A branch-migration based fluorescent probe for straightforward, sensitive and specific discrimination of DNA mutations

Genetic mutations are important biomarkers for cancer diagnostics and surveillance. Preferably, the methods for mutation detection should be straightforward, highly specific and sensitive to low-level mutations within various sequence contexts, fast and applicable at room-temperature. Though some of the currently available methods have shown very encouraging results, their discrimination efficiency is still very low. Herein, we demonstrate a branch-migration based fluorescent probe (BM probe) which is able to identify the presence of known or unknown single-base variations at abundances down to 0.3%-1% within 5 min, even in highly GC-rich sequence regions. The discrimination factors between the perfect-match target and single-base mismatched target are determined to be 89–311 by measurement of their respective branch-migration products via polymerase elongation reactions. The BM probe not only enabled sensitive detection of two types of EGFR-associated point mutations located in GC-rich regions, but also successfully identified the BRAF V600E mutation in the serum from a thyroid cancer patient which could not be detected by the conventional sequencing method. The new method would be an ideal choice for high-throughput in vitro diagnostics and precise clinical treatment.