Method for dissociating biotin complexes

• US 6,022,688 A
• Filed: 05/13/1996
• Issued: 02/08/2000
• Est. Priority Date: 05/13/1996
• Status: Expired due to Term

• Current Assignee: Agena Bioscience Inc. (Mesa Laboratories Inc.)
• Original Assignee: Sequenom Incorporated (Laboratory Corporation Of America Holdings)
• Inventors: Koster, Hubert, Jurinke, Christian, Van den Boom, Dirk
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US08/649,876
• Time in Patent Office: 1,366 Days
• Field of Search: 435/6, 435/7.1, 435/7.2, 436/501, 530/300, 530/350, 530/387.1, 536/25.3
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: G01N 33/53 Immunoassay; Biospecific binding assay; Materials therefor

Abstract

A method for dissociating a complex of a biotin compound and a biotin-binding compound, by contacting the complex with an amine, is disclosed.

Claims

1. A method for dissociating a biotin compound:

biotin-binding compound complex, the method comprising;

contacting the complex with an effective amount of an amine, under conditions such that the complex is dissociated;

thereby releasing a biotin compound and a biotin-binding compound.

2. The method of claim 1, wherein the complex is contacted with an amine at a temperature of between about 25°

C. and about 100°

C.

3. The method of claim 1, wherein the biotin compound is a biotinylated macromolecule.

4. The method of claim 3, wherein the biotinylated macromolecule is selected from the group consisting of a biotinylated nucleic acid sequence, a biotinylated protein, a biotinylated carbohydrate, and a biotinylated lipid.

5. The method of claim 1, wherein the biotin-binding compound is selected from the group consisting of avidin and streptavidin.

6. The method of claim 5, wherein the biotin-binding compound is immobilized on a solid support.

7. The method of claim 6, wherein the solid support is a magnetic bead.

8. The method of claim 1, wherein, after the contacting step, the biotin compound is separated from the biotin-binding compound.

9. The method of claim 8, wherein after the separation step, the biotin compound is purified.

10. The method of claim 9, wherein the biotin compound is purified by a method selected from the group consisting of lyophilization, precipitation, filtration, and dialysis.

11. The method of claim 1, wherein, prior to the contacting step, the complex is purified.

12. The method of claim 1, wherein the biotin compound is immobilized on a solid support.

13. The method of claim 1, wherein, after dissociation of the complex, at least one of the biotin compound and the biotin-binding compound is analyzed by mass spectrometry.

14. The method of claim 1, wherein, after dissociation of the complex, the biotin-binding compound retains biotin-binding activity.

15. The method of claim 1, wherein, after dissociation of the complex, the biotin moiety of the biotin compound remains substantially intact.

16. The method of claim 1, wherein the amine is ammonia.

17. The method of claim 1, wherein the amine is a primary amine.

25. The method of claim 6, wherein, prior to the contacting step, the complex is purified.

33. The method of claim 1, wherein the complex is contacted with an amine at a pH of between about 7 and about 14.

34. The method of claim 1, wherein the complex is contacted with an amine at a concentration of between about 5% and about 28%.

35. The method of claim 1, wherein:

the amine has the formula NR'"'"'₃ or N⁺ R'"'"'₄, in which each R'"'"' is independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl and aryl.

18. A method for analyzing a biotinylated nucleic acid, comprising:

contacting the biotinylated nucleic acid with a biotin-binding compound, thereby forming a biotinylated nucleic acid;

biotin-binding compound complex;

contacting said complex with an effective amount of an amine, under conditions such that the complex is dissociated;

thereby releasing a biotinylated nucleic acid and a biotin-binding compound; and

analyzing the biotinylated nucleic acid.

19. The method of claim 18, wherein the nucleic acid is DNA.

20. The method of claim 18, wherein the biotin-binding compound is immobilized on a solid support.

21. The method of claim 18, wherein the biotinylated nucleic acid is analyzed by mass spectrometry.

22. The method of claim 18, wherein the amine is ammonia.

23. The method of claim 18, wherein, after the first contacting step, the biotinylated nucleic acid is subjected to an enzymatic reaction.

24. The method of claim 23, wherein the enzymatic reaction comprises treatment with an enzyme selected from the group consisting of endonucleases and exonucleases.

26. The method of claim 18, wherein, after the second contacting step, the biotinylated nucleic acid is subjected to an enzymatic reaction.

27. The method of claim 18, wherein, before the first contacting step, the biotinylated nucleic acid is subjected to an enzymatic reaction.

28. The method of claim 26, wherein the enzymatic reaction comprises treatment with an enzyme selected from the group consisting of endonucleases and exonucleases.

29. The method of claim 27, wherein the enzymatic reaction comprises treatment with an enzyme selected from the group consisting of endonucleases and exonucleases.

30. The method of claim 20, wherein, after the first contacting step, the biotinylated nucleic acid is subjected to an enzymatic reaction.

31. The method of claim 20, wherein, after the second contacting step, the biotinylated nucleic acid is subjected to an enzymatic reaction.

32. The method of claim 20, wherein, before the first contacting step, the biotinylated nucleic acid is subjected to an enzymatic reaction.

36. The method of claim 18, wherein:

the amine has the formula NR'"'"'₃ or N⁺ R'"'"'₄, in which each R'"'"' is independently selected from hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl and aryl.

37. The method of claim 30, wherein the enzymatic reaction comprises treatment with an enzyme selected from the group consisting of endonucleases and exonucleases.

38. The method of claim 31, wherein the enzymatic reaction comprises treatment with an enzyme selected from the group consisting of endonucleases and exonucleases.

39. The method of claim 32, wherein the enzymatic reaction comprises treatment with an enzyme selected from the group consisting of endonucleases and exonucleases.