Development Of An Assay For The Deletion Mutation Of EGFR In Lung Cancer And Its Application In Circulating DNA

Aims: We aim to develop a mutation sensitive switch with qPCR based assay for thedetection of the two common epidermal growth factor receptor (EGFR) mutations(in-frame deletion at exon19) in lung cancer. To investigate the biomedical applications ofbeta-gal based blue/white colony screening assay in noninvasive diagnosis of lung cancerrelated EGFR deletion mutations. Using the blocking3’ modified oligonucleotidecombined the switch qPCR or the blue/white colony screening assay to improve thespecificities of assay identified in circulating DNA from patients with lung cancer.Methods: Two hotspots deletions in exon19of EGFR mutations were chosen as thedetection targets of this study. They are15-bp deletion of E746–A750and18-bp deletionof L747–S752ins S. Allelic specific primers targeting wild type and mutation type weredesigned with the primers’3’ terminal phosphorothioate modification. Two-directionalprimers extension was performed using exo+polymerases. The negative and positivecontrol templates of wild type and deletion mutations were prepared using recombinantplasmids containing relevant DNA fragments respectively. Genomic DNAs were extractedfrom lung cancer tissues. The assays for EGFR deletion detection were evaluated for theirsensitivities and specificities in recombinant plasmids and genomic DNA respectively.The blue/white screening methods for EGFR deletion mutations in a samplecomprising a mixture of human DNA, comprising the steps of:(a) obtaining DNA fromselected samples;(b) amplifying the fragment harboring hot spot mutations:(c) cloning theamplified fragment into blue-white selection vector; transforming the ligated products;(d)numbering blue and white colonies from the transformation;(e) determining the presenceor absence of EGFR deletion mutation; and (f) subsequently sequencing the mutantcolonies to obtain the sequence information of the deletion.Adding the blocking3’ modified oligonucleotide targeting the WT sequence into thePCR reaction, the new assay was evaluated the sensitivities and specificities in circulating DNA from patients with lung cancer.Results: Two assays for EGFR15-bp and18-bp deletion mutations analysis havebeen developed with the mutation sensitive switch. Amplified by exo+polymerase, allelicspecific primers perfectly matching deletion mutated allele were extended while noproducts were yielded from primers targeting wild type allele. The mutation sensitiveswitch was able to detect10copies for the deletion mutation. In screening circulating DNAfrom24cases of lung cancer, five15-bp deletions were identified (5/24). When comparedwith the sequencing results of the tumor samples, the direct sequencing analysis is notsensitive enough to identify the mutation in this case.Disclosed is a genetic diagnostic assay to achieve a determination of DNA mutationsusing LacZ based blue-white seletion. The preferred method uses an artificially stop codonresiding in the fragment of EGFR gene to interrupt the expression of lacZ gene. Wild typegene yields white colonies and EGFR mutants with deletions yield blue colonies. Bycoloring the colonies or the number of different colored colonies, target DNAs can bedetermined with or without EGFR deletions.Conclusion: In addition to point mutation analysis, the mutation sensitive switch issensitive and specific for the detection of EGFR19exon deletion mutations in bothcirculating DNA samples and tumor samples.This work successfully investigated the biomedical applications of beta-gal basedblue/white screening assay in the diagnosis DNA with or without the deletion of EGFRexon19. Besides, additional blocker primer can improve the specificity of detection assay.