荧光定量PCR仪说明书

A
A B A B A B A B A B AB A B A B
Melt curve
55 Primer Conc. 200 nM 70
B = NTC
data from Walling lab
A = Template + primers
B = Template only (NO primers)
iQ5 Features for Reaction Optimization
Analysis at the end of the qPCR run to determine the melting temperature (Tm) of PCR products
Tm of amplicon
Plot rate of change of fluorescence vs. temperature
iQ5 & MYQ™ Real-Time PCR
Why Real Time PCR?
• More accurate than RT PCR • Publications are requiring real time PCR data rather than RT PCRs • Ease and speed of quantification
CT Values Determine the Copy Number
Standard Dilution Series can be used to……
…determine the Copy Number or Concentration of an Unknown
Example of Good Efficiency
Quantitative PCR
Through the use of fluorescent dyes that detect PCR products, real-time PCR measures the reaction directly during amplification
iQ5™ Optical System
90%
Primer Dimers Raise the Apparent Efficiency
Optimization of RT-qPCR: Annealing Temperature
A
A B A B A B A B A B AB A B A B
Melt curve dimers
55 Primer Conc. 200 nM 70
• Can do many technical replicates without exhausting cDNA template
Real-Time PCR: Applications
Real-Time qPCR provides information for relative or absolute measurements of starting material.
Correlation Coefficient >0.9
Efficiency >90%
Example of Unreasonably High Efficiency
How do you get an efficiency of >200%?
Real-time PCR Efficiencies
Efficiency =10
3’
5’
Taq
3’
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3’
3’
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5’
5’
5’
3’

5’
R
5’
Q
3’
Annealing
Cleavage-based assay: TaqManTM
Q
3’
R
5’ 3’ 5’
5’
3’
Extension Step
Q
3’
R
Taq
5’
5’
3’
R Q
3’
Taq
R
5’ 3’
5’
Taq
3’
Q
5’ 3’
5’
Taq
Genomic DNA cDNA product
data from Walling lab
- DNAse treatment will reduce contamination - Also design primers across intron/exon boundaries
Melt Curve Analysis
-1/slope
% Efficiency=10 • • •
-1/slope
- 1 X 100
Primer-dimer! Pipette calibration Dilution Errors
110%
|
Target Range
• • • Secondary structures Pipette calibration Dilution errors
B = NTC
data from Walling lab
A = Template + primers
B = Template only (NO primers)
What Are Primer Dimers?
Primer Concentration Affects qPCR Efficiency
Optimization of RT-qPCR: Primer Concentration
Why Do Melt Curve?
• Identification of non-specific products • Mutation detection/allelic discrimination
Primer Dimers Raise the Apparent Efficiency
Optimization of RT-qPCR: Annealing Temperature
In some cases, can eliminate extension step since all amplification has already occurred during annealing
3’
R
Q
5’ 3’
5’
Threshold Cycle (CT)
- Assigned point at which the fluorescence rises appreciably above background -The CT can be placed anywhere in the exponential (log-linear) phase
iQ5™ Utilizes Filter Wheels
Emission Filter Detector
Excitation Filter
Light Source (Simple halogen bulb)
Emission Filter Windows
490/530 Cy5
0 5000
575/620
n
Observed
But the exponential phase becomes linear as reactants become rate-limiting
Cycle #
RT PCR is an End-Point Analysis
96 identical reactions will have very different final amounts of fluorescence at endpoint due to differences in kinetics and pipetting

Results are More Accurate in the Log Phase
96 identical reactions will have nearly identical CT values when measured at the linear phase of amplification
2 NTC
1
1: 100 nM 2: 200 nM
data from Walling lab
Primer Concentration
Primer-dimers can be influenced by template concentration
10,000 copies template 2,000 copies
CT
Threshold Cycle (CT)
- At the CT, the fluorescence correlates well with the starting copy number - BioRad claims a dynamic range of 9 orders of magnitude - Each 10x dilution equals 3.3 cycles
• Continuous Data Collection • Thermal Gradient • Melt Curve
Continuous Data Collection
• iCycler iQ features a continuous data collection that is designed to focus analytical scrutiny on subsets of the data gathered
3’ 5’
3’
5’ 3’
3’
5’
Add iQ Supermix, Hybridization Probe and sample
3’
3’
3’
5’
5’
5’
5’
3’
3’
Primers
d.NTPs Thermal Stable DNA Polymerase