Q-PCR Bige Vardar -01780333. OUTLINE What is PCR and purpose of it? What is Q-PCR and purpose of it?...
-
Upload
damon-copeland -
Category
Documents
-
view
221 -
download
0
Transcript of Q-PCR Bige Vardar -01780333. OUTLINE What is PCR and purpose of it? What is Q-PCR and purpose of it?...
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
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)~1mi
n Annealing(55oC)~45sec Extension(72oC)~2min Cycle thorugh 25-35
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
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
Research Objectives
Gene validationPrimary validationConfirmation of microarray data
Viral detectionBacterial detection and
identificationGene 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
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
Types of Q-PCR
Hydrolyzation based Assays Taqman, Beacons, Scorpions
DNA-binding agents SYBR Green
Hybridization based Assay Light cycler(Roche)
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
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
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
Amplicon
molecular beaconsmolecular beacons
A
B
C
FRET
Reporter
Non-fluorescent Quencher
Excitation
ANNEALING
SYBR GreenSYBR Green I 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
http://www.youtube.com/watch?v=5ZEySHfCWAU&feature=related
Taqman vs. SYBR Green ITaqMan 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.
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 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.
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 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
Quantification using real-time PCR technology<http://www.wzw.tum.de/gene-quantification/klein-2002.pdf>
Real-Time PCR (qPCR) Basics <http://www.primerdesign.co.uk/Download%20material/Beginners%20guide%20to%20real-time%20PCR.pdf>
QUESTIONS?