Single molecule sequencing of captured nucleic acids

1. A method for obtaining sequence information from a nucleic acid, the method comprising the steps of:

capturing target nucleic acids with a sequence-specific capture probe to produce a target/probe duplex;

isolating said target nucleic acids from said target/probe duplexes;

attaching said target nucleic acids to a surface such that at least a portion of said target nucleic acids are individually optically resolvable;

exposing said target nucleic acids to a nucleic acid primer capable of forming a target/primer duplex;

introducing a polymerase and at least one nucleotide species comprising an optically-detectable label under conditions sufficient for template-dependent nucleotide addition to said primer;

removing unincorporated nucleotide;

identifying nucleotide species incorporated into said primer, thereby obtaining sequence information from said target.

2. The method of claim 1 , further comprising the step of removing said optically-detectable label from nucleotide incorporated into said primer.

3. The method of claim 1 , wherein said capturing step comprises hybridizing said target nucleic acids to capture probes immobilized in a polyacrylamide gel.

4. The method of claim 1 , wherein said capture probes are attached to a bead.

5. The method of claim 1 , wherein said capture probes are immobilized on a chromatography column.

6. The method of claim 1 , wherein said optically-detectable label is a fluorescent label.

7. The method of claim 1 , wherein said introducing, removing, and identifying steps are repeated at least once.

8. The method of claim 1 , wherein said surface comprises an epoxide.

9. The method of claim 1 , wherein said attaching step comprises modifying said target nucleic acid to introduce a 3′ or 5′ amine.

10. The method of claim 1 , wherein said primer is attached to said surface.

11. The method of claim 1 , wherein said surface is steptavidinated.

12. The method of claim 11 , wherein said target and/or said primer comprise a biotin end label.

13. A method for obtaining sequence information from a selected nucleic acid, the method comprising the steps of:

capturing target nucleic acids with a sequence-specific capture probes to produce a plurality of target/probe duplexes;

attaching members of said plurality of duplexes to a surface such that at least some members of said plurality of duplexes are individually optically resolvable;

introducing a polymerase and at least one nucleotide species comprising an optically-detectable label under conditions sufficient for template-dependent nucleotide addition to said probe;

removing unincorporated nucleotide;

identifying nucleotide species incorporated into said probe, thereby obtaining sequence information from said target.

14. The method of claim 13 , wherein said introducing, removing, and identifying steps are repeated at least once.

15. The method of claim 14 , wherein said surface is an epoxide surface.

16. The method of claim 15 , wherein said epoxide surface is derivatized for attachment of said duplex.

17. The method of claim 13 , wherein said label is a fluorescent label.

18. A method for obtaining sequence information from a selected nucleic acid, the method comprising the steps of:

capturing target nucleic acids with sequence-specific capture probes attached to a surface to form a target/probe duplex;

introducing a polymerase and at least one nucleotide species comprising an optically-detectable label under conditions sufficient for template-dependent nucleotide addition to said probe;

removing unincorporated nucleotide;

identifying nucleotide species incorporated into said probe, thereby obtaining sequence information from said target.

19. The method of claim 18 , wherein said surface is an epoxide surface.

20. The method of claim 18 , wherein said introducing, removing, and identifying steps are repeated at least once.