Modified nucleotides
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.
1. A method of controlling the incorporation of a nucleotide having a deoxyribose sugar moiety into a growing polynucleotide that is complementary to a target single-stranded polynucleotide, the method comprising:
(a) incorporating the nucleotide into the growing complementary polynucleotide, thereby preventing introduction of subsequent nucleotide molecules into the growing polynucleotide, wherein the nucleotide comprises a fluorescent detectable label linked by a phosphine-cleavable azide-containing linker, and a protecting group is attached to the 3′ oxygen atom of the deoxyribose sugar moiety; and
(b) reacting the nucleotide with a water-soluble phosphine to cleave the linker and the protecting group in a single step.
2. The method of claim 1 , wherein the method is used in a sequencing reaction.
3. The method of claim 1 , wherein the method is used in a synthesis reaction.
4. The method of claim 1 , wherein the incorporating step comprises contacting the growing complementary polynucleotide with a polymerase.
5. The method of claim 1 , further comprising repeating the incorporating and reacting steps one or more times, thereby incorporating multiple nucleotides into the growing complementary polynucleotide.
6. The method of claim 1 , wherein the linker comprises a group having formula C(R′) 2 N 3 , wherein each R′ is independently hydrogen, alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, or amido group, or (R′) 2 represents an alkylidene group of formula ═C(R′″) 2 , wherein each R′″ is independently selected from hydrogen, halogen and alkyl.
7. The method of claim 6 , wherein each R′ is hydrogen.
8. The method of claim 1 , wherein the phosphine is derivatized with one or more sulfonate, amine, hydroxyl or carboxylate residues to render it water-soluble.
9. The method of claim 8 , wherein the derivatized phosphine is selected from a triaryl phosphine and a trialkyl phosphine.
10. The method of claim 9 , wherein the triaryl phosphine is 3,3′,3″-phosphinidynetris(benzenesulfonic acid) or a salt thereof.
11. The method of claim 9 , wherein the trialkyl phosphine is tris(2-carboxyethyl)phosphine or a salt thereof.