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. 2015 Sep 28;10(9):e0139074. doi: 10.1371/journal.pone.0139074

Fig 2. Four different KRAS multiplex digital PCR assays combining G12C, G12V and G12D mutant assays and corresponding duplex assays.

Fig 2

KRAS G12C mutant droplet populations are indicated by a red dashed square, KRAS G12D mutant populations by a blue dashed square and KRAS G12V mutant populations by a yellow dashed square. Each multiplex assay, combining all relevant FAM and HEX assays, KRAS WT gDNA and G12V, D and C mutant gDNA, is shown in the top panel. The corresponding duplex assay for each mutation, using the same FAM and HEX assay concentration with KRAS WT DNA and the appropriate KRAS mutant DNA present, is shown in the panels below each multiplex assay. Multiplex 1 (top left panel) is an assay combination of 900 nM primers and 500 nM G12C probe, 562.5 nM primers and 312.5 nM G12D probe and 225 nM primers and 125 nM G12V probe with 450 nM primers and 250 nM WT for G12C probe. Multiplex 2 uses the same concentration of G12V and WT for G12C assay as Multiplex 1 but also contains 450 nM primers and 250 nM G12C probe and 675 nM primers and 375 nM G12D probe. Multiplex 3 uses the same concentration of KRAS mutant assays as in Multiplex 2 but with the WT for G12V probe assay present, used at the same concentration as the WT for G12C assay in Multiplex 1. Multiplex 4 is an assay combination of the G12C assay as in Multiplex 2 with 225 nM primers and 125 nM G12D probe and 675 nM primers and 375 nM G12V probe with the WT for G12D assay at the same concentration as the WT for G12C assay in Multiplex 1. Key: black- empty droplets, blue- mutant DNA FAM positive droplets, green- wild-type DNA HEX positive droplets, brown—wild-type and mutant DNA double positive droplets. The same scale is used for all panels (HEX amplitude up to 7000 and FAM amplitude up to 22000).