An improved digital polymerase chain reaction protocol to capture low-copy KRAS mutations in plasma cell-free DNA by resolving 'subsampling' issues.

Genetic alterations responsible for the initiation of cancer may serve as immediate biomarkers for early diagnosis. Plasma levels of cell-free DNA (cfDNA) in patients with cancer are higher than those in healthy individuals; however, the major technical challenge for the widespread implementation of cfDNA genotyping as a diagnostic tool is the insufficient sensitivity and specificity of detecting early-stage tumors that shed low amounts of cfDNA. To establish a protocol for ultrasensitive droplet digital polymerase chain reaction (ddPCR) for quantification of low-frequency alleles within a limited cfDNA pool, two-step multiplex ddPCR targeting eight clinically relevant mutant KRAS variants was examined.