# US 2014/0194296 A1 · Multiplex Capture of Nucleic Acids > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2014/0194296 A1 - Document ID: US20140194296A1 - Status: Published application - Kind: A1 - Application number: 14/105,080 - Filed: 2013-12-12 - Published: 2014-07-10 - Earliest claimed: 2005-05-09 - Assignees: Affymetrix, Inc. - Applicants: Affymetrix, Inc. - Inventors: Yuling Luo; Yunqing Ma; Gary McMaster; Quan Ngoc Nguyen; Frank Witney; Wen Yang - Web page: https://patentgrep.com/patent/US20140194296A1 ## CPC - C12Q 1/6874: involving nucleic acid arrays, e.g. sequencing by hybridisation - C12Q 1/6832: Enhancement of hybridisation reaction - C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase - C12Q 1/6837: using probe arrays or probe chips (C12Q1/6874 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 Methods of capturing two or more nucleic acids simultaneously from a single sample are provided. Different nucleic acids are captured through cooperative hybridization events on different subsets of particles or at different selected positions on a spatially addressable solid support. Methods of capturing one or more long nucleic acids and methods of capturing one or more nucleic acid for sequencing are also provided. Compositions, kits, and systems related to the methods are also described. ## Claims ### Claim at position 1 1-61. (canceled) ### Claim 62 A method of capturing ten or more different target nucleic acids, the method comprising: providing a sample comprising or suspected of comprising the ten or more different target nucleic acids; providing a solid support having associated therewith a support capture probe; providing ten or more subsets of n different target capture probes, wherein n is at least two, wherein a different subset of target capture probes is provided for each different target nucleic acid, wherein the target capture probes in each subset are capable of hybridizing to nonoverlapping polynucleotide sequences in the corresponding target nucleic acid, and wherein the target capture probes in each subset are capable of hybridizing to the support capture probe; hybridizing any target nucleic acid present in the sample to its corresponding subset of n target capture probes and hybridizing the subset of n target capture probes to the support capture probe, thereby capturing the target nucleic acid on the solid support, wherein the hybridizing the subset of n target capture probes to the support capture probe is performed at a hybridization temperature which is greater than a melting temperature Tm of a complex between each individual target capture probe and the support capture probe; after hybridizing any target nucleic acid present in the sample to its corresponding subset of n target capture probes and hybridizing the subset of n target capture probes to the support capture probe, removing the captured target nucleic acids from the solid support; and after removing the captured target nucleic acids from the solid support, manipulating the captured target nucleic acids. ### Claim 63 (depends on 62) The method of claim 62, wherein manipulating the captured target nucleic acids comprises determining a polynucleotide sequence of one or more of the captured target nucleic acids or a portion thereof. ### Claim 64 A method of capturing and sequencing nucleic acids, the method comprising: providing a sample comprising or suspected of comprising ten or more different target nucleic acids; providing a solid support having associated therewith a support capture probe; providing ten or more subsets of n different target capture probes, wherein n is at least two, wherein a different subset of target capture probes is provided for each different target nucleic acid, wherein the target capture probes in each subset are capable of hybridizing to nonoverlapping polynucleotide sequences in the corresponding target nucleic acid, and wherein the target capture probes in each subset are capable of hybridizing to the support capture probe; hybridizing any target nucleic acid present in the sample to its corresponding subset of n target capture probes and hybridizing the subset of n target capture probes to the support capture probe, thereby capturing the target nucleic acid on the solid support, wherein the hybridizing the subset of n target capture probes to the support capture probe is performed at a hybridization temperature which is greater than a melting temperature Tm of a complex between each individual target capture probe and the support capture probe; and after capturing the target nucleic acids on the solid support, determining a polynucleotide sequence of one or more of the captured target nucleic acids or a portion thereof. ### Claim 65 (depends on 64) The method of claim 64, comprising separating the target capture probes from the captured target nucleic acids prior to determining the polynucleotide sequence. ### Claim 66 (depends on 65) The method of claim 65, wherein separating the target capture probes from the captured target nucleic acids comprises separating the target capture probes and the captured target nucleic acids based on their relative sizes. ### Claim 67 (depends on 65) The method of claim 65, wherein separating the target capture probes from the captured target nucleic acids comprises selectively degrading the target capture probes. ### Claim 68 (depends