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

QQ

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