Single-strand conformation polymorphism analysis by capillary and microchip electrophoresis: A fast, simple method for detection of common mutations in BRCA1 and BRCA2

As a result of intensive studies on hereditary breast and ovarian cancers, two breast cancer susceptibility genes, BRCA1 and BRCA2, have been identifi ed. In each gene, a small number of specific mutations have been found at r elatively high frequency in certain ethnic populations, The mutations, 185d elAG and 5382insC in BRCA1 and 6174delT in BRCA2, have been identified as c ommon mutations in the Ashkenazi Jewish population, with a combined frequen cy of 2.0 to 2.5%, Women who have one of the above three common mutations a re at a high risk of developing breast or ovarian cancer. Consequently, acc urate and cost-effective detection of these three mutations may have import ant implications for risk assessment in susceptible women and men. In this report, we describe a fast and simple capillary electrophoresis (CE)-based method using a polymer network for screening the three common mutations in BRCA1 and BRCA2, Fluorescent dye-labeled primers (B-FAM-tagged) were used t o amplify three DNA fragments of 258, 296, and 201 bp for detection of the 185delAG, 5382insC, and 6174delT mutations, respectively. After the PCR pro ducts were denatured, a single-strand conformation polymorphism (SSCP) prof ile could be obtained for each mutation in less than 10 min by CE in a poly mer network. We demonstrate the potential provided by translating this assa y to the microchip format where the SSCP analysis is complete in 120 s, rep resenting only a fraction of the reduction ill analysis time that can be ac hieved with microchip technology. The speed and simplicity of the SSCP meth odology for detection of these mutations make it attractive for use in the clinical diagnostic laboratory. (C) 2000 Academic Press.