Conclusion Ø MP PCR is an alternative real-time PCR technique § LOD, inter- and Conclusion Ø 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 © Copyright 2009 by the American Association for Clinical Chemistry

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