Nucleotide analogs
View Patent ↗The disclosure provides nucleotide analogs and methods of their use. Analogs of the invention comprise a reporter molecule (label) attached via the N4, N6, O4, or O6 position of the nitrogenous base portion of the analog. In a preferred embodiment, nucleotide analogs of the invention comprise a label attached to the nitrogenous base portion of the analog via a cleavable linker at the N4, O4, N6 or O6 position.
1. A nucleotide analog, the analog comprising:
a nitrogenous base;
a linker attached at the O4 or O6 of the nitrogenous base; and
a moiety attached to the nitrogenous base via the linker.
2. The analog according to claim 1 , wherein the linker comprises a cleavable bond.
3. The analog according to claim 1 , wherein the moiety is a label.
4. The analog according to claim 3 , wherein the label is a detectable label.
5. The analog according to claim 4 , wherein the detectable label is an optically detectable label.
6. The analog according to claim 5 , wherein the optically detectable label is a fluorescent label.
7. The analog according to claim 6 , wherein the fluorescent label is selected from the group consisting of: Cy-5, Cy-3, Joe, Rox, and TAMRA.
8. The analog according to claim 1 , wherein the moiety is a blocking group.
9. The analog according to claim 1 , further comprising a mono-, di-, tri-, or bis-phosphate.
10. The analog according to claim 1 , further comprising a ribose sugar or a deoxyribose sugar.
11. A nucleotide analog, the analog comprising:
a nitrogenous base;
a linker attached at the N4 or N6 of the nitrogenous base; and
a moiety attached to the nitrogenous base via the linker, wherein the linker comprises a cleavable bond such that a native nucleotide monophosphate is regenerated upon incorporation of the analog into a nucleic acid duplex.
12. The analog according to claim 11 , wherein the moiety is a label.
13. The analog according to claim 12 , wherein the label is an optically detectable label.
14. The analog according to claim 13 , wherein the optically detectable label is a fluorescent label.
15. The analog according to claim 14 , wherein the fluorescent label is selected from the group consisting of: Cy-5, Cy-3, Joe, Rox, and TAMRA.
16. The analog according to claim 11 , wherein the moiety is a blocking group.
17. The analog according to claim 11 , further comprising a mono-, di-, tri-, or bis-phosphate.
18. The analog according to claim 11 , further comprising a ribose sugar or a deoxyribose sugar.
19. A method for sequencing a nucleic acid, the method comprising the steps of:
attaching directly or indirectly a nucleic acid duplex to a surface, the duplex comprising a template portion and a primer portion hybridized thereto;
exposing the duplex to an analog of claim 1 or claim 11 in the presence of a polymerase capable of catalyzing addition of the analog to the primer portion in a template-dependent manner;
removing unincorporated analog and polymerase;
detecting incorporation of analog into said primer; and
repeating the exposing, removing, and detecting steps at least once.
20. The method according to claim 19 , further comprising the step of cleaving the molecule from the analog after the detecting step.