# US 2014/0155275 A1 · Semi-digital ligation assay > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0155275 A1 - Document ID: US20140155275A1 - Status: Published application - Kind: A1 - Application number: 14/130,776 - Filed: 2012-07-06 - Published: 2014-06-05 - Earliest claimed: 2011-07-06 - Assignees: The Johns Hopkins University - Inventors: Bert Vogelstein; Kenneth W. Kinzler - Web page: https://patentgrep.com/patent/US20140155275A1 ## CPC - C12Q 1/6827: for detection of mutation or polymorphism - C12Q 1/6883: for diseases caused by alterations of genetic material - C12Q 1/6886: for cancer (immunoassay for cancer G01N33/575) - C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES ## 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. ## Claims ### Claim 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. ### Claim 2 (depends on 1) The method of claim 1 the test sample is an amplification product. ### Claim 3 (depends on 1) The method of claim 1 further comprising the step of: asymmetrically amplifying an analyte nucleic acid with a first and second primer, wherein the first primer is in excess of a second primer, to form the test sample. ### Claim 4 (depends on 1) The method of claim 1 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 oligocale algorithm. ### Claim 5 (depends on 1) The method of claim 1 wherein the probe complementary to the mutant sequence comprises one or more locked nucleic acid nucleotides. ### Claim 6 (depends on 1) The method of claim 1 wherein the probe complementary to the mutant sequence comprises three locked nucleic acid nucleotides. ### Claim 7 (depends on 1) The method of claim 1 wherein the probe complementary to the mutant sequence comprises three locked nucleic acid nucleotides at positions -2,-3, and -7, wherein position 0 is the selected location. ### Claim 8 (depends on 1) The method of claim 1 wherein the probes complementary to the wild-type and mutant sequences are labeled with distinct fluorescent moieties. ### Claim 9 (depends on 1) The method of claim 1 wherein the probes complementary to the wild-type and mutant sequences are of distinct lengths. ### Claim 10 (depends on 1) The method of claim 1 wherein the probes complementary to the wild-type and mutant sequences have distinct fluorescent moieties and distinct lengths. ### Claim 11 (depends on 8) The method of claim 8 wherein the mutation is detected if the fluorescent moiety with which the probe complementary to the mutant sequence is labeled is detected. ### Claim 12 (depends on 10) The method of claim 10 Wherein the mutation is detected if the fluorescent moiety with which the probe complementary to the mutant sequence is labeled is detected. ### Claim 13 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. ### Claim 14 (depends on 13) The method of claim 13 wherein the probes complementary the wild-type and mutant sequences are labeled with distinct fluorescent moieties. ### Claim 15 (depends on 13) The method of claim 13 wherein the probes complementary to the wild-type and mutant sequences are of distinct lengths. ### Claim 16 (depends on 13) The method of claim 13 wherein the probes complementary to the wild-type and mutant sequences have distinct fluorescent moieties and distinct lengths. ### Claim 17 (depends on 14) The method of claim 14 wherein the mutation is detected if the fluorescent moiety with which the probe complementary to the mutant sequence is labeled is detected. ### Claim 18 (depends on 16) The method of claim 16 wherein the mutation is detected if the fluorescent moiety with which the probe complementary to the mutant sequence is labeled is detected.