Use of denaturing gradient gel blots to screen for point mutations in the factor VIII gene.
Denaturing gradient gel electrophoresis (DGGE) is commonly used to search for point mutations in DNA fragments amplified in vitro by the polymerase chain reaction (PCR). For the complete detection of mutations in large genes with many exons, the DGGE-PCR approach, or any other PCR-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 genes and sequence polymorphisms in human genes without prior PCR amplification. To determine if human point mutations could be detected using denaturing gradient gels (DGG blots), genomic DNA samples from hemophilia 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 FVIII cDNA probes revealed mutant and polymorphic DNA sequence differences. Among 26 affected families that were not carriers of intron 22 inversion mutations, 18 family-specific DNA fragment polymorphisms and one multiexon deletion were mapped. DNA sequencing of eight patient-specific polymorphic DNA fragments revealed six single base change mutations, one 4 bp deletion, and one 13 bp duplication.
Laprise
S L
SL
Department of Anatomy and Cellular Biology, Tufts University School of Medicine, Boston, MA, USA.
Mak
E K
EK
Killoran
K A
KA
Layman
L C
LC
Gray
M R
MR
D015139:Blotting, Southern; D004247:DNA / Q000032:analysis; D004252:DNA Mutational Analysis / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005091:Exons / Q000235:genetics; D005169:Factor VIII / Q000235:genetics*; D005260:Female; D020440:Gene Duplication; D006467:Hemophilia A / Q000235:genetics; D006801:Humans; D008297:Male; D016254:Mutagenesis, Insertional; D009691:Nucleic Acid Denaturation; D010375:Pedigree; D017354:Point Mutation / Q000235:genetics*; D011110:Polymorphism, Genetic / Q000235:genetics; D017384:Sequence Deletion
D005169:Factor VIII; D004247:DNA