Analysis of duplex DNA by triple helix formation: application to detection of a p53 microdeletion.
Conventional methods for mutation detection include Southern hybridization, direct sequencing of PCR products and single-strand conformation polymorphism analysis. We present an additional screening method that employs oligonucleotide-directed DNA triple helix formation to detect mutations within homopurine sequences. The proposed strategy is simple and may be of particular value when screening many DNA samples for changes involving particular homopurine sites. We have applied the method to the analysis of a clinically relevant 8-bp micro-deletion in the human p53 tumor suppressor gene. Affinities of oligonucleotide probes toward radiolabeled wild-type and mutant p53 DNA duplexes were quantitated by electrophoretic mobility shift assays. Recombinant plasmids carrying wild-type or microdeleted forms of the p53 homopurine sites of interest were created. Dimethyl sulfate footprinting was used to verify intended probe specificities. Duplex PCR products amplified from plasmid constructs were directly probed by incubation with labeled oligonucleotides. After electrophoresis and autoradiography, patterns of triple helix formation allowed discrimination between the mutant and wild-type p53 sequences. Direct DNA analysis by triple helix formation may simplify other procedures that normally require DNA denaturation and hybridization.
Olivas
W M
WM
Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha.
Maher
L J
LJ
D001483:Base Sequence; D004247:DNA / Q000032:analysis*; D017353:Gene Deletion; D016158:Genes, p53; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D015345:Oligonucleotide Probes
D015345:Oligonucleotide Probes; D004247:DNA