© copyright 2009 by the american association for clinical chemistry mediator probe pcr: a novel...
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© 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?
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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
*
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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
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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)
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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)
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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
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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