Quantitative "Real Time"
RT-PCR as a " + / - " test
Progress of DNA amplification during a Polymerase
Chain Reaction (PCR) can be
monitored in "real time" by
measuring the release of fluorescent dyes
during amplification. Reaction rates can be measured continuously, or
determined at a fixed timepoint during the
exponential amplification phase. A computer measures the rate of dye
release in 96 simultaneous experimental PCR reactions relative to a control reaction
in the same tube (top, right). A
"positive"
result ("red"
signals, below right) indicates that the
target DNA sequence is
present. Becasue amplification is measured against a control, a "negative" ("black" background signal) is a
definite indication of the absence of the
target allele.
Unlike ordinary preparative PCR, RT-PCR
allows the success of multiple PCR reaction to be determined
automatically after only
a few cycles, without separate analysis of each reaction, and avoids
the
problem of "false negatives".
All material ©2005 by Steven M. Carr
