Sensitive detection of EGFR mutations using a competitive probe to suppress background in the SMart Amplification...

In a previous study, a single nucleotide polymorphism (SNP) diagnostic system named the SMart Amplification Process version 2 (SMAP 2) was reported, which enabled rapid gene diagnostics from crude samples such as whole blood. The asymmetric primer design and use of Taq MutS were reported as innovative background suppression technologies employed by SMAP 2, but Taq MutS is known to display differential affinities for various mismatch combinations, and hence may not be entirely effective for all possible applications. To address this issue we developed another approach using a competitive probe (CP) to enhance background suppression technology instead of Taq MutS. CP is a 3'-end aminated oligonucleotide that competes with 3'-end of a discrimination primer or the self-priming elongation site on intermediate product 2 (IM2) for non-target sequences, such as the alternative allele. The preferred hybridization kinetics for the full-match CP on the non-target sequence results in effective background suppression in SMAP 2 assays. By using a CP, we demonstrated the sensitive detection of EGFR gene mutations in purified genomic DNA from mixed cell populations. The CP approach is another tool enhancing the effectiveness and versatility of SMAP 2 assays, expanding its potential applications, and reinforcing its position as a highly effective technology for molecular diagnostics.