Back to results
US 2014/0155275 A1Published application
Semi-digital ligation assay
AssigneeThe Johns Hopkins University
InventorsBert Vogelstein, Kenneth W. Kinzler
Life of this patent
Estimating the term…
Abstract
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.
Claims18
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.
7 more elements
A method for detecting mutations at a selected location in a nucleotide sequence, comprising the steps of:
- asymmetrically amplifying an analyte nucleic acid with a first and second prix wherein the first primer is in excess of a second primer, to form a test sample;
- contacting to form a reaction mixture:
- (a) 200 or fewer molecules of analyte nucleic acid of the test sample;
- (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 the analyte nucleic acid adjacent to and distal to the selected location;
- and
- (e) thermotolerant DNA ligase;
- wherein the probe complementary to the mutant sequence has a Tm of 32 to 36 deg C., the probe complementary to the wild-type sequence has a Tm of 32 to 38 deg C., and the anchoring oligonucleotide has a Tm of 36 to 44 deg C. as assessed by oligocalc algorithm, wherein the probe complementary to the mutant sequence comprises one or more locked nucleic acid nucleotides, wherein the wild-type and mutant probes are labeled with distinct fluorescent moieties, or wherein the wild-type and mutant probes are of distinct lengths, or wherein the wild-type and mutant probes have distinct fluorescent moieties and distinct lengths;
- thrmocycling 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.
8 more elements
Dependencies are inferred from the claim text.