Fig. 1.

Optimization of the digital real-time PCR platform LOAA. A: Images of the LOAA cartridge and wells within it (left), and representative results including real-time amplification curves and a two-dimensional scatter chart of FAM (channel 1) and FRET (channel 2) signals (right). B–G: Digital real-time PCR amplification curves using a set of assays distinguishing the T790M mutation (FAM) from the wild-type (FRET). FAM channel (B and D) and FRET channel (C and E) with indicated conditions. Digital real-time PCR amplification curves using the following optimal concentrations of primers and probes for two assays (different quenchers): forward primer: reverse primer: FAM probe: FRET probe as 20:20:4:20 pmol per reaction (F and G). Blue lines indicate a fixed fluorescence intensity of 300.