Single molecule loading methods and compositions

• US 9,993,794 B2
• Filed: 11/04/2014
• Issued: 06/12/2018
• Est. Priority Date: 03/31/2008
• Status: Active Grant

• Current Assignee: Paciic Biosciences of California Incorporated
• Original Assignee: Paciic Biosciences of California Incorporated
• Inventors: Turner, Stephen, Flusberg, Benjamin, Sun, Lei
• Primary Examiner(s): Flinders, Jeremy C
• Application Number:
  US14/532,502
• Publication Number:
  US 20150141266A1
• Time in Patent Office: 1,316 Days
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: C12N 2310/50 Physical structure
• CPC Class Codes: C12N 2310/51 in polymeric form, e.g. multimers, concatemers
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/155 Particles of a defined size, e.g. nanoparticles
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: G01N 33/54313 the carrier being characterised by its particulate form
• CPC Class Codes: G01N 33/54366 Apparatus specially adapted for solid-phase testing

Abstract

Methods, compositions and arrays for non-random loading of single analyte molecules into array structures are provided. For example, methods are presented for providing a surface comprising the plurality of array regions by exposing the surface to a solution comprising polymerase enzymes where each polymerase enzyme is bound to a binding structure having several functional moieties. The functional moieties of the binding structure react with the binding elements on the array regions such that the functional moieties on the binding structure react with other available binding sites in an array region, preventing other polymerase-binding structures from loading, and resulting in a single polymerase molecule bound to each of these regions.

Claims

1. A method of distributing single polymerase enzyme molecules into a plurality of array regions comprising:

providing a surface comprising the plurality of array regions, each array region comprising several binding elements; and

exposing the surface to a solution comprising polymerase enzymes, each polymerase enzyme bound to a binding structure comprising several functional moieties, whereby the functional moieties of the binding structure react with the binding elements on the array regions; and

whereby in at least some of the array regions, the functional moieties on the binding structure react with other available binding sites in a given array region, depleting the binding sites and preventing other polymerase-binding structures from loading, resulting in a single polymerase enzyme bound to each of these regions.

2. The method of claim 1 wherein the array regions comprise wells.

3. The method of claim 1 wherein the array regions comprise the bottoms of zero mode waveguides (ZMWs).

4. The method of claim 1 wherein the binding structures comprise branched DNA, self-assembling multimers, dendrimers, dendrions, branched polymers, or fusion proteins.

5. The method of claim 1 wherein the binding structures comprise dendrimers and dendrons 5 nm to 25 nm in size.

6. The method of claim 4 wherein the dendrimers of dendrons have multiple terminal groups, each with the same functional moiety.

7. The method of claim 1 wherein the functional moieties comprise biotin.

8. The method of claim 6 wherein the dendrimer comprises 10 or more biotin groups.

9. The method of claim 1 wherein the binding sites comprise streptavidin.

10. The method of claim 8 wherein the binding sites which comprise streptavidin are attached to biotins on the array region.

11. The method of claim 2 whereby greater than 50% of the wells have only one polymerase.

12. The method of claim 2 whereby greater than 70% of the wells only one polymerase.

13. The method of claim 1 wherein the single polymerase molecule is associated with a nucleic acid template.

14. The method of claim 1 wherein the single polymerase molecules on the array are measured using a single molecule analyte monitoring.

15. The method of claim 1 further comprising carrying out single-molecule real time sequencing using the single polymerase molecules bound to the array regions.