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. A method for determining the sequence of a single-stranded target polynucleotide, comprising:
(a) contacting a primer polynucleotide/target polynucleotide complex with one or more of nucleoside triphosphates A, G, C and T, wherein at least one of said nucleoside triphosphate has the structure:
wherein
B is a nucleobase selected from the group consisting of a purine, a pyrimidine and a deazapurine;
Z is —CH 2 N 3 ;
X is a triphosphate group;
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 maybe present;
(b) incorporating one nucleoside triphosphate into the primer polynucleotide;
(c) performing one or more fluorescent measurements to determine the identity of the incorporated nucleoside triphosphate; and
(d) cleaving the Linker after performing the one or more fluorescent measurements.
2. The method of claim 1 , wherein R is not present and the Linker-Label comprises:
3. The method of claim 2 , 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 nucleoside triphosphate and a polymerase.
4. The method of claim 2 , wherein the Linker-Label comprises:
wherein the double asterisks ** indicates the attachment point to the nucleobase.
5. The method of claim 4 , wherein the Fluor comprises a cyanine, a rhodamine, or a coumarin dye.
6. The method of claim 5 , wherein the Fluor is a Cy3 or a Cy5 dye.
7. The method of claim 5 , wherein the Fluor is attached to the Linker by reacting an N-hydroxysuccinimide ester of the Fluor with an amino moiety of the Linker.
8. The method of claim 6 , wherein said at least one nucleoside triphosphate is a G nucleoside triphosphate and the Fluor is a Cy5 dye.
9. The method of claim 4 , further comprising (e) removing the —CH 2 N 3 to expose a 3′-OH.
10. The method of claim 9 , wherein the —CH 2 N 3 removing step (e), and the Linker cleaving step (d) are performed in a single chemical treatment step.
11. The method of claim 10 , wherein the —CH 2 N 3 is removed and the Linker is cleaved by a water soluble phosphine reagent.
12. The method of claim 11 , wherein the water soluble phosphine reagent is a trialkyl phosphine derivatized with one or more hydroxyl functionalities.
13. The method of claim 9 , wherein steps (a) through (e) are repeated multiple times to determine the sequence of the target polynucleotide.
14. The method of claim 1 , wherein the primer polynucleotide/target polynucleotide complex is formed by contacting the target polynucleotide with a single-stranded primer polynucleotide complementary to at least a portion of the target polynucleotide.
15. The method of claim 14 , wherein the primer polynucleotide/target polynucleotide complex is immobilized to a solid support.
16. The method of claim 1 , wherein said incorporating step is accomplished by a polymerase.
17. The method of claim 1 , wherein any unincorporated nucleoside triphosphates are removed prior to said determining step.
18. The method of claim 17 , wherein the unincorporated nucleoside triphosphates are removed by using a washing buffer comprising ethylenediaminetetraacetic acid (EDTA).
19. The method of claim 1 , wherein at least one of the A, G, C, or T nucleoside triphosphates has the structure:
respectively.
20. The method of claim 4 , wherein at least one nucleoside triphosphate has the structure:
or a salt thereof.