informative

1. Presentation by : Dr.Aina Khurshid
MBBS, MPhil biochemistry, CHPE.

2. CASE SCENARIO:
A 20 year old girl was presented to Nishtar hospital
accident & emergency department with complaints of
fever, cough, flu & general body aches for 10 days. The
duty doctor asked her to go to corona ward because of
suspicion of covid-19. what is the most common test for
the detection of covid-19?

3. PCR Technique

4. This Photo by Unknown Author is licensed under CC BY-SA

5. Definition:
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 to generate thousands to millions of
copies of a particular DNA sequence.
 History:
It was invented in 1983 by Dr. Kary Mullis, for which he
received the Nobel Prize in Chemistry in 1993.

6. Why “Polymerase”?
It is called “polymerase” because the only enzyme used
in this reaction is DNA polymerase.
Why “Chain”?
It is called “chain” because the products of the first
reaction become substrates of the following one, and so
on.

7. Basic requirement for PCR technique:
DNA sequence of target region must be known.
Primer --- A primer is a short nucleic acid sequence
that provides a starting point for DNA synthesis. It
consist of 20-30 nucleotide bases.
Thermostable DNA polymerase --- Taq polymerase
is derived from bacterium Thermus aquaticus &
this is not inactivated by heating to 95°c.

8. PCR machine ---- which can be programmed to carry
out heating & cooling of samples over a number of
cycles.
dNTPs - deoxynucleotide triphosphates: DNA
building blocks. It includes dATP, dGTP, dCTP, dTTP.
Mg++ ions - cofactor of the enzyme.
Buffer solution – maintains pH and ionic strength of
the reaction solution suitable for the activity of the
enzyme.

9. Primer design:
Primer sequence should be unique.
G/C content should be 45- 55%.
The primer must not be base paired with each other
to form hairpins like structure.
Primers must avoid repetitive DNA regions.
Primer always binds to 3 end of template strand.

10. How does PCR works?
Heat ( 94° c) to denature DNA strand.
Cool (54° c) to anneal primers to template.
Warm (72° c) to activate Taq polymerase , which
extends primer & replicates DNA.
Repeat multiple cycles.

12. Denaturation:
Temperature = 92°- 94° c.
Double stranded DNA melts to single stranded DNA.

13. Annealing:
Temperature = 50° – 70° c.
Primers bind to their complementary sequence.

14. Extension:
Temperature = 72° c
DNA polymerase binds to anneal primers & extend DNA at
3 end of chain.

16. Application of PCR:
Genetic matching.
Genetic engineering.
Pre-natal diagnosis.
Mutation detection.

17. Classification of organisms.
Detection of pathogens.
Genotyping.
Cancer research.

18. THANK YOU