REAL TIME PCR

Presented by: SUVODIP JANA

16/BT/06
 OUTLINE
1.What is PCR ?
2.Mechanism of PCR
3.Disadvantages of PCR
4.Real time PCR
5.Mechanism of real time PCR
6.Types of real time PCR probes
7.Advantages of Real time PCR
8.Disadvantages of real time PCR
9.Applications of real time PCR
Polymerase chain reaction (PCR) is a method widely used in molecular biology to make many copies of a specific DNA
segment. Using PCR, copies of DNA sequences are exponentially amplified to generate thousands to millions of more copies
of that particular DNA segment.

PCR was developed by Kary Mullis in 1983 while he was an employee of the Cetus Corporation. He was awarded the Nobel
Prize in Chemistry in 1993 (along with Michael Smith) for his work in developing the method.

The vast majority of PCR methods rely on thermal cycling.

A basic PCR set-up requires several components and reagents:

1. DNA template that contains the DNA target region to amplify
2. A DNA polymerase (it should remain intact during the high-temperature DNA denaturation process)
3. Two DNA primers that are complementary to the 3' (three prime) ends of each of the sense and anti-sense
strands of the DNA target
4. deoxynucleoside triphosphates, or dNTPs
5. A buffer solution providing a suitable chemical environment for optimum activity and stability of the DNA
polymerase
 Mechanism of PCR
Initialization: This step is only required for DNA polymerases that require heat activation by hot-start PCR.It consists of heating
the reaction chamber to a temperature of 94–96 °C (201–205 °F), or 98 °C (208 °F) if extremely thermostable polymerases
are used, which is then held for 1–10 minutes.

Denaturation: This step is the first regular cycling event and consists of heating the reaction chamber to 94–98 °C (201–208 °F)
for 20–30 seconds. This causes DNA melting, or denaturation, of the double-stranded DNA template by breaking the hydrogen
bonds between complementary bases, yielding two single-stranded DNA molecules.

Annealing: In the next step, the reaction temperature is lowered to 50–65 °C (122–149 °F) for 20–40 seconds, allowing
annealing of the primers to each of the single-stranded DNA templates. Two different primers are typically included in the
reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-
stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short
sequences at the 3' end of each strand.

Extension/elongation: The temperature at this step depends on the DNA polymerase used; the optimum activity temperature for
the thermostable DNA polymerase of Taq (Thermus aquaticus) polymerase is approximately 75–80 °C (167–176 °F),[16][17]
though a temperature of 72 °C (162 °F) is commonly used with this enzyme. In this step, the DNA polymerase synthesizes a new
DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that are
complementary to the template in the 5'-to-3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxy
group at the end of the nascent (elongating) DNA strand.
Animation
 Disadvantages of traditional PCR
In traditional PCR, the product is analyzed only at the end of the reaction. The most commonly
used method is agarose gel electrophoresis that separates DNA fragments on the basis of their
molecular mass. At the end of the separation, the gel is stained with a dye called ethidium
bromide that binds to DNA and can be visualized under UV light

Less sensitivity towards specific DNA sequence

It requires post PCR processing to know about the amplification of the specific sequence.
A real-time polymerase chain reaction (Real-Time PCR), also known as quantitative polymerase chain reaction
(qPCR), is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors
the amplification of a targeted DNA molecule during the PCR, i.e. in real-time, and not at its end, as in conventional
PCR. Real-time PCR can be used quantitatively (quantitative real-time PCR), and semi-quantitatively, i.e.
above/below a certain amount of DNA molecules (semi quantitative real-time PCR).

