© 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.

© 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

© Copyright 2009 by the American Association for Clinical Chemistry

BackgroundBackground

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

© Copyright 2009 by the American Association for Clinical Chemistry

Background (continued)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 probesPrimer elongation and cleavage of hydrolysis probe

Figure 2. Hydrolysis probe: structure (top) and cleavage during primer elongation (bottom).

Annealing of primers and hydrolysis probe

© Copyright 2009 by the American Association for Clinical Chemistry

QuestionQuestion

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

MethodsMethods

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

*

© Copyright 2009 by the American Association for Clinical Chemistry

Methods (continued)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

© Copyright 2009 by the American Association for Clinical Chemistry

QuestionQuestion

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© 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.

ResultsResults

© Copyright 2009 by the American Association for Clinical Chemistry© 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 Results (continued)(continued)

© Copyright 2009 by the American Association for Clinical Chemistry© 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)Results (continued)

© Copyright 2009 by the American Association for Clinical Chemistry

Results (continued)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

© Copyright 2009 by the American Association for Clinical Chemistry© 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 Results (continued)(continued)

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Results (continued)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 targets 1 4 10

Hydrolysis probe PCR 245 980 2450

MP PCR1 655 895 1500

UR 600 675 950

MP 55 220 550

1Cost of MP PCR = Cost of UR + Cost of MP

© Copyright 2009 by the American Association for Clinical Chemistry

QuestionQuestion

What is the advantage of the MP PCR over hydrolysis probe PCR?

© Copyright 2009 by the American Association for Clinical Chemistry

ConclusionConclusion 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

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