Study On Detection Method Of EGFR Gene With 19 Exon Deletion

Objective: to establish a rapid and sensitive technology to detect EGFR gene with 19 exon deletion mutations by using PCR, the restriction enzyme and blue white screening techniques with different proportions of wild-type and mutant plasmids.Methods: To construct plasmid template of mutant and wild type EGFR gene with 19 exon deletion del E746-A750 and del L747-S752 ins S by paired primers and and PCR. Then simulate the actual samples of the wild type and mutant gene ratio to amplify the insertion of plasmid template with a set of specific PCR primers. Combined with the blue/white screening technology principle, wild type gene yields white colonies because of the stop codon TAA in the reading frame and EGFR mutants with deletions yield blue colonies. By coloring the colonies or the number of different colored colonies, target DNAs can be determined with or without EGFR deletions. For improve the efficiency, the restriction enzyme can be used to digest wild fragment, to achieve indirect enrichment of mutant DNA.Results: Wild type gene yields white colonies and mutants with deletions yield blue colonies. By simulating different proportions, it is concluded that the system can still detect the presence of mutation in the presence of only 1/10000 mutant concentration(the wild template: mutant form =10000:1).Conclusion: This method has high sensitivity and specificity in the detection of different proportion of template. It has great application value in early diagnosis and monitoring of tumor recurrence, and has great economic and social benefits.