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Direct detection of mutations in the breast and ovarian cancer susceptibility gene BRCA1 by PCR-mediated site-directed mutagenesis

¹ Department of Pathology and Laboratory Medicine, CB7600, and
² Curriculum in Genetics, University of North Carolina, Chapel Hill, NC 27599.

³ Department of Medicine, Room 1009, 422 Curie Blvd., and
⁴ Department of Genetics, University of Pennsylvania, Philadelphia, PA 19104.
^a Author for correspondence. Fax 919-966-4526; e-mail lmsmol.dhl1{at}mail.unch.unc.edu

The tumor suppressor genes BRCA1 and BRCA2, which confer increased susceptibility to breast and (or) ovarian cancer, were recently identified. Mutation analysis of BRCA1 has demonstrated significant allelic heterogeneity; however, some distinct mutations have been detected in unrelated individuals. The most notable is the 185delAG mutation, which occurs at an estimated frequency of ~1% in individuals of Ashkenazi Jewish descent [1]. Although consensus has not been reached regarding clinical testing for mutations in BRCA1, a tiered strategy may be appropriate, in which direct testing for the more common mutations is one component. Specific alleles can be detected by using PCR-mediated site-directed mutagenesis (PSM), which alters the PCR products derived from either the wild-type or mutant allele to create or destroy a restriction endonuclease recognition site. Recognition sites are introduced by a base substitution in one of the primers. The alleles are then resolved by electrophoresis of the digested PCR products. We have applied this technique to the detection of four BRCA1 mutations: 185delAG, 5382insC, E1250X, and R1443X. Another mutation, 1294del40, can be resolved from the wild-type allele by high-resolution gel electrophoresis alone. The PSM technique is sensitive, does not require radioactivity, and is specific for individual mutations.

Key Words: indexing terms: polymerase chain reaction • ovarian cancer • genetics

The following articles in journals at HighWire Press have cited this article:

				P. C. R. Chan, B. Y.L. Wong, H. Ozcelik, and D. E.C. Cole Simple and Rapid Detection of BRCA1 and BRCA2 Mutations by Multiplex Mutagenically Separated PCR Clin. Chem., August 1, 1999; 45(8): 1285 - 1287. [Full Text] [PDF]

				E. M. Petty and A. A. Killeen BRCA1 Mutation Testing: Controversies and Challenges Clin. Chem., January 1, 1997; 43(1): 6 - 8. [Full Text] [PDF]