Defining Real Time PCR

By definition, a PCR reaction is being monitored in “real time,' when DNA detection and amplification (copying) are being measured simultaneously throughout the reaction process. Real time PCR utilizes laser optics and fluorescing molecules ( probes ) to measure amplification at each cycle of the PCR process rather than at the completion of the final cycle. Back to the flashlight analogy, with conventional PCR you would observe light only after all of the participants in the city turned on their flashlights. In contrast with real time PCR, you would be looking for light when a handful of friends initially turned on their flashlights, and then again each time they added more people to their group, until all of the participants in the city turned on their flashlights.

RT PCR Terminology

You might come across two different terms in your molecular biology studies, “quantitative PCR ” and “real time PCR.” These refer to the same type of PCR process and are used interchangeably. However, real time PCR is sometimes abbreviated RT-PCR, which unfortunately is the same abbreviation used for a different laboratory technique, called reverse transcription PCR. Reverse transcription PCR uses the enzymereverse transcriptase and an RNA template to create DNA molecules. In contrast, as you will discover in the next sections, real time PCR uses the enzyme, Taq polymerase, and a DNA template for creating DNA molecules (Figure 5).

Figure 5. Setting up a real time PCR reaction.