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

1. 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).

2. The method according to claim 1 , wherein the first nucleic acid sequence is in a biological sample.

3. The method according to claim 2 , wherein the biological sample is a cytology or histology sample.

4. The method according to claim 1 , 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.

5. The method according to claim 1 , wherein the step of hybridization takes less than 8 hours.

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

7. The method according to claim 1 , with the proviso that the aqueous composition does not contain formamide.

8. The method according to claim 1 , with the proviso that the first aqueous composition contain less than 10% formamide.

9. The method according to claim 1 , wherein the polar aprotic solvent in the first aqueous composition has lactone, sulfone, nitrile, sulfite, and/or carbonate functionality.

10. The method according to claim 9 , wherein the polar aprotic solvent in the first aqueous composition has a cyclic base structure.

11. The method according to claim 9 , 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 Y is O or S or C.

12. The method according to claim 1 , 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), pyridazine, 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.

13. The method according to claim 1 , wherein the polar aprotic solvent in the first aqueous composition is selected from the group consisting of:

14. The method according to claim 1 , wherein the polar aprotic solvent in the first aqueous composition is:

15. The method according to claim 1 , 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.

16. The method according to claim 15 , wherein the accelerating agent is dextran sulfate and the salts are NaCl and/or phosphate buffer.

17. The method according to claim 16 , wherein the dextran sulfate is present at a concentration of 5% to 40%, the NaCl is present at a concentration of 0 mM to 1200 mM, and/or the phosphate buffer is present at a concentration of 0 mM to 50 mM.

18. The method according to claim 17 , 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.

19. The method according to claim 15 , wherein the accelerating agent is selected from the group consisting of: formamide, DMSO, glycerol, propylene glycol, 1,2-propanediol, diethylene glycol, ethylene glycol, glycol, and 1,3 propanediol, and the buffering agent is citric acid buffer.

20. The method according to claim 19 , 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.