Fluorescence-Based Detection of KRAS Mutations in Genomic DNA Using Magnetic Bead-Coupled LDR Assay

We previously developed a bead-coupled ligase detection reaction (LDR) assay that enables simple and rapid detection of single-nucleotide variations (SNVs) using synthetic oligonucleotide templates. In the present study, this approach was extended to genomic DNA extracted from colorectal cancer cell lines to evaluate its applicability to clinically relevant samples. Targeting codon 12 of the <i>KRAS</i> gene, PCR-amplified products served as templates for bead-coupled LDR, and fluorescence excitation-emission matrix (EEM) analysis was employed for signal readout. The four fluorophores used in the assay exhibited distinct spectral properties, allowing their signals to be clearly resolved within the EEM profiles. This mapping provided characteristic fluorescence signatures that revealed the underlying genotypes, enabling not only the distinction between homozygous and heterozygous states but also the precise identification of allele compositions, as exemplified by G/A, T/T, G/G, and G/C in colorectal cancer cell lines. The single-tube workflow, integrating magnetic bead capture with fluorescence-based detection, demonstrated robustness, speed, and cost-effectiveness compared with conventional mutation detection methods. These findings confirm that the LDR-EEM platform can be successfully applied to genomic DNA analysis, underscoring its potential as an accessible and reliable tool for SNV detection in both research and diagnostic contexts.