Analysis of duplex DNA by triple helix formation

Application to detection of a p53 microdeletion

W. M. Olivas, L James Maher III

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

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 microdeletion 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.

Original languageEnglish (US)
Pages (from-to)128-132
Number of pages5
JournalBioTechniques
Volume16
Issue number1
StatePublished - 1994
Externally publishedYes

Fingerprint

DNA
Oligonucleotides
Plasmids
Nucleic Acid Denaturation
Screening
Polymerase Chain Reaction
Mutation
Oligonucleotide Probes
Electrophoretic Mobility Shift Assay
Tumor Suppressor Genes
Autoradiography
Electrophoretic mobility
Denaturation
Electrophoresis
Polymorphism
Conformations
Tumors
Assays
Genes
dimethyl sulfate

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Clinical Biochemistry

Cite this

Analysis of duplex DNA by triple helix formation : Application to detection of a p53 microdeletion. / Olivas, W. M.; Maher III, L James.

In: BioTechniques, Vol. 16, No. 1, 1994, p. 128-132.

Research output: Contribution to journalArticle

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