Amplification techniques used in molecular biology for amplifying a target DNA to produce millions of copy. An important tool in molecular study and analysis.

1. POLYMERASE
CHAIN
REACTION
BY SAJIN DANGOL

2. 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 across several orders of
magnitude, generating thousands to millions
of copies of a particular DNA sequence.

3. Developed in 1983 by Kary Mullis, PCR is
anow a common technique used in clinical
and research laboratories for a broad variety
of applications.
In 1993, Mullis was awarded the Nobel Prize
in Chemistry forhis work on PCR.

4. Components of PCR
DNA Template:
DNA template is DNA target sequence.
DNA template is the DNA molecule
containing the DBA region to be
amplified, i.e. the target sequence of
interest.

5. DNA Polymerase:
DNA polymerase helps in extension of primer, it
sequentially adds nucleotides complimentary to
template strand at 3’-OH of the primers used.
Synthesize new strand of DNA complementary
to target sequence.
Taq DNA polymerase ( from Thermus aqaticus,
a thermophilic bacterium) is used due to stability
at high temperature.

6. Primers
A pair of synthetic oligonucleotides is a
prerequisite to prime DNA synthesis. The
length of primers are about 18 – 25 nucleotides
complementary to the 3’- end of the template
strand.
Two primers are designed: the forward primer
and the reverse primer.

8. Nucleotides (dNTPS or
Deoxyribonucleotide triphosphates)
All types of nucleotides are “building
blocks” for new DNA strands and
essential for reaction. It includes
Adenine(A), Guanine(G), Cytosine(C),
Thymine(T) or Uracil(U).

9. Magnesium
Magnesium affects primer annealing and
template denaturation, as well as enzyme
activity. An excess of magnesium gives non
specific amplification products, while low
magnesium yields lesser amount of desired
products.

10. STEPS OF PCR

15. PCR Cycles
First three steps are repeated 35 – 40
times to produce millions of exact
copies of target DNA sequence.

17. ReQUIREMENTS OF PCR

18. Analyze the results of your PCR
reaction via gel electrophoresis

19. The eNd!
tHANk yOU!