REAL TIME PCR
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LIMITATIONS OF END-POINT PCR
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Poor Precision
Low sensitivity
Low resolution
Non - Automated
Size-based discrimination only
Results are not expressed as numbers
Ethidium bromide for staining is not very quantitative
Post PCR processing
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THE EVOLUTION OF PCR TO REAL-TIME
Traditional PCRs use Agarose gels for detection
of amplification at the final phase or end-point
of the PCR reaction
 Real time PCR has advanced to allow detection
before the end-point of the reaction.

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REAL-TIME PCR DETECTION
 While

the reaction is occurring
monitors the fluorescence emitted during the
reaction as an indicator of amplicon production at
each PCR cycle
 Real-Time
chemistries allow for the detection of
PCR amplification during the early phases of the
reaction
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Exponential:
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Exact doubling of product is accumulating at every cycle (assuming 100%
reaction efficiency). The reaction is very specific and precise.
Linear (High Variability):
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PCR PHASES
The reaction components are being consumed, the reaction is slowing, and
products are starting to degrade.
Plateau (End-Point: Gel detection for traditional methods):
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The reaction has stopped, no more products are being made and if left
long enough, the PCR products will begin to degrade.
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 The
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plateau effect on a fivefold dilution series:
The 5-fold dilution series, seems to plateau at the same place
even though the exponential phase clearly shows a difference
between the points along the dilution series.
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This reinforces the fact that if measurements were taken at the plateau phase, the data
would not truly represent the initial amounts of starting target material.
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REAL-TIME PRINCIPLES
Three general methods for the quantitative detection:

1.
Hydrolysis probes (TaqMan, Beacons).
2.
Hybridisation probes (Light Cycler).
3.
DNA-binding agents (SYBR Green).
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TAQMAN CHEMISTRY

Real-time systems using fluorogenic-labeled probes that
use the 5´ nuclease activity of Taq DNA polymerase.
Detecting only specific amplification products.
 Eliminate post-PCR processing for the analysis of probe
degradation
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A fluorogenic probe enables the detection of a specific PCR product
as it accumulates during PCR.
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The TaqMan® Probe is designed with a high-energy dye termed a
Reporter at the 5 end, and a low-energy molecule termed a Quencher
at the 3 end.
While the probe is intact, the proximity of the quencher dye greatly
reduces the fluorescence emitted by the reporter dye by fluorescence
resonance energy transfer (FRET) through space.
When reporter and quencher are separated the reporter fluoroscence
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HOW TAQMAN SEQUENCE DETECTION
CHEMISTRY WORKS
If the target sequence is present, the probe anneals
downstream from one of the primer sites
During extension the probe is cleaved by the 5´
nuclease activity of Taq DNA polymerase.
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2.
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3.
The reporter dye is separated from the quencher dye,
increasing the reporter dye signal.
The probe is removed from the target strand, allowing
primer extension to continue to the end of the template
strand..
Additional reporter dye molecules are cleaved from
their respective probes with each cycle.
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The fluorescence intensity is increased proportional to the
amount of amplicon produced.
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MOLECULAR BEACONS
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HYBRIDIZATION PROBES
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Fluorescence Resonance Energy Transfer (FRET) Concept
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Donor fluorophore is excited by appropriate wavelength
Donor energy is transferred non-radiatively to the acceptor
fluorophore
Excited acceptor emits at longer wavelength
Increase in fluorescence with each cycle
 The signal depends only on hybridization

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HOW THE HYBRIDISATION PROBES WORK?
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.
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SYBR® GREEN I DYE CHEMISTRY

