Q-PCR

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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?