Sl. Laprise et al., USE OF DENATURING GRADIENT GEL BLOTS TO SCREEN FOR POINT MUTATIONS INTHE FACTOR-VIII GENE, Human mutation, 12(6), 1998, pp. 393-402
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.