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.

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.

Objective: To explore the association between the single nucleotide polymorphism (SNP) of rs175048 in ataxia telangiectasia mutated (ATM) gene and lung cancer susceptibility in Han population. Methods: A total of 225 cases of blood samples from lung cancer patients treated in Hospital of Traditional Chinese Medicine of Hengyang City and the Affiliated First Hospital of Nanhua University from October 2015 to August 2016 were collected as case group, and 128 cases of blood samples from healthy people were collected as the control. The polymorphisms of ATM rs175048 of above mentioned participants were detected by using the SNP sensitive On/Off Switch technique. The genotypes and allele frequencies were analyzed to compare the distribution difference between case group and control group as well as its association to the clinical features of lung cancer. Results: The genotype frequencies of AA, AT and TT of ATM rs175048 were 24.9%, 52.9%, 22.2% in case group and 42.2%, 42.2%, 15.6% in control group, respectively (all P< 0.01). Moreover, the frequencies of alleles A and T were 51.0%, 49.0% in case group, and 63.0%, 37.0% in control group (all P<0.01). Genotype TT might increase while genotype AT might decrease the risk of lung cancer. rs175048 SNP was significantly correlated with smoking, age, sex and family history (all P<0.05). Conclusion: rs175048 SNPis significantly associated with lung cancer, and TT genotype may increase the risk of lung cancer.

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.
Carcinoma, Non-Small-Cell Lung ; Drug Resistance, Neoplasm ; ErbB Receptors ; genetics ; Female ; Genes, erbB-1 ; Humans ; Lung Neoplasms ; Mutation ; Protein Kinase Inhibitors ; Uterine Cervical Neoplasms ; genetics