©2001 Timothy G. Standish V.S.RAVIKIRAN, MSc. Polymerase Chain Reaction
Polymerase chain reaction medical school
Aug 15, 2015
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
©2001 Timothy G. Standish
V.S.RAVIKIRAN, MSc.
Polymerase Chain Reaction
V.S.RAVIKIRAN, MSc., Department of Biochemistry,
ASRAM Medical college, Eluru-534005.AP, [email protected]
om
©2001 Timothy G. Standish
V.S.RAVIKIRAN, MSc., Department of Biochemistry,
ASRAM Medical college, Eluru-534005.AP, [email protected]
om
©2001 Timothy G. Standish
Polymerase Chain Polymerase Chain ReactionReaction
V.S.RAVIKIRAN
©2001 Timothy G. Standish
HistoryHistoryThe Polymerase Chain Reaction (PCR) was not
a discovery, but rather an inventionA 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
©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
©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
©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
©2001 Timothy G. Standish
How PCR WorksHow PCR Works
As in replication, PCR involves:– Melting DNA– Priming– Polymerization
©2001 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’
©2001 Timothy G. Standish
Leading StrandLeading Strand
Laging StrandLaging Strand
3’
5’3’
5’
Extension - The Replication ForkExtension - The Replication Fork5’
5’5’3’
3’
5’3’3’
5’
Single strand binding proteins
DNA Polymerase
Okazaki fragment
RNA Primers
Primase
5’3’
5’
Helicase
©2001 Timothy G. Standish
Functions And Their Functions And Their Associated EnzymesAssociated Enzymes
LigaseJoining nicks
DNA PolymerasePolymerizing DNAPrimaseProviding primer
EnzymeFunctionHelicaseSSB ProteinsTopisomerase
Melting DNA
©2001 Timothy G. Standish
Components of a PCR Components of a PCR ReactionReaction
Buffer (containing Mg++)Template DNA2 Primers that flank the fragment of
DNA to be amplifieddNTPsTaq DNA Polymerase (or another
thermally stable DNA polymerase)
©2001 Timothy G. Standish
PCRPCRMelting
94 oC
Melting
94 oC
AnnealingPrimers
50 oC
Extension
72 oCT
empe
ratu
re
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’
©2001 Timothy G. Standish
PCRPCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
5’3’
3’5’
©2001 Timothy G. Standish
PCRPCRMelting
94 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’
Heat
©2001 Timothy G. Standish
PCRPCRMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
3’5’
5’3’5’
5’
Melting94 oC
©2001 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’
Heat
Heat
5’
5’
5’
©2001 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’5’
5’
5’
5’
5’
5’
©2001 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
Heat
Heat
©2001 Timothy G. Standish
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’5’
5’
5’
5’
5’
5’
5’
5’
©2001 Timothy G. Standish
Fragments of defined length
PCRPCRMelting
94 oCMelting
94 oCAnnealing
Primers50 oC
Extension72 oC
Tem
pera
ture
100
0
50
T i m e
30x
3’5’
5’3’ 5’
5’ 5’
5’
5’
5’
5’
5’
5’
5’
©2001 Timothy G. Standish
DNA Between The Primers Doubles DNA Between The Primers Doubles With Each Thermal CycleWith Each Thermal Cycle
0Cycles
Number1
3
8
2
4
1
2
4
16
5
32
6
64
©2001 Timothy G. Standish
More Cycles = More DNAMore Cycles = More DNANumber of cycles
0 10 15 20 25 30Size
Marker
©2001 Timothy G. Standish
Theoretical Yield Of PCRTheoretical Yield Of PCRTheoretical 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
©2001 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 PolymerasePolymerizing DNA
Primers are added to the reaction mix
Providing primer
PCRFunctionHeatMelting DNA
©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
©2001 Timothy G. Standish
04/15/23 28
Multiplex PCRMultiplex PCR PCR reactions can be devised in which several
targets are amplified simultaneously — often used in diagnostic applications.
©2001 Timothy G. Standish
04/15/23 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
Rev
erse
-tra
scri
pti
on P
CR
Rev
erse
-tra
scri
pti
on P
CR
©2001 Timothy G. Standish
04/15/23 30
PCRPCR5’ 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 oC)
primer annealing (50-70 oC)
primer extension (72 oC)
Next round…..
3’
RT-PCRRT-PCR5’ 3’AAAAAn
TTTTTn
5’ 3’AAAAAnTTTTTn
first-strand cDNA synthesis by RT
5’3’
TTTTTn 5’3’5’
TTTTTn
5’ AAAAAn
RNase treatment;primer annealing (50-70 oC)
primer extension (72 oC)
mRNA
TTTTTn3’
5’
5’AAAAAn
TTTTTn
AAAAAn
Next round…..
An example was described in the article by Halpin et al. (1998).
Rev
erse
-tra
scri
pti
on P
CR
Rev
erse
-tra
scri
pti
on P
CR
Basic Reaction of PCR & RT-PCRBasic Reaction of PCR & RT-PCR
©2001 Timothy G. Standish
04/15/23 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.
THE END
THANKS FOR YOUR ATTENTION
Related Documents
