USE OF DENATURING GRADIENT GEL BLOTS TO SCREEN FOR POINT MUTATIONS INTHE FACTOR-VIII GENE

Denaturing gradient gel electrophoresis (DGGE) is commonly used to sea rch for point mutations in DNA fragments amplified in vitro by the pol ymerase chain reaction (PCR), For the complete detection of mutations in large genes with many exons, the DGGE-PCR approach, or any other PC R-based method, requires many primer sets and amplification reactions to scan the entire protein coding sequence. We previously demonstrated that DGGE analysis using DNA blots detects mutations in Drosophila ge nes and sequence polymorphisms in human genes without prior PCR amplif ication. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemoph ilia A families were analyzed for mutations in the factor VIII (FVIII) gene. Restriction enzyme digested DNA samples were subjected to DGGE and transferred to nylon blots. Hybridization of the DGG blots with FV III cDNA probes revealed mutant and polymorphic DNA sequence differenc es, Among 26 affected families that were not carriers of intron 22 inv ersion mutations, 18 family-specific DNA fragment polymorphisms and on e multiexon deletion were mapped. DNA sequencing of eight patient-spec ific polymorphic DNA fragments revealed six single base change mutatio ns, one 4 bp deletion, and one 13 bp duplication. Hum Mutat 12:393-402 , 1998, (C) 1998 Wiley Liss, Inc.