Post on 20-Aug-2018
Introduction to:Introduction to:PolymerasePolymerase Chain Chain
Reaction & Reaction & TaqManTaqMan Real Time PCRReal Time PCR
PCRPCR PolymerasePolymerase Chain Reaction (1)Chain Reaction (1)
95 °C
45-65 °C 72 °C
PCR PCR Exponential amplificationExponential amplification
25 cycles
225 copies(1,073,741,824)
PCR PCR Exponential amplificationExponential amplification
end point
real time
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..
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQ
5’
5’
3’ 5’
3’5’
forwardprimer
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQforwardprimer
5’
5’
3’ 5’
3’5’
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQforwardprimer
5’
5’
3’ 5’
3’5’
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQ
5’
5’
3’ 5’
3’5’
forwardprimer
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQ
5’
5’
3’ 5’
3’5’
forwardprimer
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQ
5’
5’
3’ 5’
3’5’
forwardprimer
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQforwardprimer
5’
5’
3’ 5’
3’5’
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• Polymerization•• PolymerizationPolymerization
RR
Denaturation Annealing
R = Reporter
Q = Quencher
QQQforwardprimer
5’
5’
3’ 5’
3’5’
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
. Strand displacement. . Strand displacementStrand displacement R = Reporter
Q = Quencher
forwardprimer
5’ 5’
3’5’
3’
5’
reverseprimer
QRR QQ
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’ 5’
3’5’
forwardprimer
R = Reporter
Q = Quencher
. Cleavage. . CleavageCleavage
RR QQQ
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’ 5’
3’5’
forwardprimer
R = Reporter
Q = Quencher
. Cleavage. . CleavageCleavage
RRQQQ
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’ 5’
3’5’
forwardprimer
R = Reporter
Q = Quencher
. Cleavage. . CleavageCleavage
RRQQQ
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’ 5’
3’5’
forwardprimer
R = Reporter
Q = Quencher
. Cleavage. . CleavageCleavage
RRQQQ
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’ 5’
3’5’
forwardprimer
R = Reporter
Q = QuencherRR
QQQ
Polymerization completedPolymerization completedPolymerization completed.
Polymerization completedPolymerization completedPolymerization completed.
5’
5’
3’
forwardprimer
5’
3’5’
RRRR
QQQ
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
R = Reporter
Q = Quencher
SYBR® green chemistry
Double stranded DNA Double stranded DNA Binding DyeBinding Dye
SYBR Green detectionSYBR Green detection
Primer
SYBR Green
template
Taq Polymerase
SYBR Green detectionSYBR Green detection
SYBR Green detectionSYBR Green detection
SYBR Green detectionSYBR Green detection
SYBR Green detectionSYBR Green detection
SYBR Green detectionSYBR Green detection
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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: mohamed.abdelfattah@analysis-ab.com