Differential detection of KRAS mutations in codons 12 and 13 with a modified loop-hybrid (LH) mobility shift assay using an insert-type LH-generator

The loop-hybrid mobility shift assay (LH-MSA) was previously developed for the rapid detection of the EGFR mutation L858R for predicting clinical responses to gefitinib in lung cancer. Recently, clinical importance of determining KRAS mutations has been demonstrated in colorectal tumors as tumors harboring mutated KRAS genes were not responsive to therapy with EGFR-targeted antibodies such as cetuximab. We developed a new version of the LH-MSA using an insert-type LH generator that was capable of detecting all 12 KRAS mutations in codons 12 and 13. Feasibility evaluation was performed with this new LH-MSA on 215 colorectal cancer specimens. KRAS codon 12 mutations were detected in 23% specimens and codon 13 mutations in 6.5% specimens by LH-MSA at a rate better than by direct sequencing. Using the new method, the G13D mutation was readily distinguishable from other KRAS mutations in codon 12 and, therefore, would be advantageous for clinical applications. ► LH-MSA is a simple method for detection of point-mutations. ► LH-MSA detected 12 mutations of KRAS in codons 12 and 13. ► The insert type LH-probe provided a way to detect these 12 mutations. ► Our method differentiated all KRAS mutations detected in colorectal tumor DNA. ► LH-MSA detected KRAS mutations in colorectal tumor DNA with 5% mutant sensitivity.