Real Time PCR

Pramod Bahadur K.C.

What is real time PCR?

 Polymerase chain reaction (PCR) is a technique used in molecular biology

to amplify a single copy or a few copies of a segment of DNA, generating
thousands to millions of copies of a particular DNA sequence.
 Its applications are vast and PCR is now an integral part of Molecular
Biology.
 Real-Time PCR a specialized technique that allows a PCR reaction to be
visualized “in real time” as the reaction progresses.
 Compared to traditional methods of DNA cloning and amplification, which
can often take days, PCR requires only a few hours.
 PCR is highly sensitive and requires minimal template for detection and
amplification of specific sequences.
 To understand real time PCR we need to first understand the principal of
basic PCR
Basic Principles

 PCR are in-vitro version of DNA replication. The following components are
needed to perform PCR in the laboratory:
1) DNA (your DNA of interest that contains the target sequence you wish to
copy)
2) A heat-stable DNA Polymerase (like Taq Polymerase)
3) All four nucleotide triphosphates (Adenine, Thymine, Cytosine, Guanine)
4) Buffers
5) Two short, single-stranded DNA molecules that serve as primers
6) Thin walled tubes
7) Thermal cycler (a device that can change temperatures dramatically in a
very short period of time)
 Real time PCR is carried out in a thermal cycler with the capacity to
illuminate each sample with a beam of light of at least one specified
wavelength and detect the fluorescence emitted by the
excited fluorophore.
 We use the reagents that can provide florescence in the presence of
amplified DNA.
 Ethidium bromide and SYBR Green 1 dye are two example of such
reagents.
 They bind to the double stranded DNA and emit light of specific
wavelength.
Methods

 Real Time PCR instrument consist of Three main components.

 Thermal Cycler (PCR machine)
 Optical Module ( for detection of fluorescence in the tubes during run)
 Computer ( to translate the fluorescence data into meaningful results)
 The PCR process generally consists of a series of temperature changes that
are repeated 25 – 50 times.
 First around 95ºC Allows separation of nucleic acid double chain
 Second around 50ºC allowing binding of the template to the DNA helixes.
 the third, at between 68 - 72 °C, facilitates the polymerization by the DNA
polymerase. Yielding double stranded DNA complex.
3’ 5’
5’ 3’

d.NTPs
Primers
Thermal Stable
DNA Polymerase

Add to Reaction Tube

Denaturation

Annealing
3’ 5’

5’ 3’

Extension

3’ Taq 5’

5’ 3’
Taq

Extension Continued

3’ Taq 5’

Taq 3’

Repeat
 In Real Time PCR, Dyes and probes are used to scan and see the
procedures as the PCR process begins.
 Dyes such as SYBR Green I, binds double-stranded DNA molecules by
intercalating between the DNA bases.
 fluorescence can be measured at the end of each amplification cycle of DNA
replication.
 Probe-based PCR uses fluorescent-labeled target-specific probes. This technique
yields increased specificity and sensitivity since only specific DNA molecules will
be labeled.
Florescent
Dyes in PCR. 3’ 5’

SYBR Green 1
binds all newly 5’ 3’

synthesized
Extension
double
stranded DNA Taq 5’
3’
ID ID
Complexes.
ID ID ID
The intensity of 5’
Taq 3’

the Apply Excitation

florescence Wavelength
(Ct value) is l
l l
measured to 3’
ID ID ID ID ID
Taq 5’
quantitate the
ID ID ID ID
newly Taq 3’

generated l
l
DNA.
Repeat
 Probes
 Probes with Q R

quencher molecule 5’ 3’
and reporter
molecule anneals to Extension
single stranded Q
R

molecule as the Taq

strand is separated.
5’ 3’
R

 As DNA polymerase Q
Hydrolysis
Taq
displaces the 5’
5’ 3’
Reporter molecule
from the probe Taq
R

during extension. 5’
Fluorescence 5’ 3’

happens. Then CT l
value is measured at R
Signal
Taq
the end of the
5’
cycle. 5’ 3’
Application of Real Time PCR

 Gene expression analysis

 Disease diagnosis and management
 Food testing
 Animal and plant breeding
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