Real-time PCR monitors DNA amplification during PCR cycles, rather than at the end. It uses probes labeled with a fluorescent reporter and quencher; as the target sequence amplifies, more reporters are released, increasing fluorescence measured in real-time by the optical module. This allows quantification of the starting DNA or RNA material. The fluorescence data can be plotted on a curve to visualize amplification over successive cycles.

2. 2
Course: Clinical Laboratory Principle
Real-Time PCR

3. 3
Real-Time Polymerase Chain Reaction
 It monitors the amplification of a targeted DNA
molecule during the PCR, not at its end, as in
conventional PCR
Known as Quantitative- PCR

4. 4
PCR relies on a
Thermostable DNA polymerase (Taq
polymerase)
DNA primers (18-22 bases)
PCR Buffer
dNTPs (dATPs, dGTPs, dCTPs, dTTPs)
Cations (Mg++)
Samples (DNA/RNA)
Probes
Real-Time Polymerase Chain Reaction

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How does Real Time PCR work?
Probes :
A probe is an oligonucleotide that
is labeled with a fluorescent
Reporter and a Quencher
 The Quencher decreases the
fluorescent intensity

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Probes :
 During The PCR Cycle the Probe
denatures and anneals to the
target sequence
 For every amplification of the
target sequence, a fluorescence
Reporter is released from the
probe

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Real – Time PCR
 Real –Time Instruments
composed of
1- Thermal Cycler
2- Optical Module
Optical Module
Thermal Cycler

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Thermal Cycler (PCR - Machine)
Denaturation
Annealing
Elongation

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Elongation

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The Reporter molecule is only released when a DNA
strand is completely polymerized by Taq Polymerase

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2- Optical Module :
To detect fluorescence in the tubes
during the PCR run
This component measures the
fluorescence intensity in the tubes

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2- Optical Module :
Cycle after cycle; the fluorescence intensity
increases indicating the amplification of
specific region in a real-time
PCR cycle Number = 10 PCR cycle Number = 35

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The fluorescence of the cycles are measured and
calculated into an amplification curve/plot

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Reference:
Tietz Fundamentals of Clinical Chemistry,
Sixth Edition. Chapter 17