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 ribose or deoxyribose sugar moiety, and said nucleotide is complementary to a second nucleotide in a target single-stranded polynucleotide in a synthesis or sequencing reaction, comprising
incorporating into the growing complementary polynucleotide said nucleotide, the incorporation of said nucleotide preventing or blocking introduction of subsequent nucleoside or nucleotide molecules into said growing complementary polynucleotide; wherein a protecting group is attached to the 3′ or the 2′ oxygen atom of the ribose or deoxyribose sugar moiety of said nucleotide; and said nucleotide comprises a detectable label linked by a linker; and
reacting the nucleotide with a water-soluble phosphine to cleave the linker;
wherein said detectable label is a fluorophore; and
wherein said water-soluble phosphine also cleaves said protecting group.
2. The method of claim 1 , wherein said linker contains a disulfide linkage.
3. The method of claim 1 , wherein said linker is a phosphine-cleavable azide-containing linker.
4. The method of claim 1 , further comprising repeating said incorporating and reacting steps one or more times, thereby incorporating multiple nucleotides into the growing complementary polynucleotide.
5. The method of claim 1 , wherein said incorporating comprises contacting the growing complementary polynucleotide with a polymerase.
6. The method of claim 1 , wherein said protecting group is attached to the 3′ oxygen atom of the ribose or deoxyribose sugar moiety of said nucleotide.
7. The method of claim 1 , wherein said protecting group is attached to the 2′ oxygen atom of the ribose or deoxyribose sugar moiety of said nucleotide.
8. A method of controlling the incorporation of a nucleotide having a ribose or deoxyribose sugar moiety, and said nucleotide is complementary to a second nucleotide in a target single-stranded polynucleotide in a synthesis or sequencing reaction, comprising
incorporating said nucleotide into the growing complementary polynucleotide with a polymerase, the incorporation of said nucleotide preventing or blocking introduction of subsequent nucleoside or nucleotide molecules into said growing complementary polynucleotide; wherein a protecting group is attached to the 3′ oxygen atom of the ribose or deoxyribose sugar moiety of said nucleotide; and said nucleotide comprises a detectable label linked by a linker; and
reacting the nucleotide with a water-soluble phosphine to cleave the linker;
wherein said detectable label is a fluorophore; and
wherein said water-soluble phosphine also cleaves said protecting group.
9. The method of claim 8 , wherein said linker contains a disulfide linkage.
10. The method of claim 8 , wherein said linker is a phosphine-cleavable azide-containing linker.
11. The method of claim 8 , further comprising repeating said incorporating and reacting steps one or more times, thereby incorporating multiple nucleotides into the growing complementary polynucleotide.