1. Q-PCR
Bige Vardar

2. OUTLINE What is PCR and purpose of it?
What is Q-PCR and purpose of it?
How does Q-PCR work?
Types of Q-PCR probes and comparison of types
Advantages and Disadvantages of Q-PCR vs. PCR
Questions

3. What is PCR? Stands for Polymerase Chain Reaction
#copies of DNA fragment(X)=Xo(1+E)n where n=#of cycles in PCR reaction and E=efficiency
Steps in PCR
Denaturation(95oC)~1min
Annealing(55oC)~45sec
Extension(72oC)~2min
Cycle thorugh 25-35

4. Purpose of PCR
Easy to sequence some of million copies and detect rather than trying to sequence and detect a single copy of a gene
Can calculate which sample is biggest when comparing two or more DNA fragments
It is used to clone specific genes

5. What is Q-PCR? Stands for Quantitative Polymerase Chain Reaction
Assay that monitors accumulation of DNA from a PCR reaction
Important technique to quantify RNA(mRNA) levels and DNA gene levels in biological samples
Templates : DNA, cDNA,RNA
Similar to PCR except the progress is monitored by a camera or detector
Uses fluorescence-based probes to detect DNA or RNA
Data collection start at early exponential phase and examined at the same time with detection

7. Bacterial detection and identification
Research Objectives
Gene validation
Primary validation
Confirmation of microarray data
Viral detection
Bacterial detection and identification
Gene duplication or DNA quantification

8. Applications Pathogen detection GMO analysis Quality control Forensics
Methylation studies
Detect proteins with Q-PCR
DNA/RNA quantification
Protein stability testing
Drug therapy efficacy / drug monitoring

9. Q-PCR
Assay uses a standard curve to quantitate the amount of target present using a fluorescence- labeled probe for detection.
Each technique uses some kind of fluorescent marker which binds to the DNA

10. Types of Q-PCR Hydrolyzation based Assays DNA-binding agents
Taqman, Beacons, Scorpions
DNA-binding agents
SYBR Green
Hybridization based Assay
Light cycler(Roche)

11. Taqman Probes Fluorescence-labeled oligonucleotides (TaqMan® probes)
TaqMan probes are complementary to a region of the target gene
The 5' to 3' exonuclease activity of the polymerase cleaves the probe, releasing the fluorophore into solution

12. Characteristics of Taqman Probes
Oligonucleotides longer than the primers ( bases long with a Tm value of 10 oC higher) that contain a fluorescent dye usually on the 5' base, and a quenching dye typically on the 3' base
The excited fluorescent dye transfers energy to the nearby quenching dye molecule rather than fluorescing(FRET)
Uses universal thermal cycling parameters and PCR reaction conditions
One specific requirement for fluorogenic probes is that there be no G at the 5' end

13. Molecular Beacons
Contain fluorescent and quenching dyes at either end but they are designed to adopt a hairpin structure while free in solution to bring the fluorescent dye and the quencher in close proximity for FRET to occur
Have two arms with complementary sequences that form a very stable hybrid or stem

14. molecular beacons A Excitation B Reporter C Non-fluorescent Quencher
FRET
ANNEALING
MB have three main components to the hairpin structure:
A. A sequence specific loop
B. An homologous stem structure
3. A 5’ reporter and a 3’ quencher molecule
Amplicon

15. SYBR Green I
A fluorogenic minor groove binding dye that exhibits little fluorescence when in solution but emits a strong fluorescent signal upon binding to double-stranded DNA
Binds to the minor groove of the DNA double helix with a higher affinity for dsDNA than for single-stranded DNA (ssDNA)
Fluorescence is greatly enhanced (1000-fold) upon DNA-binding making this dye a sensitive indicator for the quantity of dsDNA

17. Taqman vs. SYBR Green I SYBR Green TaqMan Probe Advantages:
Relative low cost of primers.
No fluorescent-labeled probes required.
Disadvantages:
Less specific – only primers determine specificity.
Specific and non-specific double-stranded PCR products generate the same fluorescence signal upon binding SYBR Green I dye.
Not possible to multiplex multiple gene targets.
TaqMan Probe
Advantages:
Increased specificity
Use when the most accurate quantitation of PCR product accumulation is desired.
Option of detecting multiple genes in the same well (multiplexing).
Disadvantages:
Relative high cost of labeled probe.

18. Q-PCR vs. PCR Poor Precision
Some of the problems with End-Point Detection:
Poor Precision
Low sensitivity
Short dynamic range < 2 logs
Low resolution
Non - Automated
Size-based discrimination only
Results are not expressed as numbers
Ethidium bromide for staining is not very quantitative
Post PCR processing

19. Eventually the reactions begin to slow down and stop all together or plateau.Each tube or reaction will plateau at a different point, due to the different reaction kinetics for each sample. These differences can be seen in the plateau phase. The plateau phase is where traditional PCR takes its measurement, also known as end-point detection.

20. Hard to differentiate between the 5-fold change on the Agarose gel
Hard to differentiate between the 5-fold change on the Agarose gel. Q-PCR is able detect a two-fold change (i.e. 10 Vs. 20 copies).

21. BioRad iCycler

22. References
Dorak MT (Ed): Real-Time PCR (Advanced Methods Series). Oxford: Taylor & Francis,
SYBR Green Quantitative PCR Protocol
Quantification using real-time PCR technology< quantification/klein-2002.pdf>
Real-Time PCR (qPCR) Basics < time%20PCR.pdf>

23. QUESTIONS?