Methods and devices for nucleic acid sequence determination
View Patent ↗Methods of the invention comprise methods and devices for nucleic acid sequence determination. Generally, the invention relates to preparing a substrate for sequencing a target nucleic acid.
1. A method for conducting a chemical reaction on a surface, the method comprising the steps of:
forming a plurality of nucleic acid template primer duplexes, wherein for each duplex, the nucleic acid comprises a homopolymer tail and the primer hybridizes to the tail of the nucleic acid;
directly or indirectly attaching the duplexes to a surface comprising epoxides or epoxide derivatives;
blocking unreacted epoxides or epoxide derivatives on the surface;
conducting a chemical reaction between the duplexes and optically labeled reversible chain elongation interfering nucleotides wherein the conducting step comprises exposing the duplex to at least one nucleotide under conditions sufficient to extend the primer by at least one base; and
observing labels incorporated into the one or more duplexes on the surface.
2. The method according to claim 1 , wherein the nucleic acids are attached to the surface through a reactive amino addition or via a bi-functional linkage.
3. The method according to claim 1 , wherein at least some of the nucleic acids are individually optically resolvable.
4. The method according to claim 1 , wherein the nucleic acids are DNA or RNA.
5. The method according to claim 1 , wherein the optically detectable label is a fluorescent molecule.
6. The method according to claim 5 , wherein the fluorescent molecule is Cyanine-3 or Cyanine-5.
7. The method according to claim 1 , further comprising exposing the surface to a drying agent.
8. The method according to claim 7 , wherein the drying agent is selected from the group consisting of PRB, EtOH, air, and N 2 .
9. The method according to claim 1 , further comprising determining nucleic sequences of one or more of the nucleic acids.
10. The method according to claim 9 , where determining comprises conducting sequencing by synthesis.
11. The method according to claim 1 , wherein the surface is selected from the group consisting of a glass, a fused silica, a plastic, and a gel.
12. The method according to claim 1 , wherein the epoxide derivatives are streptavidnated epoxides.
13. The method according to claim 2 , wherein the epoxides are unreacted epoxides.
14. The method according to claim 1 , wherein the label is the chain elongation interfering moiety of the nucleotide.
15. A method for conducting a chemical reaction on a surface, the method comprising the steps of:
forming a plurality of nucleic acid template primer duplexes, wherein for each duplex, the nucleic acid comprises a homopolymer tail and the primer hybridizes to the tail of the nucleic acid;
directly or indirectly attaching the duplexes to a surface;
blocking the surface using a blocking solution, wherein the blocking solution generates a net negative charge on the surface;
conducting a chemical reaction between the duplexes and optically labeled reversible chain elongation interfering nucleotides wherein the conducting step comprises exposing the duplex to at least one nucleotide under conditions sufficient to extend the primer by at least one base; and
observing labels incorporated into the one or more duplexes on the surface.
16. The method according to claim 15 , further comprising exposing the surface to a drying agent.
17. The method according to claim 15 , further comprising determining nucleic sequences of one or more of the nucleic acids.
18. A method for conducting a chemical reaction on a surface, the method comprising the steps of:
forming a plurality of nucleic acid template primer duplexes, wherein for each duplex, the nucleic acid comprises a homopolymer tail and the primer hybridizes to the tail of the nucleic acid;
directly or indirectly attaching the duplexes to a surface;
blocking the surface using a blocking solution;
conducting a chemical reaction between the duplexes and optically labeled reversible chain elongation interfering nucleotides wherein the conducting step comprises exposing the duplex to at least one nucleotide under conditions sufficient to extend the primer by at least one base;
rinsing unincorporated labeled nucleotides from the surface using a rinse solution comprising an active rinse agent; and
observing labels incorporated in the one or more duplexes on the surface.
19. The method according to claim 18 , further comprising exposing the surface to a drying agent.
20. The method according to claim 18 , further comprising determining nucleic sequences of one or more of the nucleic acids.
21. The method according to claim 18 , wherein the active rinse agent comprises at least one hydrophobic compound.
22. The method according to claim 18 , wherein the active rinse agent is acetonitrile.
23. The method according to claim 18 , wherein acetonitrile makes-up from about 10% to about 50% of the rinse solution.