Impact of DNA polymerases and their buffer systems on quantitative real-time PCR

Abstract

An investigation of the influence of five DNA polymerase-buffer systems on real-time PCR showed that the choice of both DNA polymerase and the buffer system affected the amplification efficiency as well as the detection window. The analytical repeatability of the data for different systems changed clearly, leading us to conclude that basing quantitative measurements on single-data-set standard curves can lead to significant errors.

FIG. 1.

Standard curves generated for five different DNA polymerase-buffer systems after two different types of analysis of the same independent triplicate data. Panels A to E show standard curves and corresponding equations when all three sets of data for each DNA polymerase were used for one analysis of DyNazyme II (A), LCTaq (B), rTth (C), Taq (D), and Tth (E). Panels F to J show standard curves and corresponding equations when the three sets of data were used for independent analysis of DyNazyme II (F), LCTaq (G), rTth (H), Taq (I), and Tth (J).

FIG. 2.

Detection probability using different DNA polymerase-buffer systems. The detection probability for each DNA concentration was determined by checking the amount of data points/total amount of analysis. Data were determined after independent triplicate experiments. —•—, DyNazyme II; --□--, LCTaq; —▴—, rTth; ——, Taq; and ---×: Tth.