Hybridization compositions and methods using formamide

• US 10,662,465 B2
• Filed: 09/28/2012
• Issued: 05/26/2020
• Est. Priority Date: 09/30/2011
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Matthiesen, Steen Hauge
• Primary Examiner(s): Crow, Robert T.
• Application Number:
  US14/348,134
• Publication Number:
  US 20140234844A1
• Time in Patent Office: 2,797 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2523/113 Denaturating agents
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/137 Concentration of a component of medium

Abstract

The invention provides methods and compositions for hybridizing at least one molecule to a target. The composition comprises at least one nucleic acid sequence, formamide, and a hybridization solution, wherein the concentration of formamide is less than or equal to 25%.

Claims

1. A method of hybridizing nucleic acid sequences comprising:

providing a first nucleic acid sequence,

providing a second nucleic acid sequence,

providing a hybridization composition comprising formamide, and

combining the first and the second nucleic acid sequence and the hybridization composition for at least a time period sufficient to hybridize the first and second nucleic acid sequences, wherein the time period is less than 4 hours, and wherein the hybridization is before a stringent wash,

wherein the concentration of formamide is 10% to 25% v/v;

wherein the hybridization composition further comprises 15% to 80% w/v of an accelerating agent and 400 mM to 1200 mM of a salt.

3. The method according to claim 1, wherein a sufficient amount of energy to hybridize the first and second nucleic acids is provided.

4. The method according to claim 3, wherein the energy is provided by heating the hybridization composition and nucleic acid sequence.

5. The method according to claim 4, wherein the heating step is performed by the use of microwaves, hot baths, hot plates, heat wire, peltier element, induction heating or heat lamps.

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

7. The method according to claim 1, further comprising a denaturation step, wherein the denaturation and the combining occur separately.

8. The method according to claim 1, wherein the combining includes steps of heating and cooling the hybridization composition and nucleic acid sequences.

9. The method according to claim 1, wherein the time period sufficient to hybridize the first and second nucleic acid sequences is less than 2 hours.

10. The method according to claim 9, wherein the time period sufficient to hybridize the first and second nucleic acid sequences is less than 1 hour.

11. The method according to claim 10, wherein the time period sufficient to hybridize the first and second nucleic acid sequences is less than 30 minutes.

12. The method according to claim 11, wherein the time period sufficient to hybridize the first and second nucleic acid sequences is less than 5 minutes.

13. The method according to claim 1, further comprising a denaturation step performed at 72 to 92°

C.

14. The method according to claim 1, further comprising a denaturation step performed at 75 to 95°

C.

15. The method according to claim 1, further comprising a denaturation step performed at 85°

C.

16. The method according to claim 1, further comprising a denaturation step performed at 82°

C.

17. The method according to claim 1, further comprising a denaturation step which takes less than 15 minutes.

18. The method according to claim 17, further comprising a denaturation step which takes less than 5 minutes.

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

20. The method according to claim 19, wherein the biological sample is a cytology or histology sample.

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

22. The method according to claim 1, wherein the concentration of formamide is less than or equal to 20% v/v.

23. The method according to claim 1, wherein the concentration of formamide is 10% to 15% v/v.

24. The method according to claim 1, wherein the concentration of formamide is 10% to 20% v/v.

25. The method according to claim 1, wherein the concentration of formamide is 15% to 20% v/v.

26. The method according to claim 1, wherein the salt is NaCl.

27. The method according to claim 26, wherein the NaCl is present at a concentration of 400 to 700 mM.

28. The method according to claim 26, wherein the NaCl is present at a concentration of about 600 mM.

29. The method according to claim 1, further comprising at least one additional component selected from the group consisting of:

buffering agents, chelating agents, detergents, blocking agents, and combinations thereof.

30. The method according to claim 27, wherein the at least one additional component is a blocking agent selected from the group consisting of:

total human DNA, COT1 DNA, blocking PNA, herring sperm DNA, salmon sperm DNA, and calf thymus DNA.

31. The method according to claim 1, wherein the blocking agent is present at a concentration of 0.01 to 10 ug/uL.

32. The method according to claim 1, wherein the accelerating agent is dextran sulfate and, wherein the dextran sulfate is present at a concentration of about 20% to about 40% w/v.

33. The method according to claim 1, wherein the accelerating agent is dextran sulfate and wherein the dextran sulfate is present at a concentration of about 20% w/v.

2. A method of hybridizing nucleic acid sequences comprising:

providing a first nucleic acid sequence in an in situ biological sample,

applying a hybridization composition comprising a second nucleic acid sequence and formamide to said first nucleic acid sequence for at least a time period sufficient to hybridize the first and second nucleic acid sequences, wherein the time period is less than 4 hours,

wherein the formamide is present in a concentration of 10% to 25% v/v;

wherein the hybridization composition further comprises 15% to 80% w/v of an accelerating agent and 400 mM to 1200 mM of a salt.

34. A method of hybridizing nucleic acid sequences comprising:

providing a first nucleic acid sequence,

providing a second nucleic acid sequence,

providing a hybridization composition comprising formamide, and

combining the first and the second nucleic acid sequence and the hybridization composition for at least a time period sufficient to hybridize the first and second nucleic acid sequences, wherein the time period is less than 4 hours,

wherein the concentration of formamide is 10% to 25% v/v;

wherein the hybridization composition further comprises 15% to 80% of an accelerating agent and 400 mM to 1200 mM of a salt.