Labelled nucleotides

• US 10,487,102 B2
• Filed: 07/06/2016
• Issued: 11/26/2019
• Est. Priority Date: 08/23/2002
• Status: Expired due to Term

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Liu, Xiaohai, Milton, John, Ruediger, Silke
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/203,562
• Publication Number:
  US 20170002408A1
• Time in Patent Office: 1,238 Days
• Field of Search: 536 41, 536 231, 536 266, 435 61
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• 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: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterized in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a C_1-10 substituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a C_1-10 substituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).

Claims

1. A nucleoside or nucleotide comprising a base attached to a detectable label via a phosphine-cleavable linker comprising a disulfide, wherein the nucleoside or nucleotide comprises a ribose or deoxyribose moiety with a hydroxyl protecting group attached to the 2′

or 3′

oxygen atom, and wherein the protecting group is azidomethyl.

2. The nucleoside or nucleotide of claim 1, wherein the linker and protecting group are cleavable under the same conditions.

3. The nucleoside or nucleotide of claim 1, wherein the base is a purine, pyrimidine or deazapurine.

4. The nucleoside or nucleotide of claim 1, wherein the detectable label is a fluorophore.

18. The nucleoside or nucleotide of claim 1, wherein the nucleoside or nucleotide is a nucleotide, and wherein the nucleotide comprises a deoxyribose moiety with an azidomethyl protecting group attached to the 3′

oxygen atom.

19. The nucleoside or nucleotide of claim 18, wherein the base is a pyrimidine or a diazapurine.

20. The nucleoside or nucleotide of claim 19, wherein the detectable label is a fluorophore.

5. An oligonucleotide comprising a nucleotide residue, wherein said nucleotide residue comprises a base attached to a detectable label via a phosphine-cleavable linker comprising a disulfide, wherein the nucleotide residue comprises a ribose or deoxyribose moiety with a hydroxyl protecting group attached to the 2′

or 3′

oxygen atom, and wherein the protecting group is azidomethyl.

6. The oligonucleotide of claim 5, wherein the oligonucleotide is attached to a solid support.

7. A solid support comprising the oligonucleotide of claim 5.

21. The oligonucleotide of claim 5, wherein the nucleotide residue comprises a deoxyribose moiety with an azidomethyl protecting group attached to the 3′

oxygen atom.

8. A composition comprising four nucleosides or four nucleotides, wherein each nucleoside or nucleotide comprises a base attached to a detectable label via a phosphine-cleavable linker comprising a disulfide, wherein each nucleoside or nucleotide comprises a ribose or deoxyribose moiety with a hydroxyl protecting group attached to the 2′

or 3′

oxygen atom, wherein the protecting group is azidomethyl, and wherein each nucleoside or nucleotide carries a different detectable label.

9. The composition of claim 8, wherein the linker and protecting group are cleavable under the same conditions.

22. The composition of claim 8, wherein said composition comprises four nucleotides, and wherein each nucleotide comprises a deoxyribose moiety with an azidomethyl protecting group attached to the 3′

oxygen atom.

10. A method for determining the identity of a nucleotide in a target single-stranded polynucleotide, comprising:

(a) providing one or more of the nucleotides A, G, C and T or U in which each of said nucleotides has a base that is attached to a distinct detectable label via a phosphine-cleavable linker comprising a disulfide, wherein each nucleotide comprises a ribose or deoxyribose moiety with a hydroxyl protecting group attached to the 2′

or 3′

oxygen atom, and wherein the protecting group is azidomethyl; and

a polynucleotide complementary to the target polynucleotide, one of said provided nucleotides being suitable for incorporation into said complementary polynucleotide;

(b) incorporating the nucleotide suitable for incorporation into said complementary polynucleotide; and

(c) detecting the detectable label, thereby determining the identity of a nucleotide.

11. The method of claim 10, wherein steps (a) and (b) are repeated one or more times so as to determine the identity of a plurality of bases in the target polynucleotide.

12. The method of claim 10, wherein step (a) comprises providing four nucleotides and contacting them with the target simultaneously.

13. The method of claim 10, wherein any unincorporated nucleotides are removed prior to the provision of further nucleotide(s).

14. The method of claim 10, further comprising cleaving the linker after detecting the label.

15. The method of claim 14, wherein said cleavage is carried out with a phosphine.

16. The method of claim 15, wherein said phosphine comprises one or more sulfonate, amine, hydroxyl or carboxylate residues.

17. The method of claim 13, wherein any unincorporated nucleotides are removed prior to detecting step (c).

23. The method of claim 10, wherein each nucleotide comprises a deoxyribose moiety with an azidomethyl protecting group attached to the 3′

oxygen atom.