lec16 realtime pcr
TRANSCRIPT
Realtime PCR
FISH 507a
What is it good for?
Real-Time qPCR
Fluorescence detection ofamplification product5’
Taq
Template
Cycle 1
Cycle 2
5’Taq
5’Taq
5’Taq
5’Taq
5’Taq
30-40 cycles
2X Template
4X Template Linear ViewLog ViewLinear View
Linear Ground Phase
CT
Log-LinearPhase
Early ExponentialPhase
Data Analysis
Real-Time qPCR Chemistries• Fluorescence-based
– After absorbance of certain wavelengths of light (excitation),the fluorophore emits light at a longer wavelength (emission)
– Fluorescence proportional to amplified product
• Two commonly used chemistries:– SYBR
® Green I
– Hybridization Probes:Displacement probesCleavage probes: TaqMan
®
Fluorophore
Wavelength
FluorescenceExcitationEmission
Excite Detect
Real-Time Chemistry: SYBRGreen I
• SYBR Green I binds dsDNA• SGI fluorescence increases
when bound to dsDNA• As dsDNA accumulates, more
SGI binds the DNA and thefluorescence increase
λ
λ
λ
λ
PCR Reaction
Progression
λ
λ
λ
λλ
λ
λ
λ λ
λ λ
λ
SYBR Green Melt CurveAnalysis
Due to increasingtemperature
Tm
DNAdenatured
SYBR Green Melting Curve Analysis• Plot the negative of the rate of change of
fluorescence vs. temperature (-dI/dT)• For new amplicons, perform melting curve followed
by gel analysis (and sequencing) to validatereaction specificity
SYBR Green I Chemistry
• Advantages– Experiment only requires primers– Post-amplification melting curve analysis
• Disadvantages– Potential contribution to fluorescence from
non-specific products (primer-dimers)– No multiplexing
Actual Research Question- Is increased fish mortality associated with a parasitic worm?
Actual Research Question- Is increased fish mortality associated with a parasitic worm?
Biology
Extract Genomic DNADesign Primers
(NCBI)
Run PCR
Run PCR on gelor Real-time
Actual Research Question- Is increased fish mortality associated with a parasitic worm?
Biology
Extract Genomic DNADesign Primers
(NCBI)
Run PCR
Run PCR on gelor Real-time
SYBR Green continued
- Multiplexing- High Specificity- High Sensitivity
Taqman Probe
- Monoplexing- Cost saving- Fast initial screening
Sybr Green I®
Hallmarks of a Good qPCR AssayOne Specific Product
10,000 copies 2,000
400 0 (NTC)Primer-dimers
Primer-Dimer Formation inNon-Optimized Assay
Primer Interactions (Primer Dimers)
3’ 5’
3’5’3’ 5’
3’5’
Stable Interaction
Amplification
Primer Dimers
For traditional PCR,primer-dimers areusually tolerated
A B CNTC
Primer-dimers
Product
Primer-Dimers in Real-Time qPCR
• Can contribute to reaction fluorescencewhen using SYBR Green I– Miscalculated Ct values
• Amplifying primer-dimers affectsreaction efficiency– Lose sensitivity of detection– Poor reproducibility
One Step or Two Step RT-PCR?
RNAOne Step RT-PCR
(One tube)Two Step RT-PCR
(Two tubes)
Amplicon Amplicon
cDNA
GS primersOligo dT
Random Primers(GS Primers)
1 target
1 amplicon
X targets
X amplicons
Target pool
Two-Step RT-PCR• Separate conditions for cDNA
synthesis & PCR• Flexible choice of primers• Ideal for quantification of
multiple genes from a limitednumber of RNA samples
One-Step RT-PCR• Highly defined conditions to
support RT and Taq• Requires gene specific primer• Ideal for quantification of 1 or 2
messages from a large numberof RNA samples
One Step or Two Step RT-PCR?
Perfect for:- Lot of samples- Small amount of targets
Perfect for:- Few samples- Large amount of targets
Two-step RT-PCR is more convenient and cost effective
Real-Time Chemistry:TaqMan®
• Target specific hybridization probe• 5’ reporter and 3’ quencher• Utilizes FRET quenching
R
Q
Reporter
QuencherR Q
* heat for BHQs
Energy
Light*Light
Taq
TaqMan® Chemistry
TaqR
λ
R QR
Q
1. During PCR, probe hybridizesto target sequence
2. Probe is partiallydisplaced during extension
3. Probe cleaved by 5’- 3’nuclease activity of polymerase
4. Illuminated free reporterexhibits unquenched fluorescence
TaqMan Primers
* equal Tm (58-600 C)
* 15-30 bases in length
* G+C content 30-80%
* no runs of four or more Gs (any nucleotide)
* no more than two G+C at the 3’ end
* no G at the 5' end
* amplicon size 50-150 bp (max 400)
* span exon-exon junctions in cDNA
ABI Primer Express Software Tutorial (www)
TaqMan® Chemistry
• Advantages– Fluorescence is target
specific– Multiplexing
• Disadvantages– High initial cost– Assay design not trivial
Worksheet
What you need for a good assay
Quality RNA
NO DNA …. How would you check this?
Good Replication
An appropriate normalizing targetsomething that does not change
Contributors to PoorReproducibility
• Laboratory technique• Specificity issues
– Cross homology of primers– Primer-dimer
• Reaction efficiency– Secondary structure of amplicon– Primer-dimers
Improving Reproducibility
• Laboratory Techniques– Use clean bench (hood)– Use aerosol resistant tips– Use calibrated micropipettors– Use large volumes (5µL and up)– Pipette into each reaction vessel
once
Good Technique Poor Technique
Cycle Cycle
Same Reagents, DifferentHands
MINER