FLUORESCENT CHEMICAL CLEAVAGE OF MISMATCHES FOR EFFICIENT SCREENING OF THE FACTOR-VIII GENE

The detection of mutations in large and complex genes represents a pra ctical challenge in research and diagnostic laboratories. Available me thods are either time-consuming or lack sensitivity. Mutation detectio n in the factor VIII gene, responsible for haemophilia A, is hampered by its large size, its many exons, and the high frequency of de novo m utations that result in different mutations in unrelated patients. For an exhaustive analysis of mutations in the factor VIII gene, we estab lished a nonradioactive screening method based on chemical cleavage of mismatches (CCM). PCR-fragments of similar to 1 kb were generated fro m genomic DNA (exon 14) or after reverse transcription from mRNA isola ted from blood cells. Some modifications have been made to improve the CCM strategy. First, using a fluorescent tag, the method gains safety and flexibility. Second, fluorescent detection allows an accurate siz ing of digested fragments when measured on an automated DNA sequencer. Third, by labelling both 5' ends of the PCR-fragment, the detection r ate is virtually 100%. Finally, in the case of an X-linked disease, sa mples from two patients can be mixed, which reduces the workload witho ut losing information. In a pilot experiment, mutations were detected in 20 of 20 patients. In this series, three small insertions, two smal l deletions, one nonsense mutation, 13 missense mutations, and one spl ice mutation were found. Fifteen of these mutations are new. Thus virt ually all kind of mutations are detectable by this method, Moreover, t he analysis of the gene can be completed in 2 days. (C) 1998 Wiley-Lis s, Inc.