Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA sequences, allowing for thousands to millions of copies from a single or few DNA fragments. The basic PCR setup requires a DNA template, DNA polymerase, primers, nucleotides, a buffer solution, and cations, and the process includes steps of denaturation, annealing, and elongation across multiple cycles to achieve amplification. Real-time PCR enables quantification of the DNA product via fluorescence detection, providing insight into the amplification process during its early phases.

1. 1
Polymerase chain reaction ( PCR )
◘ Definition :-
- is a technique used in molecular biology to amplify a
single copy or a few copies of a piece of DNA across
several orders of magnitude , generating thousands to
millions of copies of a particular DNA sequence .
- It is In vitro ( Test Tube ) amplification to a specific
sequence .
- It is easy and cheap tool to amplify a focused segment of
DNA .
◘ Useful for such purposes as :- ( Applications )
- Quantification of viral genetic material
- Gene expression
- Gene typing and Allelic Discrimination .
- Diagnosis
- Monitoring of genetic diseases .
- Identification of criminals ( In the field of forensics )
- Studying the function of a targeted segment of DNA .

2. 2
◘ A basic PCR set-up requires several components and
reagents including: :-
1- a DNA template that contains the DNA target region to
amplify.
2- a DNA polymerase, an enzyme that polymerizes new DNA
strands; heat-resistant Taq polymerase is especially common
- Taq polymerase isolated from Thermus aquaticus , is the first
isolated and best known enzyme.
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; (without primers there is no double-stranded
initiation site at which the polymerase can bind)
4- deoxynucleoside triphosphates, or dNTPs
; (nucleotides containing triphosphate groups), the building blocks
from which the DNA polymerase synthesizes a new DNA strand
5- a buffer solution providing a suitable chemical environment
for optimum activity and stability of the DNA polymerase
6- bivalent cations, typically magnesium (Mg)
or manganese (Mn) ions; Mg2+
is the most common
- Mg : is a co-factor , and make chelating for DNase .
7- monovalent cations, typically potassium (K) ions
♠ Master Mix : all of the components required for PCR, except
DNA template and primers.)‫االستخدام‬ ‫قبل‬ ‫باردة‬ ‫حرارة‬ ‫درجة‬ ‫تحت‬ ‫بيكون‬ (

3. 3
◘ Steps :
1- Denaturation
2- Annealing ‫األساسية‬ ‫الخطوات‬ ‫دول‬
3- Extension / elongation ‫دورة‬ ‫لكل‬
♠ Procedures :-
1- 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, which is then held for 1–10 minutes.
2- Denaturation: This step is the first regular cycling event and
consists of heating the reaction chamber to 90–95 °C 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.
92C
3’5’
3’ 5’
+
5’3’
5’ 3’

4. 4
3- Annealing: In the next step, the reaction temperature is lowered
to 45–75 °C 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.
- This temperature must be low enough to allow
for hybridization of the primer to the strand, i.e., the primer should
bind only to a perfectly complementary part of the strand, If the
temperature is too low, the primer may bind imperfectly. If it is
too high, the primer may not bind at all.
- A typical annealing temperature is about 3–5 °C below
the Tm of the primers used.
5’3’
5’ 3’
Forward primer
Reverse primer

5. 5
4- Extension/elongation: The temperature at this step depends on
the DNA polymerase used; the optimum activity temperature
for Taq polymerase is approximately 72–73 °C .
- 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.
Taq
5’
3’
Taq5’

6. 6
♠ The processes of denaturation, annealing and elongation constitute a
single cycle.
♠ Multiple cycles are required to amplify the DNA target to millions of
copies.
♠ The formula used to calculate the number of DNA copies formed after a
given number of cycles is 2n
, where n is the number of cycles.
5- Final elongation: This single step is optional, but is performed at a
temperature of 70–74 °C (the temperature range required for optimal
activity of most polymerases used in PCR) for 5–15 minutes after the last
PCR cycle to ensure that any remaining single-stranded DNA is fully
elongated.
6- Final hold: The final step cools the reaction chamber to 4–15 °C for an
indefinite time, and may be employed for short-term storage of the PCR
products.

7. 7
Theoretically ( E= 100 % )
◘ Efficiency
Practically ( E < 100 % )
(Theoretically)
Target
Cycles
◘ ( Linear Shape )
◘ The formula used to calculate the number of DNA
copies formed after a given number of cycles is 2n
,
where n is the number of cycles.

8. 8
(Practically)
Target
Cycles
◘ Exponential curve
◘ Efficiency < 100 %

9. 9
the entire PCR process can further be divided into stages
based on reaction progress:
Target Plateau phase
Base phase
= Lag phase Log – Linear Phase
= Stochastic
phase
Exponential phase
No. of cycles
• Base phase : No Increase .
• Exponential phase : Sudden Increase .
• Log – Linear Phase : Slight Increase .
• Plateau phase : No Increase .

