Beta-gal Based Blue/White ScreeningAssay And Its Bioligical And Biomedical Applications

Objective：To investigate the bioligical and biomedical applications of beta-galbased blue/white colony screening assay: including (1) analysis of the activity andoff-target effects of TALENs and (2) noninvasive diagnosis of lung cancer relatedEGFR deletion mutations.Methods: Two blue/white colony-based TA cloning vectors were evaluated in theircolor selection by inserting in-frame and frame-shifted inserts shorter than150bp. TheTwo blue/white-based TAcloning vectors（pGEM-T Easy vector and pTrueBlue vector）have different cloning sites. Colonies yielded in these assays were further confirmed bysequencing analysis. The blue/white screening method for genetic editing efficiencyevaluation deploys three plasmids: two plasmids coding for the two TALENs subunitsand one coding for report gene. The evaluation of TALENs’ editing efficiency dependson successful transformation of three plasmids simultaneously. The blue/whitescreening methods for EGFR deletion mutations in a sample comprising a mixture ofhuman DNA, comprising the steps of:(a) obtaining DNA from selected samples;(b)amplifying the fragment harboring hot spot mutations:(c) cloning the amplifiedfragment into blue-white selection vector; transforming the ligated products;(d)numbering blue and white colonies from the transformation;(e) determining thepresence or absence of EGFR deletion mutation; and (f) subsequently sequencing themutant colonies to obtain the sequence information of the deletion.Results： Although the pTrueBlue was claimed to have a100%accuracy, ourresults with inserts shorter than150p showed no differences between the pGEM-T easyand the pTrueBlue vectors. Both of them had blue colonies when in-frame inserts werecloned and white colonies with frame-shifted inserts. These results were further confirmed by sequencing analysis. Based on the comparison of the two vectors, our dataconvincingly demonstrated that the final effect on the reading frame is probably theonly factor deciding the color of the colonies following transformation for inserts of150nucleotides or shorter. The successful transformation of three plasmids (two plasmidscoding for the two TALENs subunits and one coding for report gene) simultaneously isthe key to evaluate the activity of TALENs. TALENs targeting at CCR5gene speciallycut the target sequences and lead to the frame-shift of the alpha complementary peptide,which eventually cause the changes of color in colonies. This new assay provides a newtechnology for optimizing the highly effective and specific TALENs. Disclosed is agenetic diagnostic assay to achieve a determination of DNA mutations using LacZbased blue-white seletion. The preferred method uses an artificially stop codon residingin the fragment of EGFR gene to interrupt the expression of lacZ gene. Wild type geneyields white colonies and EGFR mutants with deletions yield blue colonies. By coloringthe colonies or the number of different colored colonies, target DNAs can be determinedwith or without EGFR deletions.Conclusion：This work successfully investigated the bioligical and biomedicalapplications of beta-gal based blue/white screening assay. This method not onlyprovides a new technology for optimizing the highly effective and specific TALENs, butalso diagnose DNA with or without the deletion of EGFR exon19. Besides, this methoddoes not require the differentiation of normal and cancer DNA. It can be applied toDNAsamples isolated from tumor tissues, plasma DNA, or DNAfrom tissue fluids.