Rapid Identification of Family-Specific Mutations in the Factor VIII Gene by One-Step DGGE.
A one-step denaturing gradient gel electrophoresis (DGGE) strategy for the rapid detection of mutations in the factor VIII gene of haemophilia A patients is described. All coding (except the middle part of exon 14) and flanking intronic regions of the gene corresponding to approximately 6.6 kb were amplified in 27 fragments using four PCR programs. Heteroduplex formation was performed for each fragment. A common denaturant gradient gel (35-65%) was chosen that allowed the simultaneous analysis of all PCR amplified regions on a single gel and run for 3.5 h at 160 V. This method was implemented for a patient whose family was seeking carrier determinations. An abnormal pattern was detected in exon 23 and the family-specific mutation was found by subsequent DNA sequencing. One-step DGGE is a promising rapid method for the carrier detection and prenatal diagnosis in haemophilia A families when immediate results are required and when polymorphic markers fail to give information.
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