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, comprising:
A nitrogenous base;
A linker attached at the N4, N6, O4, or O6 of said nitrogeneous base; and
A detectable label attached to said nitrogenous base via said linker,
wherein said linker comprises a cleavable bond such that a native nucleotide monophosphate is regenerated upon incorporation of said analog into a nucleic acid duplex.
2. The nucleotide analog of claim 1 , further comprising a mono-, di-, tri-, or bis-phosphate.
3. The nucleotide analog of claim 1 , further comprising a ribose sugar or a deoxyribose sugar.
4. The nucleotide analog of claim 1 , wherein said analog has the structure:
Wherein P is a monophosphate, diphosphate, bisphosphate, triphosphate, or halogen-substituted phosphate, and wherein X and Y are independently selected from the group consisting of a cleavable bond, an alkyl, an esther, an ether, or a combination of foregoing.
5. The nucleotide analog of claim 4 , wherein X is an alkyl group or an aryl group.
6. The nucleotide analog of claim 4 , wherein said alkyl group comprises between about 2 and about 10 atoms.
7. The nucleotide analog of claim 1 , wherein said label is selected from cyanine-5, Cy-5, Cy-3, Joe, Rox, and TAMRA.
8. The nucleotide analog of claim 1 , wherein said analog has the structure:
Wherein P is a monophosphate, diphosphate, bisphosphate, triphosphate, or halogen-substituted phosphate, and wherein X and Y are independently selected from the group consisting of a cleavable bond, an alkyl, an esther, an ether, a halogen, or a combination of the foregoing.
9. The nucleotide analog of claim 8 , wherein X is (CH 2 ) n , and wherein n is between about 2 and about 10.
10. The nucleotide analog of claim 8 , wherein Y is a carbonyl.
11. The nucleotide analog of claim 8 , wherein said label is an optically-detectable label.
12. The nucleotide analog of claim 11 , wherein said detectable label is selected from cyanine-5, Cy-5, Cy-3, Joe, Rox, and TAMRA.
13. A method for sequencing a nucleic acid, the method comprising the steps of:
anchoring a nucleic acid duplex to a surface, the duplex comprising a template portion and a primer portion hybridized thereto:
Exposing said duplex to an analog of claim 4 or claim 10 in the presence of a polymerase capable of catalyzing addition of said analog to said primer portion in a template-dependent manner;
Removing unincorporated analog and polymerase;
Detecting incorporation of analog into said primer; and
Repeating said exposing, removing, and detecting steps at least once.
14. The method of claim 13 , further comprising the step of cleaving detectable label from said analog after said detecting step.