Labelled nucleotides

• US 7,795,424 B2
• Filed: 07/24/2008
• Issued: 09/14/2010
• Est. Priority Date: 08/23/2002
• Status: Expired due to Term

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Milton, John, Liu, Xiaohai, Ruediger, Silke
• Primary Examiner(s): Riley; Jezia
• Application Number:
  US12/220,682
• Publication Number:
  US 20090062145A1
• Time in Patent Office: 782 Days
• Field of Search: 536/23.1, 536/25.3, 536/26.6, 435/6
• US Class Current: 536/26.6
• 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 composition comprising a phosphine cleavage reagent and a nucleotide or oligonucleotide having a base attached to a detectable label via a cleavable linker, wherein the cleavable linker comprises an azide (N₃) moiety.

2. The composition according to claim 1, wherein the phosphine cleavage reagent comprises one or more sulfonate, amine, hydroxyl or carboxylate residues.

3. The composition according to claim 1, wherein the phosphine cleavage reagent is a derivitised tri-alkyl phosphine.

4. The composition according to claim 1, wherein the phosphine cleavage reagent is a derivitised tri-aryl phosphine.

5. The composition according to claim 1, wherein the phosphine cleavage reagent is Tris (2-carboxyethyl)phosphine (TCEP).

6. The composition according to claim 1, wherein the phosphine cleavage reagent is 3,3′

,3″

-phosphinidynetris(benzenesulfonic acid).

7. The composition according to claim 1, wherein the phosphine cleavage reagent is Ph₂P(CH₂)n-R and wherein n is 1 to 5 and R is a phosphate, sulphate, carboxylate or hydroxyl group.

8. The composition according to claim 1, wherein the nucleotide or oligonucleotide further comprises a hydroxyl protecting group attached to the 2′

or 3′

oxygen atom.

9. The composition according to claim 8, wherein the hydroxyl protecting group comprises an azide or an allyl moiety.

10. The composition according to claim 9, wherein the hydroxyl protecting group is azidomethyl.

20. The composition according to claim 1, wherein the nucleotide or oligonucleotide is attached to a solid support.

11. A composition comprising a thiol cleavage reagent and a nucleotide or oligonucleotide having a base attached to a detectable label via a cleavable linker, wherein the cleavable linker comprises an azide (N₃) moiety.

12. The composition according to claim 11, wherein the thiol cleavage reagent is dithiothreitol (DTT).

13. The composition according to claim 11, wherein the nucleotide or oligonucleotide further comprises a hydroxyl protecting group attached to the 2′

or 3′

oxygen atom.

14. A composition comprising a phosphine cleavage reagent, a palladium cleavage reagent and a nucleotide or oligonucleotide having a base attached to a detectable label via a cleavable linker, wherein the cleavable linker comprises an allyl moiety.

15. The composition according to claim 14, wherein the phosphine cleavage reagent comprises one or more sulfonate, amine, hydroxyl or carboxylate residue.

16. The composition according to claim 14, wherein the phosphine cleavage reagent is a derivitised tri-alkyl phosphine or a derivitised tri-aryl phosphine.

17. The composition according to claim 14, wherein the phosphine cleavage reagent is Tris (2-carboxyethyl)phosphine (TCEP), 3,3′

,3″

-phosphinidynetris(benzenesulfonic acid) or Ph₂P(CH₂)n-R, wherein n is 1 to 5 and R is a phosphate, sulphate carboxylate or hydroxyl group.

18. The composition according to claim 14, wherein the nucleotide or oligonucleotide further comprises a hydroxyl protecting group attached to the 2′

or 3′

oxygen atom.

19. The composition according to claim 18, wherein the hydroxyl protecting group comprises an allyl moiety.