The reproducible quantitation of amplification products has long been the goal for many scientists and researchers. The traditional process requires the end-point analysis of amplification products via gel electrophoresis. This method allows for the identification of target and competitor product sizes, estimation of purity, and subjective measuring of band intensities. However, the reproducibility of amplification end products is highly variable due to limiting reagents, which compound the difficulties with this process.
It is the exponential phase of amplification that provides the most useful and reproducible data. There is a quantitative relationship between the amount of starting target DNA and the amount of amplification product during the exponential phase of a cycling program. This is the very basis for Real-Time Amplification. Aided by the help of DNA intercalating dyes and probe specific chemistries, the study of the amplification process has improved by quantum leaps as a result of real-time detection.
Today’s real-time instruments are comprised of a fluorometer and a thermal cycler for the detection of fluorescence during the cycling process. A computer that communicates with the real-time machine collects fluorescence data. This data is displayed in a graphical format through software developed for real-time analysis.
Fluorescent data is collected at least once during each cycle of amplification allowing for real-time monitoring of amplification. A user is able to determine which samples are amplifying on a cycle-by-cycle basis. This instant data allows them to see how individual samples amplify in relation to known standards, positive controls and negative controls. Not only is the user able to monitor the whole reaction during the amplification process, but they can truly optimize their protocols based on the information they receive. This leads to increased sensitivity, specificity and efficiency.
After raw data is collected, the analysis can begin. The software for the real-time instrument normalizes the data to account for differences in background fluorescence. Once normalization is complete, a threshold level can be set. This is the level at which fluorescence data is analyzed