Direct genomic multiplex PCR for BRAC1 and application to mutation detection by single-strand conformation and heteroduplex analysis

Most mutation detection methods are based on analysis of PCR amplified segm ents and the application of multiplex PCR is one central approach to improv ing screening efficiency. Genes like the breast-ovarian cancer susceptibili ty gene BRCA1 pose a difficult challenge to efficient mutation screening be cause of large coding regions, numerous exons, and complex mutational spect ra. The application to BRCA1 of a general approach to effective multiplex: PCR is described here, Fifteen tripler PCRs and a single PCR reaction condi tion were used for amplification of all BRCA1 coding regions and the BRCA1- specific segments from the duplicated promoter region. SSCP/HDX gel analysi s of the multiplex products detected mobility distinctions for 34/34 sets o f allelic BRCA1 fragments. A novel polymorphism was found, CTTCT4CT10CT12>C T4CT11, a compound deletion in a region beginning at the +33 position of IV S7 and resulting in a net deletion of 15 bp. This change was shown to be on e of the common polymorphisms that define the two major haplotypes of the B RCA1-RNU2 region in a large proportion of the world population, A tripler P CR for SSCP detection of this deletion and two other distantly located comm on polymorphisms may be used to screen haplotype content and facilitate com parison of samples with similar haplotypes in subsequent mutation screening . The approach for robust multiplex amplification is generally applicable a nd allows rapid development of efficient testing for a wide variety of muta tions in any gene(s) encompassing a large coding region or numerous exons a nd including as many as 50 different genomic PCR products, Hum Mutat 16:334 -344, 2000, (C) 2000 Wiley-Liss, Inc.