Labelled nucleotides
The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised 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. An oligonucleotide comprising a nucleotide residue, wherein said nucleotide residue comprises the structure:
wherein Z is —CH 2 N 3 ;
B is a nucleobase selected from the group consisting of a purine, a pyrimidine and a deazapurine; and
Linker-Label comprises
wherein R is an optionally present electron-withdrawing group; Fluor is a fluorophore; and the dotted line connecting the Fluor and the benzene ring indicates that substitution may be at any free position on the benzene ring and further atoms not shown may be present.
2. The oligonucleotide of claim 1 , wherein R is not present.
3. The oligonucleotide of claim 2 , wherein Linker-Label comprises
4. The oligonucleotide of claim 3 , wherein the dotted line connecting the Fluor and the benzene ring comprises one or more spacer units such that the Fluor is held a sufficient distance from the nucleobase so as not to interfere with any interaction between the nucleotide and an enzyme.
5. The oligonucleotide of claim 4 , wherein the spacer unit comprises —O—, —NH—, or —N(alkyl)-.
6. The oligonucleotide of claim 3 , wherein the Linker-Label comprises:
wherein the double asterisks ** indicates the attachment point to the nucleobase.
7. The oligonucleotide of claim 6 , wherein the Fluor comprises a cyanine, a rhodamine, or a coumarin dye.
8. The oligonucleotide of claim 7 , wherein the Fluor is a rhodamine dye.
9. The oligonucleotide of claim 7 , wherein the Fluor is a Cy5 dye.
10. The oligonucleotide of claim 7 , wherein the Fluor is attached to the Linker by reacting an N-hydroxysuccinimide ester of the Fluor with an amino moiety of the Linker.
11. The oligonucleotide of claim 6 , wherein the nucleobase is
wherein the asterisk * indicates the attachment point to the sugar moiety of the nucleotide residue.
12. The oligonucleotide of claim 11 , wherein the nucleobase is
and the Fluor is a Cy5 dye.
13. The oligonucleotide of claim 11 , wherein the nucleobase is
and the Fluor is a rhodamine dye.
14. The oligonucleotide of claim 6 , wherein the nucleotide residue comprises the structure:
15. The oligonucleotide of claim 6 , wherein the oligonucleotide is in contact with an aqueous solution comprising ethylenediaminetetraacetic acid (EDTA).
16. The oligonucleotide of claim 6 , wherein the oligonucleotide is in contact with a mutant polymerase.
17. The oligonucleotide of claim 6 , wherein the oligonucleotide is in contact with a water soluble phosphine.
18. The oligonucleotide of claim 6 , wherein the oligonucleotide is hybridized to a target polynucleotide, and wherein the target polynucleotide is immobilized to a solid support.
19. The oligonucleotide of claim 18 , wherein the solid support comprises a plurality of immobilized target polynucleotides, wherein at least one immobilized target polynucleotide is hybridized to a second oligonucleotide, and wherein the second oligonucleotide comprises a different fluorophore than the Fluor.
20. The oligonucleotide of claim 19 , the solid support comprises a planar surface.