PCR polymerase chain reaction What is PCR? History of PCR Requirements for PCR Procedure for PCR

2. Anoop Kumar
8th semester
Presentation Topic : PCR
Submitted to : Dr Hussain Ahmad Sir

4. PCR

5. Contents
• What is PCR?
• History of PCR
• Requirements for PCR
• Procedure for PCR

6. What is PCR ?

7. What is PCR ?
• PCR is a scientific technique & Enzymatic process
• Used in molecular biology
• In which a specific region of DNA is replicated over
and over again to yield many copies of a particular
sequence.
• In vitro technique

8. • PCR is a method widely used to rapidly make millions
to billions of copies of dna from specific DNA sample
• which are either complete or partial
• Scientists to take a very small sample of DNA and
amplify it (or a part of it) to a large enough amount to
study
What is PCR ?

9. • PCR was invented in 1983 by American
biochemist Kary Mullis at Cetus Corporation
History
of PCR

10. • PCR was invented in 1983 by American
biochemist Kary Mullis at Cetus Corporation
of PCR

11. • 1985: First publication of PCR by Cetus
Corporation appears in Science.
• 1986: Purified Taq polymerase is first used
in PCR
• 1989: Science declares Taq polymerase
"molecule of the year.
History
of PCR

12. • 1993:Dr. Kary Mullis awarded Nobel Prize
in Chemistry for conceiving PCR
technology.
History
of PCR

13. • 1993:Dr. Kary Mullis awarded Nobel Prize
in Chemistry for conceiving PCR
technology.
of PCR

14. • 1993:Dr. Kary Mullis awarded Nobel Prize
in Chemistry for conceiving PCR
technology.
of PCR

15. Requirements for PCR

16. Requirements for PCR
DNA
• Do
•
•
•
•
•

17. DNA Template
• Double stranded target
region of DNA
• 18
• Pro
com
• Als
DN
• GC
• Me
64°
Requirements for PCR

18. DNA Template
• Double stranded target
region of DNA
Primer
• 18 and 30 bases long
• Produced by commercial
companies
• Also be prepared using a
DNA synthesizer
• GC content 40 and 60%
• Melting temperature 60-
64°C
Ta
p
•
Requirements for PCR

19. DNA Template
• Double stranded target
region of DNA
Primer
• 18 and 30 bases long
• Produced by commercial
companies
• Also be prepared using a
DNA synthesizer
• GC content 40 and 60%
• Melting temperature 60-
64°C
dTNPs
(deoxyribonucleoside
triphosphates)
• Building blocks for new DNA
strands
• Adenosine triphosphate
(dATP)
• Guanosine triphosphate
(dGTP)
• Cytidine triphosphate (dCTP)
• Thymidine triphosphate
(dTTP)
Taq P
• Th
polym
• Whic
by
Requirements for PCR

20. DNA Template
• Double stranded target
region of DNA
Primer
• 18 and 30 bases long
• Produced by commercial
companies
• Also be prepared using a
DNA synthesizer
• GC content 40 and 60%
• Melting temperature 60-
64°C
dTNPs
(deoxyribonucleoside
triphosphates)
• Building blocks for new DNA
strands
• Adenosine triphosphate
(dATP)
• Guanosine triphosphate
(dGTP)
• Cytidine triphosphate (dCTP)
• Thymidine triphosphate
Taq Polymerase
• Thermostable DNA
polymerase that is used in
PCR.
• Which is not inactivated
by heating to 95°C.
• Comp
pota
magn
• The p
betw
• It pro
cond
the D
funct
Requirements for PCR

21. DNA Template
• Double stranded target
region of DNA
Primer
• 18 and 30 bases long
• Produced by commercial
companies
• Also be prepared using a
DNA synthesizer
• GC content 40 and 60%
• Melting temperature 60-
64°C
dTNPs
(deoxyribonucleoside
triphosphates)
• Building blocks for new DNA
strands
• Adenosine triphosphate
(dATP)
• Guanosine triphosphate
(dGTP)
• Cytidine triphosphate (dCTP)
• Thymidine triphosphate
Taq Polymerase
• Thermostable DNA
polymerase that is used in
PCR.
• Which is not inactivated
by heating to 95°C.
Buffer
• Composed of Tris-HCl,
potassium chloride, and
magnesium chloride .
• The pH of the buffer falls
between 8.0 to 9.5
• It provide suitable
conditions under which
the DNA polymerase can
function properly.
• It a
en
• Bo
am
• He
the
DN
• It i
tem
Requirements for PCR

22. DNA Template
• Double stranded target
region of DNA
Primer
• 18 and 30 bases long
• Produced by commercial
companies
• Also be prepared using a
DNA synthesizer
• GC content 40 and 60%
• Melting temperature 60-
64°C
dTNPs
(deoxyribonucleoside
triphosphates)
• Building blocks for new DNA
strands
• Adenosine triphosphate
(dATP)
• Guanosine triphosphate
(dGTP)
• Cytidine triphosphate (dCTP)
• Thymidine triphosphate
Taq Polymerase
• Thermostable DNA
polymerase that is used in
PCR.
• Which is not inactivated
by heating to 95°C.
Buffer
• Composed of Tris-HCl,
potassium chloride, and
magnesium chloride .
• The pH of the buffer falls
MgCl2
• It acts as a cofactor that
enhances
• Boosting DNA
amplification
• Helps primers anneal to
the correct location on a
DNA template
• It increases the melting
temperature
• U
re
• P
re
• D
a
Requirements for PCR

