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Principles of Real-Time Quantitative PCR Techniques
Molecular Diagnostics Molecular Diagnostics Principles of Real-Time Quantitative PCR Techniques 1
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What is Wrong with Agarose Gels?
2 Molecular Diagnostics Molecular Diagnostics What is Wrong with Agarose Gels? Poor precision Low sensitivity Short dynamic range < 2 logs Low resolution Non-automated Size-based discrimination only Results are not expressed as numbers Ethidium bromide staining is not very quantitative
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3 Molecular Diagnostics Molecular Diagnostics Real-Time PCR Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection based on the detection and quantitation of a fluorescent reporter the first significant increase in the amount of PCR product correlates to the initial amount of target template
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Molecular Diagnostics
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Real-Time PCR Chmistries
5 Molecular Diagnostics Molecular Diagnostics Real-Time PCR Chmistries Three general methods for the quantitative assays: Hydrolysis probes (TaqMan, Beacons) Hybridization probes (Light Cycler) DNA-binding agents (SYBR Green)
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6 Molecular Diagnostics Molecular Diagnostics SYBR Green I technique SYBR Green I fluorescence is enormously increased upon binding to double-stranded DNA. During the extension phase, more and more SYBR Green I will bind to the PCR product, resulting in an increased fluorescence. Consequently, during each subsequent PCR cycle more fluorescence signal will be detected.
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Hydrolysis probe technique
7 Molecular Diagnostics Molecular Diagnostics Hydrolysis probe technique A quencher fluorochrome absorbs the fluorescence of the reporter fluorochrome as long as the probe is intact. Upon amplification, the hydrolysis probe is displaced and subsequently hydrolyzed by the Taq polymerase. The reporter and quencher fluorochrome are separated and consequently the fluorescence of the reporter fluorochrome becomes detectable.
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8 Molecular Diagnostics Molecular Diagnostics Molecular Beacons
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Hybridization probes technique
9 Molecular Diagnostics Molecular Diagnostics Hybridization probes technique One probe is labelled with a donor fluorochrome at the 3’ end and a second –adjacent- probe is labelled with an acceptor fluorochrome. When the two fluorochromes are in close vicinity (1–5 nucleotides apart), the emitted light of the donor fluorochrome will excite the acceptor fluorochrome (FRET). This results in the emission of fluorescence, which subsequently can be detected during the annealing phase and first part of the extension phase of the PCR reaction. FRET = Förster/fluorescence resonance energy transfer
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Increase in Reporter Signal “Reports” on Amplification
Molecular Diagnostics Increase in Reporter Signal “Reports” on Amplification Cycle number
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PCR Phases Problems with Linear and Plateau Phases
Molecular Diagnostics PCR Phases Log [DNA] Cycle # Geometric Plateau Linear Ethidium-Gel detection Geometric Phase y = 2X Variable Linear Phase Plateau Effect Problems with Linear and Plateau Phases
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12 Molecular Diagnostics Molecular Diagnostics Definitions Rn (normalized reporter): The fluorescence emission intensity of the reporter dye divided by the fluorescence emission intensity of the passive reference dye Rn+: The Rn value of a reaction containing all components, including the template Rn-:The Rn value of an un-reacted sample. The Rn-value can be obtained from: The early cycles of a real -time PCR run (those cycles prior to a detectable increase in fluorescence) A reaction that does not contain any template
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13 Molecular Diagnostics Molecular Diagnostics Definitions ΔRn (delta Rn): The magnitude of the signal generated by the given set of PCR conditions. The ΔRn = (Rn+) – (Rn-) Threshold: The average standard deviation of Rn for the early PCR cycles, multiplied by an adjustable factor. CT (Threshold cycle value): is the cycle at which a significant increase in DRn is first detected
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Absolute Quantitation
Molecular Diagnostics Absolute Quantitation A number of dilutions in triplicates are amplified If the CT values for each of the dilutions are plotted against concentrations, the result should be a linear graph with a high correlation coefficient (> 0.99). CT value of 40 or more means no amplification and cannot be included in the calculations theoretically a single copy of the target should create a CT value of 40 (if efficiency is 100%), which is the y-intercept in a standard curve experiment Log (ΔRn)
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Amplification Efficiency
Molecular Diagnostics Amplification Efficiency The slope of a standard curve is a reflection of the amplification efficiency The efficiency of the reaction can be calculated by the following equation: Eff=10(-1/slope) –1 The efficiency of the PCR should be % (ideal slope = 3.32) A number of variables can affect the efficiency of the PCR. These factors can include: length of the amplicon secondary structure primer design
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Molecular Diagnostics
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Using the PCR Equation Xn X0 Xn = X0(1 + E)n
17 Molecular Diagnostics Molecular Diagnostics Using the PCR Equation Xn = X0(1 + E)n Xn = PCR product after cycle n X0 = initial copy number E = amplification efficiency n = cycle number Xn E IS MAXIMALLY 1.0, BUT USUALLY AROUND 0.9. X0 cycle number 17
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Effect of Amplification Efficiency
18 Molecular Diagnostics Molecular Diagnostics Effect of Amplification Efficiency Xn = X0(1+E)n Case 1: E = Case 2: E = 0.8 Xn = 100 (1+0.9)30 Xn = 100 (1+0.8)30 Xn = 2.3 x 1010 Xn = 4.6 x 109 CALCULATE EFFICIENCIES FROM THE SLOPE OF YOUR DOSE RESPONSE CURVES Result A difference of 0.1 in amplification efficiencies created a five-fold difference in the final ratio of PCR products after 30 cycles 18
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