ADVANCES IN MOLECULAR

BIOLOGY AND BIOINFORMATICS

UIMLT

Prepared by :Syeda Rida Shah

Senior lecturer
THE UNIVERSITY OF LAHORE
REAL TIME PCR
INTRODUCTION

Real Time PCR is a advanced biotechnological

instrument. The term real time denote it can
monitor the progress of the amplification when
the process is going on.
 HISTORY:
1993 – First real-time PCR detection experiments
to show utility for DNA quantization reaction
took place using EtBr detection
(illumination,CCD camera detection).
1996 – TaqMan detection methods used, instead of
EtBr, for real-time detection of PCR.
1996-7 – ABI introduces first real-time qPCR
(“Sequence Detection System”) instrument (the
ABI 7700).
Role of Real Time PCR

Beside normal amplification process performed

by normal PCR, Real Time PCR can perform
detection, analysis and quantification of the
sample.
Detection: Find out the presence of targeted gene
sequence which is assured by the presence
of the amplification curve.
Quantification: Quantification of targeted DNA in
a sample can be done by using the
cycle no. needed to obtain the threshold
value of detector and PCR efficiency.
Analysis: Analysis of the variants can be done by
studying the melting curve or comparing
the melting temperature with the sequences
of the database.
 Advantage over normal PCR
Real Time PCR has many advantages over normal
PCR:

It does not require gel preparation like

traditional PCR.

It is not time consuming like normal PCR.

Less complexity at the quantification of

sample.etc.
It gives a look in to the reaction that
is help to decide which reactions
have worked well and which have
failed.
The efficiency of the reaction can be
precisely calculated.
There is no need to run the PCR
product out on a gel after the
reaction as the melt curve analysis
serve the purpose.
The real-time PCR data can be used
to perform truly quantitative analysis
of gene expression. In comparison,
old fashioned PCR was only ever
semi-quantitative at best.
Faster than normal PCR.
Less complexity at the quantification
of sample.etc.
What is real time PCR?
It is a technique used to monitor the
progress of a PCR reaction in real-
time.
At the same time, a relatively small
amount of PCR product (DNA, cDNA
or RNA) can be quantified.
It is based on the detection of the
fluorescence produced by a reporter
molecule which increases, as the
reaction proceeds.
It is also known as a quantitative
polymerase chain reaction
(qPCR), which is a laboratory
technique of molecular biology
based on the
polymerase chain reaction (PCR).
qPCR is a powerful technique that
allows exponential amplification of
DNA sequences.
A PCR reaction needs a pair of
primers that are complementary to
the sequence of interest. Primers are
extended by the DNA polymerase.
After amplification, however, gel
electrophoresis is used to analyze the
amplified PCR products and this makes
conventional PCR time consuming; since
the reaction must finish before
proceeding with the post-PCR analysis.
Real-Time PCR overcomes this problem.
The copies produced after the extension,
so-called amplicons, are re-amplified
with the same primers leading thus to
exponential amplification of the DNA
molecules.
The term “real-time” denotes that it
can monitor the progress of the
amplification when the process is
going on in contrast to the
conventional PCR method where
analysis is possible only after the
process is completed.
PCR Terminology
Polymerase chain
PCR
reaction
Reverse transcription-
polymerase chain RT-PCR
reaction
Real-time polymerase
qPCR
chain reaction
RT-PCR / qPCR
qRT-PCR
combined technique
Principle of Real Time PCR

 Thissame principle of amplification of

PCR is employed in real-time PCR. But
instead of looking at bands on a gel at the
end of the reaction, the process is
monitored in “real-time”. The reaction is
placed into a real-time PCR machine that
watches the reaction occur with a camera
or detector.
Principle of Real Time PCR
 Although many different techniques are
used to monitor the progress of a PCR
reaction, all have one thing in common.
They all link the amplification of DNA to
the generation of fluorescence which can
simply be detected with a camera during
each PCR cycle. Hence, as the number of
gene copies increases during the reaction,
so does the fluorescence, indicating the
progress of the reaction.
MECHANISMS OF REAL TIME PCR
Flourescent Dye –based RealTime PCR
Flourescent Dye –based RealTime PCR
 Dye-based quantitative PCR (qPCR) uses
real-time fluorescence of a double-
stranded DNA (dsDNA) binding dye (e.g.,
SYBR® Green) to measure DNA
amplification as it occurs during PCR.
 The dye displays weak background
fluorescence that increases dramatically
upon binding to dsDNA. Thus,
amplification of the target sequence results
in an increase of fluorescence that is
directly proportional to the amount of
dsDNA present at each PCR cycle.
Flourescent Dye –based RealTime PCR
 This type of qPCR assay requires only
two sequence-specific primers, making
it a rapid and cost-effective way to
interrogate a large number of
samples/targets.
 One disadvantage of intercalating dye-
based methods is that they detect any
dsDNA produced in the reaction. This
includes off target amplification
products and primer-dimers, which
results in inaccurate quantification.
 Flourescent Dye –based RealTime PCR
 Therefore, it is imperative to perform a
denaturation (melt) curve after each qPCR
experiment to verify reaction specificity
and ensure that only the desired target was
amplified.
 Additionally, unlike with
probe-based qPCR that permits
multiplexing, only one target can be
interrogated at a time in a dye-based
qPCR experiment.
DNA Probe –based RealTime PCR
 DNA Probe –based RealTime PCR
 Probe-based quantitative PCR (qPCR)
uses real-time fluorescence from 5ʹ-3ʹ
exonuclease cleavage of a fluorescently-
labeled, target-specific probe to measure
DNA amplification at each cycle of a
PCR. Because probe-based qPCR is
typically more specific than dye-based
qPCR
 DNA Probe –based RealTime PCR
 Probe designs vary but the most common
type, hydrolysis (e.g., TaqMan®) probes,
incorporate a 5’ reporter fluorophore and a 3’
quencher on a short oligonucleotide
complementary to the target sequence.
 Fluorescence resonance energy transfer
(FRET) prohibits emission of the fluorophore
while the oligo probe is intact. During each
PCR cycle, the 5’ flap endonuclease domain
of Taq DNA polymerase hydrolyzes the probe
as the primer is extended and the target
sequence is amplified.
 DNA Probe –based RealTime PCR
 Thiscleavage event separates the reporter
fluorophore from the quencher and results
in an amplification-dependent increase in
fluorescence.
 Probe-based qPCR allows multiple targets
to be quantified in a single reaction
(multiplexing) by using a unique
fluorescent dye for each amplicon-specific
probe.
 Alberts B, Johnson A, Lewis J, Raff M, Roberts K,
Walter P. Molecular Biology of the Cell: 5th Ed;
Taylor & Francis Group; 2007.
 2.Karp G. Cell and Molecular Biology: Concepts and
Experiments: 6th Ed; John Wiley & Sons; 2009.
 3. WatsonJD, James D, Myers RM, Caudy AA,
Witkowski JA. Recombinant DNA: genes and
genomes: a short course. 3rd ed; Macmillan; 2007.
 4.Baxevanis AD. Current Protocols in
Bioinformatics: Volumes 1-3; John Wiley & Sons:
2003.
 David P. Calrk . Molecular Biology