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