Polymerase chain reaction (PCR) is a powerful tool that is an essential part of many research and clinical protocols. Successful PCR depends on accurately controlling the solution temperature during temperature cycling. Due to limitations associated with directly contacting the solution, temperature measurements are typically made externally to the sample. This compromises solution temperature accuracy, particularly during rapid temperature transitions, where the solution temperature will lag behind instrument readings. Typically, this temperature lag results in the actual solution temperature being lower than the desired denaturation temperature and higher than the desired annealing temperature. This can lead to reduced PCR product yield, low efficiency, and the amplification of non-specific products.
To achieve more rapid temperature cycling, alternative methods of accurately tracking the solution temperature are required. We propose to extend the use of fluorescence, already an integral part of real-time PCR and melting analysis, to further serve as a monitor and control of solution temperature. Through adding a temperature-sensitive passive reference dye to the PCR reaction, changes in temperature may be correlated to variations in fluorescence, so that the solution temperature may be non-invasively monitored in real-time. Furthermore, this approach allows for temperature cycling to be controlled through actual solution temperature measurements for improved temperature accuracy. To meet these goals, three specific aims will be pursued: 1) Correlation of temperature and the fluorescence of a passive reference dye during PCR and melting curve analysis, 2) Utilization of ratios on multiple spectral bands to improve solution temperature accuracy, and 3) Demonstration of fluorescence-based temperature control on prototype real-time instruments.