title: Convenient, nonradioactive, heteroduplex-based methods for identifying recurrent mutations in the BRCA1 and BRCA2 genes.
abstract: The ability to identify individuals who are predisposed to specific malignant tumors is a promising molecular diagnostic by-product of over two decades of intensive research into the genetic pathogenesis of human cancer. Approximately 2% of Ashkenazi Jews carry recurrent germline mutations in either the BRCA1 or BRCA2 genes that may predispose these individuals to the development of breast and ovarian cancer. We have developed a nonisotopic method, based on the formation of heteroduplexes between polymerase chain reaction (PCR) amplified wild-type and mutant alleles, which can be used to identify the BRCA1 185delAG and the BRCA2 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 amplification, heteroduplex formation, acrylamide gel electrophoresis, and ethidium bromide staining/UV-fluorescence photography) can be readily and reproducibly performed in the course of a single day, making this a useful method for the routine identification of these mutations.
authors.affiliation: Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, New York 10032, USA.
authors.forename: M M
authors.initials: MM
authors.lastname: Mansukhani
authors.forename: K L
authors.initials: KL
authors.lastname: Nastiuk
authors.forename: H
authors.initials: H
authors.lastname: Hibshoosh
authors.forename: P
authors.initials: P
authors.lastname: Kularatne
authors.forename: D
authors.initials: D
authors.lastname: Russo
authors.forename: J J
authors.initials: JJ
authors.lastname: Krolewski
mesh_terms: D000483:Alleles; D024682:BRCA2 Protein; D001943:Breast Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D004273:DNA, Neoplasm / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005784:Gene Amplification; D019398:Genes, BRCA1 / Q000235:genetics*; D006801:Humans; D019656:Loss of Heterozygosity; D008856:Microscopy, Fluorescence; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics*; D010051:Ovarian Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D024682:BRCA2 Protein; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D014157:Transcription Factors