Biomedical Engineering Reference
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Fig. 1. Schematic representation of TaqMan PCR assay.
(A)
TaqMan probe with
fl uorescent reporter dye FAM (6-carboxyfl uorescein) attached to the 5
end and a
quencher dye TAMRA (6-carboxytetramethylrhodamine) attached to the 3
end.
(B)
During polymerization the TaqMan probe containing a reporter dye (R) and a quencher
dye (Q) specifi cally anneals to one strand of the DNA template between forward and
reverse PCR primers. During amplifi cation, the fl uorogenic probe is displaced by
Taq polymerase and then cleaved
(C)
, releasing the reporter dye. After cleavage, the
shortened probe dissociates from the template, allowing polymerization of the strand to
complete. Fluorescence is proportional to the amount of product accumulated.
template between forward and reverse PCR primers
(Fig. 1B)
. When the
probe is intact the reporter fl uorophore emission is suppressed by a quencher
fl uorophore. The 5
-nuclease activity of Taq polymerase releases the reporter
dye from the vicinity of the quencher dye, resulting in the generation of
reporter fl uorescence
(Fig. 1C)
. The remaining probe fragment dissociates
from the target sequence, allowing polymerization to continue. The intensity of
fl uorescence is proportional to the amount of DNA amplifi ed, and this reaction
occurs during every cycle of PCR. The fl uorescent signal is captured using an
ABI Prism 7700 sequence detection system (PE Applied Biosystems) which
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