# US 10,308,979 B2 · Target enrichment and labeling for multi-kilobase DNA > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,308,979 B2 - Document ID: US10308979B2 - Status: Granted - Kind: B2 - Application number: 13/830,408 - Filed: 2013-03-14 - Published: 2013-12-05 - Granted: 2019-06-04 - Earliest claimed: 2012-06-01 - Assignees: Agilent Technologies, Inc. - Applicants: Agilent Technologies, Inc. - Inventors: Brian Jon Peter; Robert A. Ach; Zoltan Timar; Joel Myerson; Jeffrey Robert Sampson; Holly Hogrefe - Web page: https://patentgrep.com/patent/US10308979B2 ## CPC - C12Q 1/6813: Hybridisation assays - C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) - 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 This disclosure provides a method comprising: a) clamping the top and bottom strands of a double stranded DNA molecule to produce a duplex in which the top and bottom strands are linked; b) denaturing the duplex to produce a denatured product; and c) renaturing the denatured product in the presence of a labeled oligonucleotide that is complementary to a sequence of nucleotides in the double stranded DNA molecule, thereby producing a D-loop-containing product. Kits for performing the method and products made by the method are also provided. ## Claims ### Claim 1 A method comprising: a) clamping the top and bottom strands of a double stranded DNA molecule to produce a duplex in which the top and bottom strands are linked; b) denaturing the duplex to produce a denatured product; and c) renaturing the denatured product in the presence of a labeled oligonucleotide, wherein the labeled oligonucleotide hybridizes to a sequence of nucleotides on one strand in the double stranded DNA molecule, thereby producing a renatured, double-stranded product that comprises a triplex region comprising the labeled oligonucleotide. ### Claim 2 (depends on 1) The method of claim 1, wherein the clamping step comprises covalently linking the top and bottom strands of the double stranded DNA molecule to produce a duplex in which the top and bottom strands are covalently linked. ### Claim 3 (depends on 2) The method of claim 2, wherein the covalently linking is done by ligating a hairpin adaptor to one or both ends of the double stranded DNA. ### Claim 4 (depends on 3) The method of claim 3, wherein the hairpin adaptor comprises a hairpin region that has a Tm over 70° C. ### Claim 5 (depends on 2) The method of claim 2, wherein the covalently linking is done by ligating a double stranded adaptor to one or both ends of the double stranded DNA, and covalently cross-linking the top and bottom strands of the double stranded adaptor. ### Claim 6 (depends on 2) The method of claim 2, wherein the covalently linking is done by ligating two molecules of a hairpin adaptor to the double stranded DNA, and wherein the hairpin adaptor comprises a nucleotide sequence that produces a restriction site if an adaptor dimer is produced. ### Claim 7 (depends on 1) The method of claim 1, wherein the labeled oligonucleotide comprises an affinity tag. ### Claim 8 (depends on 7) The method of claim 7, wherein the affinity tag is a biotin moiety. ### Claim 9 (depends on 7) The method of claim 7, further comprising: d) contacting the renatured, double-stranded product of step (c) with a solid support comprising a surface tethered capture agent for the affinity tag, thereby binding the renatured, double-stranded product to the solid support and isolating the renatured, double-stranded product from other nucleic acids in the sample, thereby producing an isolated renatured double-stranded product. ### Claim 10 (depends on 9) The method of claim 9, wherein the method comprises genotyping the double stranded DNA molecule in the isolated renatured double-stranded product. ### Claim 11 (depends on 9) The method of claim 9, wherein in step (c) the labeled oligonucleotide hybridizes to more than one region of the double stranded DNA molecule to produce a topologically distinct region, and the method comprises supercoiling the renatured product after it is contacted with the solid support. ### Claim 12 (depends on 9) The method of claim 9, wherein the covalently linking is done by ligating an adaptor to the double stranded DNA, wherein the adaptor comprises at least one cleavable linkage so that it can be removed from the renatured product after the renatured product has been isolated. ### Claim 13 (depends on 9) The method of claim 9, wherein step (c) further comprises renaturing the denatured product in the presence of multiple different labeled oligonucleotides, wherein the multiple different labeled oligonucleotides hybridize to different sequences of nucleotides in the double stranded DNA molecule. ### Claim 14 (depends on 1) The method of claim 1, wherein the labeled oligonucleotide comprises an optically detectable label. ### Claim 15 (depends on 1) The method of claim 1, wherein the labeled oligonucleotide is made of locked nucleic acid (LNA) or unstructured nucleic acid (UNA). ### Claim 16 (depends on 1) The method of claim 1, wherein the method does not comprise studying DNA methylation, and the denatured product of step (c) is not bisulfite-treated. ### Claim 17 (depends on 1) The method of claim 1, wherein the double stranded DNA molecule of (a) is in the range of 20-200 kb in length.