10. 10
Qualitative ( Semi-
Quantitative ) PCR
Quantitative PCR
(q PCR)
( Kinetic PCR )
Conventional = Regular PCR Automated PCR
Detection only
Traditional PCR methods is
use
gel electrophoresis for the
detection of PCR amplification
in the final phase or at end-
point of the PCR reaction.
Detection& Quantification
real time PCR allows for the
detection of PCR product (
DNA )during the early phases
of the reaction.
End point PCR Real Time PCR
Two Steps One step
Principle:
- Detection According to Gel
( Gel Based PCR )
Principle:
-Detection & Quantification
According to Fluorescence
(Fluorescence based PCR )
Discrimination of product by
Size ( Gel electrophoresis )
Discrimination according to
Fluorescence Intensity
Simple Data analysis Complex Data analysis
Inexpensive Expensive

11. 11
◘ ΔRn is plotted against PCR cycle number, Where ΔRn is
magnitude of Fluorescence intensity
ΔRn = Fluorescence emission of target minus ( - ) Fluorescence of
Reference dye ( Ref )
◘ Real-time PCR, also called quantitative PCR or qPCR, can
determining the amount of a target sequence or gene that is
present in a sample.

12. 12
◘ Ct (threshold cycle) is the intersection between
an amplification curve and a threshold line
- Ct : is defined as the fractional PCR cycle number
at which the reporter fluorescence is greater than
the threshold .
- Ct : is a basic principle of real time PCR and is
essential component in producing accurate and
reproducible data .
- The number of cycles at which the fluorescence
exceeds the threshold is called the threshold cycle .
- A signal that is detected above the threshold is
considered a real signal that can be used to define
the threshold cycle for a sample .

13. 13
How Real-Time PCR Works) ‫(للتوضيح‬
To understand how real-time PCR works, let’s start by examining a
sample amplification plot
-- In this plot, the PCR cycle number is shown on the x-axis,
and the fluorescence from the amplification reaction,is shown on the
y-axis.
… which is proportional to the amount of amplified product in the
tube
- The amplification plot shows two phases, an exponential phase
followed by a non-exponential plateau phase.
- During the exponential phase, the amount of PCR product
approximately doubles in each cycle. As the reaction proceeds,
however, reaction components are consumed, and ultimately one or
more of the components becomes limiting. At this point, the reaction
slows and enters the plateau phase (cycles 28–40 in Figure).

14. 14
◘ Quantitative PCR methods ( Real time PCR ) :
• Quantitative PCR methods use fluorescent dyes, such as SYBR
Green ,or Taq Man probe Technique ( Hydrolysis ) to measure
the amount of amplified product in real time.
1- SYBR Green (DNA-binding agents)
Step-by-step process
1.When SYBR® dye is added to a sample, it immediately
binds to all double-stranded DNA present in the sample.
2.During PCR, DNA polymerase amplifies the target
sequence which creates the PCR products.
3.SYBR® dye then binds to each new copy of double-
stranded DNA.
4.As the PCR progresses, more PCR product is created.
SYBR® dye binds to all double-stranded DNA, so the
result is an increase in fluorescence intensity proportioned
to the amount of PCR product produced.

15. 15
- No fluorescent-labeled probes required.
- Increased fluorescence when bound to double-stranded DNA
- the amount of signal is dependent on the mass of double-
stranded DNA produced in the reaction.

16. 16
2- Taq Man probe Technique
- TaqMan probes are hydrolysis probes that are designed to
increase the specificity of quantitative PCR
- TaqMan probe is a short DNA sequence with a high energy dye
called reporter dye at the 5´ end and a low energy dye called
quencher at the 3´ end.
- TaqMan probes consist of a fluorophore covalently attached to
the 5’-end of the oligonucleotide probe and a quencher at the 3’-
end
- As long as the fluorophore ( reporter ) and the quencher are in
proximity ) ‫بعض‬ ‫من‬ ‫بالقرب‬ (, quenching inhibits any fluorescence
signals.
- the 5' to 3' exonuclease activity of the Taq polymerase degrades
the probe that has annealed to the template. Degradation of the
probe releases the fluorophore (the reporter ) from it and breaks
the close proximity to the quencher, thus relieving the quenching
effect and allowing fluorescence of the fluorophore.
- Hence, fluorescence detected in the quantitative PCR thermal
cycler is directly proportional to the fluorophore released and the
amount of DNA template present in the PCR.

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‫انتهت‬‫وراثة‬ ‫محاضرة‬ ‫أخر‬♥
Q.A ☺ ♥