Tagging DNA mismatches by selective 2 '-amine acylation

Background: Widespread characterization of genetic variation and disease at the gene-sequence level has inaugurated a new era in human biology. Techni ques for the molecular analysis of these variations and their linkage with measurable phenotypes will profoundly affect diverse fields of biological c hemistry and biology. Results: A chemical tagging method has been developed to detect point mutat ions and other defects in nucleic acid sequences. The method employs oligod eoxynucleotide probes in which one 2'-ribose position (-H) is substituted w ith an amine (-NH2) group. 2'-Amine-substituted nucleotides are specificall y 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-pa ired nucleotides. 2'-Amine acylation is governed primarily by local, rather than global, differences in nucleotide dynamics, such that site-specific t agging of DNA mismatches does not require discriminatory hybridization cond itions to be determined. Conclusions: 2'-Amine mismatch tagging offers an approach for chemically in terrogating the base-paired state of individual nucleotides in a hybridized duplex and for quantifying nucleicacid hybridization with single-base spec ificity.