Figure 3.

Comparison of melt and annealing curves of target and primer L-DNAs during a single thermal cycle for the two-channel (A) and single-channel (B) systems. (A) In the original two-channel system, the melting of HEX-labeled L-DNA targets was monitored during heating on the HEX channel (yellow curve, from 0–25 s), then the annealing of Texas Red-labeled L-DNA primers was monitored during cooling on the Texas Red channel (red curve, from 25–85 s). The original Adaptive PCR instrument was programmed to switch to cooling based on the proportion of L-DNA targets melted in the HEX channel and switch to heating based on the proportion of L-DNA primers annealed in the Texas Red channel, as indicated on the graph. (B) In the single-channel system, both the L-DNA target melt probes and the L-DNA primer anneal probes are labeled with Texas Red and monitored on the Texas Red channel (red curve), which results in stair-stepped fluorescence curves. The Adaptive RT-PCR instrument was programmed to switch between heating and cooling based on the second steps of the curve, which correspond to L-DNA target melting (from 15–25 s) and L-DNA primer annealing (from 55–85 s), respectively, while ignoring the first steps of the curve, which correspond to L-DNA primer melting (from 0–15 s) and L-DNA target annealing (from 25–55 s), respectively.