Association of EGFR gene G719S and T790M mutations with cervical cancer / 中华医学遗传学杂志

OBJECTIVE@#To establish a rapid and accurate "on/off" switch technique consisted of 3'-phosphorothioate-modified allele-specific primers and exo+ polymerase to screen the G719S and T790M mutations of epidermal growth factor receptor (EGFR) gene. The switch was used to identify cervical cancer patients who are sensitive to tyrosine kinase inhibitor (TKI).@*METHODS@#Allele-specific primers targeting recombinant wild-type and mutation-type templates were designed with 3' terminal phosphorothioate modification. Two-directional primer extension was carried out using Pfu polymerase. The G719S and T790M mutations were detected by the technique among cervical cancer tissues. The results were verified by Sanger sequencing.@*RESULTS@#No mutation was detected among the 80 cervical cancer cases, and the results were consistent with that of Sanger sequencing. No significant difference was found between the frequencies of the G719S and T790M mutations between the patient and the control groups (P> 0.05).@*CONCLUSION@#A sensitive "on/off" switch technique for detecting the two EGFR mutations was established. The G719S and T790M mutations are not associated with cervical cancer.

Construction of EGFR gene G719S and T790M mutation vector and preliminary clinical application / 临床检验杂志

Objective To construct mutant recombinant vector of epidermal growth factor receptor ( EGFR) gene G719S and T790M sites associated with cervical cancer, lay the foundation for the detection of EGFR gene mutation in cervical cancer. And using it to es-tablish a molecular switch platform to detect cervical cancer EGFR gene mutations. Methods Using the wild-type recombinant plasmid as template, the mutant fusion target fragment were amplified by overlap PCR, then connect this target fragment into the vector pMD19-T. The constructed mutant recombinant plasmid was finally transformed into competent cells E.coli DH5αfurther identified by PCR with bacterial solution and genome sequencing. Establishing the molecular switch for the detection of clinical cervical cancer samples. Re-sults The G719S and T790M mutations were successfully certified by genome sequencing, and the site-directed mutant vector was successfully constructed. In addition, a molecular switch detection platform was also successfully established for the detection of cervical cancer tissue DNA. Conclusion We successfully constructed an EGFR gene mutant recombinant vector by overlap PCR technique, which providing a new technical means for gene site-directed mutagenesis. And the molecular switch detection platform was successfully established based on it, which furnishing a new method for clinical detection of EGFR gene mutations.

Construction of fused recombinant vector of EGFR gene containing G719S and T790M by overlap PCR / 临床检验杂志

Objective To construct the recombinant pMD19-exon18-exon20 plasmid containing locus G719S and T790M of EGFR gene associated with cervical cancer,which may provide a template for preparing the mutant recombinant vector of EGFR gene.Methods Using the healthy human genome DNA as templates,the segments of exon 18 and exon 20 of EGFR gene were amplified by two pairs of specific primers which were designed based on the sequences of overlapping and complementary area.The amplified segments were linked by overlap PCR.The products of linked exon18-exon20 were further inserted into the vector pMD19-T.The constructed recombi nant pMD19-exon18-exon20 plasmid was finally transformed into competent cells E.coli DH5α and then identified by PCR with bacterial solution and genome sequencing.Results The amplified fragments of exon18 and exon20 were clearly appeared at 778 bp and 596 bp and the fused product of exon18-exon20 was showed at 1 374 bp on agarose gel electrophoresis.The recombinant plasmid of fusion EGFR gene was consistent with the expected results via bacterial PCR assay and DNA sequencing.Conclusion We successfully fused the segments of exon18 with exon20 and constructed the recombinant expression vector of EGFR gene by using overlap PCR method.