# US 2020/0399689 A1 · Methods of detecting nucleic acid sequences with high specificity > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2020/0399689 A1 - Document ID: US20200399689A1 - Status: Published application - Kind: A1 - Application number: 16/892,826 - Filed: 2020-06-04 - Published: 2020-12-24 - Earliest claimed: 2009-09-28 - Assignees: Advanced Cell Diagnostics, Inc. - Applicants: Advanced Cell Diagnostics, Inc. - Inventors: Yuling LUO; John James FLANAGAN; Nan SU; Huei-Yu Fay WANG - Web page: https://patentgrep.com/patent/US20200399689A1 ## CPC - C12Q 1/6841: In situ hybridisation - 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 The invention relates to methods of detecting nucleic acids, including methods of detecting one or more target nucleic acid sequences in multiplex branched-chain DNA assays, are provided. Nucleic acids captured on a solid support or suspending cells are detected, for example, through cooperative hybridization events that result in specific association of a label with the nucleic acids. The invention further relates to methods to improve probe hybridization specificity and their application in genotyping. The invention also relates to in situ detection of mis-joined nucleic acid sequences. The invention relates to reducing false positive signals and improve signal-to-background ratio in hybridization-based nucleic acid detection assay. The invention further relates to method to improve specificity in hybridization based nucleic acid using co-location probes. Compositions, tissue slides, sample of suspended cells, kits, and systems related to the methods are also described. ## Claims ### Claim at position 1 1-256. (canceled) ### Claim 257 A composition comprising: (a) a target nucleic acid; (b) at least one set of two or more capture probes hybridized to the target nucleic acid; (c) at least one set of two or more preamplifiers hybridized to the two or more capture probes; (d) at least one set of two or more linker capture probes hybridized to the two or more preamplifiers; (e) at least one amplifier hybridized to the two or more linker capture probes; and (f) at least one label molecule hybridized to a section of the at least one amplifier; wherein each capture probe comprises a T section complementary to a region of the target nucleic acid and an L section complementary to a region of one of the two or more preamplifiers, wherein each T section is complementary to a non-overlapping region of the target nucleic acid and each L section is complementary to a non-overlapping region of one of the two or more preamplifiers; and wherein the at least one set of two or more linker capture probes cannot bind stably to either the two or more preamplifiers or the at least one amplifier alone. ### Claim 258 (depends on 257) The composition of claim 257, wherein each of the two or more linker capture probes hybridizes to different preamplifiers. ### Claim 259 (depends on 258) The composition of claim 258, wherein each of the two or more linker capture probes comprises a region complementary to a region of one of the two or more preamplifiers. ### Claim 260 (depends on 257) The composition of claim 257, wherein both of the two or more linker capture probes hybridize to the same amplifier. ### Claim 261 (depends on 260) The composition of claim 260, wherein each of the two or more linker capture probes comprises a region complementary to non-overlapping regions of the same amplifier. ### Claim 262 (depends on 257) The composition of claim 257, further comprising at least a second set of two or more linker capture probes hybridized to the two or more preamplifiers. ### Claim 263 (depends on 262) The composition of claim 262, wherein each of the two or more linker capture probes in the second set hybridizes to different preamplifiers. ### Claim 264 (depends on 263) The composition of claim 263, wherein each of the two or more linker capture probes in the second set comprises a region complementary to a region of one of the two or more preamplifiers. ### Claim 265 (depends on 263) The composition of claim 263, wherein each of the two or more linker capture probes in the second set comprises a region complementary to non-overlapping regions of the same amplifier. ### Claim 266 (depends on 257) The composition of claim 257, wherein the at least one set of two or more linker capture probes is integrated into the at least one amplifier. ### Claim 267 (depends on 257) The composition of claim 257, wherein the T section of at least one of the two or more capture probes is 3′ of its L section. ### Claim 268 (depends on 257) The composition of claim 257, wherein the T section of at least one of the two or more capture probes is 5′ of its L section. ### Claim 269 (depends on 257) The composition of claim 257, wherein the target nucleic acid is RNA. ### Claim 270 (depends on 257) The composition of claim 257, further comprising a cell comprising or suspected of comprising the target nucleic acid. ### Claim 271 (depends on 257) The composition of claim 257, wherein the T sections of the two or more capture probes are at least 20 nucleotides in length. ### Claim 272 (depends on 257) The composition of claim 257, wherein the L sections of the two or more capture probes are at least 13 nucleotides in length. ### Claim 273 (depends on 257) The composition of claim 257, wherein each T section of the two or more capture probes comprises a nucleotide sequence having a melting temperature that is above the melting temperature of its corresponding L section. ### Claim 274 A kit comprising: (a) at least one set of two or more capture probes designed to hybridize to a target nucleic acid; (b) at least one set of two or more preamplifiers designed to hybridize to the two or more capture probes; (c) at least one set of two or more linker capture probes designed to hybridize to the two or more preamplifiers; (e) at least one amplifier designed to hybridize to the two or more linker capture probes; and (f) at least one label molecule designed to hybridize to a section of the at least one amplifier; wherein each capture probe comprises a T section complementary to a region of the target nucleic acid and an L section complementary to a region of one of the two or more preamplifiers, wherein each T section is complementary to a non-overlapping region of the target nucleic acid and each L section is complementary to a non-overlapping region of one of the two or more preamplifiers; and wherein the at least one set of two or more linker capture probes cannot bind stably to either the two or more preamplifiers or the at least one amplifier alone. ### Claim 275 A method of in situ detection of a target nucleic acid in a sample of cells, the method comprising: (a) contacting the sample, wherein the cells of the sample are fixed and permeabilized, with at least one set of two or more capture probes designed to hybridize to the target nucleic acid; (b) contacting the sample with at least one set of two or more preamplifiers designed to hybridize to the two or more capture probes; at least one set of two or more linker capture probes designed to hybridize to the two or more preamplifiers; at least one amplifier designed to hybridize to the two or more linker capture probes; and a plurality of label molecules designed to hybridize to a section of the at least one amplifier; wherein each capture probe comprises a T section complementary to a region of the target nucleic acid and an L section complementary to a region of one of the two or more preamplifiers, wherein each T section is complementary to a non-overlapping region of the target nucleic acid and each L section is complementary to a non-overlapping region of one of the two or more preamplifiers; and wherein the at least one set of two or more linker capture probes cannot bind stably to either the two or more preamplifiers or the at least one amplifier alone; (c) removing unbound probes, preamplifiers, amplifiers, and label molecules from the sample; and (d) detecting in situ signal generated from the plurality of label molecules, thereby detecting the target nucleic acid. ### Claim 276 (depends on 275) The method of claim 275, wherein step (a) is performed at a hybridization temperature that is higher than the melting temperature of each of the T sections of the two or more capture probes.