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US 10,480,025 B2Granted
Labelled nucleotides
AssigneeIllumina Cambridge Limited
InventorsShankar Balasubramanian, Colin Lloyd Barnes, Xiaohai Liu, John Milton, Xiaolin Wu, Harold Swerdlow
Life of this patent
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Abstract
Nucleosides and nucleotides are disclosed that are linked to detectable labels via a cleavable linker group.
Claims62
A nucleotide or nucleoside molecule having a ribose or deoxyribose sugar moiety and a base linked to a detectable label via a cleavable linker, wherein the sugar moiety comprises a protecting group attached via a 3′ oxygen atom, and wherein said protecting group comprises an azido group that can be modified or removed to expose a 3′ OH group.
A kit comprising a plurality of nucleotides, each having a ribose or deoxyribose sugar moiety and a base linked to a detectable label via a cleavable linker, wherein the sugar moiety comprises a protecting group attached via the 3′ oxygen atom, and said protecting group comprises an azido group that can be modified or removed to expose a 3′ OH group, and wherein the detectable label linked to each nucleotide can be distinguished upon detection from the detectable labels used for structurally different nucleotides.
A nucleotide molecule, wherein:
- the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
- the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
- the nucleotide molecule comprises a 7-deazaadenine base,
- the 7-deazaadenine base of the nucleotide molecule is substituted with a linker at the 7-deaza position,
- the linker is bound to a fluorophore,
- the linker comprises a cleavable moiety located between the 7-deazaadenine base and the fluorophore,
- the cleavable moiety comprises a disulfide moiety or an azido moiety,
- the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
- upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the 7-deazaadenine base, and
- upon cleavage of the azidomethyl moiety, a 3-OH group is formed and the formation of the 3-OH group allows addition of a nucleotide to the 3′ oxygen atom.
7 more elements
A 7-substituted 7-deaza-2′-deoxy-adenosine triphosphate nucleotide molecule, wherein:
- the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
- the 7-deazaadenine base of the molecule is linked to a fluorophore via a cleavable linker at the 7-deaza position of the base,
- the linker comprises a cleavable moiety located between the 7-deazaadenine base and the fluorophore,
- the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
- upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the 7-deazaadenine base, and
- upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.
3 more elements
A nucleotide molecule having the structure:
- wherein:
- X is triphosphate,
- R1 is —OCH2N3,
- R2 is hydrogen,
- Linker is a cleavable linker comprising a cleavable moiety,
- Label is a fluorophore, and
- upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.
4 more elements
A nucleotide molecule, wherein:
- the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
- the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
- the nucleotide molecule comprises a 7-deazaguanine base,
- the 7-deazaguanine base of the nucleotide molecule is substituted with a linker at the 7-deaza position,
- the linker is bound to a fluorophore,
- the linker comprises a cleavable moiety located between the 7-deazaguanine base and the fluorophore,
- the cleavable moiety comprises a disulfide moiety or an azido moiety,
- the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
- upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the 7-deazaguanine base, and
- upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′ oxygen atom.
7 more elements
A 7-substituted 7-deaza-2′-deoxy-guanosine triphosphate nucleotide molecule, wherein:
- the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
- the 7-deazaguanine base of the molecule is linked to a fluorophore via a cleavable linker at the 7-deaza position of the base,
- the linker comprises a cleavable moiety located between the 7-deazaguanine base and the fluorophore,
- the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
- upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the 7-deazaguanine base, and
- upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.
3 more elements
A nucleotide molecule having the structure:
- wherein:
- X is triphosphate,
- R1 is —OCH2N3,
- R2 is hydrogen,
- Linker is a cleavable linker comprising a cleavable moiety,
- Label is a fluorophore, and
- upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.
4 more elements
A nucleotide molecule, wherein:
- the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
- the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
- the nucleotide molecule comprises a cytosine base,
- the cytosine base of the nucleotide molecule is substituted with a linker at the 5 position of the base,
- the linker is bound to a fluorophore,
- the linker comprises a cleavable moiety located between the cytosine base and the fluorophore,
- the cleavable moiety comprises a disulfide moiety or an azido moiety,
- the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
- upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the cytosine base, and
- upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′ oxygen atom.
7 more elements
A 5-substituted 2′-deoxy-cytidine triphosphate nucleotide molecule, wherein:
- the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
- the cytosine base of the molecule is linked to a fluorophore via a cleavable linker at the 5 position of the base,
- the linker comprises a cleavable moiety located between the cytosine base and the fluorophore,
- the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
- upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the cytosine base, and
- upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.
3 more elements
A nucleotide molecule having the structure:
- wherein:
- X is triphosphate,
- R1 is —OCH2N3,
- R2 is hydrogen,
- Linker is a cleavable linker comprising a cleavable moiety,
- Label is a fluorophore, and
- upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.
4 more elements
A nucleotide molecule, wherein:
- the nucleotide molecule comprises an azidomethyl moiety attached to the 3′ oxygen atom such that the azidomethyl moiety prevents addition of a nucleotide to the 3′ oxygen atom,
- the nucleotide molecule comprises a 2′ deoxyribose, wherein said 2′ deoxyribose comprises a triphosphate at the 5′ position,
- the nucleotide molecule comprises a uracil base,
- the uracil base of the nucleotide molecule is substituted with a linker at the 5 position of the base,
- the linker is bound to a fluorophore,
- the linker comprises a cleavable moiety located between the uracil base and the fluorophore,
- the cleavable moiety comprises a disulfide moiety or an azido moiety,
- the cleavable moiety and the azidomethyl moiety are cleavable chemically under identical conditions,
- upon cleavage of the cleavable moiety, the fluorophore is removed from the nucleotide molecule and part of the linker remains attached to the uracil base, and
- upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′ oxygen atom.
7 more elements
A 5-substituted 2′-deoxy-uridine triphosphate nucleotide molecule, wherein:
- the 3′-O is substituted with an azidomethyl moiety that prevents addition of a nucleotide to the 3′-O,
- the uracil base of the molecule is linked to a fluorophore via a cleavable linker at the 5 position of the base,
- the linker comprises a cleavable moiety located between the uracil base and the fluorophore,
- the cleavable linker and the azidomethyl moiety are cleavable in a chemical reaction under identical conditions,
- upon cleavage of the cleavable moiety, the fluorophore is removed from the molecule and part of the linker remains attached to the uracil base, and
- upon cleavage of the azidomethyl moiety, a 3′-OH group is formed and the formation of the 3′-OH group allows addition of a nucleotide to the 3′-O.
3 more elements
A nucleotide molecule having the structure:
- wherein:
- X is triphosphate,
- R1 is —OCH2N3,
- R2 is hydrogen,
- Linker is a cleavable linker comprising a cleavable moiety,
- Label is a fluorophore, and
- upon cleavage of the cleavable moiety, Label is removed and part of Linker remains attached to the nucleobase.
4 more elements
Dependencies are inferred from the claim text.