# US 10,119,165 B2 · Nucleic acid constructs and methods of use > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,119,165 B2 - Document ID: US10119165B2 - Status: Granted - Kind: B2 - Application number: 15/349,927 - Filed: 2016-11-11 - Published: 2017-03-02 - Granted: 2018-11-06 - Earliest claimed: 2009-04-30 - Assignees: Prognosys Biosciences, Inc. - Applicants: Prognosys Biosciences, Inc. - Inventors: Mark S. Chee - Web page: https://patentgrep.com/patent/US10119165B2 ## CPC - C12Q 1/6869: Methods for sequencing - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - C12Q 1/6809: Methods for determination or identification of nucleic acids involving differential detection - C12Q 1/6827: for detection of mutation or polymorphism - C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - C12Q 1/6876: Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes - 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 present invention provides oligonucleotide constructs, sets of such oligonucleotide constructs, and methods of using such oligonucleotide constructs to provide validated sequences or sets of validated sequences corresponding to desired ROIs. Such validated ROIs and constructs containing these have a wide variety of uses, including in synthetic biology, quantitative nucleic acid analysis, polymorphism and/or mutation screening, and the like. ## Claims ### Claim 1 A method for determining sequences of a plurality of nucleic acid molecules in a set, comprising: (a) tagging one or both ends of each nucleic acid molecule comprising a region of interest in the set of nucleic acid molecules, by ligation with a unique identifier to form a tagged construct, wherein the unique identifier distinguishes the tagged construct from other tagged constructs in the set; (b) selectively amplifying regions of interest and unique identifiers using PCR to form an amplified set of tagged constructs; (c) sequencing the amplified set of tagged constructs; and (d) determining sequence of the amplified tagged constructs using the unique identifiers and sequence information from step (c) to generate a consensus sequence among molecules that are identical by descent. ### Claim 2 (depends on 1) The method of claim 1, wherein each nucleic acid molecule is tagged with two unique identifiers. ### Claim 3 (depends on 2) The method of claim 2, wherein the two unique identifiers are both on one end of the nucleic acid molecule. ### Claim 4 A method for determining sequences of a plurality of nucleic acid molecules in a set, comprising: (a) tagging one or both ends of each nucleic acid molecule comprising a region of interest in the set of nucleic acid molecules, by ligation with two unique identifiers to form a tagged construct, wherein the unique identifiers distinguish the tagged construct from other tagged constructs in the set; (b) selectively amplifying regions of interest and unique identifiers using PCR to form an amplified set of tagged constructs; (c) sequencing the amplified set of tagged constructs; and (d) determining sequence of the amplified tagged constructs using the unique identifiers and sequence information from step (c) to generate a consensus sequence among molecules that are identical by descent, wherein the two unique identifiers are one on each end of the nucleic acid molecule. ### Claim 5 (depends on 1) The method of claim 1, wherein the nucleic acid molecules are isolated from a biological sample. ### Claim 6 (depends on 1) The method of claim 1, wherein the determining further comprises determining allelic variants of a region of interest in the set of nucleic acid sequences. ### Claim 7 (depends on 6) The method of claim 6, wherein the allelic variants comprise one or more haplotypes. ### Claim 8 (depends on 1) The method of claim 1, wherein the set of nucleic acid sequences comprises at least two tagged constructs with substantially the same region of interest and different unique identifiers. ### Claim 9 (depends on 1) The method of claim 1, wherein the sequencing comprises performing massively parallel next generation sequencing. ### Claim 10 A method for determining sequences of a plurality of nucleic acid molecules in a set, comprising: (a) tagging one or both ends of each nucleic acid molecule comprising a region of interest in the set of nucleic acid molecules, by ligation with a unique identifier to form a tagged construct, wherein the unique identifier distinguishes the tagged construct from other tagged constructs in the set; (b) selectively amplifying regions of interest and unique identifiers using PCR to form an amplified set of tagged constructs; (c) sequencing the amplified set of tagged constructs; (d) determining sequence of the amplified tagged constructs using the unique identifiers and sequence information from step (c) to generate a consensus sequence among molecules that are identical by descent, and (e) identifying a mutation in a region of interest if it appears in substantially all of the tagged constructs having an identical descent. ### Claim 11 (depends on 1) The method of claim 1, further comprising amplifying the tagged constructs with universal primers. ### Claim 12 (depends on 1) The method of claim 1, wherein further comprising amplifying the tagged constructs with subset-specific primers. ### Claim 13 (depends on 1) The method set of claim 1, wherein the nucleic acid comprises DNA. ### Claim 14 (depends on 1) The method of claim 1, wherein the nucleic acid comprises RNA. ### Claim 15 (depends on 13) The method of claim 13, wherein the DNA is genomic DNA.