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Fluorescent chemical cleavage of mismatches for efficient screening of the factor VIII gene
Author(s) -
Freson Kathleen,
Peerlinck Kathelijne,
Aguirre Tania,
Arnout Jef,
Vermylen Jozef,
Cassiman JeanJacques,
Matthijs Gert
Publication year - 1998
Publication title -
human mutation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 162
eISSN - 1098-1004
pISSN - 1059-7794
DOI - 10.1002/(sici)1098-1004(1998)11:6<470::aid-humu8>3.0.co;2-a
Subject(s) - biology , exon , nonsense mutation , genetics , gene , microbiology and biotechnology , mutation , missense mutation , mutation rate , gene mutation , genomic dna
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 ˜ 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. Hum Mutat 11:470–479, 1998. © 1998 Wiley‐Liss, Inc.