Abstract: Objective To construct and optimize a high-fidelity polymerase-mediated mutation sensitive molecular switch technique platform for detecting exon 19 del18bp of EGFR gene. Methods Using genome DNA from a healthy volunteer as PCR template, the wild-type and mutant-type DNA fragment containing mutation site were amplified by common PCR and overlap PCR respectively, which then were connected with the vector pMD19-T. The constructed wild-type and mutant-type recombinant plasmid was transformed into competent cells E.coli DH5α further appraised by bacteria solution PCR and genome sequencing. The mutation-specific upstream detection primer was designed with 3′phosphorothioate modification, and two-directional primers extension was performed using high- fidelity polymerase. The annealing temperature, primer concentration, template concentration, cycle number, and other high-fidelity polymerase-mediated PCR conditions were all optimized through an orthogonal test protocol to establish a molecular switch detection platform for exon 19 del18bp of EGFR gene. Results The molecular switch detection platform for exon 19 del18bp of EGFR gene was successfully established. The optimal PCR condition of detecting EGFR gene exon 19 del18bp using mutation-sensitive molecular switch technique was as follows: 57 ℃ optimal annealing temperature, 0.16 μmol/L primer concentration, 1.48×10^－2 μmol/L template concentration and 25 PCR cycles. And the allele-specific primer for del18bp can detect the perfectly matched template at a sensitivity of about 10^－3 μmol/L. Conclusions Mutation-sensitive molecular switch technology composed of high-fidelity polymerase with 3′ phosphorothioate-modified prime provides a new technology which can implement EGFR mutation test for personalized targeted meditation for treatment of lung cancer.

Key words: lung cancer, epidermal growth factor receptor(EGFR), high-fidelity polymerase, molecular switch