on 65) The method of claim 65, wherein the target capture probes comprise an affinity tag, and wherein separating the target capture probes from the captured target nucleic acids comprises removing the target capture probes from the captured target nucleic acids by affinity purification. ### Claim 69 (depends on 68) The method of claim 68, wherein the target capture probes comprise a biotin tag, and wherein removing the target capture probes from the captured target nucleic acids by affinity purification comprises removing the target capture probes through binding of the biotin tag to avidin or streptavidin. ### Claim 70 (depends on 64) The method of claim 64, comprising: after capturing the target nucleic acids on the solid support and before determining the polynucleotide sequence of one or more of the captured target nucleic acids or a portion thereof, ligating oligonucleotide adapters to the one or more of the captured target nucleic acids. ### Claim 71 (depends on 70) The method of claim 70, comprising removing the captured target nucleic acids from the solid support prior to ligating the oligonucleotide adapters to the one or more of the captured target nucleic acids. ### Claim 72 (depends on 64) The method of claim 64, wherein determining the polynucleotide sequence comprises determining the polynucleotide sequence by sequencing by synthesis, sequencing by cyclic reversible termination, sequencing by single nucleotide addition, real-time sequencing, dideoxy sequencing, or sequencing by ligation. ### Claim 73 (depends on 64) The method of claim 64, wherein determining the polynucleotide sequence comprises a chromosome walk, resequencing, or sequencing two or more members of a gene or protein family. ### Claim 74 (depends on 64) The method of claim 64, wherein the ten or more target nucleic acids are ten or more fragments of genomic DNA. ### Claim 75 (depends on 64) The method of claim 64, wherein the ten or more different target nucleic acids represent ten or more different exons. ### Claim 76 (depends on 64) The method of claim 64, wherein the ten or more different target nucleic acids are ten or more different RNAs. ### Claim 77 (depends on 64) The method of claim 64, wherein the ten or more target nucleic acids comprise 20 or more, 30 or more, 40 or more, or 50 or more target nucleic acids, and wherein the ten or more subsets of n target capture probes comprise 20 or more, 30 or more, 40 or more, or 50 or more subsets of n target capture probes. ### Claim 78 (depends on 64) The method of claim 64, wherein the solid support comprises particles. ### Claim 79 (depends on 64) The method of claim 64, wherein the solid support comprises particles of a single type, which particles bear the support capture probe. ### Claim 80 (depends on 64) The method of claim 64, wherein the solid support comprises a surface of a well of a multiwell plate ### Claim 81 (depends on 64) The method of claim 64, wherein the solid support comprises a surface of a well of a multiwell plate, which surface bears the support capture probe. ### Claim 82 (depends on 64) The method of claim 64, wherein n is at least three. ### Claim 83 (depends on 64) The method of claim 64, wherein n is at most ten. ### Claim 84 (depends on 64) The method of claim 64, wherein each target capture probe comprises a polynucleotide sequence U-1 that is complementary to a polynucleotide sequence U-2 in the support capture probe, and wherein U-1 and U-2 are 20 nucleotides or less in length. ### Claim 85 (depends on 84) The method of claim 84, wherein U-1 and U-2 are between 9 and 17 nucleotides in length. ### Claim 86 (depends on 64) The method of claim 64, wherein the hybridization temperature is about 5° C. or more greater than the Tm. ### Claim 87 A method of identifying the presence of target nucleic acids in a sample, the method comprising: providing a sample comprising or suspected of comprising ten or more different target nucleic acids; providing a solid support having associated therewith a support capture probe; providing ten or more subsets of n different target capture probes, wherein n is at least two, wherein a different subset of target capture probes is provided for each different target nucleic acid, wherein the target capture probes in each subset are capable of hybridizing to nonoverlapping polynucleotide sequences in the corresponding target nucleic acid, and wherein the target capture probes in each subset are capable of hybridizing to the support capture probe; hybridizing any target nucleic acid present in the sample to its corresponding subset of n target capture probes and hybridizing the subset of n target capture probes to the support capture probe, thereby capturing the target nucleic acid on the solid support, wherein the hybridizing the subset of n target capture probes to the support capture probe is performed at a hybridization temperature which is greater than a melting temperature Tm of a complex between each individual target capture probe and the support capture probe; and after hybridizing any target nucleic acid present in the sample to its corresponding subset of n target capture probes and hybridizing the subset of n target capture probes to the support capture probe, identifying the captured target nucleic acids, thereby determining which of the ten or more different target nucleic acids were present in the sample.