Single molecule sequencing of captured nucleic acids

• US 9,868,978 B2
• Filed: 02/02/2016
• Issued: 01/16/2018
• Est. Priority Date: 08/26/2005
• Status: Active Grant

• Current Assignee: Standard Biotools Incorporated
• Original Assignee: Fluidigm Corporation
• Inventors: Lapidus, Stanley N.
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/013,915
• Publication Number:
  US 20160362741A1
• Time in Patent Office: 714 Days
• Field of Search: None
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target

Abstract

The invention provides methods and devices for detecting, enumerating or identifying target nucleic acid molecules using immobilized capture probes and single molecule sequencing techniques.

Claims

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.