Polymerase chain reaction or PCR is a laboratory technique that has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing.

1. Polymerase Chain Reaction
Jasleen Saini

2. Polymerase Chain Reaction (PCR)
• PCR is a technique that is used to amplify few copies of DNA to
generate millions of copies of DNA segment.
• It is the most common technique used in medical and biological
research for a variety of applications.
• These applications include
Cloning of DNA
DNA based phylogeny studies
Functional analysis of genes
Diagnosis of hereditary diseases
Genetic fingerprinting for forensic science and paternity testing
Detection and diagnosis of infectious disease.

3. Principle of PCR
• It relies on the principle of of thermal cycling, consisting of cycles of
repeated heating and cooling of the reaction for DNA denaturing and
enzymatic replication of the DNA.
• To specifically amplify a specific segment of DNA, oligonucleotide primers
(short DNA fragments ranging from 15 to 40 base pairs) containing
sequences complementary to the target segment are added to a reaction
along with DNA polymerase and other components necessary for DNA
polymerization.
• As PCR progresses through cycle after cycle, the DNA generated at each
cycle is itself used as a template for another round of replication, resulting
in a chain reaction in which the DNA segment is exponentially amplified.

4. Components of PCR
1. DNA template – This is the target DNA to be amplified.
2. Primers – These are the short DNA fragments ranging from 15 to 40
nucleotides that are complementary to the 3' ends of each of the DNA
strands.
3. Taq polymerase or another thermostable DNA polymerase – This
catalyzes the primer-dependent incorporation of nucleotides into duplex
DNA in the 5′→3′ direction in the presence of Mg2+.
4. Deoxynucleotide triphosphates (dNTPs) - These are the building blocks
from which the DNA polymerase synthesizes a new DNA strand.
5. Buffer solution - This contains salts, providing a suitable chemical
environment for optimum activity and divalent cation, magnesium
(Mg2+) which is required by Taq polymerase work properly.

5. 5’
5’
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Initiation and Denaturation
Annealing
Extension
Final extension
Steps
of PCR

6. Steps of PCR
• Initial denaturation/Initiation: This step consists of heating the reaction to a
temperature of 94–96°C, which is held for 1–9 minutes. This step ensures denaturation
of the double stranded DNA into two single strands.
• Denaturation: This step is the first regular cycling step and consists of heating the
reaction to 94–98°C for 20–60 seconds. It causes DNA denaturation by disrupting the
hydrogen bonds between complementary bases, yielding single stranded DNA
molecules.
• Annealing: The reaction temperature is lowered to 50–68 °C for 20–60 seconds allowing
annealing of the DNA oligonucleotide primers to the single stranded DNA template.
While the annealing temperature is specific to the oligonucleotide primers and salt
concentration, typically the annealing temperature is about 3-5 degree Celsius below the
melting temperature of the primers. Stable DNA-DNA hydrogen bonds are formed when
the primer sequence very closely matches the template sequence. The DNA polymerase
binds to the primer template hybrid and begins DNA synthesis.

7. • Extension/elongation step: The temperature at this step depends on the
DNA polymerase. Taq polymerase has its optimum activity at 75–80 °C, and
commonly 72°C is used. At this step, the DNA polymerase synthesizes a
new DNA strand complementary to the DNA template strand by adding
dNTPs that are complementary to the template in 5' to 3' direction. The
extension time with vary depending both on the DNA polymerase used and
on the length of the DNA fragment to be amplified. As a rule of thumb, at
its optimum temperature, the Taq DNA polymerase will polymerize
approximately 1000 bases per minute.
• Final elongation: This single step is occasionally performed at a
temperature of 70–74 °C for 5–15 minutes after the last PCR cycle to
ensure that any remaining single stranded DNA is fully extended.
• Final hold: At this stage, the thermal cycler will cycle to 4°C for an
indefinite time. For example, if you chose to run the PCR reaction
overnight, you may not be there when it finishes. Thus, the thermocycler
will cool to 4oC and remain at that temperature until you return to remove
your sample.

8. How to set up a PCR reaction?
• Add the following reagents to a PCR tube according to the Taq manufacturer’s
protocol: (The one shown below is by NEB)
• Mix gently by vortex and centrifuge briefly.
• Place the tube in the thermocycler, set up the required protocol and start the run.
Reagent 25 ul reaction 50 ul reaction Final concentration
10X Buffer 2.5 ul 5 ul 1 X
dNTPs 0.5 ul 1 ul 200 uM
Forward Primer 0.5 ul 1 ul 0.05-1 uM
Reverse Primer 0.5 ul 1 ul 0.05-1 uM
DNA template Variable Variable <1000 ng
Taq Polymerase 0.125 ul 0.25 ul 1.25 units/50 ul
Nuclease-free water to 25 ul to 50 ul
https://www.neb.com/protocols/0001/01/01/taq-dna-polymerase-with-standard-taq-buffer-m0273