Abstract

KRAS mutations are among the most common drivers of malignant tumors in lung, colorectal, and pancreatic cancers. Identifying specific mutations in solid tumors guides treatment selection, while quantifying mutant-to-wild-type alleles in liquid biopsies helps assess therapeutic response and recurrence risk. Accurate mutation profiling is essential for developing KRAS-targeted therapies.

However, standard qPCR assays detect mutations only at ~1% mutant allele frequency (MAF) and require multiple reactions, increasing cost and reducing scalability. Digital PCR (dPCR) improves the sensitivity to ~0.2% MAF but often groups different targets into shared channels, limiting specificity. For low-abundance, limited-input samples such as cell-free DNA (cfDNA), a more sensitive and efficient assay is urgently needed that addresses both sensitivity and specificity challenges.

Here, we present a robust PCR-based method for simultaneous quantification of multiple KRAS mutations in a single reaction.