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Details of the system
The ABI 7300 Sequence Detection System allows fewer than 10 copies to over 10,000,000 copies to be quantitated accurately after less than 2 hours of thermocycling and a few minutes of analysis. Researchers can set up their own samples.
In brief, the 7300 detection system (Applied Biosystems) consists of a 96 well thermal cycler connected to a light source for excitation and an optics system for detection. The amount of a gene sequence of interest can be compared among samples by monitoring the amount of fluorescence obtained during amplification.
The 5' Nuclease chemistry is the key to the detection system. A probe (TaqMan) is designed to anneal to the target sequence between the traditional forward and reverse primers. The probe is labeled at the 5' end with a reporter fluorochrome (usually 6-carboxyfluorescein, 6-FAM) and a Black Hole Quencher at the 3' end. The probe is designed to have a higher Tm than the primers and during the extension phase a requirement is that the probe is 100% hybridized. As long as the probe is intact, the quencher molecule stops all fluorescence by the reporter (which is triggered by the excitation of the light source). However, as the TAQ polymerase extends the primer, an intrinsic nuclease activity of this TAQ degrades the probe, releasing the reporter which now fluoresces. Thus, the amount of fluorescence released during the amplification cycle is proportional to the amount of product generated in each cycle.
Want to run your own samples?
You can book time on two real-time PCR machines, the 7300-A and the 7300-B, using the online booking facility at http://tanager.biotec.psu.edu/ (you will need to create an account if you don't already have one).