pe biosystems sequence detection systems real time detection pcr for nucleic acids quantitation
TRANSCRIPT
PE BiosystemsSequence Detection Systems
Real time Detection PCRReal time Detection PCR
forfor
Nucleic Acids QuantitationNucleic Acids Quantitation
PE BiosystemsSequence Detection Systems
OutlineOutline
What is Real Time detection in PCR?What is Real Time detection in PCR? What instrument you need to perform What instrument you need to perform
RTD?RTD? Why is RTD better for quantitative Why is RTD better for quantitative
analysis?analysis?
PE BiosystemsSequence Detection Systems
TaqMan™ Fluorogenic TaqMan™ Fluorogenic ProbeProbe
Quencher(Rhodamine)
Reporter(Fluoresceine)
Phosphate Group
3’5’
Laser Excitation
Energy Transfer
Fam Vic Tet
PE BiosystemsSequence Detection Systems
TaqMan™ Fluorogenic TaqMan™ Fluorogenic ProbeProbe
Quencher(Rhodamine)
Reporter(Fluoresceine)
Phosphate Group
3’5’
Laser Excitation
Fam Vic Tet
Cleaved Probe
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• PolymerizationPolymerization• PolymerizationPolymerization
5’
5’
3’5’
3’5’
forwardprimer
RR
R = Reporter
Q = Quencher
Denaturation Annealing
QQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• PolymerizationPolymerization• PolymerizationPolymerization
5’
5’
3’5’
3’5’
forwardprimer
RR
R = Reporter
Q = Quencher
Denaturation Annealing
QQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• PolymerizationPolymerization• PolymerizationPolymerization
5’
5’
3’5’
3’5’
forwardprimer
RR
R = Reporter
Q = Quencher
Denaturation Annealing
QQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• PolymerizationPolymerization• PolymerizationPolymerization
5’
5’
3’5’
3’5’
forwardprimer
RR
R = Reporter
Q = Quencher
Denaturation Annealing
QQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• PolymerizationPolymerization• PolymerizationPolymerization
5’
5’
3’5’
3’5’
forwardprimer
RR
R = Reporter
Q = Quencher
Denaturation Annealing
QQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• PolymerizationPolymerization• PolymerizationPolymerization
5’
5’
3’5’
3’5’
forwardprimer
RR
R = Reporter
Q = Quencher
Denaturation Annealing
QQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• PolymerizationPolymerization• PolymerizationPolymerization
5’
5’
3’5’
3’5’
forwardprimer
RR
R = Reporter
Q = Quencher
Denaturation Annealing
QQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
• PolymerizationPolymerization• PolymerizationPolymerization
5’
5’
3’5’
3’5’
forwardprimer
RR
R = Reporter
Q = Quencher
Denaturation Annealing
QQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
R = Reporter
Q = Quencher
. . Strand displacementStrand displacement . . Strand displacementStrand displacement
5’
5’
3’5’
3’5’
forwardprimer
reverseprimer
QRR QQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’5’
3’5’
forwardprimer
R = Reporter
Q = Quencher
. . CleavageCleavage. . CleavageCleavage
RRQQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’5’
3’5’
forwardprimer
R = Reporter
Q = Quencher
. . CleavageCleavage. . CleavageCleavage
RRQQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’5’
3’5’
forwardprimer
R = Reporter
Q = Quencher
. . CleavageCleavage. . CleavageCleavage
RRQQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’5’
3’5’
forwardprimer
R = Reporter
Q = Quencher
. . CleavageCleavage. . CleavageCleavage
RRQQQ
PE BiosystemsSequence Detection Systems
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
5’
5’
3’5’
3’5’
forwardprimer
R = Reporter
Q = Quencher
RRQQQ
Polymerization completedPolymerization completedPolymerization completedPolymerization completed.
PE BiosystemsSequence Detection Systems
Polymerization completedPolymerization completedPolymerization completedPolymerization completed.
5’
5’
3’
forwardprimer
RR
QQQ
5’
3’5’
Taqman PCR ChemistryTaqman PCR ChemistryTaqman PCR ChemistryTaqman PCR Chemistry
R = Reporter
Q = QuencherRR
PE BiosystemsSequence Detection Systems
ß-Actinß-Actin∆R∆Rnn vs Cycle Number vs Cycle Number
00.20.40.60.8
11.21.41.61.8
22.2
0 10 20 30 40
cycle number
∆R
n
no template
20000
10000
5000
2000
1000
500
100
PE BiosystemsSequence Detection Systems
Why Real Time Detection allows Why Real Time Detection allows
Accurate Quantitation?Accurate Quantitation?
PE BiosystemsSequence Detection Systems
Problems with End-Point Problems with End-Point QuantitationQuantitation
Problems with End-Point Problems with End-Point QuantitationQuantitation
96 Replicates
-1.00E-01
4.00E-01
9.00E-01
1.40E+00
1 6
11
16
21
26
31
36
Cycle Number
Flu
ore
scen
ce (
delt
a R
n)
96 replicates
Cycle Number
Flu
or
(delt
a R
n)
Ct 30
PE BiosystemsSequence Detection Systems
Real-Time Quantitation:Real-Time Quantitation:PCR Uniformity in Log PhasePCR Uniformity in Log Phase
Real-Time Quantitation:Real-Time Quantitation:PCR Uniformity in Log PhasePCR Uniformity in Log Phase
-2.00E-02
0.00E+00
2.00E-02
4.00E-02
6.00E-02
8.00E-02
1.00E-01
20 22 24 26 28 30
Threshold = 0.05
96 replicates
Cycle Number
Flu
or
(Delt
a R
n)
geometric
PE BiosystemsSequence Detection Systems
AccuracyAccuracyAccuracyAccuracy How well a measured sample How well a measured sample matches the true value = matches the true value = the measure of exactness.the measure of exactness.
Important for absoluteImportant for absolutequantitation.quantitation.
PE BiosystemsSequence Detection Systems
PrecisionPrecisionPrecisionPrecisionThe degree that multiple samplingsThe degree that multiple samplingsof the same homogenous sourceof the same homogenous sourcegive similar values = give similar values = degree of degree of agreementagreement..
Precision is critical for quatification.Precision is critical for quatification.