Use of denaturing gradient gel blots to screen for point mutations in the factor VIII gene

Abstract

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. Hum Mutat 12:393–402, 1998. © 1998 Wiley‐Liss, Inc.

Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A

“…On the other hand, there are many short homopolymeric tracts of As and Ts in exon 14, as well as highly recombinogenic palindromic sequences in 200-400 bp distance of one another, which are molecular features very likely to promote slipped mispairing and intragenic recombinations. The last could be true for the observed duplication of 56 bp in exon 21, there is only one similar duplication of 13 bp found in exon 14 of the gene [Laprise et al, 1998]. …”

Covalent genomic DNA modification patterns revealed by denaturing gradient gel blots

“…DGG blots and agarose Southern blots were prepared as previously described (Laprise et al, 1998). Southern blots were also made from 4.5% non-denaturing polyacrylamide gels in TBE buffer (0.089 M Tris-borate, 0.089 M boric acid, 2 mM EDTA, pH 8.0) by electrotransfer to nylon membrane as previously described for DGG blots (Laprise et al, 1998).…”

“…Southern blots were also made from 4.5% non-denaturing polyacrylamide gels in TBE buffer (0.089 M Tris-borate, 0.089 M boric acid, 2 mM EDTA, pH 8.0) by electrotransfer to nylon membrane as previously described for DGG blots (Laprise et al, 1998). Blots were hybridized as described previously (Laprise et al, 1998;Reindollar et al, 2000) with several genomic and cDNA clones: plasmid p625.8, containing sequences 30 kb downstream from the human F8 gene Wood et al, 1984); mouse F8 cDNA (Elder et al, 1993); human ADRB2 cDNA (Kobilka et al, 1987); human GCCR genomic fragments (Reindollar et al, 2000); human MYB genomic and cDNA fragments (Franchini et al, 1983); human COL2AI genomic fragments (Cheah et al, 1985); human TP53 cDNA (Nogueira, 1997); human AMH cDNA (Cate et al, 1986); and mouse Th genomic fragments (Iwata et al, 1992).…”

“…All differences seen on DGGE, whether shifts of sharply focused bands or qualitative transitions to diffuse bands, can be attributed to either sequence changes or DNA modification differences (Fischer and Lerman, 1983;Gray et al, 1991;Abrams and V.P. Stanton, 1992;Gray, 1992;Laprise et al, 1998).…”

“…Denaturing gradient gel blots (DGG blots) were used in previous experiments to identify single base change mutations and polymorphisms in genomic fragments from several genes as sequence restriction fragment melting polymorphisms (RMFPs) (Gray et al, 1991;Gray, 1992;Laprise et al, 1998). In an earlier study, genomic DNA fragments from paired human sperm and leukocyte samples were compared using DGG blots (Reindollar et al, 2000).…”