Multiplex Real-Time PCR Assays that Measure the Abundance of Extremely Rare Mutations Associated with Cancer
(A) To mimic assays initiated with DNA fragments isolated from blood plasma, we utilized SuperSelective primer EGFR L858R 24-14/14-5:1:1 in a set of eight PCR assays that were initiated with different quantities of digested human genomic DNA encoding the EGFR L858R mutation (DNA from 0; 10; 30; 100; 300; 1,000; 3,000; or 10,000 cells) in the presence of digested human genomic DNA from 10,000 wild-type human cells. The threshold cycle measured for each reaction that contained mutant templates is plotted as a function of the logarithm of the number of mutant templates initially present in each reaction. The dotted orange line indicates the threshold cycle of the reaction containing only wild-type templates. (B) Comparison of the number of EGFR T790M mutant target molecules present in human genomic DNA samples (as determined by droplet digital PCR) to the Ct values obtained for the same samples in real-time PCR assays that utilize SuperSelective primer EGFR T790M 24-14/14-4:1:1. The dotted orange line indicates the threshold cycle of a reaction containing only wild-type templates.