Why we need PCR Invention Of PCR What is PCR Principle of PCR Application of PCR Advantages of PCR Limitation of PCR Conclusion

1. CR Group
Members:
 H. M. Adil Saleem
 CH. M. Mustaqeem
 Syed Qamar
 Samreen Fatima
 Laiba Naseem
Department:
BS. Biochemistry
Cholistan University of
Veterinary and Animal

2. PCR
Polymerase Chain
Reaction

3. Content Of Presentation
1. Why we need PCR
2. Invention Of PCR
3. What is PCR
4. Principle of PCR
5. Application of PCR
6. Advantages of PCR
7. Limitation of PCR
8. Conclusion

4. Why we Need PCR
Because of following reason we need PCR
• How do we identify and detect a specific sequence in a genome?
TWO BIG ISSUES
1. There are a LOT of other sequences in a genome that we’re not
interested in detecting. (SPECIFICITY)
2. The amount of DNA in samples we’re interested in is VERY small.
(AMPLIFICATION)
Specificity
Pine: 68 billion bp
Corn: 5.0 billion bp
Soybean: 1.1 billion bp
Human: 3.4 billion bp
Housefly: 900 million bp
Rice: 400 million bp
E. coli: 4.6 million bp
HIV: 9.7 thousand bp

5. Amplification
How many molecules do we need to be able to see
them?
1. To be visible on an agarose gel, need around 10 ng
DNA for fluorescent stain (or around 25ng for
FastBlast).
2. For a 500-bp product band, weighing 660 g/mol.bp,
therefore need 10e-9 / (500*660) = 3.03e-14 moles.
3. Avogadro’s number = 6.02e23.
4. Therefore need 1.8e10 copies!
5. In other words, to “see” a single “gene”, the DNA in a
sample of 100 cells would have to be multiplied 180
million times!!!!!
PCR solves BOTH of these
issues!!!

6. Invention Of PCR
 Mullis succeeded in demonstrating PCR
on December 16, 1983.
 But He was not alone in this Invention
Michael Smith also involved in this
important invention.
Noble Prize Winning
In 1983 Karry B. Mullis Awarded
Noble Prize in Chemistry along with
Michael Smith for his work on PCR

7. Kary
B.Mullis
Noble Prize
Pics

8. What is PCR?
A technique used to make numerous copies of a specific
segment of DNA quickly and accurately. The polymerase chain
reaction enables investigators to obtain the large quantities of
DNA that are required for various experiments and procedures
in molecular biology, forensic analysis, evolutionary biology,
and medical diagnostics.

10. Principle of PCR
1. Denaturation step:
This step is the first regular cycling event and consists of heating the
reaction to 94-98°C. It causesDNA melting of the DNA template by disrupting
the hydrogen bonds between complementary bases, yielding singlestranded
DNA molecules.
2. Annealing step:
The reaction temperature is lowered to 50-65°C for 20-40 seconds
allowing annealing of the primers to thesingle-stranded DNA template.
3. Extension/elongation step:
The temperature at this step depends on the DNA polymerase used;
Taq polymerase has itsoptimum activity temperature at 75-80°C, and commonly
a temperature of 72°C is used with this enzyme. At this step theDNA polymerase
synthesizes a new DNA strand complementary to the DNA template strand by
adding dNTPs that arecomplementary to the template in 5′ to 3′ direction,
condensing the 5′-phosphate group of the dNTPs with the 3′-hydroxylgroup at
the end of the nascent (extending) DNA strand.

12. Applications of PCR

13. Advantages of PCR
• Small amount of DNA is required per test
• Result obtained more quickly - usually within
1 day for PCR
• Usually not necessary to use radioactive
material (32P) for PCR.
• PCR is much more precise in determining the
sizes of alleles - essential for some disorders.
• PCR can be used to detect point mutations.

14. Limitations of PCR
 Prior Sequence knowledge.
 Short size range of amplification Products.
100bp_ 500bp
 Chances Of Contamination.
 Traditional PCR
 Real Time PCR

15.  In conclusion the polymerase chain reaction is
a very helpful way to replicate DNA and
diagnose certain diseases.
 It is a very realizable way to test and analyze
the DNA and is also very efficient in the sense
that it only takes a few hours.
Conclusion

16. The end