Labelled nucleotides

• US 20100311178A1
• Filed: 06/21/2010
• Published: 12/09/2010
• Est. Priority Date: 08/23/2002
• Status: Active Grant

• Current Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Original Assignee: Illumina Cambridge Ltd. (California) (Illumina Incorporated)
• Inventors: Milton, John, Liu, Xiaohai, Ruediger, Silke
• Granted Patent: US 8,084,590 B2
• Time in Patent Office: Days
• US Class Current: 436/94
• 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, 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).

Claims

1-60. -60. (canceled)

61. A nucleoside or nucleotide comprising a base attached to a detectable label via a cleavable linker, wherein the cleavable linker comprises an azido group.

62. The nucleoside or nucleotide of claim 61, wherein the nucleoside or nucleotide further comprises a dideoxyribose moiety.

63. The nucleoside or nucleotide of claim 61, wherein the nucleoside or nucleotide further comprises a ribose or deoxyribose moiety with a hydroxyl protecting group attached to the 2′

or 3′

oxygen atom.

64. The nucleoside or nucleotide of claim 63, wherein the linker and protecting group are cleavable under the same conditions.

65. The nucleoside or nucleotide of claim 63, wherein the protecting group is azidomethyl.

66. The nucleoside or nucleotide of claim 61, wherein the base is a purine, pyrimidine or deazapurine.

67. The nucleoside or nucleotide of claim 61, wherein the detectable label is a fluorophore.

68. An oligonucleotide comprising a nucleoside or nucleotide of claim 61.

69. The oligonucleotide of claim 68, wherein the oligonucleotide is attached to a solid support.

70. A solid support comprising the oligonucleotide of claim 68.

71. A composition comprising four nucleosides or nucleotides, wherein each nucleoside or nucleotide comprises a base attached to a detectable label via a cleavable linker, wherein the cleavable linker comprises an azido group and each nucleoside or nucleotide carries a different detectable label.

72. 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 comprising an azido group; and

a polynucleotide complementary to the target polynucleotide, one of said provided nucleotides being suitable for incorporation into said complementary polynucleotide;

(b) incorporating the nucleotide suitable for incorporation into said complementary polynucleotide; and

(c) detecting the detectable label, thereby determining the identity of a nucleotide.

73. The method according to claim 72, 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.

74. The method according to claim 72, wherein step (a) comprises providing four nucleotides and contacting them with the target simultaneously.

75. The method according to claim 72, wherein any unincorporated nucleotides are removed prior to the provision of further nucleotide(s).

76. The method according to claim 72, wherein the linker is cleaved after detecting the label.

77. The method according to claim 76, wherein said cleavage is carried out with a phosphine.

78. The method according to claim 77, wherein said phosphine comprises one or more sulfonate, amine, hydroxyl or carboxylate residues.

79. The method of claim 75, wherein any unincorporated nucleotides are removed prior to detecting step (c).