Realtime PCR

When generating a DNA profile, the PCR products are normally analysed at the end point after 28-34 cycles. It is, however, possible to monitor the generation of PCR products as they are generated - real time. This was first developed using ethidium bromide: as PCR products are generated in each cycle, more ethidium bromide intercalates with the double-stranded DNA molecule and fluoresces under UV light. The increase in fluorescence can be detected using a suitable 'camera' [38]. Increasingly sensitive

Figure 4.6 (a) The TaqMan® quantification system consists of two PCR primers and an internal probe that hybridizes within the region that is the amplified region; (b) as the primer extends it encounters the probe, the 5' exonuclease activity of the Taq polymerase degrades the probe: (c) the reporter molecule is no longer in proximity to the quencher and fluoresces

Figure 4.6 (a) The TaqMan® quantification system consists of two PCR primers and an internal probe that hybridizes within the region that is the amplified region; (b) as the primer extends it encounters the probe, the 5' exonuclease activity of the Taq polymerase degrades the probe: (c) the reporter molecule is no longer in proximity to the quencher and fluoresces assays have been developed, such as SYBR® Green and the TaqMan1® system. Using SYBR® Green, as PCR products are generated, the dye binds to the double-stranded product and the fluorescence increases. The TaqMan1® system uses a different approach, with two primers and a probe; the probe is within the region defined by the primers and is labelled on the 5' end with a fluorescent molecule and on the 3' end with a molecule that quenches the fluorescence. As the primers are extended by the Taq polymerase, one of them meets the probe, which is degraded by the polymerase, releasing the probe and the quencher into solution - efficient quenching of the fluorescent molecules only occurs when they are in close proximity on the probe molecule (Figure 4.6).

As more PCR products are generated, more fluorescent molecules are released and the fluorescence from the sample increases. Real-time assays are highly sensitive, human specific and are not labour intensive.

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