TaqMan® Principles
ABI PRISM® 7700 Sequence Detection System
Integrates a PCR-based assay with laser scanning technology to excite
fluorescent dyes present in the specially designed TaqMan® probes. It is a fully integrated system for real-time
detection of PCR. The system includes a built-in thermal cycler, a laser to
induce fluorescence, CCD (charge-coupled device) detector, real-time sequence
detection software, and TaqMan® reagents for the fluorogenic 5' nuclease
assay. The cycle-by-cycle detection of the increase in the amount of PCR product
is quantified in real time as the special probes, "reporter
dye", fluoresces when the "quencher"
is removed from the fragment during the PCR extension cycle.
Principle
Application
of the "TaqMan ®
Probe" to Gene expression problems
To induce fluorescence during PCR, laser light is distributed to
the 96 sample wells via a multiplexed array of optical fibers. The resulting
fluorescent emission returns via the fibers and is directed to a spectrograph
with a charge-coupled device (CCD) camera.
TaqMan®
detection of renin mRNA.
Amplification plots with (left to right) duplicate
samples of 250,50,10 and 2 pg of renin cRNA.
Quantitative curve showing the difference between RNA
from Agt +/- (open arrow) and Agt +/+ (solid arrow). Shows the threshold cycles, 20.50 and 21.62, respectively,
observed with mice having one (mutant) or two (wild type) copies of
the Agt gene. From this we calculate that the
mutant RNA is 2.20 x wild type [2 21.62-20.50 = 2.20].
Key Features
* Continuous wavelength detection from 500-660 nm allows the use of multiple
fluorophores in a single reaction
* The ability to perform multiple target quantitation in a single tube results
in reduced sample and reagent costs
* Multiple chemistries enable versatile assay development
* Proven assay development guidelines save time and money
* Multiple applications including real-time quantitation, SNP detection, and
+/- assay with an internal positive control provide the ultimate flexibility
for your research needs
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