This document discusses real-time quantitative PCR (qPCR) and its applications. It describes how qPCR works by amplifying and simultaneously quantifying a targeted DNA sequence. Two common chemistries are explained: SYBR Green I which binds double stranded DNA and fluoresces, and TaqMan probes which utilize the 5' nuclease activity of Taq polymerase and fluorescence resonance energy transfer. Considerations for proper experimental design such as primer specificity, reaction efficiency, and reproducibility are also covered.

1. Realtime PCR
FISH 507a

2. What is it good for?

3. Real-Time qPCR
Template
Taq Fluorescence detection of
Cycle 1 5’
5’ amplification product
Taq
2X Template
Taq
5’
5’
Taq
Cycle 2 Taq
5’
5’
Taq
4X Template
Linear View
Log View
30-40 cycles

4. Data Analysis
Early Exponential
CT
Phase
Log-Linear
Phase
Linear Ground Phase

5. 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 probes
®
Cleavage probes: TaqMan Excite Detect
Excitation
Fluorescence
Emission Fluorophore
Wavelength

6. Real-Time Chemistry: SYBR
Green I
• SYBR Green I binds dsDNA
λ λ • SGI fluorescence increases
when bound to dsDNA
• As dsDNA accumulates, more
λ λ SGI binds the DNA and the
PCR fluorescence increase
Reaction
Progression
λ
λ
λ
λ λ λ
λ
λ
λ λ
λ λ

7. SYBR Green Melt Curve
Analysis
Due to increasing DNA
temperature denatured
Tm

8. 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 validate
reaction specificity

9. 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

10. Actual Research Question-
Is increased fish mortality associated with a parasitic worm?

11. Actual Research Question-
Is increased fish mortality associated with a parasitic worm?
Biology
Extract Genomic DNA
Design Primers
(NCBI)
Run PCR
Run PCR on gel
or Real-time

12. Actual Research Question-
Is increased fish mortality associated with a parasitic worm?
Biology
Extract Genomic DNA
Design Primers
(NCBI)
Run PCR
Run PCR on gel
or Real-time

13. SYBR Green continued
- Monoplexing - Multiplexing
- Cost saving - High Specificity
- Fast initial screening - High Sensitivity
Sybr Green I® Taqman Probe

14. Hallmarks of a Good qPCR Assay
One Specific Product

15. Primer-Dimer Formation in
Non-Optimized Assay
10,000 copies 2,000
400 Primer- 0 (NTC)
dimers

16. Primer Interactions (Primer Dimers)
Amplification 5’ 3’
5’ 3’
3’ 5’ 3’ 5’
Stable Interaction Primer Dimers
For traditional PCR,
primer-dimers are
usually tolerated Product
Primer-dimers
NTC A B C

17. Primer-Dimers in Real-Time qPCR
• Can contribute to reaction fluorescence
when using SYBR Green I
– Miscalculated Ct values
• Amplifying primer-dimers affects
reaction efficiency
– Lose sensitivity of detection
– Poor reproducibility

18. One Step or Two Step RT-PCR?
One Step RT-PCR Two Step RT-PCR
(One tube) RNA (Two tubes)
1 target X targets
Oligo dT
GS primers Random Primers
(GS Primers)
cDNA Target pool
1 amplicon Amplicon Amplicon X amplicons

19. One Step or Two Step RT-PCR?
One-Step RT-PCR Two-Step RT-PCR
• Highly defined conditions to • Separate conditions for cDNA
support RT and Taq synthesis & PCR
• Requires gene specific primer • Flexible choice of primers
• Ideal for quantification of 1 or 2 • Ideal for quantification of
messages from a large number multiple genes from a limited
of RNA samples number of RNA samples
Perfect for: Perfect for:
- Lot of samples - Few samples
- Small amount of targets - Large amount of targets
Two-step RT-PCR is more convenient and cost effective

20. Real-Time Chemistry:
TaqMan ®
• Target specific hybridization probe
• 5’ reporter and 3’ quencher
• Utilizes FRET quenching
Light*
Light
Energy
R Reporter
R Q
Q Quencher
* heat for BHQs

21. TaqMan® Chemistry
λ
1. During PCR, probe hybridizes
to target sequence
R 2. Probe is partially
R Q
Taq Taq displaced during extension
3. Probe cleaved by 5’- 3’
nuclease activity of polymerase
4. Illuminated free reporter
exhibits unquenched fluorescence

22. 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)

23. TaqMan® Chemistry
• Advantages
– Fluorescence is target
specific
– Multiplexing
• Disadvantages
– High initial cost
– Assay design not trivial

26. Worksheet

27. What you need for a good assay
Quality RNA
NO DNA …. How would you check this?
Good Replication
An appropriate normalizing target
something that does not change

28. Contributors to Poor
Reproducibility
• Laboratory technique
• Specificity issues
– Cross homology of primers
– Primer-dimer
• Reaction efficiency
– Secondary structure of amplicon
– Primer-dimers

29. 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

30. Same Reagents, Different
Hands
Cycle Cycle
Good Technique Poor Technique

31. MINER