What is Wrong with Agarose Gels? * 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 staining is not very quantitative
q Real-time PCR • Real-time PCR is a form of polymerase chain reaction (PCR) that enables detection of product as the reaction proceeds also continuous data collection enables one of the principal applications of real-time PCR, target quantitation. Because quantitation is among the most common uses for real-time PCR, it is often referred to as quantitative PCR or qrt-PCR.
PCR Phases A basic PCR run can be broken up into three phases: • Exponential: Exact doubling of product is accumulating at every cycle (assuming 100% reaction efficiency). The reaction is very specific and precise. • Linear (High Variability): The reaction components are being consumed, the reaction is slowing. • Plateau (End-Point: Gel detection for traditionalmethods): The reaction has stopped, no more products are being made.
Fig. :PCR—kinetic vs. endpoint detection A plot of the quantity of amplicon DNA over time; in real-time PCR we are only concerned with amplification during the exponential phase of amplification, as accurate quantification of DNA is not possible at the plateau.
Principle of Real-Time PCR Real-time PCR monitors the fluorescence emitted during the reaction as an indicator of amplicon production at each PCR cycle (in real time) as opposed to the endpoint detection
Real-time PCR Principles * based on the detection and quantification of a fluorescent reporter * the first significant increase in the amount of PCR product (CT - threshold cycle) correlates to the initial amount of target template
Real-Time PCR Principles Three general methods for the quantitative assays: 1. Hydrolysis probes (TaqMan) 2. Hybridization probes (Beacons) 3. DNA-binding agents (SYBR Green)
Principle of SYBR Green • SYBR Green technique: SYBR Green I fluorescence is enormously increased upon binding to double-stranded DNA. During the extension phase, more and more SYBR Green I will bind to the PCR product, resulting in an increased fluorescence. Consequently, during each subsequent PCR cycle more fluorescence signal will be detected.
TaqMan • Hydrolysis probe technique: The hydrolysis probe is conjugated with a quencher fluorochrome, which absorbs the fluorescence of the reporter fluorochrome as long as the probe is intact. However, upon amplification of the target sequence, the hydrolysis probe is displaced and subsequently hydrolyzed by the Taq polymerase. This results in the separation of the reporter and quencher fluorochrome and consequently the fluorescence of the reporter fluorochrome becomes detectable. During each consecutive PCR cycle this fluorescence will further increase because of the progressive and exponential accumulation of free reporter fluorochromes.
Hybridization Probe In hybridization probes: the donor and the acceptor are placed on different oligonucleotides. The detection of the PCR product is based on fluorescence resonance energy transfer (FRET) from the donor to the acceptor, when the two probes bind to the target sequence. The more target that is present, the higher the signal from the acceptor will be.