Gene Expression Analysis
The TaqMan® technology uses a fluorogenic probe to enable the detection of a specific PCR product as it accumulates during PCR cycles.
- An oligonucleotide probe is constructed containing a reporter fluorescent dye on the 5´ end and a quencher dye on the 3´ end. While the probe is intact, the proximity of the quencher dye greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer (FRET) through space.
- If the target sequence is present, the probe anneals downstream from one of the primer sites and is cleaved by the 5´ nuclease activity of Taq DNA polymerase as this primer is extended.
- This cleavage of the probe a) separates the reporter dye from the quencher dye, increasing the reporter dye signal and b) removes the probe from the target strand, allowing primer extension to continue to the end of the template strand. Thus, inclusion of the probe does not inhibit the overall PCR process.
- Additional reporter dye molecules are cleaved from their respective probes with each cycle, resulting in an increase in fluorescence intensity proportional to the amount of amplicon produced.
For more on Taq®Man technology and applications visit the Applied Biosystems web site.