Quantitative "Real Time" RT-PCR as a " + / - " test

    Progress of DNA amplification during a Polymerase Chain Reaction (PCR) can be monitored in "real time" (RT-PCR) by measuring the release of fluorescent "flashes" during amplification.  Reaction rates can be measured continuously, or determined at a fixed time-point during the exponential amplification phase. A computer measures the rate of "flashing"  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. Because 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