real time pcr principle & applications - assiut university pcr/assyout workshop dec.05.pdf ·...
TRANSCRIPT
Real Time PCR
Principle & Applications
By Dr. Raghad Safwat
CliniLab
Why Real Time PCR?
PCR Cycle - Step 1 - Denaturation by Heat
cDNA
RNA
DNA
DNA
Target RNA Sequence Target DNA Sequence
PCR Cycle - Step 2 - Primer Pair Anneals to Ends of Target Sequence
Target Sequence
Target Sequence
Primer 1Primer 2
5’
3’
5’
5’
3’
5’
3’
3’
PCR Cycle - Step 3 - Taq DNA Polymerase Catalyses Primer Extension as Complementary Nucleotides are Incorporated
Target Sequence
Target Sequence
Primer 1
Primer 2
5’
3’
5’
5’
3’
5’
3’
3’
Taq DNAPolymerase
End of the 1st PCR Cycle - Results in Two Copies of Target Sequence
Target Sequence
Target Sequence
Target Amplification
Cycle 1 = 2 Amplicons
Cycle 2 = 4 Amplicons
Cycle 3 = 8 Amplicons
Cycle 4 = 16 Amplicons
Cycle 5 = 32 Amplicons
Cycle 6 = 64 Amplicons
Cycle 7 = 128 Amplicons
20١،٠٤٨،٥٧٦
664
532
416
38
25١،٠٧٣،٧٤١،٨٢٤
24
12
No. of CyclesNo. AmpliconCopies
PCRTheoretical Concept
N = N0 x 2n
N: numberof amplified molecules
N0: initialnumber of molecules
n: number of amplification cycles
PCRTheoretical Concept - Example
N = N0 x 2n
20 cycles 1,048,576 = 1 x 220
1,048,576 = ? x 220
20١،٠٤٨،٥٧٦664532
41638
25١،٠٧٣،٧٤١،٨٢٤
24
12
No. of CyclesNo. AmpliconCopies
PCRTheory and Practice!
Real
Theory
N = N0 x (Econst)n
N = N0 x 2n
N = N0 x (Evar)n
N
n
N
n
0
end-point-PCR
log-phase-PCR
N: number of amplified molecules n:number of amplification cyclesN0: initialnumber of molecules E:amplification efficiency
Problems with End-Point QuantificationEfficiency
log-phase analysis
N
n
end-point analysis
high concentration /high efficiencyhigh concentration /low efficiencylow concentration /high efficiency
N :number of amplified moleculesn :number of amplification cycles
Problems with End-Point QuantificationLatent Cycles & Plateau Effect
Latent cycles
N
n
Plateau Effect
Factors Contributing to the Plateau Effect
Progressive inactivation of Taq polymerase
Progressive reduction in the efficiency of the primer - template complex formation, due to product re-annealing
Consumption of reagents become limiting facators
Why Real Time Detection Allows Accurate Quantification?
RTD turns the question HOW MUCH?
into the questionWHEN?
End Point vs Real Time Quantification
end point
real time
TaqMan Chemistry
5’ Nuclease Assay
Detection of Specific PCR Product by utilizing the 5’ to 3’ Exonuclease Activity of Thermus Aquaticus
DNA Pol.
Holland, Abramson, Watson, & GelfandAugust 1991 PNAS 88: 7276-7280
3’ EXO
5’ EXO Synthetic
Thermostable DNA Polymerase Domains
Proofreading activity
Cleavage Synthesis
Taq 5’ - 3’ Exonuclease Activity
5’
3’Degradation of DNA “hinders” ahead
Fluoresceins Rhodamines
FRETFRET
Fluorescence Resonance Energy Transfer (FRET)
TaqMan PCR Cheminstry
Quencher(Rhodamine)
Reporter(Fluoresceine)
Energy Transfer
5’ 3’Fam
VicTet
Phosphate Group
Laser Excitation
TaqMan PCR Chemistry
Reporter(Fluoresceine)
Quencher(Rhodamine)
3’5’
FamVic
Tet
Cleaved Probe
Phosphate Group
Laser Excitation
TaqMan PCR Chemistry
TaqMan ProbeForwardPrimer
5’3’
5’5’3’
5’
ReversePrimer
R Qp
TaqMan Probe Format Overview
reporter quencher
TaqMan PCR ChemistrySummary
Denaturation Annealing R = Reporter
Q = QuencherPolymerization
QQQRRforwardprimer
5’5’
3’5’
3’
5’
TaqMan PCR ChemistrySummary
R = Reporter
