Solid phase nucleic acid labeling by transamination

• US 6,194,563 B1
• Filed: 03/26/1999
• Issued: 02/27/2001
• Est. Priority Date: 03/26/1999
• Status: Expired due to Term

• Current Assignee: Abbott Molecular Incorporated (Abbott Laboratories)
• Original Assignee: Vysis, Inc. (Abbott Laboratories)
• Inventors: Cruickshank, Kenneth A.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US09/277,087
• Time in Patent Office: 704 Days
• Field of Search: 435/6, 536/22.1, 536/23.1, 536/25.3, 536/25.4, 536/25.32
• US Class Current: 536/25.3
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

The invention relates to a method for linking a detectable label to a nucleic acid by (1) providing a nucleic acid bound to a solid support, the nucleic acid having a cytidine base; (2) transaminating the cytidine base with a reactive group to form a covalent linkage between the cytidine base and the reactive group; and (3) linking a detectable label to the reactive group. The invention also includes compositions containing a labeled nucleic acid produced by the methods of the invention immobilized on a solid support, and a kit containing a solid support, a bisulfite, a reactive group, and a detectable label.

Claims

1. A method for linking a detectable label to a nucleic acid, the method comprising

providing a nucleic acid bound to a solid support, the nucleic acid comprising a cytidine base;

transaminating the cytidine base with a reactive group to form a covalent linkage between the cytidine base and the reactive group; and

linking a detectable label to the reactive group.

2. The method of claim 1, wherein the solid support is a bead.

3. The method of claim 1, wherein the solid support is a chromium-plated glass slide.

4. The method of claim 1, wherein the transaminating step is catalyzed by a bisulfite.

5. The method of claim 1, wherein the reactive group comprises the formula:

wherein

R is an alkylene radical containing 2 to 14 carbon atoms inclusive;

X is a divalent radical selected from the group consisting of

R₁ and R₂ are each independently selected from the group consisting of hydrogen and a lower alkyl.

6. The method of claim 5, wherein the reactive group is ethylenediamine.

7. The method of claim 1, wherein the reactive group is dissolved in a solvent comprising a trihaloacetate chaotrope anion.

8. The method of claim 7, wherein the trihaloacetate chaotrope anion is trifluoroacetate.

9. The method of claim 1, wherein the detectable label is a fluorescent molecule.

10. The method of claim 1, wherein the nucleic acid is about 1 to 2 kilobases in length.

11. The method of claim 1, further comprising releasing the nucleic acid from the solid support.

12. The method of claim 1, wherein the nucleic acid is covalently bound to the solid support.

13. The method of claim 12, wherein the nucleic acid is bound to the solid support by reacting a terminal phosphate of the nucleic acid and an amino activated solid support with 1-ethyl-3-(dimethylaminopropyl)carbodiimide.

24. The method of claim 1, wherein the reactive group comprises H₂N(CH₂CH₂X)_nCH₂CH₂NH₂, wherein n=1-4, X is O, SO, SO₂, Si, or NR₃, wherein R₃ is a lower alkyl.

14. A composition comprising

a solid support;

a nucleic acid bound to the solid support, the nucleic acid comprising a cytidine base;

a reactive group covalently linked to the cytidine base; and

a detectable label linked to the reactive group.

15. The composition of claim 14, wherein the solid support is a bead.

16. The composition of claim 14, wherein the solid support is a chromium-plated glass slide.

17. The composition of claim 14, wherein the reactive group comprises the formula:

wherein

R is an alkylene radical containing 2 to 14 carbon atoms inclusive;

X is a divalent radical selected from the group consisting of

R₁ and R₂ are each independently selected from the group consisting of hydrogen and a lower alkyl.

18. The composition of claim 17, wherein the reactive group is ethylenediamine.

19. The composition of claim 14, wherein the detectable label is a fluorescent molecule.

20. The composition of claim 14, wherein the nucleic acid is about 1 to 2 kilobases in length.

21. The composition of claim 14, wherein the nucleic acid is covalently bound to the solid support.

22. The composition of claim 21, wherein the nucleic acid is bound to the solid support by reacting a terminal phosphate of the nucleic acid and an amino activated solid support with 1-ethyl-3-(dimethylaminopropyl)carbodiimide.

25. The composition of claim 14, wherein the reactive group comprises H₂N(CH₂CH₂X)_nCH₂CH₂NH₂, wherein n=1-4, X is O, SO, SO₂, Si, or NR₃, wherein R₃ is a lower alkyl.

23. A method for linking a detectable label to a plurality of nucleic acids, each of the plurality having a unique sequence, the method comprising

providing the plurality of nucleic acids bound to a solid support, each of the plurality comprising a cytidine base;

transaminating each cytidine base with reactive groups to form a covalent linkage between each cytidine base and a reactive group; and

linking a detectable label to the reactive groups.

26. A kit comprising

a solid support;

a bisulfite;

a reactive group; and

a detectable label.

27. The kit of claim 26, wherein the solid support is a bead.