replication-pcr use for laboratory 1.ppt

laboratory use

1. ©2000 Timothy G. Standish
DNA ReplicationDNA Replication
and theand the
Polymerase Chain ReactionPolymerase Chain Reaction
Md Tausif Raza, M.Sc., Ph. D.
Microbiologist
Sadar Hospital, Siwan, Bihar
[email protected]
9650876720

2. ©2000 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

3. ©2000 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
Defective genes can be amplified to diagnose any number of illnesses
Genes from pathogens can be amplified to identify them (i.e., HIV)
Amplified fragments can act as genetic fingerprints

4. ©2000 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
As in replication, PCR involves:
–Melting DNA
–Priming
–Polymerization

5. ©2000 Timothy G. Standish
Initiation - Forming the Initiation - 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’

6. ©2000 Timothy G. Standish
Leading Strand
Lagging 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

7. ©2000 Timothy G. Standish
Functions And Their Functions And Their
Associated EnzymesAssociated Enzymes
Ligase
Joining nicks
DNA Polymerase
Polymerizing DNA
Primase
Providing primer
EnzymeFunction
Helicase
SSB Proteins
Topisomerase
Melting DNA

8. ©2000 Timothy G. Standish
Components of a PCR Components 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)

9. ©2000 Timothy G. Standish
PCRPCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
T
e
m
p
e
r
a
t
u
r
e
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’

10. ©2000 Timothy G. Standish
PCRPCR
Melting
94
o
C
T
e
m
p
e
r
a
t
u
r
e
100
0
50
T i m e
5’3’
3’5’

11. ©2000 Timothy G. Standish
PCRPCR
Melting
94
o
C
T
e
m
p
e
r
a
t
u
r
e
100
0
50
T i m e
3’5’
5’3’
Heat

12. ©2000 Timothy G. Standish
PCRPCR
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
T
e
m
p
e
r
a
t
u
r
e
100
0
50
T i m e
3’5’
5’3’
5’
5’
Melting
94
o
C

13. ©2000 Timothy G. Standish
PCRPCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
T
e
m
p
e
r
a
t
u
r
e
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’

14. ©2000 Timothy G. Standish
PCRPCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
T
e
m
p
e
r
a
t
u
r
e
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’

15. ©2000 Timothy G. Standish
PCRPCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
T
e
m
p
e
r
a
t
u
r
e
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
Heat
Heat

16. ©2000 Timothy G. Standish
PCRPCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
T
e
m
p
e
r
a
t
u
r
e
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’

17. ©2000 Timothy G. Standish
Fragments of
defined length
PCRPCR
Melting
94
o
C
Melting
94
o
C
Annealing
Primers
50
o
C
Extension
72
o
C
T
e
m
p
e
r
a
t
u
r
e
100
0
50
T i m e
30x
3’5’
5’3’
5’
5’
5’
5’
5’
5’
5’
5’
5’
5’

18. ©2000 Timothy G. Standish
DNA Between The Primers Doubles DNA 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

19. ©2000 Timothy G. Standish
More Cycles = More DNAMore Cycles = More DNA
Number of cycles
0 10 15 20 25 30
Size
Marker

20. ©2000 Timothy G. Standish
Theoretical Yield Of PCRTheoretical Yield Of PCR
Theoretical yield = 2
n
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?
2
n
x y
= 2
30
x 100
= 1,073,741,824 x 100

21. ©2000 Timothy G. Standish
How The Functions Of Replication How The Functions Of Replication
Are Achieved During PCRAre Achieved During PCR
N/A as fragments are shortJoining nicks
Taq DNA PolymerasePolymerizing DNA
Primers are added to the
reaction mix
Providing primer
PCRFunction
Heat
Melting DNA

22. ©2000 Timothy G. Standish