Prediction of DNA single-strand conformation polymorphism: analysis by capillary electrophoresis and computerized DNA modeling.
We have analyzed previously three representative p53 single-point mutations by capillary-electrophoresis single-strand conformation polymorphism (CE-SSCP). In the current study, we compared our CE-SSCP results with the potential secondary structures predicted by an RNA/DNA-folding algorithm with DNA energy rules, used in conjunction with a computer analysis workbench called STRUCTURELAB. Each of these mutations produces measurable shifts in CE migration times relative to wild type. Using computerized folding analysis, each of the mutations was found to have a conformational difference relative to wild type, which accounts for the observed differences in CE migration. Additional properties exhibited in the CE electropherograms were also explained using the computerized analysis. These include the appearance of secondary peaks and the temperature dependence of the electrophoretic patterns. The results yield insight into the mechanism of SSCP and how the conditions of this measurement, especially temperature, may be optimized to improve the sensitivity of the SSCP method. The results may also impact other diagnostic methods, which would benefit by a better understanding of DNA single-strand conformation polymorphisms to optimize conditions for enzymatic cleavage and DNA hybridization reactions.
Atha
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DH
Biotechnology Division, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA. donald.atha@nist.gov
Kasprzak
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W
O'Connell
C D
CD
Shapiro
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BA
D001483:Base Sequence; D019295:Computational Biology / Q000379:methods; D003205:Computing Methodologies; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D019075:Electrophoresis, Capillary; D005091:Exons / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D013696:Temperature; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D016159:Tumor Suppressor Protein p53; D004247:DNA