Isothermal amplification reaction of Temp(GGG-CCC) RNA (initial concentration of 10 or 8 pmol) with NV3Dpol(11 or 4 pmol). Time-course of RNA synthesis reaction with NV3Dpol. RNA template (10 pmol) was incubated with NV3Dpol (11 pmol) at 30°C. The reaction in each aliquot was stopped by adding EDTA, and analyzed on an 8 M urea denaturing 12% PAGE (A) or a non-denaturing 12% PAGE (B). M; 10 bp DNA step ladder (Promega). The RNAs appeared in the 80–90 nts (in a case of (A)) or in the 40–50 bp (in a case of (B)) mobility were tRNA from the cell-free translation system. (C) Closed square: Quantification of each bands in (A) (= the amount of ssRNA). Closed circle: Quantification of template-excess experiments (n = 2) (RNA template 8 pmol and NV3Dpol 4 pmol in 32 μL reaction volume). Same lot of the enzyme was used. Quantification of each bands was performed by a calibration curve of Temp(GGG-CCC) RNA (2, 5, 7 and 10 pmol) on the denaturing PAGE.