Study On The Correlation Between EGFR Gene G719S And T790M Mutation And Cervical Cancer

Objective : Using the high-fidelity DNA polymerase mutant sensitive molecular switch combined with thiophosphorylation modified specific detection primers,we established two rapid and accurate techniques to screen the EGFR gene G719 S and T790 M mutations in cervical cancer.Then use the techniques to investigate the presence or absence of two related mutations of EGFR gene in cervical cancer,and to screen out patients with cervical cancer sensitive to TKI,therefore guiding the rational use of EGFR-TKI in patients.Methods: Fristly,the blood genomic DNA was extracted respectively.Two primers were designed according the 18 th exon of G719 S and 20 th exon of T790 M mutations in EGFR gene.Additionally,the primer of18 bp overlapping regions was designed by Primer Premier 6.0 software.The PCR primer was extended through high fidelity of DNA polymerase chain reaction.Moreover,the template of recovered DNA fragment was used for the extension of primer and subsequent the production of fusion fragment by low-fidelity DNA polymerase chain reaction.The fusion fragment was cloned into PMD19-T vector,then a wild type was produced.And the wild type was further sequenced.Furthermore,The mutated target product was obtained by overlapping extension PCR.The extended reaction product was cloned into T vector,the positive mutant template plasmid was produced by ampicillin with blue and white screening.Two specific mutant base forward primer and reverse primer two primers were modified by 3 ’end vulcanization.by high fidelity DNA polymerase-mediated mutant-sensitive molecular switches.At the end of the study,G719 S and T790 M mutation were detected by high-fidelity DNA polymerase-mediated mutation-sensitive molecular switching technique in EGFR gene of cervical cancer tissue.Finally,we screened out cervical cancer patients sensitive to TKI,and leading to a rational use of EGFR-TKI in patients.Results: DNA sequencing results confirmed that plasmid templates containing two site-directed mutation sites were successfully obtained by overlapping extension PCR.Mutation Sensitive Molecular Switch was used to detect DNA samples from 80 cases of clinical cervical cancer.The results showed that mutations were not detected by PCR and sequencing.The genotypes of G719 S and T790 M were GG and CC homozygous respectively,and there was no significant difference compared with the control group(P> 0.05).Conclusion: The combination of high fidelity DNA polymerase-mediated mutant sensitivity molecule switch and agarose gel electrophoresis had high specificity and sensitivity to cervical cancer EGFR gene.In this study,the mutant sensitive molecular switch system could be used to detect two mutations sites of cervical cancer EGFR gene.More importantly,we screened out EGFR-TKI gene mutation sites,which might guide the rational use of drugs for clinicians and provide a new target for drug discovery.