BIOCHEMISTRY

1. ©2001 Timothy G. Standish
V.S.RAVIKIRAN, MSc.
Polymerase Chain Reaction

2. V.S.RAVIKIRAN, MSc.,
Department of Biochemistry,
ASRAM Medical college,
Eluru-534005.AP, India.
vsravikiran2013@gmail.com

3. ©2001 Timothy G. Standish
V.S.RAVIKIRAN, MSc.,
Department of Biochemistry,
ASRAM Medical college,
Eluru-534005.AP, India.
vsravikiran2013@gmail.com

4. ©2001 Timothy G. Standish
Polymerase ChainPolymerase Chain
ReactionReaction
V.S.RAVIKIRAN

5. ©2001 Timothy G. Standish
HistoryHistory
The Polymerase Chain Reaction (PCR)
was not a discovery, but rather an
invention
A special DNA polymerase (Taq) is used
to make many copies of a short length of
DNA (100-10,000 bp) defined by primers
Kary Mullis, the inventor of PCR, was
awarded the 1993 Nobel Prize in
Chemistry

6. ©2001 Timothy G. Standish
What PCR Can DoWhat PCR Can Do
PCR can be used to make many copies of any
DNA that is supplied as a template
Starting with one original copy an almost infinite
number of copies can be made using PCR
“Amplified” fragments of DNA can be sequenced,
cloned, probed or sized using electrophoresis

7. ©2001 Timothy G. Standish
What PCR Can DoWhat PCR Can Do
Defective genes can be amplified to diagnose any
number of illnesses
Genes from pathogens can be amplified to identify
them (ie. HIV)
Amplified fragments can act as genetic fingerprints

8. ©2001 Timothy G. Standish
How PCR WorksHow PCR Works
PCR is an artificial way of doing DNA
replication
Instead of replicating all the DNA
present, only a small segment is
replicated, but this small segment is
replicated many times

9. ©2001 Timothy G. Standish
How PCR WorksHow PCR Works
As in replication, PCR involves:
–Melting DNA
–Priming
–Polymerization

10. ©2001 Timothy G. Standish
Initiation - Forming theInitiation - Forming the
Replication EyeReplication Eye
3’ 5’
3’5’
5’
5’
3’
3’
Origin of Replication
5’
3’
3’
5’
5’
3’
5’
5’
5’
3’
3’
3’

11. ©2001 Timothy G. Standish
Leading Strand
Laging Strand
3’
5’
3’
5’
Extension - The Replication ForkExtension - The Replication Fork
5’
5’
5’
3’
3’
5’3’
3’
5’
Single strand
binding
proteins
DNA
Polymerase
Okazaki
fragment
RNA
Primers
Primase
5’
3’
5’
Helicase

12. ©2001 Timothy G. Standish
Functions And TheirFunctions And Their
Associated EnzymesAssociated Enzymes
LigaseJoining nicks
DNA PolymerasePolymerizing DNA
PrimaseProviding primer
EnzymeFunction
Helicase
SSB Proteins
Topisomerase
Melting DNA

13. ©2001 Timothy G. Standish
Components of a PCRComponents of a PCR
ReactionReaction
Buffer (containing Mg++
)
Template DNA
2 Primers that flank the fragment of
DNA to be amplified
dNTPs
Taq DNA Polymerase (or another
thermally stable DNA polymerase)

14. ©2001 Timothy G. Standish
PCRPCR
Melting
94 o
C
Melting
94 o
C
Annealing
Primers
50 o
C
Extension
72 o
C
Temperature
100
0
50
T i m e
30x
5’3’
3’5’
3’5’
5’
5’3’
5’
3’5’
5’
5’
5’
5’3’
3’5’
3’5’
5’3’
5’3’
5’

15. ©2001 Timothy G. Standish
PCRPCRMelting
94 o
C
Temperature
100
0
50
T i m e
5’3’
3’5’

16. ©2001 Timothy G. Standish
PCRPCRMelting
94 o
C
Temperature
100
0
50
T i m e
3’5’
5’3’
Heat

17. ©2001 Timothy G. Standish
PCRPCRMelting
94 o
C
Annealing
Primers
50 o
C
Extension
72 o
CTemperature
100
0
50
T i m e
3’5’
5’3’
5’
5’
Melting
94 o
C

18. ©2001 Timothy G. Standish
PCRPCRMelting
94 o
C
Melting
94 o
C
Annealing
Primers
50 o
C
Extension
72 o
CTemperature
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’

