Endonuclease IV based competitive DNA probe assay for differentiation of low-abundance point mutations by discriminating stable single-base mismatches

Abstract

We disclosed the unique discrimination property of Endo IV toward stable single-base mismatches located at the second nucleotide 3′ to the AP site. Coupled with thermodynamic differentiation and competitive blocker strands, a highly sensitive and specific detection system was established with discrimination factors of 510–1079 for G:X mismatches and LODs of 0.003–0.005% for KRAS G12A, KRAS G12V and KRAS G12S mutations.