Novel Multiplexing Strategies for Quantification of Rare Alleles Using ddPCR

Droplet digital PCR (ddPCR) has come to be regarded as the gold standard for the ultrasensitive detection and absolute quantification of closely related DNA sequences within complex mixtures. Most ddPCR assays to date, however, rely on sets of hydrolysis probes conjugated with dyes having different emission spectra to allow independent counting of rare mutant and wild-type alleles. Here, we describe a set of novel strategies that leverage the simultaneous detection and quantification of both mutant and wild-type alleles with a single hydrolysis probe. Variants of these strategies empower multiplexing and a more cost-effective approach for concurrent screening of multiple genetic variants.