A Modified Mutation-Sensitive On/Off Switch and Its Application in B-Raf Proto-Oncogene Hotspot Mutation Assay

Abstract

The combination of phosphorothioate-modified primer and high-fidelity DNA polymerase constitute a mutation sensitive on/off switch. Herein, a modified mutation-sensitive on/off switch was designed with an extra point mutation at 5′ far away from the 3′ of the phosphorothioate modified primer to test the power of the new enzymes: Phusion, Q5 and Phanta UC polymerases. A modified on/off switch with the introduction of an extra point mismatched nucleotide at –8 from the 3′ terminus, and the use of Phanta UC Super-fidelity DNA polymerase has been confirmed to be highly sensitive and specific as compared to those without the 5′ artificial mismatched nucleotide, when applied in circulating tumor DNA (ctDNA) analysis of BRAF hotspot mutation.