Transcript Mar. 8 Presentation Q-PCR

Q-PCR
Bige Vardar -01780333
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
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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
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Denaturation(95oC)~1min
 Annealing(55oC)~45sec
 Extension(72oC)~2min
 Cycle thorugh 25-35
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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
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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
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Uses fluorescence-based probes to detect DNA or
RNA
Data collection start at early exponential phase and
examined at the same time with detection
Research Objectives
 Gene
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validation
Primary validation
Confirmation of microarray data
 Viral
detection
 Bacterial detection and identification
 Gene duplication or DNA
quantification
www.scienceboard.net
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
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Q-PCR
Assay uses a standard curve to quantitate the
amount of target present using a fluorescencelabeled probe for detection.
 Each technique uses some kind of fluorescent
marker which binds to the DNA
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Types of Q-PCR
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Hydrolyzation based Assays
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DNA-binding agents
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Taqman, Beacons, Scorpions
SYBR Green
Hybridization based Assay
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Light cycler(Roche)
Taqman Probes
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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
Characteristics of Taqman Probes
Oligonucleotides longer than the primers (20-30
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
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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
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molecular beacons
A
Excitation
B
C
FRET
ANNEALING
Amplicon
Reporter
Non-fluorescent
Quencher
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
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http://www.youtube.com/watch?v=5ZEySHfCWAU&feature
Taqman vs. SYBR Green I
TaqMan Probe
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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).
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Disadvantages:
 Relative high cost of labeled
probe.
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SYBR Green
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.
Q-PCR vs. PCR
Some of the problems with End-Point Detection:
 Poor Precision
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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
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.
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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).
BioRad iCycler
References
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Dorak MT (Ed): Real-Time PCR (Advanced Methods Series). Oxford: Taylor
& Francis, 2006 http://dorakmt.tripod.com/genetics/realtime.html
http://www.protocolonline.org/prot/Molecular_Biology/PCR/Real-time_PCR/index.html
SYBR Green Quantitative PCR Protocol
http://www.genetics.ucla.edu/labs/lusis/greenquantitative.htm
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Quantification using real-time PCR technology<http://www.wzw.tum.de/genequantification/klein-2002.pdf>
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Real-Time PCR (qPCR) Basics
<http://www.primerdesign.co.uk/Download%20material/Beginners%20guide%20to%20realtime%20PCR.pdf>
QUESTIONS?