Feasibility of Bead-Coupled Ligase Detection Reaction and Fluorescence Excitation–Emission Matrix Spectroscopy for Minority KRAS Variant Discrimination in a Wild-Type Background

Detecting minority mutant sequences in an excess of wild-type DNA requires discrimination of weak mutant-associated signals from background. We previously demonstrated the identification of KRAS (KRas proto-oncogene, GTPase) variants in individual colorectal cancer cell-line samples using a bead-coupled ligase detection reaction (LDR) assay. In the current study, we assessed the feasibility and limitations of a mutation-focused version in defined mixtures of mutant-cell-line and wild-type (WT) amplicons. Polymerase chain reaction amplicons from each mutant cell line were mixed with WT-derived amplicons at mutant:WT ratios of 1:10, 1:100, 1:200, and 1:400. Each LDR contained hexachlorofluorescein (HEX)-, carboxytetramethylrhodamine (TAMRA)-, and carboxy-X-rhodamine (ROX)-labeled discriminating primers for c.35G>A, c.35G>T, and c.35G>C, respectively, and a biotinylated common primer. Ligation products were captured on streptavidin-coated magnetic beads, washed, eluted, and analyzed by fluorescence excitation–emission matrix (EEM) spectroscopy. Total fluorescence intensities at excitation/emission wavelength pairs selected for HEX and ROX were significantly higher than those of the WT-only control for c.35G>A and c.35G>C, respectively, at 1:10 and 1:100, but not at 1:200 or 1:400 (n = 4). For c.35G>T, no significant difference was observed at any of the four tested ratios. The WT-only HEX-region response and HEX-TAMRA overlap complicate interpretation. The results support a feasibility assessment with partial discrimination of defined mixtures by total fluorescence, but do not support multiplex dye assignment or identification of an unknown variant. Individual dye contributions and the sources of WT-only fluorescence remain unresolved.