Klinik für Innere Medizin IHämatologie, Onkologie und Stammzelltransplantation

This figure shows the amplification of samples from a series of 10-fold dilutions. Reporter signal is plotted against time.  

Two fluorescent dyes, a reporter (R) and a quencher (Q), are attached to the hydrolysis probe. The 3' end of the probe is blocked, so it is not extended during the PCR reaction.

When both dyes are attached to the probe, reporter dye emission is quenched due to fluorescence energy transfer (FRET) from the reporter dye to the quencher dye.  During each extension cycle, the probe is displaced at the 5' end by the DNA polymerase.

Taq DNA polymerase then cleaves the reporter dye from the probe via its 5'-3' exonuclease. This exonuclease functions best at 60^o, which is why the extension step is normally at that temperature.

Once separated from the quencher, the reporter dye emits its characteristic fluorescence which can then be measured on the LC 480.  The amount of fluorescence measured is proportional to the amount of PCR product made.

The design of hydrolysis probes and primers is critical to the success of the experiment.  We have a copy of  the Beacon Designer program to help you out with probe and primer design.

SYBR Green Chemistry:

It is also possible to use the DNA binding dye SYBR Green for QPCR.  This dye fluoresces only when bound to double-stranded DNA, and offers an alternative way to label PCR products, without having to construct a new hydrolysis probe for each assay. Because the dye binds all double stranded nucleic acids present, including primer dimers, it is necessary to perform a melting curve analysis at the end of each run. 

Measurement of Fluorescence:

During the cycling process, fluorescence emission is measured every cycle at the appropriate wavelength(s) within each well.  After the run, the results are analyzed and can be examined in a number of ways, one of which is the amplification plot shown below: