Direct genomic multiplex PCR for BRCA1 and application to mutation detection by single-strand conformation and heteroduplex analysis.

Most mutation detection methods are based on analysis of PCR amplified segments and the application of multiplex PCR is one central approach to improving screening efficiency. Genes like the breast-ovarian cancer susceptibility gene BRCA1 pose a difficult challenge to efficient mutation screening because of large coding regions, numerous exons, and complex mutational spectra. The application to BRCA1 of a general approach to effective multiplex PCR is described here. Fifteen triplex PCRs and a single PCR reaction condition were used for amplification of all BRCA1 coding regions and the BRCA1-specific segments from the duplicated promoter region. SSCP/HDX gel analysis of the multiplex products detected mobility distinctions for 34/34 sets of allelic BRCA1 fragments. A novel polymorphism was found, CTTCT(4)CT(10)CT(12) >CT(4)CT(11), a compound deletion in a region beginning at the +33 position of IVS7 and resulting in a net deletion of 15 bp. This change was shown to be one of the common polymorphisms that define the two major haplotypes of the BRCA1-RNU2 region in a large proportion of the world population. A triplex PCR for SSCP detection of this deletion and two other distantly located common polymorphisms may be used to screen haplotype content and facilitate comparison of samples with similar haplotypes in subsequent mutation screening. The approach for robust multiplex amplification is generally applicable and allows rapid development of efficient testing for a wide variety of mutations in any gene(s) encompassing a large coding region or numerous exons and including as many as 50 different genomic PCR products.