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Real Time PCR - Some Basic Principles

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Probe Systems - Taqman

Taqman - Hydrolysis Probe Example (Heid et al., 1996)

This animation depicts the details of how the Taqman probe system works. You may wish to view it now or come back after reading the next section.





Figure 13. Illustration of the Taqman probe design.

The Taqman system utilizes a labeled probe that contains a reporter molecule (FAM) attached at the 5’ end of the oligonucleotide (yellow ball in Figure 13). This molecule gives off fluorescence when excited at a specific wavelength of light from the Taqman machine. Adjacent to the reporter molecule, at the 3’ end of the oligonucleotide probe is a quencher molecule (TAMARA)(cloud in Figure 13). When the quencher is located near the reporter, it prevents the reporter from fluorescing. This is the state of the probe in the beginning of the PCR experiment, no fluorescence is detected.

When the PCR reaction is in the cooling stage of a cycle, the probe binds to the DNA template along with the primers in the typical complementary nature of cytosine (C) pairing with guanine (G), and adenine (A) pairing with thymine (T) (Figure 14). The DNA polymerase enzyme, Taq polymerase, binds first to the 3’ end of the primers and then systematically begins adding new complementary DNA nucleotides in a 5’ -> 3’ direction to the growing copy of the target gene (Figure 15). Taq polymerase also has an exonuclease activity as a normal function, which allows it to remove DNA nucleotides if mistakes are made during the DNA replication process. In real time PCR experiments, when Taq polymerase reaches the bound probe, this exonuclease function enables it to remove the probe nucleotide by nucleotide as it is also adding new nucleotides to the new DNA strand. As a result, this breaks apart the reporter on the 5’ end from the quencher molecule on the 3’ end, and fluorescence is given off (Figure 16). The more copies of the target DNA present, the more probe which can bind and therefore, the more fluorescence detected.
 

Figure 14. Binding of probes and primers in the annealing stage. Figure 15. The extension stage of PCR. Figure 16. The quencher and reporter are no longer located near each other and fluorescence is emitted.


 

Click below to view a video clip where Dr. Louis Busjaeger demonstrates how to run a PCR experiment using the Taqman 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.

 

a link to a video of Dr Busjaeger demonstrating the PCR experiment a link to a video of using the Taqman probe system
Taqman video for slower Internet connections. Taqman video clip with greater resolution for high speed Internet connections.

 

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