Chemical cleavage of DNA duplexes with single base mismatches as a basis for detection of random point mutations |
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Authors: | A. A. Neschastnova M. G. Yakubovskaya V. K. Gasanova G. A. Belitsky N. G. Dolinnaya |
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Affiliation: | (1) Institute of Carcinogenesis, Blokhin Cancer Research Center, Russian Academy of Medical Sciences, Moscow, 115478, Russia;(2) Department of Chemistry, Moscow State University, Moscow, 119992, Russia |
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Abstract: | The most promising approaches to detection of random point mutations are based on chemical cleavage of mismatches and other noncomplementarities. To demonstrate the specificity of this method, a model system was obtained for the first time as sets of 50-mer imperfect DNA duplexes containg all variants of mismatched and unpaired internal residues located in an invariant context and flanked by either A · T or G · C base pairs. Chemical cleavage of DNA duplexes immobilized on magnetic beads via the biotin-streptavidin interaction was accomplished using potassium permanganate or hydroxylamine, which are sensitive to the secondary DNA structure and react with thymine and cytosine, respectively. The reactivity of different mismatches was connected with the local duplex structure and depended on their type, orientation, and flanking nucleotides. The use of potassium permanganate and hydroxylamine to modify a heteroduplex mixture makes it possible to unambiguously detect a mismatch and, based on the type of reagent and the size of the cleavage products, to suppose the type and position of the mismatch and the flanking nucleotides. The model system can be used to evaluate the sensitivity of a chemical cleavage method and to control false-positive and false-negative results when different protocols are applied to the detection of DNA point mutations. |
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Keywords: | DNA structure mismatches heteroduplexes heterocyclic bases chemical modification mutation detection |
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