19. ©2001 Timothy G. Standish
PCRPCRMelting
94 o
C
Melting
94 o
C
Annealing
Primers
50 o
C
Extension
72 o
CTemperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’

20. ©2001 Timothy G. Standish
PCRPCRMelting
94 o
C
Melting
94 o
C
Annealing
Primers
50 o
C
Extension
72 o
CTemperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
Heat
Heat

21. ©2001 Timothy G. Standish
PCRPCRMelting
94 o
C
Melting
94 o
C
Annealing
Primers
50 o
C
Extension
72 o
CTemperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’

22. ©2001 Timothy G. Standish
Fragments of
defined length
PCRPCRMelting
94 o
C
Melting
94 o
C
Annealing
Primers
50 o
C
Extension
72 o
CTemperature
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’ 5’
5’
5’
5’
5’
5’
5’
5’

23. ©2001 Timothy G. Standish
DNA Between The Primers DoublesDNA Between The Primers Doubles
With Each Thermal CycleWith Each Thermal Cycle
0
Cycles
Number
1
3
8
2
4
1
2
4
16
5
32
6
64

24. ©2001 Timothy G. Standish
More Cycles = More DNAMore Cycles = More DNA
Number of cycles
0 10 15 20 25 30
Size
Marker

25. ©2001 Timothy G. Standish
Theoretical Yield Of PCRTheoretical Yield Of PCR
Theoretical yield = 2n
x y
Where y = the starting
number of copies and
n = the number of thermal cycles
= 107,374,182,400
If you start with 100 copies, how many copies are
made in 30 cycles?
2n
x y
= 230
x 100
= 1,073,741,824 x 100

26. ©2001 Timothy G. Standish
How The Functions Of ReplicationHow The Functions Of Replication
Are Achieved During PCRAre Achieved During PCR
N/A as fragments
are short
Joining nicks
Taq DNA PolymerasePolymerizing DNA
Primers are added to the reaction mix
Providing primer
PCRFunction
HeatMelting DNA

27. ©2001 Timothy G. Standish
27
Type of Gene CyclerType of Gene Cycler
Multi Block PCRStandard PCR
Gradient PCR Real-Time PCR
Gene cyclers available
from many suppliers

28. ©2001 Timothy G. Standish
05/30/15 28
Multiplex PCRMultiplex PCR
PCR reactions can be devised in which several
targets are amplified simultaneously — often used
in diagnostic applications.

29. ©2001 Timothy G. Standish
05/30/15 29
What is RT-PCRWhat is RT-PCR
RT-PCR is Reverse transcription PCR
The source of material is mRNA
First step is cDNA synthesis by reverse
transcriptase at 42ºC
Second step is Standard PCR procedure
Result is cDNA of gene target
Reverse-trascriptionPCRReverse-trascriptionPCR

30. ©2001 Timothy G. Standish
05/30/15 30
PCRPCR
5’ 3’
5’3’
5’ 3’
5’3’
5’ 3’
5’3’
5’ 3’
5’3’
5’ 3’
5’3’
5’
5’
3’
3’
5’ 3’
5’3’
5’
3’5’3’
denaturation (94 o
C)
primer annealing (50-70 o
C)
primer extension (72 o
C)
Next round…..
3’
RT-PCRRT-PCR
5’ AAAAAn
TTTTTn
5’ 3AAAAAn
TTTTTn
first-strand cDNA synthesis by RT
53’
TTTTTn 53’
5’
TTTTTn
5’ AAAAAn
RNase treatment;
primer annealing (50-70 o
C)
primer extension (72 o
C)
mRNA
TTTTTn3’
5’
5’
AAAAAn
TTTTTn
AAAAAn
Next round…..
An example was described in the article by Halpin et al. (1998).
Reverse-trascriptionPCRReverse-trascriptionPCR
Basic Reaction of PCR & RT-PCRBasic Reaction of PCR & RT-PCR

31. ©2001 Timothy G. Standish
05/30/15 31
Applications of PCRApplications of PCR
Mutation testing, e.g. cystic fibrosis.
Diagnosis or screening of acquired diseases, e.g.
AIDS.
Genetic profiling in forensic, legal and bio-
diversity applications.
Site-directed mutagenesis of genes.
Quantitation of mRNA in cells or tissues.

32. THE END
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ATTENTION