FIELD: chemistry.
SUBSTANCE: present invention relates to molecular biology. Disclosed is a method of detecting frameshift and nonsense mutations in the BRCA1 gene, which involves construction of recombinant plasmids where the amplified gene fragment is located in a single translation frame with the alkaline phosphatase gene of E.coli (phoA). A plasmid vector pPhoA-frame, which contains a DNA sequence which encodes alkaline phosphatase of E.coli was constructed. A DNA fragment containing restriction endonuclease recognition sites BgIII, StuI, Apal, SacII and intended for cloning BRCA1 gene fragments (polylinker) was inserted into said DNA sequence. The amplified BRCA1 gene fragment is inserted into the plasmid vector pPhoA-frame in a single translation frame with phoA. The occurrence of mutations which violate reading frame integrity in the investigated gene fragment is evaluated visually from the absence of colour of E.coli. cell colonies transformed by the obtained recombinant plasmid on an indicator dish containing a substrate for alkaline phosphatase. The disclosed method enables to detect only mutations that are significant for development of pathology since it avoids detection of polymorphous versions which do not lead to stop codons and most cases have not significant effect on protein function. The method enables to detect any, including unknown, mutations which violate frame integrity.
EFFECT: method can be used to detect frameshift and nonsense mutations that are responsible for the development of a range of cancerous diseases in human genes.
2 cl, 3 dwg, 2 ex
