Controllable strand scission of mini circle DNA

• US 8,551,704 B2
• Filed: 02/11/2008
• Issued: 10/08/2013
• Est. Priority Date: 02/16/2007
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Otto, Geoffrey, Lyle, John
• Primary Examiner(s): Chunduru, Prabha
• Application Number:
  US12/029,273
• Publication Number:
  US 20080199874A1
• Time in Patent Office: 2,066 Days
• Field of Search: 435/6, 435/91.2, 536/24.33
• US Class Current: 435/6.12
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2525/307 Circular oligonucleotides

Abstract

The invention provides methods and compositions for the controlled termination of polymerase mediated primer extension reactions. The methods and compositions of the invention are broadly useful, and in a preferred aspect can be used in identifying sequence elements of template nucleic acids. Control of termination not only provides temporal control over termination, but, when used in conjunction with optically confined reaction regions, also spatially controls such termination.

Claims

1. A method for the synthesis of a nucleic acid comprising:

providing a complex, wherein said complex comprises a polymerase, a first template, nucleoside triphosphates and/or analogues thereof, and a primer, wherein said first template comprises at least one selectively cleavable linkage within a nucleotide sequence of the template, and further wherein said first template is a circular template;

cleaving said selectively cleavable linkage to provide a new terminus in the template in the complex; and

synthesizing said nucleic acid by polymerase mediated, first template dependent extension of said primer.

2. The method of claim 1, wherein said cleaving said selectively cleavable linkage arrests said synthesis of said nucleic acid.

3. The method of claim 1, wherein subsequent to said cleaving of said cleavable linkage, said first template is replaced by a second template, and synthesizing said nucleic acid is resumed by a polymerase mediated, second template dependent extension of said primer.

4. The method of claim 1, wherein said cleavable linkage is selected from the group consisting of a photocleavable linkage and an enzymatically cleavable linkage.

5. The method of claim 1, wherein said selectively cleavable linkage comprises a restriction site.

6. The method of claim 1, wherein said complex is immobilized upon a surface of a substrate.

7. The method of claim 6, wherein said at least one of said polymerase, first template, nucleoside triphosphates and/or analogues thereof, and primer of said complex is immobilized upon said surface of said substrate.

8. The method of claim 7, wherein said polymerase is immobilized upon said surface of said substrate.

9. The method of claim 7, wherein said first template is immobilized upon said surface of said substrate,

10. The method of claim 7, wherein said nucleoside triphosphates and/or analogues thereof are immobilized upon said surface of said substrate.

11. The method of claim 7, wherein said printer is immobilized upon said surface of said substrate.

12. The method of claim 6, wherein said complex is immobilized in an optically confined region on said surface of said substrate.

13. The method of claim 6, wherein said substrate is transparent.

14. The method of claim 12, wherein said complex is immobilized within art illumination volume of a zero mode waveguide.

17. The method of claim 1, wherein said new terminus is a new 5′

terminus.

18. The method of claim 17, wherein said new 5′

terminus is a run-off point for the polymerase.

15. A method of identifying a base in a nucleic acid template, comprising:

providing a polymerase/template/primer complex, wherein the template comprises a cleavable linking group within a nucleotide sequence of the template, and further wherein said template is a circular template;

cleaving the cleavable linking group to arrest synthesis;

identifying one or more nucleotides or nucleotide analogues adjacent to said cleavable linking group, thereby identifying a base in said nucleic acid template.

16. The method of claim 15, wherein said cleavable linking group is selected from the group consisting of a photocleavable linking group and an enzymatically cleavable linking group.