A rapid fluorescent multiplexed-PCR analysis (FMPA) for founder mutations in the BRCA1 and BRCA2 genes

Mutations of the BRCA1 and BRCA2 genes account for approximately 80% of her editary breast/ovarian cancer families, bur the size of these two genes mak es mutation analysis time-consuming and technically challenging. In some po pulations such as the Ashkenazi Jewish and the French-Canadian, a small num ber of recurrent founder mutations account for the majority of mutations in cancer families. We have therefore developed two rapid genetic screening t ests, which allow us to detect three frequent frameshift mutations in the A shkenazi Jewish population and five frameshift mutations in the French-Cana dian population. These fluorescent non-radioactive methods permit the simul taneous detection of multiple mutations by generating multiplexed PCR-ampli fied gene fragments, and by discriminating these on the basis of their size in a denaturing polyacrylamide gel. Using these methods, we were able to c orrectly identify all mutants in a blinded analysis of 276 DNA samples, inc luding 30 derived from paraffin-embedded tumor samples and 10 from buccal-c ell brushes, with no false positive or false negative results. These techniques designed for the direct detection of recurrent mutations i n the BRCA1 and BRCA2 genes, have the advantages of being efficient, sensit ive, cost-effective, and are applicable to large scale screening for epidem iologic studies.