Research On Detection Methods Of EGFR Gene Mutations In Lung Cancer

Objective: To prove whether the inserted fragments with a stop codon will change thecolor of blue screening, then to detect if EGFR gene contains a pathogenic mutation.Detecting EGFR drug-sensitive mutations use high-fidelity DNA enzyme-mediatedswitch “on/off”, so as to guide rational use of tyrosine kinase inhibitors for lung cancerpatients.Methods: Firstly, we find the wild-type gene exon sequences of exons19of theEGFR gene in Genebank, and find the pMD19-T plasmid LacZ gene sequence.According to the reported Common pathogenic mutations “△E746–A750and△L747–P753(insS)”, we design the ordinary PCR primers and Sulfide-modified detectionprimers. We use over-lapping extension PCR to bulid the wild type and the mutationplasmids, then we use the blue screening method to dectect them. Then we screen10clinical circulating DNA samples to dectect if they have△E746–A750or△L747–P753in EGFR gene, then we sequence the blue bacterias to assess the method.Then we assess the10clinical tissue DNA samples. Finally we use fidelity polymerasemediated mutation sensitivity molecular switch which developed by our grouprespectively combine with agarose gel electrophoresis and fluorescence quantitative PCRtechnology to take a Qualitative and quantitative detection for△E746–A750or△L747–P753mutations in EGFR of the10clinical tissue DNA samples.Results: on LB solid medium, the colonies of wild plasmid EA are all white, and D15,D18mutated plasmids are all blue. The clones of recombinant DNA plasmids of10clinical blood circulating samples are largely white, a small amount of blue. Select theblue colonies to sequence, detected the absence of15bp,18bp deletion is not detected.sequencing the corresponding tissue samples, displays that the tissue samples have both wild-type EGFR gene and15bp deletion, agree with blue Filter results. Use the modifiedprimers of molecular swtich to detect10clinical tissue samples shows that No.2,4,6samples don’t exist15bp deletion, the rest are all have15bp deletion, all of the10samples don’t exist18bp deletion. agree with tissue samples sequencingresults.Molecular switch combines quantitative PCR results shows that all samplesexcept6th are appeared15bp deletion, copy number at around103-105, and it also showsthat all samples except6th exist18bp deletion, but their copy numbers are all under10.Conclusion: use the stop codon of inserted fragments of EGFR wild gene productscombines the blue screening assay is a feasible procedure for detecting the possibleEGFR pathogenic mutations; molecular switch technology combines with quantitativePCR technology for EGFR deletion mutations can do the qualitative and quantitativeanalysis, and may have a very high detection sensitivity.