Q = Quencher
Denaturation Annealing
Polymerization
QQQRRforwardprimer
5’5’
3’5’
3’
5’
TaqMan PCR ChemistrySummary
AnnealingDenaturation R = Reporter
Q = Quencher
Polymerization
QQQRRforwardprimer
5’5’
3’5’
3’
5’
TaqMan PCR ChemistrySummary
AnnealingDenaturation R = Reporter
Q = Quencher
Polymerization
QQQRRforwardprimer
5’5’
3’5’
3’
5’
TaqMan PCR ChemistrySummary
R = Reporter
Q = Quencher
AnnealingDenaturation
Polymerization
QQQRRforwardprimer
5’5’
3’5’
3’
5’
TaqMan PCR ChemistrySummary
R = Reporter
Q = Quencher
AnnealingDenaturation
Polymerization
QQQRRforwardprimer
5’5’
3’5’
3’
5’
TaqMan PCR ChemistrySummary
AnnealingDenaturation R = Reporter
Q = Quencher
Polymerization
QQQRRforwardprimer
5’5’
3’5’
3’
5’
TaqMan PCR ChemistrySummary
AnnealingDenaturation R = Reporter
Q = Quencher
Polymerization
QQQRRforwardprimer
5’5’
3’5’
3’
5’
5’
5’
3’ 5’
3’5’
forwardprimer
RR QQQ
R = Reporter
Q = Quencher
Cleavage
TaqMan PCR ChemistrySummary
5’
5’
3’ 5’
3’5’
forwardprimer
RRQQQ
R = Reporter
Q = Quencher
Cleavage
TaqMan PCR ChemistrySummary
5’
5’
3’ 5’
3’5’
forwardprimer
RRQQQ
R = Reporter
Q = Quencher
Cleavage
TaqMan PCR ChemistrySummary
5’
5’
3’ 5’
3’5’
forwardprimer
RRQQQ
R = Reporter
Q = Quencher
Cleavage
TaqMan PCR ChemistrySummary
TaqMan PCR ChemistrySummary
5’
5’
3’ 5’
3’5’
forwardprimer
RRQQQ
R = Reporter
Q = Quencher
Polymerization Completed
TaqMan PCR ChemistrySummary
R = Reporter
Q = Quencher
Polymerization Completed
QQ Q
RRRR
forwardprimer
5’
3’5’
5’
3’
5’
Applications
Quantification
Types of Quantification
I Absolute:
Results is expressed in absolute units e.g copy number.Standard curve required
Absolute Quantification Principle
5
10
15
20
25
30
35
0 2
Valu
e C
t
Ct =28
Log(Qty)4 6 8 10Log (Copy Number)
Absolute Quantification
leukemia associated with chromosomal translocations
Translocation in Leukemia t(4;14)
FISH analysis on gDNA
Absolute QuantificationMinimal Residual Disease
Time 6
Times 3-5
Time 7 & 8
Quantitative measurement of relapse ensures early treatment
Relapse
Time 2Time 1
Types of Quantification
II Relative:
Results is expressed in relation to a reference targetAmount of increase/decrease (different time points, treatments or tissues, normal vs. tumor etc.)Standard curve is not necessarily required!
Relative Quantification
Gene Expression Profiling
Target GeneReference GeneCtCt
Comparative methodComparative method
Relative Quantification(gene duplication/deletion-DNA level)
Deletion of PMP22 Gene
22 23
Nor
mal
ized
fluo
resc
ence
Threshold cycle (CT)
Normal Genome
22 22
Nor
mal
ized
fluo
resc
ence
Threshold cycle (CT)
Duplication of PMP22 Gene
2221.5
Nor
mal
ized
fluo
resc
ence
Threshold cycle (CT)
Allele 1 Allele 2Chromosome 17p11.2-12
PMP22Reference Gene
Allele 1 Allele 2Chromosome 17p11.2-12
Allele 1 Allele 2Chromosome 17p11.2-12
Relative Quantification(gene expression delta Ct method-mRNA level)
BrainBrainBrain
Ct=26 Ct=36GAPDH amplificationc-myc amplification ∆Ct = c-myc Ct - gapdh Ct
∆Ct = 10
Relative Quantification(gene expression delta Ct method-mRNA level)
Ct=26 Ct=36 Ct=26 Ct=33.5
Ct=27 Ct=32 Ct=26 Ct=32
∆Ct=10
∆Ct=5
∆Ct=7.5
∆Ct=6
Brain Kidney
Liver Lung
Real Time PCR QuantificationApplication Areas
VirologyGenotypingHaematology e.g. Minimal Residual DiseaseGenetically Modified OrganismsForensicsGenomic AmplificationGene ExpressionSingle Nucleotide Polymorphisms
.. Thank you !