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Vol 46(2012) N 5 p. 658-663;
N.I. Gutkina1, V.V. Bogachev2, S.P. Kovalenko1,3*

Detection of Nonsense and Frameshift Mutations in BRCA1 Gene Using a New Plasmid Vector pPhoA-Frame

1Institute of Molecular Biology and Biophysics, Siberian Division, Russian Academy of Sciences, Novosibirsk, 630117 Russia
2State Research Center of Virology and Biotechnology "Vector", Koltsovo, 630559 Russia
3Novosibirsk State University, Faculty of Medicine, Novosibirsk, 630090 Russia

*sp_kovalenko@yahoo.com
Received - 2012-01-06; Accepted - 2012-02-15

The technique for detecting frameshift and nonsense mutations in the human BRCA1 gene has been suggested. The technique presumes the construction of recombinant plasmids where the tested DNA fragment is placed in frame with alkaline phosphatase gene of Escherichia coli (phoA). A special plasmid pPhoA-frame was constructed for this analysis, and the plasmid contained the DNA fragment that encodes alkaline phosphatase of E. coli. The synthetic DNA fragment with BglII, StuI, ApaI and SacII sites was inserted into the DNA fragment that encodes alkaline phosphatase of E. coli between Ala218 and Gly219 codons to facilitate the cloning of BRCA1 gene fragments. The occurrence of the frameshift or nonsense mutation in the tested DNA fragment can be detected after the transformation of E. coli by the recombinant plasmid that contains the tested fragment. E. coli colonies with newly constructed recombinant plasmids are plated out on the indicator agar. In the case of the frameshift or nonsense mutation, the colonies are not colored, and DNA fragments without these mutations result in the formation of blue colonies.

alkaline phosphatase, frameshift mutations, nonsense mutations, recombinant plasmids



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