New Assays For Detection Of EGFR Exon 19 And 21 Hot Mutations

Objective: Based on the biological characteristics of EGFR mutations in exon 19 and exon 21,we enriched the mutant gene fragments by PCR and other techniques and developed rapid,sensitive and inexpensive detection methods with potential application for individual therapy of lung cancer.Methods: Zeocin resistance gene is a report gene to screen components of plasmid.When foreign genes with stop codon(TAA)are inserted into the sequence of zeocin resistance gene,the zeocin resistance gene will be inactivated.Instead,When foreign genes without termination codon are inserted,the resistance gene retain its activity.As known,the Del E746-A750,Del L747–S752 in EGFR exon19 do not contain “TAA”,while the exon19 wide-type contains “TAA” that was readjusted to form a Ocher stop codon.The EGFR exon19 wild-type and mutant DNA were inserted into sequence of zeocin resistance gene in vector p ZAmp B and then translated to E.coli.As a result,colonies formed from subcloning wild typed DNA fragments will not grow in the plate with zeocin,while colonies formed from subcloning mutant DNA fragments will grow in the plate with zeocin.The EGFR 858 wide-type DNA contains Bse GI restriction enzymes site following a point mutation plus the cancer related mutation(GGATGG),while mutant gene does not harbor a restriction site following an intentional point mutation with the sequence of GGAGGG.Mutations can be detected by genotype-switch method(PCR coupling with Bse GI).Results: With EGFR exon19 mutation as targets,1000 fold enrichment was achieved by the first new assay using mixed wide-type and mutant DNA fragments.The sensitivity of L858 R mutation detection by using genotype-switch is as high as 1 / 10000.Conclusion: Two new assays were developed: zeocin as a reporter gene to detect EGFR19 exon deletion mutation and genotype-switch to detect L858 R mutation,which are highly sensitive and have potential for cancer diagnosis.