Diagnostic strategy for analytical scanning of BRCA1 gene by fluorescence-assisted mismatch analysis using large, bifluorescently labeled amplicons

The aim of this study was to develop a protocol for reliable, sensitive, an d cost-effective mutation scanning of the BRCA1 gene, based on a modificati on of fluorescence-assisted mismatch analysis. The main features of this me thod are: (Q) robust PCR amplification and strand-specific labeling of 25 l arge amplicons using uniform conditions and universal fluorescent primers; and (b) sensitive characterization of the position of sequence changes. The diagnostic accuracy of this method was tested by scanning the large exon 1 1 in 12 DNA samples with reported mutations. In a blind test, specific patt erns of fluorescence profiles were obtained, and all were attributed correc tly, without sequencing, to each mutation or polymorphism. Seven breast-ova rian cancer families,vith high probability of BRCA1-related predisposition were screened. Three truncating mutations (of which one was novel and three were missense changes, including two novel ones) were detected. The three missense mutations affect the highly conserved BRCT domain, Scanning by FAR A appears to be free of biases for particular types of sequence changes- ex cept for exon deletions/duplications, which cannot be detected by conventio nal PCR-based methods-and allows substantial savings in the number of seque ncing reactions and in the time invested in their interpretation. Therefore , it lends itself to screening structurally complex loci in the diagnostic context and in other fields of genetic analysis.