Methods and compositions for sequencing a nucleic acid
View Patent ↗The invention provides nucleotide analogs and methods of using them in sequencing reactions.
1. A nucleotide analog monomer, wherein the monomer comprises a plurality of detectable labels attached thereto.
2. A nucleotide analog comprising a plurality of detectable labels attached via a cleavable linker to a nitrogenous base portion of said analog.
3. A nucleotide analog monomer, wherein the monomer comprises a sugar, a phosphate, and a nitrogenous base, wherein a plurality of detectable labels are attached at one or more of said sugar, phosphate, and nitrogenous base.
4. The nucleotide analog of claim 3 , wherein a first detectable label is attached to said nitrogenous base and a second detectable label is attached to said sugar or said phosphate.
5. The nucleotide analog of claim 3 , wherein said plurality of detectable labels is attached to said nitrogenous base.
6. A method for detecting a nucleotide analog of any of claims 1 - 3 , the method comprising imaging the nucleotide analog in the presence of a substance that is capable of disrupting dye:dye interactions thereby resulting in reduced or minimized dye quenching.
7. The method of the claim 6 , wherein a water miscible organic solvent is present in an imaging solution used for imaging the nucleotide analog.
8. The method of claim 7 , wherein the organic solvent is an alcohol.
9. The method of claim 8 , wherein the alcohol is methanol or ethanol.
10. The method of claim 7 , wherein the organic solvent is acetonitrile.
11. A method for sequencing a nucleic acid comprising:
directly or indirectly anchoring a nucleic acid duplex to a surface, the duplex comprising a template portion and a primer portion hybridized thereto;
exposing the duplex to nucleotide analog of any one of claims 1 - 3 or any combination thereof in the presence of a polymerase capable of catalyzing the addition of the nucleotide analog to the primer portion in a template-dependent manner;
detecting incorporation of the nucleotide analog into the primer portion; and
repeating said exposing and detecting steps at least once.
12. The method of claim 11 , further comprising:
removing unincorporated nucleotide analog and polymerase in all or some repetitions of the exposing and detecting steps.
13. The method of claim 11 , further comprising:
cleaving the label from the nucleotide analog in all or some repetitions of the exposing and detecting steps.
14. A method for sequencing a nucleic acid comprising:
anchoring a nucleic acid duplex to a surface by a polymerase adhered directly or indirectly to the surface, the duplex comprising a template portion and a primer portion hybridized thereto and the polymerase being capable of catalyzing the addition of the nucleotide analog to the primer portion in a template-dependent manner;
exposing the duplex to nucleotide analog of any one of claims 1 - 3 or any combination thereof in the presence of the polymerase;
detecting incorporation of the nucleotide analog into the primer portion; and
repeating said detecting step at least once.
15. The nucleotide analog according to claim 1 , wherein the plurality of detectable labels are all attached at a same attachment site.
16. The nucleotide analog according to claim 1 , wherein the plurality of detectable labels are all attached by a same bond.
17. The nucleotide analog according to claim 1 , wherein the plurality of detectable labels are all attached via a single cleavable linker.