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

Patent metadata

Field	Value
Publication number	US-9309562-B2
Application number	US-201013203160-A
Country	US
Kind code	B2
Filing date	Feb 26, 2010
Priority date	Feb 26, 2009
Publication date	Apr 12, 2016
Grant date	Apr 12, 2016

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

The invention claimed is:

1. A method of hybridizing nucleic acid sequences comprising:
providing a first nucleic acid sequence within a sample having a preserved cell morphology, with a first composition comprising at least one polar aprotic solvent in an amount effective to denature a double-stranded nucleotide sequence while preserving cell morphology, and at least 10% dextran sulfate,
providing a second nucleic acid composition comprising a second nucleic acid sequence and a second aqueous composition comprising at least one denaturing agent in an amount effective to denature double-stranded nucleotide sequences, and
combining the first and the second nucleic acid sequence compositions for at least a time period sufficient to hybridize the first and second nucleic acid sequences such that the cell morphology is preserved,

wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).

2. A method of hybridizing nucleic acid sequences comprising:
providing a first nucleic acid sequence, within a sample having a preserved cell morphology, with a first composition comprising at least one polar aprotic solvent in an amount effective to denature a double-stranded nucleotide sequence while preserving cell morphology, and at least 10% dextran sulfate, and
combining said first nucleic acid composition with a second nucleic acid composition comprising a second nucleic acid sequence and at least one denaturing agent in an amount effective to denature double-stranded nucleotide sequences for at least a time period sufficient to hybridize the first and second nucleic acid sequences such that the cell morphology is preserved,

wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).

3. A method of hybridizing nucleic acid sequences comprising:
providing a first nucleic acid sequence within a sample having a preserved cell morphology, with a composition comprising at least one polar aprotic solvent in an amount effective to denature a double-stranded nucleotide sequence while preserving cell morphology, and at least 10% dextran sulfate, and
combining said nucleic acid composition with a second nucleic acid sequence for at least a time period sufficient to hybridize the first and second nucleic acid sequences such that the cell morphology is preserved,

wherein the polar aprotic solvent is not dimethyl sulfoxide (DMSO).

4. The method according to claim 1 , wherein the denaturing agent in the second nucleic acid composition is a polar aprotic solvent.

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

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

7. The method according to claim 1 , wherein the first nucleic acid sequence within the sample is a single stranded sequence and
the second nucleic acid sequence is a double stranded sequence.

8. The method according to claim 1 , wherein the first nucleic acid sequence within the sample is a double stranded sequence and
the second nucleic acid sequence is a single stranded sequence.

9. The method according to claim 1 , wherein the first nucleic acid sequence within the sample is a double stranded sequence and
the second nucleic acid sequence is a double stranded sequence.

10. The method according to claim 1 , wherein the first nucleic acid sequence within the sample is a single stranded sequence and
the second nucleic acid sequence is a single stranded sequence.

11. The method according to claim 1 , wherein: (a) a sufficient amount of energy to hybridize the first and second nucleic acids within the sample is provided, (b) a sufficient amount of energy to denature the first nucleic acid within the sample is provided, and/or (c) a sufficient amount of energy to denature the second nucleic acid is provided.

12. The method according to claim 11 , wherein the energy is provided by heating the compositions.

13. The method according to claim 12 , wherein the heating step is performed by the use of microwaves, hot baths, hot plates, heat wire, peltier element, induction heating or heat lamps.

14. The method according to claim 12 , wherein the temperature for denaturing the first nucleic acid within the sample is 70° C. to 85° C.

15. The method according to claim 12 , wherein the temperature for denaturing the second nucleic acid is 70° C. to 85° C.

16. The method according to claim 12 , wherein the temperature for denaturing the first nucleic acid within the sample is 60° C. to 75° C.

17. The method according to claim 12 , wherein the temperature for denaturing the second nucleic acid is 60° C. to 75° C.

18. The method according to claim 12 , wherein the temperature for denaturing the first nucleic acid within the sample is 62° C., 67° C., 72° C., or 82° C.

19. The method according to claim 12 , wherein the temperature for denaturing the second nucleic acid is 62° C., 67° C., 72° C., or 82° C.

20. The method according to claim 1 , wherein a sufficient amount of time to denature the first nucleic acid within the sample and/or the second nucleic acid is provided.

21. The method according to claim 20 , wherein the time is 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 15 minutes, or 30 minutes.

22. The method according to claim 1 , wherein the step of hybridizing includes the steps of heating and cooling the compositions.

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

24. The method according to claim 23 , wherein the step of hybridization takes less than 1 hour.

25. The method according to claim 24 , wherein the step of hybridization takes less than 30 minutes.

26. The method according to claim 25 , wherein the step of hybridization takes less than 15 minutes.

27. The method according to claim 26 , wherein the step of hybridization takes less than 5 minutes.

28. The method according to claim 1 , further comprising a blocking step.

29. The method according to claim 1 , wherein the concentration of polar aprotic solvent in the nucleic acid composition(s) is about 1% to 90% (v/v).

30. The method according to claim 29 , wherein the concentration of polar aprotic solvent is 5% to 10% (v/v).

31. The method according to claim 29 , wherein the concentration of polar aprotic solvent is 10% to 20% (v/v).

32. The method according to claim 29 , wherein the concentration of polar aprotic solvent is 20% to 30% (v/v).

33. The method according to claim 1 , wherein the polar aprotic solvent in the nucleic acid composition(s) is non-toxic.

34. The method according to claim 1 , with the proviso that the nucleic acid composition(s) do not contain formamide.

35. The method according to claim 1 , with the proviso that the nucleic acid composition(s) contain less than 10% formamide.

36. The method according to claim 35 , with the proviso that the nucleic acid composition(s) contain less than 2% formamide.

37. The method according to claim 36 , with the proviso that the nucleic acid composition(s) contains less than 1% formamide.

38. The method according to any of claim 1 , wherein the polar aprotic solvent in the nucleic

Assignees

• Matthiesen Steen Hauge
• Dako Denmark As

Inventors

• Matthiesen Steen Hauge

Classifications

• C12Q1/6832 Primary

  Enhancement of hybridisation reaction · CPC title

• C12Q2527/137

  Concentration of a component of medium · CPC title

• C12Q1/6841 Primary

  In situ hybridisation · CPC title

• C12Q2527/125

  Specific component of sample, medium or buffer · CPC title