Granted Patent
B2
US 12,534,755 · App. 17/588,023 · Granted Jan 27, 2026
Compositions and methods for performing hybridizations with separate denaturation of the sample and probe
Assignee:
AGILENT TECHNOLOGIES, INC.
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Abstract
The invention provides methods and compositions for separately denaturing a probe and target in hybridization applications. The invention may, for example, eliminate the use of, or reduce the dependence on formamide in hybridization applications. Compositions for use in the invention include an aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature double-stranded nucleotide sequences.
Claims (22)
1 . A method of hybridizing nucleic acid sequences comprising:
combining a first nucleic acid sequence within a sample with a first aqueous composition comprising at least one polar aprotic solvent and at least one accelerating agent, wherein the polar aprotic solvent is in an amount effective to denature a double-stranded nucleotide sequence and is not dimethyl sulfoxide (DMSO); and
combining said first nucleic acid sequence which has been combined with said first aqueous composition with a second nucleic acid sequence for at least a time period sufficient to hybridize the first and second nucleic acid sequences, wherein the polar aprotic solvent is selected from the group consisting of:
2 . The method according to claim 1 , wherein the polar aprotic solvent is
3 . The method according to claim 1 , wherein (a) a sufficient amount of energy to denature the first nucleic acid sequence is provided, and/or (b) a sufficient amount of energy to denature the second nucleic acid sequence is provided, wherein the energy is provided by heating.
4 . The method according to claim 3 , wherein the heating to denature the first nucleic acid sequence and/or the second nucleic acid sequence is at a temperature of 70° C. to 85° C.
5 . The method according to claim 3 , wherein the heating to denature the first nucleic acid sequence and/or the second nucleic acid sequence is at a temperature of 67° C. to 92° C.
6 . The method according to claim 3 , wherein the first nucleic acid sequence and/or the second nucleic acid sequence is denatured for a time of 0 to 15 minutes.
7 . The method according to claim 1 , wherein the step of hybridizing includes steps of heating and cooling the compositions, and the step of hybridization is performed within 3 hours.
8 . The method according to claim 1 , wherein the step of hybridizing includes steps of heating and cooling the compositions, and the step of hybridization is performed within 2 hours.
9 . The method according to claim 1 , wherein the first aqueous composition comprises one phase at room temperature.
10 . The method according to claim 1 , wherein the second nucleic acid sequence is in a second aqueous composition, and the second aqueous composition comprises one phase at room temperature.
11 . The method according to claim 1 , wherein the first nucleic acid sequence and the second nucleic acid sequence are combined and hybridized in a volume of 10 μL to 150 μL.
12 . The method according to claim 1 , wherein the polar aprotic solvent has a concentration in the first aqueous composition of 5% to 10% (v/v).
13 . The method according to claim 1 , wherein the polar aprotic solvent has a concentration in the first aqueous composition of 10% to 20% (v/v).
14 . The method according to claim 1 , wherein the polar aprotic solvent has a concentration in the first aqueous composition of 20% to 30% (v/v).
15 . The method according to claim 1 , wherein the first aqueous composition further comprises at least one additional component selected from the group consisting of: buffering agents, salts, chelating agents, detergents, blocking agents, and combinations thereof.
16 . The method according to claim 1 , wherein the first aqueous composition further comprises a salt selected from the group consisting of: sodium chloride, sodium phosphate, magnesium phosphate, and combinations thereof.
17 . The method according to claim 1 , wherein the first aqueous composition further comprises Tris buffer.
18 . The method according to claim 1 , wherein the accelerating agent is present at a concentration of 10% to 40%.
19 . The method according to claim 1 , wherein the accelerating agent is selected from the group consisting of: FICOLL, polyvinylpyrrolidone (PVP), heparin, dextran sulfate, proteins, glycols, organic solvents, and combinations thereof.
20 . The method according to claim 1 , wherein the accelerating agent is selected from the group consisting of: FICOLL, polyvinylpyrrolidone (PVP), heparin, dextran sulfate, bovine serum albumin (BSA), formamide, DMSO, glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, 1,3-propanediol, and combinations thereof.
Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Apr 28, 2022
From: DAKO DENMARK APS
To: AGILENT TECHNOLOGIES, INC.
Reel/Frame 059759/0103 →
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded Apr 28, 2022
From: MATTHIESEN, STEEN HAUGE
To: DAKO DENMARK A/S
Reel/Frame 059765/0106 →
CHANGE OF NAME
Recorded Apr 28, 2022
From: DAKO DENMARK A/S
To: DAKO DENMARK APS
Reel/Frame 059820/0262 →
Continuity (6)
Continuation
13203160
Provisional Application
61289617
· Dec 23, 2009
Provisional Application
61155683
· Feb 26, 2009
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