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Real Time PCR - Some Basic Principles

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Probe Systems - FRET

FRET -- Hybridization Probe Example (Haugland et al., 2002)

FRET is an acronym for Fluorescence Resonance Energy Transfer. In this type of detection system, two flanking primers are needed; in addition, two oligonucleotide probes are also required (as compared to only one in the Taqman format). The probes must be located internal of the flanking primers. On the 3’ end of one chemically modified oligonucleotide is a fluorescein donor molecule. This molecule becomes excited when an LED light source from the PCR machine emits light at 493 nm. Adjacent to the donor, on the 5’ end of the other chemically modified oligonucleotide is an acceptor molecule (Figure 9), which emits fluorescence at either 640 nm or 710 nm. The acceptor must be located near (1-5 nucleotides) the donor for light energy to be transfered from the donor to the acceptor. When they are far from one another, the donor emits light at 493 nm (Figure 10).


Figure 9. Diagram of two labelled probes used in FRET systems. Oligo 1 is the donor molecule and Oligo 2 is the receptor. (Image adapted from: European Commission Joint Research Centre.) Figure 10. Fluorescence emitted in FRET system. Oligo 1 is the donor molecule and Oligo 2 is the receptor. (Image adapted from: European Commission Joint Research Centre.) Figure 11. Oligo 1 and 2 are close enough to transfer light energy to the acceptor molecule, which then fluoresces. (Image adapted from: European Commission Joint Research Centre.)

 
Figure 12. PCR machine where temperature cycles are performed and fluorescence measured.

However when the two probes are close, light is absorbed by the donor molecule and transferred to the acceptor, then the acceptor molecule will emit fluorescence at 640 - 710 nm (Figure 11). This fluorescence is measured by the PCR machine using optics and photohybrids (Figure 12). Using the FRET technology, fluorescence measurements are taken at the end of the annealing stage. This is different from the Taqman system in which measurements are made at the end of the extension stage of PCR.

Click below to view a video clip where Dr. Luke Shokere demonstrates how to run a PCR experiment using the FRET probe system. If you have slow 'dial-up' Internet service, choose the smaller video file to view. (NOTE: These videos are in Windows Media Video [.wmv] format, and require a video player to view.  

Link For slower Internet connections. Link for high speed Internet connections.
FRET video for slower Internet connections. FRET video clip with greater resolution for high speed Internet connections.

Discussion Question :
Why would no fluorescence be detected at the end of a PCR cycle when Taq polymerase has finished copying the target DNA sequences using the FRET system?

 

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