FIGURE 23.22 Realtime Fluorescent PCR with SYBR® Green

When the fluorescent probe SYBR® green is present during a PCR reaction, it binds to the double-stranded PCR product and emits light at 520 nm. The SYBR® Green dye only fluoresces when bound to DNA. Hence, the amount of fluorescence correlates with the amount of PCR product produced. This allows the accumulation of PCR product to be followed through many cycles.

Bound S SYBR Green fluoresces green

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Bound SYBR Green fluoresces green

Specific probes can be included in fluorescent PCR procedures to ensure that only specific target DNA sequences give rise to fluorescence.

During PCR the TaqMan® probe binds to the target sequence after the denaturation step that separates the two DNA strands. As the Taq polymerase extends the primer during the next PCR cycle it will eventually bump into the TaqMan® probe. The Taq polymerase is not only capable of displacing strands ahead of it but also has a 5'-nuclease activity that degrades the DNA strand of the probe.This breaks the linkage between the two fluorophores and disrupts the FRET.The short-wavelength fluorophore is now free from quenching and its fluorescence increases. In this case the increase in fluorescence is directly related to the amount of the specific target sequence that has been amplified.

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