Labelled nucleotides

• US 9,127,314 B2
• Filed: 11/06/2013
• Issued: 09/08/2015
• Est. Priority Date: 08/23/2002
• Status: Expired due to Fees

• Current Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Limited (Illumina Incorporated)
• Inventors: Liu, Xiaohai, Milton, John, Ruediger, Silke
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US14/073,593
• Publication Number:
  US 20140141414A1
• Time in Patent Office: 671 Days
• Field of Search: 435/1, 435/6.1, 435/91, 435/91.2, 435/91.1, 536/23.1, 536/24.3, 536/26.6
• US Class Current: 1/1
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2535/113 Cycle sequencing
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

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).

Claims

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 a disulfide; 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 disulfide 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.