Subject Name: Genetic Engineering-II

Subject Code: MTI-605

Unit No:5
Unit Name: Molecular Diagnostics
Faculty Name : Dr. V. Suresh Gupta

Sub Topic Name Real Time PCR

Real Time PCR

In molecular biology, real-time polymerase chain reaction,

also called quantitative real time polymerase chain reaction
(QRT-PCR) or kinetic polymerase chain reaction, is a
laboratory technique used to simultaneously quantify and
amplify a specific part of a given DNA molecule.
It is used to determine whether or not a specific sequence is
present in the sample; and if it is present, the number of
copies in the sample.
It is the real-time version of quantitative polymerase chain
reaction(Q-PCR),itself a modification of polymerase chain
reaction.

Real Time PCR

Follows principle of polymerase chain reaction; its key feature

is that the amplified DNA is detected as the reaction
progresses in realtime,a new approach compared to standard
PCR,where the product of the reaction is detected at its end.
Frequently, real-time PCR is combined with reverse
transcription to quantify messenger RNA and non-coding RNA
in cells or tissues.

Real Time PCR

NEED FOR REAL TIME PCR

WHATS WRONG WITH AGAROSE GELS?
Poor precision
Low sensitivity
Low resolution
Non-automated
Size-based discrimination only
Results are not expressed as numbers
Ethidium bromide staining is not very quantitative

Real Time PCR

Real time detection as opposed to end-point detection

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Real Time PCR

PRINCIPLES OF REAL TIME PCR

Based on the detection and quantitation of a fluorescent reporter

The first significant increase in the amount of PCR product (CTthreshold cycle) correlates to the initial amount of target
template.
There are 3 general methods for the quantitative detection:
Hydrolysis probes (TaqMan, Beacons, Scorpions)
DNA-binding agents (SYBR Green)
Hybridisation probes (Light Cycler)

Real Time PCR

Hydrolysis Probes
The hydrolysis probe is conjugated with a quencher
fluorochrome , which absorbs the fluorescence of the reporter
fluorochrome as long as the probe is intact .
However, upon amplification of the target sequence , the
hydrolysis probe is displaced and subsequently hydrolyzed by
the Taq polymerase.
This results in the separation of the reporter and quencher dyes
and consequently the fluorescence of the reporter dye becomes
detectable .
During each consecutive PCR cycle this fluorescence will
further increase because of the progressive and exponential
accumulation of free reporter dyes.
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Real Time PCR

1. TaqMan Probes
TaqMan probes are oligonucleotides that have a fluorescent reporter
dye attached to the 5 end and a quencher moiety coupled to the 3
end .
These probes are designed to hybridize to an internal region of a
PCR product.
TaqMan probes depend on the 5 to 3 exonuclease activity of the
DNA polymerase used for PCR to hydrolyze an oligonucleotide that
is hybridized to the target amplicon
In the unhybridized state, the proximity of the flour and the quench
molecules prevents the detection of fluorescent signal from the
probe.
It does this by the use of Fluorescence ( or Forester ) Resonance
Energy Transfer (FRET), which is the inhibition of one dye caused
by another without emission of a proton.
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Real Time PCR

During PCR , when the polymerase replicates a template on

which a TaqMan probe is bound , the 5 to 3 exonuclease
activity of the polymerase cleaves the probe.
This decouples the fluorescent and quenching dyes and FRET
no longer occurs.
Thus , fluorescence increases in each cycle , proportional to
the amount of probe cleavage

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Real Time PCR

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Real Time PCR

2. Molecular Beacons
Molecular Beacons also use FRET to detect and quantitate the
synthesized PCR product via a fluor coupled to the 5 end and a
quench attached to the 3 end of an oligonucleotide substrate.
They are designed to remain intact during the amplification reaction
, and must rebind to target in every cycle for signal measurement.
They form a stem-loop structure when free in solution. Thus, the
close proximity of the fluor and quench molecules prevents the
probe from fluorescing.
When a Molecular Beacon hybridizes to a target , the fluorescent
dye and quencher are separated, FRET does not occur, and the
fluorescent dye emits light upon irradiation.

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Real Time PCR

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Real Time PCR

Scorpion Probes
With Scorpion probes, sequence specific priming and PCR product detection
is achieved using a single oligonucleotide.
The probe maintains a stem-loop configuration in the unhybridized state. The
fluorophore is attached to the 5' end and the quench moiety is coupled to the
3'end.
The 3' portion of the stem also contains sequence that is complementary to
the extension product of the primer.
This sequence is linked to the 5' end of a specific primer via a nonamplifiable monomer.
After extension of the Scorpion primer, the specific probe sequence is able to
bind to its complement within the extended amplicon thus opening up the hair
pin loop.
Thus fluorescence is being quenched and a signal is observed.
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Real Time PCR

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Real Time PCR

DNA Binding Agents

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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Real Time PCR

SYBR Green Chemistry

SYBR Green provides the simplest and most economical format for
detecting and quantitating PCR products in real-time reactions.
SYBR Green binds double stranded DNA, and upon excitation emits
light. Thus, as a PCR product accumulates, fluorescence increases.
The advantages of SYBR Green are that it is inexpensive, easy to
use, and sensitive. The disadvantage is that SYBR Green will bind
to any double-stranded DNA in the reaction, which results in an over
estimation of the target concentration.
At the beginning of amplification, the reaction mixture contains the
denatured DNA, the primers, and the dye. The unbound dye
molecules weakly fluoresce, producing a minimal background
fluorescence signal which is subtracted during computer analysis.

