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How
Real-Time PCR Works
Real-time
PCR analysis detects specific nucleic acid amplification products
as
they accumulate in real-time. Real-time PCR uses a fluorescently
labeled
oligonucleotide probe, which eliminates the need for post-PCR
processing. It is
capable of screening genetic activity within hours using a minimal
amount of
sample material, and can detect a single molecule of DNA or
RNA.
During
polymerization, a reporter fluorescence dye and a quencher dye
are
attached to a TaqMan® probe. Negligible fluorescence
from the reporter dye’s
emission is observed once both dyes are attached to the probe.
Once PCR
amplification begins, DNA polymerase cleaves the probe, and the
reporter dye
is released from the probe. The reporter dye, which is separated
from the
quencher dye during every amplification cycle, generates a sequence-specific
fluorescent signal. The signal increases in real time as PCR cycles
continue;
the fluorescence intensity increases proportionally.
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