Locally averaged background correction

Equal volumes of ACB-PCR products were analyzed on non-denaturing 8% polyacrylamide gels, loading 10 μl of KRAS and BRAF ACB-PCR products or 15 μl of the PIK3CA and HRAS ACB-PCR products. The fluorescent ACB-PCR products were visualized using a PharosFX Molecular Imager with an external blue laser (Bio-Rad). Pixel intensities of the correct-sized bands were quantified using Quantity One software and a locally-averaged background correction (Bio-Rad). Although duplicate 10^− 1 to 10^− 5 MF standards were included in each experiment, the set of standards that gave the best quantification across the observable range of MFs for an entire set of unknown samples was used to construct each standard curve [10^− 5 to 10^− 1 for PIK3CA and BRAF, 10^− 5 to 10^− 3 for KRAS and HRAS]. For PIK3CA and KRAS, log-log plots relating MF to fluorescence (in pixels) were constructed and fit with a power function. For BRAF, log-linear plots relating MF to fluorescence were constructed and fit with a logarithmic function. For HRAS, linear-linear plots relating MF to fluorescence were constructed and fit with a linear function. Using the function of the standard curve and the pixel intensities of the ACB-PCR products for each mutation, the MF of each mutation within each unknown sample was calculated.