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Real Time PCR

After annealing of the primers, a few dye molecules can bind to the double
strand. DNA binding results in a dramatic increase of the SYBR Green I
molecules to emit light upon excitation.
During elongation, more and more dye molecules bind to the newly
synthesized DNA. If the reaction is monitored continuously, an increase in
fluorescence is viewed in real time. Upon denaturation of the DNA for the
next heating cycle, the dye molecules are released and the fluorescence signal
falls.

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Real Time PCR

In this technique one probe is labelled with a donor

fluorochrome at the 3end and a secondadjacentprobe is
labelled with an acceptor fluorochrome.
When the two fluorochromes are in close vicinity (15
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. After
each subsequent PCR cycle more hybridisation probes can
anneal, resulting in higher fluorescence signals.

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Real Time PCR

A Light Cycler FRET probe system is a pair of single-stranded

fluorescently-labeled oligonucleotides.
Probe 1 (the donor probe) is labeled at its 3 end with a donor
fluorophore (generally fluorescein) and Probe 2 (the acceptor probe)
is labeled at its 5 end with one of four available fluorophores.
The free 3 hydroxyl group of Probe 2 must be blocked with a
phosphate group (P) to prevent Taq DNA polymerase extension.
During the annealing step of real-time PCR, the PCR primers and
the Light Cycler probes hybridize to their specific target regions
causing the donor dye to come into close proximity to the acceptor
dye. When the donor dye is excited by light from the Light Cycler
instrument (h1), energy is transferred by FRET from the donor to
the acceptor dye.
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Real Time PCR

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Real Time PCR

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Real Time PCR

The first step is reverse transcription (RT), in which RNA is

reverse transcribed to cDNA using reverse transcriptase and
primers.
The RT step can be performed either in the same tube with
PCR (one-step PCR) or in a separate one (two-step PCR) using
a temperature between 40 C and 50 C, depending on the
properties of the reverse transcriptase used.
The next step involves the denaturation of the dsDNA
followed by annealing.
The final step of PCR amplifications DNA extension from the
primers.
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Real Time PCR

USES OF REAL TIME PCR

Highly sensitive technique in which a very low copy number

of RNA molecules can be detected.
Diagnosis of genetic diseases and determination of the specific
different RNA molecules within a cell or tissue as measure of
gene expression.
RT- PCR is commonly used in studying the genomes of viruses
whose genomes are composed of RNA, such as Influenza virus
A and retroviruses like HIV

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Real Time PCR

ADVANTAGES OF Q-PCR

Not influenced by non-specific amplification

Amplification can be monitored real-time
No post PCR processing of products (high through put, low
contamination risk)
Ultra-rapid cycling ( 30 minutes to 2 hours)
Wider dynamic range of up to 10 10 fold
Requirement of 1000-fold less RNA than conventional assays
Detection is capable down to a 2-fold change
Confirmation of specific amplification by melting point analysis
Most specific, sensitive and reproducible
Not much more expensive than conventional PCR

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Real Time PCR

APPLICATIONS OF PCR / Q-PCR

Detection of the presence/absence of pathogens in a host

Genetic fingerprinting
Paternity testing
Detection of hereditary diseases
Cloning genes
Mutagenesis
Analysis of ancient DNA
Genotyping of specific mutations
Comparison of gene expression
Determination of viral load
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Real Time PCR

http://en.wikipedia.org/wiki/Real-time polymerase chain reaction

http://www.bio.davidson.edu/Courses/Molbio/Molstudents/spring2003/Pierce
/realtimepcr.htm
http://pathmicro.med.sc.edu/pcr/realtime-home.htm
www.biosearchtechnologies.com
http://www.invitrogen.com/site/us/en/home/Products-andServices/Applications/PCR/real-time-pcr.html
Real-Time Pcr: An Essential Guide-Kirstin Jane Edwards, Julie (Julie M. J.)
Logan, Nick (Nick A. ) Saunders
http://in.docsity.com/endocs/Real_Time_PCR-Genetic_EngineeringLecture_Slides_
https://www.youtube.com/watch?v=WuLtPNBc1U8

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Real Time PCR

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Thank You