Tagging DNA mismatches by selective 2′-amine acylation

Abstract

Background: Widespread characterization of genetic variation and disease at the gene-sequence level has inaugurated a new era in human biology. Techniques for the molecular analysis of these variations and their linkage with measurable phenotypes will profoundly affect diverse fields of biological chemistry and biology. Results: A chemical tagging method has been developed to detect point mutations and other defects in nucleic acid sequences. The method employs oligodeoxynucleotide probes in which one 2′-ribose position (−H) is substituted with an amine (−NH 2) group. 2′-Amine-substituted nucleotides are specifically acylated by succinimidyl esters to form a 2′-amide product. The mutation detection method exploits our observation that 2′-amine groups at the site of a mismatch are acylated more rapidly than amine substitutions at base-paired nucleotides. 2′-Amine acylation is governed primarily by local, rather than global, differences in nucleotide dynamics, such that site-specific tagging of DNA mismatches does not require discriminatory hybridization conditions to be determined. Conclusions: 2′-Amine mismatch tagging offers an approach for chemically interrogating the base-paired state of individual nucleotides in a hybridized duplex and for quantifying nucleicacid hybridization with single-base specificity.