Rapid photometric detection of thymine residues partially flipped out of double helix as a method for direct scanning of point mutations and apurinic DNA sites
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A spectroscopic assay for detection of extrahelical thymine residues in DNA heteroduplexes under their modification by potassium permanganate has been developed. The assay is based on increase in absorbance at 420 nm due to accumulation of thymidine oxidation intermediates and soluble manganese dioxide. The analysis was carried out using a set of 19-bp DNA duplexes containing unpaired thymidines opposite tetrahydrofuranyl derivatives mimicking a widespread DNA damage (apurinic (AP) sites) and a library of 50-bp DNA duplexes containing all types of base mismatches in different surroundings. The relation between the selectivity of unpaired T oxidation and the thermal stability of DNA double helix was investigated. The method described here was shown to discriminate between DNA duplexes with one or two AP sites and to reveal thymine-containing mismatches and all noncanonical base pairs in AT-surroundings. Comparative results of CCM analysis and the rapid photometric assay for mismatch detection are demonstrated for the first time in the same model system. The chemical reactivity of target thymines was shown to correlate with local disturbance of double helix at the mismatch site. As the spectroscopic assay does not require the DNA cleavage reaction and gel electrophoresis, it can be easily automated and used for primary screening of somatic mutations.
Key wordsDNA structure noncanonical pairs heteroduplexes chemical modification of heterocyclic bases detection of mutations apurinic sites
Chemical Cleavage of Mismatches
melting temperature; prefix “d” in designation of sequences of oligodeoxyribonucleotides is omitted
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