DETERMINING MUTATIONS BY SELECTIVE REACTION OF THE 2'-RIBOSE POSITION IN HYBRIDIZED OLIGONUCLEOTIDES

摘要

New chemical tagging methods and kits of the present invention utilize the discovery that chemical modification (e.g., acylation) of 2'-substituted ribonucleotides or deoxyribonucleotides by reactive compounds (e.g., activated esters) is sensitive to the base-paired state of the nucleotide. Perfectly base-paired positions are generally unreactive, while mismatched or unmatched bases are reactive under a wide variety of reaction conditions. The methods of the invention include a method of detecting a mutation in a nucleic acid molecule suspected of containing a mutation. The nucleic acid is hybridized to an oligonucleotide having a sequence complementary to the sequence the nucleic acid would have if a mutation were not present. The oligonucleotide comprises at least one nucleotide with a substitution at the 2'-ribose position. After the oligonucleotide is hybridized to the nucleic acid molecule, the hybridized oligonucleotide is then contacted with a reactive compound comprising a reporter moiety. Detection of the binding of the reporter moiety to the hybridized oligonucleotide indicates a mismatch i.e., a mutation) in the nucleic acid molecule. The methods and kits of the present invention are useful in detecting point mutations and other defects in nucleic acid sequences. These methods and kits are also useful for detecting single nucleotide polymorphisms (SNPs) and mutations responsible for cancer and other genetic diseases in humans; for quantifying amounts of nucleic acids; and for detecting conformational changes in nucleic acid structures, including those found in the products of in vitro selection experiments i.e., aptamers).