Patent ID: 6025139
Filing Date: 2000-02-15
Classification: C12Q

Abstract:
A method for detection of mutations within a target gene or gene fragment in a sample, said target gene or gene fragment having a known wild-type sequence, comprising the steps of:(a) combining the sample with at least three species of oligonucleotide probes, a labeled 3'-probe, a labeled 5'-probe and at least one intermediate probe, under hybridization conditions, said species of oligonucleotide probes having sequences which are perfectly complementary to and hybridize with sequential and contiguous portions of the wild-type sequence, to preferentially form duplex structures between the target gene or gene fragment and those species of oligonucleotide probes which are perfectly complementary to the target gene or gene fragment, said at least three oligonucleotide probes including probes which are different in sequence from one another;(b) ligating the hybridized oligonucleotide probes to join those oligonucleotide probes which hybridize to the gene fragment in ligatable proximity to one another to form one or more species of ligation products; and(c) evaluating the species of ligation products by separating the ligation products into subgroups based upon the size of the ligation product and detecting the labels associated with the labeled 3'-probe and the labeled 5'-probe, wherein the ligation reaction is conducted under conditions such that variations between the wild-type sequence and the target sequence which are intermediate in location between the 5' and 3'-end bases of the probes result in a different product mixture than those obtained in the absence of such variations, and wherein a difference in length or quantity between the ligation products formed and standard values obtained using a wild-type target is indicative of the presence of a mutation in the gene or gene fragment in the sample.