Figure 5.

PG–RCA under optimized reaction condition. (A) Fluorescent intensity of PG–RCA reaction at the optimized reaction condition (0.4 µM dNTP, 0.05 U Vent (exo-) DNA polymerase, 1 U Nb.BsmI and 7.5 nM circular probe II) was monitored in real time. Sample DNA concentration in each reaction was prepared by 10-fold serial dilution from 5 fmol to 0.5 ymol and their signal amplification curves were indicated by colored lines (dark blue, blue, light blue, purple, dark green, green, brown, orange, yellow, red and gray, respectively) (n=2). Negative controls are indicated by black lines (n=2). (B) Threshold time (T_T) was plotted against the sample DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 5 fmol and 0.5 zmol sample DNA and its formulation is T_T=−19.2 log₁₀(S)+75.6 (R²=0.998). Perforated line indicates average T_T value of the negative controls. Limit of detection is 84.5 ymol or 50.7 molecules of sample DNA by calculation from the intersection of both lines.