Labelled nucleotides
Nucleosides and nucleotides are disclosed that are linked to detectable labels via a cleavable linker group.
1. A method of labeling a nucleic acid molecule, the method comprising incorporating into the nucleic acid molecule a nucleotide or nucleoside molecule, wherein the nucleotide or nucleoside molecule has a base that is linked to a detectable label via a cleavable linker and the nucleotide or nucleoside molecule has a ribose or deoxyribose sugar moiety, wherein the ribose or deoxyribose sugar moiety comprises a protecting group attached via the 2′ or 3′ oxygen atom, and said protecting group can be modified or removed to expose a 3′ OH group and the protecting group comprises an azido group.
2. The method of claim 1 , wherein said incorporating is accomplished via a terminal transferase, a polymerase or a reverse transcriptase.
3. The method of claim 1 , wherein the base is a deazapurine.
4. The method of claim 1 , wherein the nucleotide is a deoxyribonucleotide triphosphate.
5. The method of claim 1 , wherein the label is a fluorophore.
6. The method according to claim 1 , wherein the protecting group is CH 2 N 3 .
7. A method of labeling a nucleic acid molecule, the method comprising incorporating into the nucleic acid molecule a nucleotide or nucleoside molecule, wherein the nucleotide or nucleoside molecule has a base that is linked to a detectable label via a cleavable linker and the nucleotide or nucleoside molecule has a ribose or deoxyribose sugar moiety, wherein the ribose or deoxyribose sugar moiety comprises a protecting group attached via the 2′ or 3′ oxygen atom, and said protecting group can be modified or removed to expose a 3′ OH group and the protecting group and cleavable linker are removable under identical conditions.
8. The method according to claim 1 , further comprising detecting the detectable label and cleaving the cleavable linker.
9. A method of labeling a nucleic acid molecule, the method comprising:
a) incorporating into the nucleic acid molecule a nucleotide or nucleoside molecule, wherein the nucleotide or nucleoside molecule has a base that is linked to a detectable label via a cleavable linker, wherein the nucleotide or nucleoside molecule has a ribose or deoxyribose sugar moiety that comprises a protecting group attached via the 2′ or 3′ oxygen atom, and said protecting group can be modified or removed to expose a 3′ OH group and the protecting group comprises an azido group;
b) detecting the detectable label and cleaving the cleavable linker, wherein the protected hydroxyl functionality and the protecting group are cleavable under identical conditions; and
c) repeating steps a) and b) at least once.
10. The method according to claim 7 , wherein the cleavable linker and the protecting group both comprise an allyl moiety.
11. The method of claim 7 , wherein said incorporating is accomplished via a terminal transferase, a polymerase or a reverse transcriptase.
12. The method of claim 7 , wherein the base is a deazapurine.
13. The method of claim 7 , wherein the nucleotide is a deoxyribonucleotide triphosphate.
14. The method of claim 7 , wherein the label is a fluorophore.
15. A method of labeling a nucleic acid molecule, the method comprising:
a) incorporating into the nucleic acid molecule a nucleotide or nucleoside molecule, wherein the nucleotide or nucleoside molecule has a base that is linked to a detectable label via a cleavable linker, wherein the nucleotide or nucleoside molecule has a ribose or deoxyribose sugar moiety that comprises a protecting group attached via the 2′ or 3′ oxygen atom, and said protecting group can be modified or removed to expose a 3′ OH group and the protecting group comprises an allyl moiety;
b) detecting the detectable label and cleaving the cleavable linker, wherein the protected hydroxyl functionality and the protecting group are cleavable under identical conditions; and
c) repeating steps a) and b) at least once.