Modified nucleotides
0 Assignments
0 Petitions
Accused Products
Abstract
The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3′-OH blocking group covalently attached thereto, such that the 3′ carbon atom has attached a group of the structure —O-Z wherein Z is any of —C(R′)2-O—R″, —C(R′)2-N(R″)2, —C(R′)2-N(H)R″, —C(R′)2-S—R″ and —C(R′)2-F, wherein each R″ is or is part of a removable protecting group; each R′ is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group; or (R′)2 represents an alkylidene group of formula ═C(R′″)2 wherein each R′″ may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R″ is exchanged for H or, where Z is —C(R′)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3′OH; with the proviso that where Z is —C(R′)2-S—R″, both R′ groups are not H.
-
Citations
55 Claims
-
1-33. -33. (canceled)
-
34. A method for determining the sequence of a target single-stranded polynucleotide, comprising monitoring the sequential incorporation of complementary nucleotides, wherein at least one incorporation is of a nucleotide having a removable 3′
- -OH blocking group covalently attached thereto, such that the 3′
carbon atom has attached a group of the structure
—
O-Zwherein Z is any of allyl, —
C(R′
)2—
N3, —
C(RIV)2—
O—
R″
, —
C(R′
)2—
N(R″
)2, —
C(R′
)2—
N(H)R″
, or —
C(RIV)2—
S—
R″
,wherein —
C(RIV)2—
O—
R″
is of the formula —
CR4(R5)—
O—
CR4(R5)—
OR6 or of the formula —
CR4(R5)—
O—
CR4(R5)—
SR6; and
wherein —
C(RIV)2—
S—
R″
is of the formula —
CR4(R5)—
S—
CR4(R5)—
OR6 or of the formula —
CR4(R5)—
S—
CR4(R5)—
SR6;each R″
is or is part of a removable protecting group;each R′
is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group;
or (R′
)2 represents an alkylidene group of formula ═
C(R′
″
)2 wherein each R′
″
may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups;each R4 and R5 is independently a hydrogen atom or an alkyl group; and R6 is alkyl, cycloalkyl, alkenyl, cycloalkenyl or benzyl, and wherein the blocking group is removed prior to introduction of the next complementary nucleotide. - View Dependent Claims (35, 37, 38, 39, 51, 52, 53, 54, 55)
- -OH blocking group covalently attached thereto, such that the 3′
-
36. A method for determining the sequence of a target single-stranded polynucleotide, comprising:
-
(a) providing a plurality of different nucleotides wherein each of said plurality of different nucleotides has a removable 3′
-OH blocking group covalently attached thereto, such that the 3′
carbon atom has attached a group of the structure
—
O-Zwherein Z is any of allyl, —
C(R′
)2—
N3, —
C(RIV)2—
O—
R″
, —
C(R′
)2—
N(R″
)2, —
C(R′
)2—
N(H)R″
, or —
C(RIV)2—
S—
R″
,wherein —
C(RIV)2—
O—
R″
is of the formula —
CR4(R5)—
O—
CR4(R5)—
OR6 or of the formula —
CR4(R5)—
O—
CR4(R5)—
SR6; and
wherein —
C(RIV)2—
S—
R″
is of the formula —
CR4(R5)—
S—
CR4(R5)—
OR6 or of the formula —
CR4(R5)—
S—
CR4(R5)—
SR6;each R″
is or is part of a removable protecting group;each R′
is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy or amido group, or a detectable label attached through a linking group;
or (R′
)2 represents an alkylidene group of formula ═
C(R′
″
)2 wherein each R′
″
may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups;each R4 and R5 is independently a hydrogen atom or an alkyl group; and R6 is alkyl, cycloalkyl, alkenyl, cycloalkenyl or benzyl, and wherein each type of nucleotide comprises a base linked to a detectable label and the detectable label linked to each type of nucleotide can be distinguished upon detection from the detectable label used for other types of nucleotides; (b) incorporating the nucleotide into the complement of the target single-stranded polynucleotide; (c) detecting the label of the nucleotide of (b), thereby determining the type of nucleotide incorporated; (d) removing the label of the nucleotide of (b) and the blocking group; and (e) optionally repeating steps (b)-(d) one or more times; thereby determining the sequence of a target single-stranded polynucleotide. - View Dependent Claims (40, 41, 42, 43, 44, 45)
-
-
46-50. -50. (canceled)
Specification