Fluorescent chemical cleavage of mismatches for efficient screening of the factor VIII gene.
The detection of mutations in large and complex genes represents a practical challenge in research and diagnostic laboratories. Available methods are either time-consuming or lack sensitivity. Mutation detection 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 mutations that result in different mutations in unrelated patients. For an exhaustive analysis of mutations in the factor VIII gene, we established a nonradioactive screening method based on chemical cleavage of mismatches (CCM). PCR-fragments of approximately 1 kb were generated from genomic DNA (exon 14) or after reverse transcription from mRNA isolated 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 sizing of digested fragments when measured on an automated DNA sequencer. Third, by labelling both 5' ends of the PCR-fragment, the detection rate is virtually 100%. Finally, in the case of an X-linked disease, samples from two patients can be mixed, which reduces the workload without losing information. In a pilot experiment, mutations were detected in 20 of 20 patients. In this series, three small insertions, two small deletions, one nonsense mutation, 13 missense mutations, and one splice mutation were found. Fifteen of these mutations are new. Thus virtually all kind of mutations are detectable by this method. Moreover, the analysis of the gene can be completed in 2 days.
Freson
K
K
Center for Human Genetics, University of Leuven, Belgium.
Peerlinck
K
K
Aguirre
T
T
Arnout
J
J
Vermylen
J
J
Cassiman
J J
JJ
Matthijs
G
G
D004247:DNA / Q000235:genetics; D005169:Factor VIII / Q000235:genetics*; D005456:Fluorescent Dyes; D006467:Hemophilia A / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009692:Nucleic Acid Heteroduplexes; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics
D005456:Fluorescent Dyes; D009692:Nucleic Acid Heteroduplexes; D012313:RNA; D005169:Factor VIII; D004247:DNA