Composition for detecting nucleic acid mutations, comprises two groups of nucleic acid attached to flanking sequences that can form distinguishable hetero- and homo-hybrids

摘要

Composition (A) comprising two groups of nucleic acids (NA1, NA2) each comprising linkers (L) that flank sequences characteristic of the groups and are identical for many molecules of a given group. The linker for NA1 is selected so that hybrids are formed between NA1 and NA2, that optionally after enzymatic treatment, represent a substrate for a double-strand-specific exonuclease, is new. Composition (A) comprising two groups of nucleic acids (NA1, NA2) each comprising linkers (L) that flank sequences characteristic of the groups and are identical for many molecules of a given group. The linker for NA1 is selected so that hybrids (heterohybrids; H1) are formed between NA1 and NA2, that optionally after enzymatic treatment, represent a substrate for a double-strand (ds)-specific exonuclease (exo), so that only NA1 of H1 is exonucleolytically shortened, is new. Homohybrids (H2) formed between two nucleic acids of the same group are not digested by exo. Independent claims are also included for the following: (1) analyzing sequence differences between molecules of NA1 and NA2, comprising: (a) forming H1; (b) optionally removing H2; (c) exonucleolytic digestion of NA1 in H1, using Exo; and (d) differentiation between perfectly matched H1 and those where one or more mismatches are present at the 3'-end of NA1; (2) similar composition in which NA2 contains degradation-resistant nucleotides (drnt); (3) similar composition in which NA1, of length n, contains a small proportion of drnt at random positions so that exonucleolytic digestion stops at drnt, resulting in a mixture, in equal proportions, of digestion products of all sizes between 1 and n-1 nucleotides; (4) method similar to (1) but using compositions of (2) or (3); (5) composition (B) comprising a mixture of more than two different NA from each of NA1 and NA2, containing H1, where NA1 is shortened, at either end, so that for each NA1 differently shortened variants exist; and (6) method similar to (1) but using (B).