23. DNA Template
• Double stranded target
region of DNA
Primer
• 18 and 30 bases long
• Produced by commercial
companies
• Also be prepared using a
DNA synthesizer
• GC content 40 and 60%
• Melting temperature 60-
64°C
dTNPs
(deoxyribonucleoside
triphosphates)
• Building blocks for new DNA
strands
• Adenosine triphosphate
(dATP)
• Guanosine triphosphate
(dGTP)
• Cytidine triphosphate (dCTP)
• Thymidine triphosphate
Taq Polymerase
• Thermostable DNA
polymerase that is used in
PCR.
• Which is not inactivated
by heating to 95°C.
Buffer
• Composed of Tris-HCl,
potassium chloride, and
magnesium chloride .
• The pH of the buffer falls
MgCl2
• It acts as a cofactor that
enhances
• Boosting DNA
amplification
• Helps primers anneal to
the correct location on a
DNA template
• It increases the melting
temperature
H2O
• Used as a solvent for the
reaction
• Provide a medium for the
reaction
• Dissolve the DNA template
and primers
P
Requirements for PCR

24. DNA Template
• Double stranded target
region of DNA
Primer
• 18 and 30 bases long
• Produced by commercial
companies
• Also be prepared using a
DNA synthesizer
• GC content 40 and 60%
• Melting temperature 60-
64°C
dTNPs
(deoxyribonucleoside
triphosphates)
• Building blocks for new DNA
strands
• Adenosine triphosphate
(dATP)
• Guanosine triphosphate
(dGTP)
• Cytidine triphosphate (dCTP)
• Thymidine triphosphate
Taq Polymerase
• Thermostable DNA
polymerase that is used in
PCR.
• Which is not inactivated
by heating to 95°C.
Buffer
• Composed of Tris-HCl,
potassium chloride, and
magnesium chloride .
• The pH of the buffer falls
MgCl2
• It acts as a cofactor that
enhances
• Boosting DNA
amplification
• Helps primers anneal to
the correct location on a
DNA template
• It increases the melting
temperature
H2O
• Used as a solvent for the
reaction
• Provide a medium for the
reaction
• Dissolve the DNA template
and primers
PCR Tube P
Requirements for PCR

25. DNA Template
• Double stranded target
region of DNA
Primer
• 18 and 30 bases long
• Produced by commercial
companies
• Also be prepared using a
DNA synthesizer
• GC content 40 and 60%
• Melting temperature 60-
64°C
dTNPs
(deoxyribonucleoside
triphosphates)
• Building blocks for new DNA
strands
• Adenosine triphosphate
(dATP)
• Guanosine triphosphate
(dGTP)
• Cytidine triphosphate (dCTP)
• Thymidine triphosphate
Taq Polymerase
• Thermostable DNA
polymerase that is used in
PCR.
• Which is not inactivated
by heating to 95°C.
Buffer
• Composed of Tris-HCl,
potassium chloride, and
magnesium chloride .
• The pH of the buffer falls
MgCl2
• It acts as a cofactor that
enhances
• Boosting DNA
amplification
• Helps primers anneal to
the correct location on a
DNA template
• It increases the melting
temperature
H2O
• Used as a solvent for the
reaction
• Provide a medium for the
reaction
• Dissolve the DNA template
and primers
PCR Tube
PCR Machine
Requirements for PCR

26. Requirements for PCR
DNA Template
• Double stranded target
region of DNA
Primer
• 18 and 30 bases long
• Produced by commercial
companies
• Also be prepared using a
DNA synthesizer
• GC content 40 and 60%
• Melting temperature 60-
64°C
dTNPs
(deoxyribonucleoside
triphosphates)
• Building blocks for new DNA
strands
• Adenosine triphosphate
(dATP)
• Guanosine triphosphate
(dGTP)
• Cytidine triphosphate (dCTP)
• Thymidine triphosphate
Taq Polymerase
• Thermostable DNA
polymerase that is used in
PCR.
• Which is not inactivated
by heating to 95°C.
Buffer
• Composed of Tris-HCl,
potassium chloride, and
magnesium chloride .
• The pH of the buffer falls
MgCl2
• It acts as a cofactor that
enhances
• Boosting DNA
amplification
• Helps primers anneal to
the correct location on a
DNA template
• It increases the melting
temperature
H2O
• Used as a solvent for the
reaction
• Provide a medium for the
reaction
• Dissolve the DNA template
and primers
PCR Tube
PCR Machine

27. Procedure for PCR

28. Procedure for PCR
1.Denaturation
2.Annealing
3.Extension

29. Procedure for PCR
1. Denaturation
Separation of the two strands of the DNA molecule.
This is accomplished by heating the starting material to temperatures of about 95 °C
(203 °F).

30. Procedure for PCR
1. Denaturation
Separation of the two strands of the DNA molecule.
This is accomplished by heating the starting material to temperatures of about 95 °C
(203 °F).

31. Procedure for PCR
2. Annealing
which involves cooling the mixture to about 50–65 °C (122–149 °F) to allow the
primers to form hydrogen bonds with their complementary sequences on the single-
stranded DNA templates

32. Procedure for PCR
2. Annealing
which involves cooling the mixture to about 50–65 °C (122–149 °F) to allow the
primers to form hydrogen bonds with their complementary sequences on the single-
stranded DNA templates

33. Procedure for PCR
3. Extension
Which involves heating the mixture to about 72 °C (162 °F) for DNA polymerase
to extend the primers along the single-stranded DNA templates

34. Procedure for PCR
3. Extension
Which involves heating the mixture to about 72 °C (162 °F) for DNA polymerase
to extend the primers along the single-stranded DNA templates

35. Procedure for PCR

37. Any Question ?

38. Thank You