SEMI-DIGITAL LIGATION ASSAY

摘要

Assays for detecting mutant sequences at particular locations, especially against a background of non-mutant sequences, employ thermocycling ligase reactions. Differentially labeled or sized probes can be used to distinguish wild-type and mutant sequences. Physico-chemical properties of the probes can be critical to successful detection. Mutation detection can be used for diagnosis, monitoring, or prognosticating diseases such as cancers.

主权项

1. A method for detecting mutations at a selected location in a nucleotide sequence, comprising the steps of:
contacting to form a reaction mixture:(a) a test sample comprising 200 or fewer molecules of analyte nucleic acid;(b) a probe complementary to a wild-type sequence at the selected location and adjacent to and proximal to the selected location;(c) a probe complementary to a mutant sequence at the selected location and adjacent to and proximal to the selected location;(d) an anchoring oligonucleotide which is complementary to e analyte nucleic acid adjacent to and distal to the selected location; and(e) thermotolerant DNA ligase;wherein the probes complementary to the wild-type and mutant sequences are labeled with distinct fluorescent moieties, or wherein the probes complementary to the wild-type and mutant sequences are of distinct lengths, or wherein the probes complementary to the wild-type and mutant sequences have distinct fluorescent moieties and distinct lengths;
thermocycling the reaction mixture such that anchoring oligonucleotides are ligated to an appropriate probe reflecting hybridization of the appropriate probe to the analyte nucleic acid, thereby forming ligation products; separating the ligation products on a gel, or detecting the distinct fluorescent moieties, or separating the ligation products on a gel and detecting the distinct fluorescent moieties on the separated ligation products on the gel.