Temperature-Shift Speed Dependence of Nonspecific Amplification of Polymerase Chain Reaction Examined by 1480 nm Photothermal Transition Speed Controllable High-Speed Polymerase Chain Reaction System

摘要

We have examined the contribution of temperature shift speed from denaturation to extension for the reduction of nonspecific amplification caused by the mismatched primer-target attachment. We have newly developed the photothermal quantitative polymerase chain reaction (qPCR) system, in which the direct absorption of a 1480 nm infrared laser beam was controlled by a rotating gradient neutral density (ND) filter to acquire the precise control of the desired speed of temperature shift between 60 and 95 ℃ up to 1 s. The results showed that a quick shift of the temperature during the qPCR procedure reduced nonspecific amplicons with a significant reduction of qPCR time when we have chosen proper primer sets, whereas the non-proper primer set amplified nonspecific amplicons in the fast qPCR. The results indicate that the potential of quick qPCR using proper primers can reduce nonspecific amplification and the required time for qPCR measurement, and the necessity of more precise check of the matching of the primer template adequate for the fast temperature shift and for quick qPCR analysis.