Isolation of polymerase-nucleic acid complexes

• US 10,000,805 B2
• Filed: 09/20/2016
• Issued: 06/19/2018
• 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): Thomas, David C
• Application Number:
  US15/270,727
• Publication Number:
  US 20170107570A1
• Time in Patent Office: 637 Days
• Field of Search: None
• 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 isolating DNA having a modified or unnatural base. Circular DNA fragments, each comprising a double stranded DNA central region and single stranded regions on the ends of the double stranded regions, are obtained. Some of the fragments have one or more modified or unnatural base. The DNA fragments are treated with a primer and a polymerase such that the polymerase extends the primer to copy at least one of the strand of the double stranded region. This results in rendering the other strand single stranded. A binding protein or antibody that is specific to the modified or unnatural base is then used to isolate strands containing the modified or unnatural bases. Methods for loading such complexes onto substrates and for single molecule sequencing of such complexes are also provided.

Claims

1. A method for isolating DNA having a modified or unnatural base comprising:

obtaining a library of circular DNA fragments each comprising a double stranded DNA central region and single stranded regions on the ends of the double stranded regions wherein at least some of the fragments comprise one or more modified or unnatural base;

treating the DNA fragments with a primer and a polymerase under conditions where the polymerase extends the primer to copy at least a portion of one strand of the double stranded region so as to render the other strand of the double-stranded portion single stranded;

using a binding protein or antibody that is specific to the one or more modified or unnatural base on the portion of the circular DNA fragments that is rendered single stranded to isolate strands containing one or more modified or unnatural base from those that do not contain an unnatural or modified base.

2. The method of claim 1 wherein the one or more modified or unnatural base includes methyl-C, hydroxymethyl-C, or oxo-G.

3. The method of claim 1 wherein the one or more modified or unnatural base includes 5-methyl-C.

4. The method of claim 1 wherein the library of circular DNA fragments is produced by ligating hairpin oligonucleotides to a library of double-stranded DNA fragments.

5. The method of claim 1 wherein the polymerase is a phi29 polymerase.

6. The method of claim 1 wherein the polymerase extension is halted.

7. The method of claim 6 wherein the polymerase extension is halted by a change in reaction conditions.

8. The method of claim 6 wherein the polymerase extension is halted by the addition of a strontium containing buffer.

9. The method of claim 6 where the polymerase extension is halted at a reversible pause point in a single stranded region.

10. The method of claim 9 wherein the pause point comprises a non-native nucleotide.

11. The method of claim 9 wherein the pause point comprises a nucleotide with a photolabile group.

12. The method of claim 9 wherein the pause point comprises a strand binding moiety.

13. The method of claim 9 wherein the pause point comprises a transcription factor.

14. The method of claim 1 wherein the binding protein comprises a polymerase, a reverse transcriptase, a histone, a nuclease, a restriction enzyme, replication protein A (RPA), single-stranded binding protein (SSB), an RNA-binding protein, a DNA damage-binding agent, or a microRNA-containing ribonucleoprotein complex.

15. The method of claim 1 wherein the one or more modified base or unnatural base includes a methylated base and the binding protein comprises a member of the MBD family of human proteins.

16. The method of claim 15 wherein the member of the MBD family comprises MECP2, MBD1, MBD2, or MBD4.

17. A method of DNA sequencing comprising carrying out the method of claim 1, and subsequently sequencing at least some of the isolated strands.

18. The method of claim 17 wherein the isolated strands are sequenced using single-molecule sequencing.

19. The method of claim 17 wherein the isolated strands are sequenced using single-molecule real-time sequencing.

20. The method of claim 17 where the at least some of the isolated strands are loaded onto a substrate prior to sequencing.

21. The method of claim 17 wherein modified bases are identified using observed changes in polymerase kinetics.

22. The method of claim 17 wherein the sequencing is carried out in a zero mode waveguide.