CONVENIENT, NONRADIOACTIVE, HETERODUPLEX-BASED METHODS FOR IDENTIFYING RECURRENT MUTATIONS IN THE BRCA1 AND BRCA2 GENES

The ability to identify inviduals who are predisposed to specific mali gnant tumors is a promising molecular diagnostic byproduct of over two decades of intensive research into the genetic pathogenesis of human cancer. Approximately 2% of Ashkenazi Jews carry recurrent germline mu tations in either the BRCA1 or BRCA2 genes that may predispose these i ndividuals to the development of breast and ovarian cancer. We have de veloped a nonisotopic method, based on the formation of heteroduplexes between polymerase chain reaction (PCR) amplified wild-type and mutan t alleles, which can be used to identify the BRCA1 185delAG and the BR CA2 6174delT mutations. The same assay can also be used to verify the loss of heterozygosity in a tumor sample arising in an individual with a germline mutation. The four steps described in this report (PCR amp lification, heteroduplex formation, acrylamide gel electrophoresis, an d ethidium bromide staining/UV-fluorescence photography) can be readil y and reproducibly performed in the course of a single day, making thi s a useful method for the routine identification of these mutations.