Revealing the secrets of PCR

The polymerase chain reaction (PCR) method and its quantitative variant (qPCR) were a landmark discovery for detection and quantification of small amounts of unambiguous deoxyribonucleic acids (DNA) due to its enormous sensitivity and specificity. The current methods for qPCR protocol optimization provide no information of the PCR propagation during the cycles as only single data point is extracted at the end of each thermal cycles, limiting a thorough understanding of reaction details. In this study, we utilized the continuous fluorescence monitoring method to observe progress of the reaction in real time with over 100 data points per each cycle, thus gaining a profound insight into the PCR itself. This provided information about the real-time PCR status, dominating reactions and their completion/incompletion during each cycle as well as their reaction kinetics. We then adjusted the duration of either annealing or elongation steps to ensure their completion within each cycle, resulting in the protocol optimization with complete amplification, enhancing PCR efficiency and taking < 20 min to obtain maximum product amount. The proposed method was verified using DNA with lengths of 177 base pairs (bp), 250 bp, and 400 bp. It can also be adopted for helping with qPCR troubleshooting as well as protocol optimizing just by reprogramming commercial real-time PCR cyclers.