Peptide nucleic acid clamping to improve the sensitivity of Ion Torrent-based detection of an oncogenic mutation in KRAS

Detecting oncogenic changes in the genome of cancer patients is crucial for targeted therapy. Such changes include alterations to KRAS, a GTPase located upstream of several signalling transduction pathways implicated in cancer formation. While next-generation sequencing (NGS) allows for comprehensive analysis of a genome, the technology can struggle to detect low frequency variants. To improve the sensitivity of NGS for detecting mutations we created a peptide nucleic acid (PNA) clamp, validated by qPCR, designed to bind wild-type KRAS (WT KRAS) across codon 12 during the PCR amplification stage of a NGS library preparation. We tested the effect of clamping the wild-type KRAS sequence in a reference standard with a KRAS c.35G>A mutation (KRASG12D) at an allelic frequency (AF) of 1.3% and on circulating-free DNA from a patient harbouring a KRASG12D mutation (at an AF of 3.2%). Runs were conducted using 10, 5, 2.5 and 1 ng of DNA input. The PNA increased the number of mutant reads and their frequency relative to wild-type calls, allowing for more sensitive detection at all tested concentrations of DNA input.