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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.

Development of sensitive assay for detection of hotspot mutations of cancer driving gene is crucial for circulating tumor DNA analysis. This study tested the possibilities of applying restriction enzyme digestion and dephosphorylation coupled with blue/white screening technology for analyzing a hotspot point mutation in codon 13 of KRAS gene. The present study has documented that the combination of PCR with restriction digestion, dephosphorylation, blue/white screening and Sanger's sequencing can identify rare mutations with sensitivities at 0.003%. This novel assay with high sensitivity may have application in the diagnosis of early cancer targeting ctDNAs.