Figure 3.

Characterization of molecular zipper in RAM reaction. (A) Molecules (1 × 10⁷) of the C-probe were hybridized to 1 × 10¹⁰ molecules of the DNA target in a 20 μl reaction at 65°C for 15 min in the presence or absence of DNA ligase. The ligation products (2 μl) were used to initiate 25 μl RAM reactions in the presence of 0.4 μM molecular zipper and the change in fluorescence was analyzed with a Smart Cycler. (B) After completion of the RAM reaction, the denaturation curves were determined by measuring the fluorescence change as a function of temperature change (up to 95°C). (C) Known concentrations of ligated C-probes (10⁸–10 molecules) were used to initiate the RAM reaction in the presence of 0.4 μM molecular zipper in 50 μl RAM reactions and the kinetics of fluorescence change were determined in a fluorometric thermal cycler (iCycler). Each concentration was carried out in triplicates. For negative control (gray line), no ligated C-probe was added. (D) R_t values obtained from Figure 3C are plotted against initial copy number of the C-probe (three replicates per concentration). The linear curve was added from a linear fitting.