# US 10,655,177 B2 · Hybridization compositions and methods > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 10,655,177 B2 - Document ID: US10655177B2 - Status: Granted - Kind: B2 - Application number: 14/352,815 - Filed: 2012-10-22 - Published: 2014-08-28 - Granted: 2020-05-19 - Earliest claimed: 2011-10-21 - Assignees: Agilent Technologies, Inc. - Applicants: Dako Denmark A/S - Inventors: Steen Hauge Matthiesen - Web page: https://patentgrep.com/patent/US10655177B2 ## CPC - C12Q 1/6881: for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes - C12Q 1/6832: Enhancement of hybridisation reaction - C12Q 1/6841: In situ hybridisation - C12Q 2523/113: Denaturating agents - 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 provides methods and compositions for hybridizing at least one molecule to a target. The invention may, for example, utilize a of cyclic and/or non-cyclic solvent that is non-toxic and may eliminate or reduce the amount of formamide in the hybridization composition. ## Claims ### Claim 1 A hybridization composition comprising at least one nucleic acid sequence, at least one solvent in an amount effective to denature double-stranded nucleotide sequences, and a hybridization solution, wherein the solvent is chosen from tetrahydrothiophene 1-oxide, valerolactam, 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone, N,N-dimethyl-acetamide and isobutyramide. ### Claim 2 (depends on 1) The hybridization composition according to claim 1, wherein the solvent is a non-cyclic solvent. ### Claim 3 (depends on 1) The hybridization composition according to claim 1, wherein the solvent is chosen from N,N-dimethyl-acetamide, isobutyramide, and 1,3-dimethyl-3,4,5,6-tetrahydro-2(1H)-pyrimidinone. ### Claim 4 (depends on 1) The hybridization composition according to claim 1, wherein the concentration of solvent is about 1% to 95% (v/v). ### Claim 5 (depends on 1) The hybridization composition according to claim 1, wherein the concentration of solvent is 5% to 10% (v/v). ### Claim 6 (depends on 1) The hybridization composition according to claim 1, wherein the concentration of solvent is 10% to 20% (v/v). ### Claim 7 (depends on 1) The hybridization composition according to claim 1, wherein the concentration of solvent is 20% to 30% (v/v). ### Claim 8 (depends on 1) The hybridization composition according to claim 1, wherein the solvent is non-toxic. ### Claim 9 (depends on 8) The hybridization composition according to claim 8, wherein the composition contains less than 10% (v/v) formamide. ### Claim 10 (depends on 9) The hybridization composition according to claim 9, wherein the composition contains less than 2% (v/v) formamide. ### Claim 11 (depends on 10) The hybridization composition according to claim 10, wherein the composition contains less than 1% (v/v) formamide. ### Claim 12 (depends on 1) The hybridization composition according to claim 1, further comprising at least one additional component selected from the group consisting of: buffering agents, salts, accelerating agents, chelating agents, detergents, and blocking agents. ### Claim 13 (depends on 12) The hybridization composition according to claim 12, wherein the at least one additional component is an accelerating agent, a salt, and/or a buffering agent; and wherein the accelerating agent is dextran sulfate, the salt is NaCl, or the buffering agent is phosphate buffer. ### Claim 14 (depends on 13) The hybridization composition according to claim 13, wherein: the NaCl is present at a concentration of up to 1200 mM, and/or the phosphate buffer is present at a concentration of up to 50 mM. ### Claim 15 (depends on 14) The hybridization composition according to claim 14, wherein: the NaCl is present at a concentration of 300 mM to 600 mM, and/or the phosphate buffer is present at a concentration of 5 mM to 20 mM. ### Claim 16 (depends on 12) The hybridization composition according to claim 12, wherein the accelerating agent is selected from the group consisting of: formamide, glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol, and the buffering agent is citric acid buffer. ### Claim 17 (depends on 16) The hybridization composition according to claim 16, wherein: the formamide is present at a concentration of 0.1-5% (v/v), the glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, or 1,3 propanediol are present at a concentration of 0.1% to 10% (v/v), and the citric acid buffer is present at a concentration of 1 mM to 50 mM. ### Claim 18 (depends on 1) The hybridization composition according to claim 1, comprising 40% (vN) of the solvent, dextran sulfate, 300 mM NaCl, and 5 mM phosphate buffer. ### Claim 19 (depends on 1) The hybridization composition according to claim 1, comprising 15% (vN/V) of the solvent, dextran sulfate, 600 mM NaCl, and 10 mM phosphate buffer. ### Claim 20 (depends on 1) The hybridization composition according to claim 1, comprising 15% (vN) of the solvent, dextran sulfate, 600 mM NaCl, and 10 mM citric acid buffer pH 6.2.