Compositions and methods for performing hybridizations with separate denaturation of the sample and probe

Patent metadata

Field	Value
Publication number	US-2016304945-A1
Application number	US-201615096036-A
Country	US
Kind code	A1
Filing date	Apr 11, 2016
Priority date	Feb 27, 2009
Publication date	Oct 20, 2016
Grant date	—

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.

First claim

1 - 2 . (canceled)

3 . A method of hybridizing nucleic acid sequences comprising:
combining a first nucleic acid sequence with a first aqueous composition comprising at least one polar aprotic solvent in an amount effective to denature a double-stranded nucleotide sequence, and combining said first nucleic acid sequence 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 not dimethyl sulfoxide (DMSO).

4 . (canceled)

5 . The method according to claim 3 , wherein the first nucleic acid sequence is in a biological sample.

6 . The method according to claim 5 , wherein the biological sample is a cytology or histology sample.

7 - 10 . (canceled)

11 . The method according to claim 3 , wherein (a) a sufficient amount of energy to hybridize the first and second nucleic acids is provided; (b) a sufficient amount of energy to denature the first nucleic acid is provided; and/or (c) a sufficient amount of energy to denature the second nucleic acid is provided.

12 - 25 . (canceled)

26 . The method according to claim 3 , wherein the step of hybridization takes less than 8 hours.

27 . The method according to claim 3 , wherein the step of hybridization takes less than 1 hour.

28 - 36 . (canceled)

37 . The method according to claim 3 , with the proviso that the aqueous composition does not contain formamide.

38 . The method according to any one of embodiments 1-36, with the proviso that the first aqueous composition contain less than 10% formamide.

39 - 40 . (canceled)

41 . The method according to any of embodiments 1-40, wherein the polar aprotic solvent in the first aqueous composition has lactone, sulfone, nitrile, sulfite, and/or carbonate functionality.

42 . (canceled)

43 . The method according to claim 41 , wherein the polar aprotic solvent in the first aqueous composition has a cyclic base structure.

44 . The method according to claim 41 , wherein the polar aprotic solvent in the first aqueous composition is selected from the group consisting of:

where X is O and R1 is alkyldiyl, and

where X is optional and if present, is chosen from O or S, where Z is optional and if present, is chosen from O or S, where A and B independently are O or N or S or part of the alkyldiyl or a primary amine, where R is alkyldiyl, and where V is O or S or C.

45 . The method according to claim 3 , wherein the polar aprotic solvent in the first aqueous composition is selected from the group consisting of: acetanilide, acetonitrile, N-acetyl pyrrolidone, 4-amino pyridine, benzamide, benzimidazole, 1,2,3-benzotriazole, butadienedioxide, 2,3-butylene carbonate, γ-butyrolactone, caprolactone (epsilon), chloro maleic anhydride, 2-chlorocyclohexanone, chloroethylene carbonate, chloronitromethane, citraconic anhydride, crotonlactone, 5-cyano-2-thiouracil, cyclopropylnitrile, dimethyl sulfate, dimethyl sulfone, 1,3-dimethyl-5-tetrazole, 1,5-dimethyl tetrazole, 1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone, 1,2-dinitrobenzene, 2,4-dinitrotoluene, dipheynyl sulfone, epsilon-caprolactam, ethanesulfonylchloride, ethyl ethyl phosphinate, N-ethyl tetrazole, ethylene carbonate, ethylene trithiocarbonate, ethylene glycol sulfate, glycol sulfite, furfural, 2-furonitrile, 2-imidazole, isatin, isoxazole, malononitrile, 4-methoxy benzonitrile, l-methoxy-2-nitrobenzene, methyl alpha bromo tetronate, 1-methyl imidazole, N-methyl imidazole, 3-methyl isoxazole, N-methyl morpholine-N-oxide, methyl phenyl sulfone, N-methyl pyrrolidinone, methyl sulfolane, methyl-4-toluenesulfonate, 3-nitroaniline, nitrobenzimidazole, 2-nitrofuran, l-nitroso-2-pyrolidinone, 2-nitrothiophene, 2-oxazolidinone, 9,10-phenanthrenequinone, N-phenyl sydnone, phthalic anhydride, picolinonitrile (2-cyanopyridine), 1,3-propane sultone, β-propiolactone, propylene carbonate, 4H-pyran-4-thione, 4H-pyran-4-one (γ-pyrone), pyrazine, 2-pyrrolidone, saccharin, succinonitrile, sulfanilamide, sulfolane, 2,2,6,6-tetrachlorocyclohexanone, tetrahydrothiapyran oxide, tetramethylene sulfone (sulfolane), thiazole, 2-thiouracil, 3,3,3-trichloro propene, 1,1,2-trichloro propene, 1,2,3 trichloro propene, trimethylene sulfide-dioxide, and trimethylene sulfite.

46 . The method according to claim 3 , wherein the polar aprotic solvent in the first aqueous composition is selected from the group consisting of:

47 . The method according to claim 3 , wherein the polar aprotic solvent in the first aqueous composition is:

48 . The method according to claim 3 , wherein the first aqueous composition further comprise at least one additional component selected from the group consisting of: buffering agents, salts, accelerating agents, chelating agents, detergents, and blocking agents.

49 . The method according to claim 48 , wherein the accelerating agent is dextran sulfate and the salts are NaCl and/or phosphate buffer.

50 . The method according to claim 49 , wherein the dextran sulfate is present at a concentration of 5% to 40%, the Ilea is present at concentration of 0 mM to 1200 mM, and/or the phosphate buffer is present at a concentration of 0 mM to 50 mM.

51 . The method according to claim 50 , wherein the dextran sulfate is present at a concentration of 10% to 30%, 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.

52 . The method according to claim 48 , wherein the accelerating agent is selected from the group consisting of: formamide, DMSO, glycerol, propylene glycol, 1,2-propanediol, diethyl ene glycol, ethylene glycol, glycol, and 1,3 propanediol, and the buffering agent is citric acid buffer.

53 . The method according to claim 52 , wherein the formamide is present at a concentration of 0.1-5%, the DMSO is present at a concentration of 0.01% to 10%, the glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol are present at a concentration of 0.1% to 10%, and the citric acid buffer is present at a concentration of 1 mM to 50 mM.

54 - 76 . (canceled)

Assignees

• Dako Denmark As

Inventors

• Matthiesen Steen Hauge

Classifications

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• C12Q1/6832

  Enhancement of hybridisation reaction · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title

• C12Q2527/137

  Concentration of a component of medium · CPC title