Modified nucleotides

摘要

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.

主权项

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.