Allele-Specific PCR Assay for Mutation Detection

The updated MUT-MAP panel was run on the BioMark platform (Fluidigm Corp.) using a 96.96 dynamic array as described previously [11] (link) with a few alterations. Preamplified DNA combined with qPCR reagents and 10× assays mixed with the Fluidigm 20× sample loading reagent (Fluidigm Corp.) were loaded onto the chip as per the manufacturer's protocol. All newly added assays were allele-specific PCR (AS-PCR) assays which utilized an engineered Thermus specie Z05 DNA polymerase (AS1) and primers to allow for allelic discrimination between the wild-type and mutant sequence. [12] , [13] An exon specific probe was used in all assays.