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 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 linker, said linker being cleavable with a water-soluble phosphine and said cleavable linker comprising an azido group; and a nascent polynucleotide complementary to the target polynucleotide, one of said provided nucleotides being suitable for incorporation into said nascent polynucleotide;
(b) incorporating the nucleotide suitable for incorporation into said nascent polynucleotide;
(c) detecting the detectable label of the incorporated nucleotide to identify the incorporated nucleotide; and
(d) cleaving the azido linker of the incorporated nucleotide with a water-soluble phosphine.
2. The method as claimed in claim 1 , 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.
3. The method as claimed in claim 1 , wherein step (a) comprises contacting the provided nucleotides with the target sequentially.
4. The method as claimed in claim 1 , wherein step (a) comprises at least one substep of providing one of the four said nucleotides.
5. The method as claimed in claim 4 , wherein step (a) further comprises, after said substep, providing the other three nucleotides simultaneously or sequentially.
6. The method as claimed in claim 5 , wherein said other three nucleotides are added sequentially, either by providing them one at a time; or two simultaneously and then the remaining one; or one of the three and then the remaining two simultaneously.
7. The method as claimed in claim 1 , wherein step (a) comprises at least a substep of providing two of the four said nucleotides.
8. The method as claimed in claim 7 , wherein step (a) further comprises, after said substep, providing the other two nucleotides simultaneously or sequentially.
9. The method as claimed in claim 1 , wherein step (a) comprises at least a substep of providing three of the four said nucleotides.
10. The method as claimed in claim 9 , wherein step (a) further comprises, after said substep, providing the remaining nucleotide of the four said nucleotides.
11. The method as claimed in claim 1 , wherein step (a) comprises providing all four of the said nucleotides and contacting them with the target simultaneously.
12. The method as claimed in claim 1 , wherein any unincorporated nucleotides are removed prior to the provision of further nucleotide(s) and/or the effecting of step (c).
13. The method as claimed in claim 12 , wherein step (c) is effected after provision of said nucleotide suitable for incorporation, and without providing nucleotides which are not suitable for incorporation.