Two common methods for the detection of PCR products in real-time PCR are:
(1) non-specific fluorescent dyes that intercalate with any double-stranded DNA, and
(2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which
permits detection only after hybridization of the probe with its complementary sequence.
Non-specific detection: Real-time PCR with double-stranded
DNA-binding dyes as reporters
A DNA-binding dye binds to all double-stranded (ds) DNA in PCR, causing fluorescence of the
dye. An increase in DNA product during PCR therefore leads to an increase in fluorescence
intensity measured at each cycle. However, dsDNA dyes such as SYBR Green will bind to all
dsDNA PCR products, including nonspecific PCR products (such as Primer dimer). This can
potentially interfere with, or prevent, accurate monitoring of the intended target sequence.

In real-time PCR with dsDNA dyes the reaction is prepared as usual, with the addition of
fluorescent dsDNA dye. Then the reaction is run in a real-time PCR instrument, and after each
cycle, the intensity of fluorescence is measured with a detector; the dye only fluoresces when
bound to the dsDNA (i.e., the PCR product). This method has the advantage of only needing a
pair of primers to carry out the amplification, which keeps costs down; multiple target
sequences can be monitored in a tube by using different types of dyes.
 Analysis of Real-Time PCR data
As the number of cycle progresses, the amount of double-stranded DNA increases and so does the fluorescence. To
quantitate the amount of DNA template, the software of themachine plots the Number of Cycles versus Laser reading of
Fluorescence Intensity.

This graph is known as ‘Amplification Curve’ A threshold is decided depending upon the range of detection of amplification.
As soon as the fluorescence crosses that threshold, the software is confident that amplification has occurred. The cycle at
which fluorescence crosses threshold value is called ‘Cycle threshold’ or ‘Ct’.

The more the Ct value, the lower the

viral genome in the serum sample.
The SYBR Green molecule can also bind to these nonspecific double-stranded DNAand
primer-dimer complexes

This problem was countered by the introduction of ‘dissociation-curve analysis’.

The melting temperature of DNA depends upon the length of the strand and its GC content.
These properties can be exploited to distinguish between the specific and non-specific products.
Thus, an extra step was added to the PCR reaction. After the reaction is complete, the
amplification products are cooled to 25oC and then the temperature of the solution is slowly but
progressively increased again.

The laser now detects the loss of fluorescence as more of SYBR Green is released when DNA
melts at high temperature.
 A ‘Dissociation curve’ is a graph of ‘Temperature vs Fluorescence’.

To ascertain whether the peak obtained is of desired product, the value can be
compared with standards
 Specific detection: fluorescent reporter probe method
The method relies on a DNA-based probe with a fluorescent reporter at one end and a quencher
of fluorescence at the opposite end of the probe.

The close proximity of the reporter to the quencher prevents detection of its fluorescence;
breakdown of the probe by the 5' to 3' exonuclease activity of the Taq polymerase breaks the
reporter-quencher proximity and thus allows unquenched emission of fluorescence, which can
be detected after excitation with a laser.

An increase in the product targeted by the reporter probe at each PCR cycle therefore causes a
proportional increase in fluorescence due to the breakdown of the probe and release of the
reporter.
 Applications of PCR
There are numerous applications for quantitative polymerase chain reaction in the
laboratory. It is commonly used for both diagnostic and basic research.
1. Quantification of gene expression

2. Diagnostic uses

Diagnostic example, infectious qualitative PCR is applied to rapidly detect nucleic acids that are
diagnostic of, for diseases, cancer and genetic abnormalities. The introduction of qualitative PCR
assays to the clinical microbiology laboratory has significantly improved the diagnosis of
infectious diseases, and is deployed as a tool to detect newly emerging diseases, such as new
strains of flu, in diagnostic tests.
Microfluidics, high throughput and the associated automation are possibly some of the key areas of hardware
development driving the industry forward. Newer technologies that allow automated processing and simplify the data
analysis will go hand-in-hand

Quantitative PCR (qPCR) has revolutionised the way pharmaceutical laboratories discover biomarkers. In general,
qPCR diagnosis is completed in an hour or even less (15 minutes in some specific conditions), which is considerably
faster than conventional PCR and other detection methods (ELISA)
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