Isolation of polymerase-nucleic acid complexes

• US 8,658,364 B2
• Filed: 03/22/2012
• Issued: 02/25/2014
• Est. Priority Date: 03/23/2011
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Pham, Thang, Bhamidipati, Arunashree, Travers, Kevin, Olivares, Eric, Clark, Tyson A., Korlach, Jonas
• Primary Examiner(s): Woolwine, Samuel
• Assistant Examiner(s): Zhang, Kaijiang
• Application Number:
  US13/427,725
• Publication Number:
  US 20120322666A1
• Time in Patent Office: 705 Days
• Field of Search: 435/6.1, 506/2, 506/9
• US Class Current: 435/6.11
• CPC Class Codes: B01L 9/523 for multisample carriers, e.g. used for microtitration plates
• 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 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2523/303 Applying a physical force on a nucleic acid
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 2333/922 Ribonucleases (RNAses); Deoxyribonucleases (DNAses)
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393

Abstract

Compositions, methods and systems are provided for the isolation of polymerase-nucleic acid complexes. Complexes can be separated from free enzyme by using hook molecules to target single stranded regions on the nucleic acid. Active complexes can be isolated from mixtures having both active and inactive complexes by initiating nucleic acid synthesis so as to open up a portion of a double stranded region rendering that region single stranded. Hook molecules are targeted to bind the sequences that are thus exposed. The hook molecules bound to active polymerase-nucleic acid complex are isolated, and the active polymerase-nucleic acid complexes released.

Claims

1. A method for isolating a polymerase-nucleic acid complex comprising:

forming a polymerase-nucleic acid complex by mixing;

(a) a polymerase enzyme comprising strand displacement activity, and (b) a nucleic acid comprising a double stranded portion comprising a first strand and a complementary second strand;

initiating nucleic acid synthesis by the polymerase enzyme to produce a nascent strand complementary to the first strand, thereby displacing a portion of the second strand;

halting or reducing the rate of nucleic acid synthesis;

hybridizing a hook oligonucleotide to the complex through a capture region on the hook oligonucleotide that is complementary to at least some of the displaced portion of the second strand; and

isolating the complex using the hook oligonucleotide.

2. The method of claim 1 further comprising loading the isolated complex onto a substrate.

3. The method of claim 2 further comprising carrying out single-molecule nucleic acid sequencing with the polymerase-nucleic acid complex on the substrate.

4. The method of claim 1 wherein the laic acid is a circular nucleic acid.

5. The method of claim 4 wherein the nucleic acid comprises a double stranded central region and single stranded hairpin end regions.

6. The method of claim 1 wherein the nucleic acid comprises a linear nucleic acid comprising a linear double-stranded adaptor.

7. The method of claim 1 wherein the hook oligonucleotide also has a retrieval region having a sequence that allows the hook oligonucleotide to be bound and retrieved from a reaction mixture.

8. The method of claim 7 wherein the retrieval region is complementary to a sequence attached to beads, and the beads are used for the isolation of the nucleic acid.

9. The method of claim 8 wherein the beads are magnetic beads.

10. The method of claim 8 wherein the retrieval region comprises poly(A), poly(dA), poly(T) or poly(dT).

11. The method of claim 1 wherein the hook oligonucleotide comprises a member of a binding pair providing for isolation of the complex comprising the hook oligonucleotide bound to the nucleic acid.

12. The method of claim 11 wherein the binding pair comprises biotin, digoxigenin, a protein, or an antibody.

13. The method of claim 1 wherein halting or rate reduction comprises adding a limiting amount of polymerase synthesis reagents.

14. The method of claim 13 wherein the limiting reagents comprise nucleotides or nucleotide analogs.

15. The method of claim 1 wherein halting or rate reduction comprises adding reagents to stop the polymerization reaction after a time.

16. A method comprising;

fragmenting a double stranded DNA sample into double stranded fragments;

ligating to each end of the double stranded fragments a hairpin to produce a population of circular DNA templates having a central double stranded region and hairpin regions on each end;

exposing the population of circular DNA templates to a primer complementary to the single stranded portion of a hairpin region of the template and to a DNA polymerase enzyme having strand displacement activity under conditions in which a population of polymerase-template-primer complexes are formed;

initiating polymerase mediated DNA synthesis to extend the primer, whereby the primer is extended into the double stranded region, displacing the portion of the double strand,

halting or reducing the rate of synthesis of DNA;

adding to the population of complexes a hook oligonucleotide comprising a capture region complementary to a displaced portion of the double strand under conditions under which hybridization between the hook oligonucleotide and the DNA templates occurs;

using the hook oligonucleotide to isolate the complexes to which it hybridized;

thereby isolating complexes having active polymerase enzyme from complexes that are not active.

17. The method of claim 16 wherein the hook oligonucleotide also has a retrieval region having a sequence that allows the hook oligonucleotide to be bound and retrieved from a reaction mixture.

18. The method of claim 17 wherein the retrieval region is complementary to a sequence attached to beads, and the beads are used for the isolation of the nucleic acid.

19. A method for DNA sequencing comprising isolating active complexes by the method of claim 16, loading the active complexes onto a substrate such that single complexes can be individually optically resolved comprising;

exposing the active complexes to a plurality of differentially labeled nucleotide analogs whose labels are cleaved upon incorporation, and

initiating DNA synthesis and observing each complex to determine the time sequence of nucleotides that are incorporated.