Small molecules that bind to doublestranded DNA can be divided into two
classes:
Intercalators
 Minor-groove binders
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There are two requirements for a DNA
binding dye for real-time detection of
PCR:
Increased fluorescence when bound to
double-stranded DNA
 No inhibition of PCR
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HOW THE SYBR GREEN I DYE CHEMISTRY
WORKS
When SYBR Green I dye is added to a sample, it
immediately binds to all double-stranded DNA present
in the sample.
● During the PCR, the DNA Polymerase amplifies the
target sequence, and creates the PCR products.
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● The SYBR Green I dye binds to each new copy of
double-stranded DNA.
● As the PCR progresses, more amplicons are created.
The result is an increase in fluorescence intensity
proportionate to the amount of PCR product produced.
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Advantages of SYBR Green I Dye
It can be used to monitor the amplification of any doublestranded DNA sequence.
 No probe is required, which reduces assay setup and running
costs.
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Disadvantage of SYBR Green I Dye
SYBR Green I dye chemistry may generate false positive
signals
 SYBR Green I dye binds to nonspecific double-stranded DNA
sequences.
 Needs fully optimized PCR system -no primer dimers or non-specific
amplicons.
 When not to choose SYBR Green
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Multiplex reactions.
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TERMS USED IN QUANTITATION ANALYSIS
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Amplification plot: The plot of fluorescence signal versus cycle
number
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Baseline: The initial cycles of PCR, in which there is little change in
fluorescence signal
Passive reference: A dye that provides an internal reference to which the
reporter dye signal can be normalized during data analysis.
Rn (normalized reporter): The fluorescence emission intensity of the
reporter dye divided by the fluorescence emission intensity of the passive
reference dye.
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Normalization is necessary to correct for fluctuations caused by changes in
concentration or volume.
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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), OR
 A reaction that does not contain any template
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ΔRn (delta Rn): The magnitude of the signal generated by the given
set of PCR conditions. [ΔRn = (Rn+) – (Rn-)]
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Threshold: The average standard deviation of Rn for the early PCR
cycles, multiplied by an adjustable factor. The threshold should be set
in the region associated with an exponential growth of PCR product.
 Ct (threshold cycle): The fractional cycle number at which the
fluorescence passes the fixed threshold
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QUANTITATION
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By Real time PCR
ABSOLUTE QUANTITATION
Used to quantitate unknown samples by interpolating
their quantity from a standard curve.
 Standard: A sample of known concentration used to
construct a standard curve.
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From a standard curve you can extrapolate the quantity of an
unknown sample.
Unknown: A sample containing an unknown quantity of
template. This is the sample whose quantity you want to
determine
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The
standard curve resulting from Ct value and standard
concentrations should be a linear graph with a high correlation
coefficient (> 0.99).
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
 CT
value of 40 or more means no amplification and cannot be included in
the calculations
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RELATIVE QUANTITATION

A relative quantitation assay is used to analyze changes
in gene expression in a given sample relative to another
reference sample (such as an untreated control sample).
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Relative quantitation might be used to measure gene
expression in response to a chemical (drug).
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RELATIVE QUANTITATION
STANDARD CURVE METHOD
 Using
a separate standard curve for the
target and endogenous control, in separate
tubes.
 Requires the least amount of optimization
and validation.
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RELATIVE QUANTITATION
COMPARATIVE CT METHOD
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The need for a standard curve is eliminated.
The target and endogenous control are amplified in the
same tube.
Increased throughput:
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wells no longer need to be used for the standard curve samples.
the adverse effect of any pipetting and dilution errors made in
creating the standard curve samples are eliminated.
A validation experiment must be run to show that the
efficiencies of the target and endogenous control
amplifications are approximately equal.
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ENDOGENOUS/INTERNAL CONTROL
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Usually an abundantly and constantly expressed
housekeeping gene
Best to run a validity test for the selected endogenous
control
Combination may be used
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UNDERSTANDING CT
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CT (threshold cycle) is the
intersection between an
amplification curve and a threshold
line.
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It is a relative measure of the
concentration of target in the PCR
reaction.
The CT values from PCR reactions
run under different conditions or
with different reagents cannot be
compared directly.
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EFFECT OF EFFICIENCY OF A PCR
REACTION ON CT
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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 90-110% (ideal
slope = 3.32)
The PCR efficiency is dependent on:
the assay and the master mix performance
sample quality.
 length of the amplicon
 secondary structure
 primer design
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USING
THE
PCR EQUATION
Xn = X0(1 + E)n
Xn
Xn = PCR product after cycle n
 X0 = initial copy number
 E = amplification efficiency
 n = cycle number

X0
cycle number
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EFFECT OF AMPLIFICATION EFFICIENCY
Xn = X0(1+E)n
Case 1: E = 0.9
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
Result
A difference of 0.1 in amplification
efficiencies created a five-fold difference in the
final ratio of PCR products after 30 cycles
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REAL-TIME PCR ADVANTAGES
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Not influenced by non-specific amplification
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Amplification can be monitored in real-time
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No post-PCR processing of products (high throughput, low contamination
risk)
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Ultra-rapid cycling (30 minutes to 2 hours)
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Most specific, sensitive and reproducible
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Not much more expensive than conventional PCR (except equipment cost)
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