PCR is a biochemical technology in molecular biology. This technique used in DNA cloning for sequencing, gene cloning and manipulating, diagnosis of hereditary diseases.

1. POLYMERASE CHAIN
REACTION (PCR)
By
Miss Rasika R. Ingale.
Biochemical Engineering and Biotechnology

2. Polymerase chain Reaction (PCR):
• It is a biochemical technology used in molecular
biology research labs. PCR is used to amplify a
single copy or few copies of DNA piece
• By using PCR technique , It is possible to generate
thousand of copies of a desired section of DNA
from very small amount of DNA.
• It is used in initial stages of DNA sequencing, for
determination of particular gene which is
responsible for disease.

3. A basic PCR set up requires several components
and reagents. These components include:
• DNA template that contains the DNA region to be
amplified.
• Two primers
• Taq polymerase or another DNA polymerase with
optimum temperature 70ᵒ C
• DNA nucleotide bases (dNTPs)
• Buffer solution used for suitable environment to
maintain stability and optimum activity of enzyme
DNA polymerase.
• Bivalent cations Mg2+ and Mn2+
• Monovalent cations potassium Ions

5. PCR consists of a series of 20-30 repeated changes in the
temperature cycles. Following steps are involved in PCR:
• Initialization
• Denaturation
• Annealing
• Elongation
• Final Elongation
Procedure:

6. Initialization Step:
This step consists of heating reaction to a temperature of 94-98ᵒ
C, which is held for 1-8 minutes. It is only required for DNA
polymerase enzyme activation.
Denaturation Step:
It causes DNA melting of DNA template by disrupting the
hydrogen bonds between complementary bases, yielding single
stranded molecules
Annealing:
The reaction temperature is decrease to 50-60 ᵒ C allowing
annealing of the primers to the single stranded DNA template.
The polymerase binds to the primer template hybrid and starts
DNA formation.

7. Elongation:
The temperature at this step depends upon the DNA polymerase
used. Taq polymerase has its optimum activity temperature at
75-80ᵒ C and commonly 72ᵒ C temperature is used with this
enzyme.
In this step the DNA polymerase synthesizes a new DNA strand
complementary to the DNA template by adding dNTP’s in 5'-3'
direction.
Final Elongation:
Sometimes final elongation step is performed at a temperature
of 70-74ᵒ C for 5-10 minutes after the last PCR cycle to make
sure that any ssDNA is fully extended.

8. PCR Cycle