Detection of nonsense and frameshift mutations in BRCA1 gene using a new plasmid vector pPhoA-frame

Abstract

The technique for the detection of frame shift and nonsense mutations in BRCA1 gene was suggested. The technique presumes the construction of recombinant plasmids where the tested DNA fragment placed in-frame with alkaline phosphatase gene of Escherichia coli (phoA). A plasmid pPhoA-frame was constructed for such analysis, the plasmid contains DNA fragment coding for alkaline phosphatase of E. coli. Synthetic DNA fragment with BglII, StuI, ApaI and SacII sites was inserted into the DNA fragment coding for alkaline phosphatase of E. coli between Ala218 and Gly219 codons to facilitate the cloning of BRCA1 gene fragments. Occurrence of the frame shift or nonsense mutation in the tested DNA fragment can be detected after transformation of E. coli by the recombinant plasmid containing the tested fragment. E. coli colonies with the newly constructed recombinant plasmids are plated out on the indicator agar. In the case of frame shift or nonsense mutation the colonies are not colored, DNA fragments without such mutations result in the formation of the blue colonies.