Detection of germline BRCA1 mutations by multiple-dye cleavase fragment length polymorphism (MD-CFLP) method

We describe the Multiple-Dye Cleavase Fragment Length Polymorphism (MD-CFLP ) method set up for a sensitive and preliminary rapid screening of BRCA1 mu tations. We analysed exons 11 and 16, which are known to cover slightly mor e than 70% of the whole coding region of the gene, subdivided into 4 amplic ons and labelled with different fluorescent dUTPs. MD-CFLP was first utilis ed on a panel of 30 DNA samples in which the presence of single-base substi tutions or small deletions/insertions had been previously identified by dir ect sequencing as gold standard, in order to define the optimal conditions in terms of PCR amplification and temperature of digestion. In a second ste p, we blindly analysed 21 DNA samples by MD-CFLP to verify its reliability. The sensitivity and specificity of MD-CFLP were both 100% in the first stu dy, and 80% and 94%, respectively, in the blind sample assay. Our results d emonstrate the capability of the MD-CFLP method to detect DNA sequence alte rations in fragments of more than 1 kb. We conclude that CFLP is a powerful tool in mutational analysis, offering reliable results in a shorter time a nd at a lower cost than conventional methods, and its potential can be enha nced when internal fluorescent labelling and laser detection are used. (C) 2001 Cancer Research Campaign.