Real-Time Polymerase Chain Reaction

1. Prinsip Real-Time PCR
Syamsi Rizal, MSi
Product Manager Life Science

2. PCR and RT-PCR
PCR, or the Polymerase Chain Reaction, is a process for the amplification of specific
fragments of DNA tell us what
Real-Time PCR a specialized technique that allows a PCR reaction to be visualized “in
real time” as the reaction progresses tell us how much
As we will see, Real-Time PCR allows us to measure minute amounts of DNA sequences in a
sample!

3. Definition
Real-time PCR is the continuous collection
of fluorescent signal from one or more
polymerase chain reactions over a range of
cycles.
Quantitative real-time PCR is the
conversion of the fluorescent signals
fromeach reaction into a numerical value
for each sample

4. PCR Amplification of DNA
In theory, product accumulation is proportional to 2n, where n is the number of
amplification cycle repeats.

5. PCR, Theory vs Reality
In Theory, PCR reactions amplify
exponentially, doubling every cycle and
grow forever.
In Reality, The reactant for polymeration is
limited.
Threshold Cycle
• threshold cycle or the CT value is the cycle at which a
significant increase in DRn is first detected.
• it is the parameter used for quantitation
* CT value of 40 or more means no amplification and cannot be
included in the calculations

6. In real-time PCR reactions, fluorescent molecules are used to
monitor the reaction while amplification is taking place
Dual hybridization probes (FRET), Eclipse probes, Amplifluor assays,
Scorpions PCR primers, LUX PCR primers, QZyme PCR primers

7. How do we detect and measure DNA?
1. Non-specific fluorescent dyes:
- intercalation with any dsDNA
- SYBR green, EVAGREEN
2. Sequence specific primers & DNA probes:
- oligonucleotides labeled with fluorescent reporter
(a) Hydrolisis probe: TaqMan, Molecular Beacon, Scorpion
(b) Hybridizing probe: FRET

8. TaqMan probe-based assays are widely used
in quantitative PCR in research and medical
laboratories:
• Gene expression assays
• Pharmacogenomics
• Human Leukocyte Antigen (HLA) genotyping
• Determination of viral load in clinical specimens
(HIV, Hepatitis)
• Bacterial Identification assays
• DNA quantification
• SNP genotyping
• Verification of microarray results

9. LAMPUNG
0% 10% 20% 30% 40% 50% 60% 70% 80%
Taqman Probes
Molecular Beacon
FRET Probes
LUX Fluorogenic Primer
MGB Eclipse Probes
Other
Scorpion Probes
Molecular Probe