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Introduction to:Introduction to:PolymerasePolymerase Chain Chain
Reaction & Reaction & TaqManTaqMan Real Time PCRReal Time PCR
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PCRPCR PolymerasePolymerase Chain Reaction (1)Chain Reaction (1)
95 °C
45-65 °C 72 °C
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PCR PCR Exponential amplificationExponential amplification
25 cycles
225 copies(1,073,741,824)
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PCR PCR Exponential amplificationExponential amplification
end point
real time
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Characteristic Amplification CurvesCharacteristic Amplification Curvesusing five quantitation standardsusing five quantitation standards
Delta Rn
Delta Delta RnRn: : Fluorescence intensity divided by theFluorescence intensity divided by the background background signal signal NTCNTC: : nonnon--template controltemplate control..
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQ
5’
5’
3’ 5’
3’5’
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQforwardprimer
5’
5’
3’ 5’
3’5’
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQforwardprimer
5’
5’
3’ 5’
3’5’
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQ
5’
5’
3’ 5’
3’5’
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQ
5’
5’
3’ 5’
3’5’
forwardprimer
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQ
5’
5’
3’ 5’
3’5’
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQforwardprimer
5’
5’
3’ 5’
3’5’
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQforwardprimer
5’
5’
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
. Strand displacement. . Strand displacementStrand displacement R = Reporter
Q = Quencher
forwardprimer
5’ 5’
3’5’
3’
5’
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QRR QQ
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
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R = Reporter
Q = Quencher
. Cleavage. . CleavageCleavage
RR QQQ
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’ 5’
3’5’
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R = Reporter
Q = Quencher
. Cleavage. . CleavageCleavage
RRQQQ
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’ 5’
3’5’
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R = Reporter
Q = Quencher
. Cleavage. . CleavageCleavage
RRQQQ
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
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3’ 5’
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Q = Quencher
. Cleavage. . CleavageCleavage
RRQQQ
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Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’ 5’
3’5’
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R = Reporter
Q = QuencherRR
QQQ
Polymerization completedPolymerization completedPolymerization completed.
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Polymerization completedPolymerization completedPolymerization completed.
5’
5’
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5’
3’5’
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QQQ
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
R = Reporter
Q = Quencher
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SYBR® green chemistry
Double stranded DNA Double stranded DNA Binding DyeBinding Dye
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SYBR Green detectionSYBR Green detection
Primer
SYBR Green
template
Taq Polymerase
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SYBR Green detectionSYBR Green detection
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SYBR Green detectionSYBR Green detection
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SYBR Green detectionSYBR Green detection
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SYBR Green detectionSYBR Green detection
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SYBR Green detectionSYBR Green detection
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CARRYCARRY--OVER PREVENTION WITH UNGOVER PREVENTION WITH UNG
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CARRYCARRY--OVER PREVENTION WITH UNGOVER PREVENTION WITH UNG
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CARRYCARRY--OVER PREVENTION WITH UNGOVER PREVENTION WITH UNG
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CARRYCARRY--OVER PREVENTION WITH UNGOVER PREVENTION WITH UNG
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CARRYCARRY--OVER PREVENTION WITH UNGOVER PREVENTION WITH UNG
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CARRYCARRY--OVER PREVENTION WITH UNG / AmpliTaq GoldOVER PREVENTION WITH UNG / AmpliTaq GoldTMTM ACTIVATIONACTIVATION
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CARRYCARRY--OVER PREVENTION WITH UNG / AmpliTaq GoldOVER PREVENTION WITH UNG / AmpliTaq GoldTMTM CYCLINGCYCLING
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CARRYCARRY--OVER PREVENTION WITH UNG / AmpliTaq GoldOVER PREVENTION WITH UNG / AmpliTaq GoldTMTM CYCLINGCYCLING
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CARRYCARRY--OVER PREVENTION WITH UNG / AmpliTaq GoldOVER PREVENTION WITH UNG / AmpliTaq GoldTMTM CYCLINGCYCLING
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For Further Information & scientific support please contact:
Dr. Mohamed Abdel FattahDr. Mohamed Abdel FattahProduct Specialist
Molecular BiologyAnalysis AB
Mobile: 012 27 906 74E-mail: [email protected]