Methods and compositions for enriching compositions for polymerase enzyme complexes

• US 10,544,457 B2
• Filed: 06/09/2017
• Issued: 01/28/2020
• Est. Priority Date: 06/14/2016
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Miller, Erik, Bjornson, Keith, Nyquist, Kristofor, Kamtekar, Satwik
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US15/618,535
• Publication Number:
  US 20170356041A1
• Time in Patent Office: 963 Days
• Field of Search: None
• CPC Class Codes: C12N 9/1241 Nucleotidyltransferases (2.7.7)
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/125 incorporating agents resulting in resistance to degradation
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/127 the enzyme inhibitor or activator used
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

The present invention provides methods, compositions, and systems for enriching compositions for polymerase enzyme complexes. In particular, the methods, compositions, and systems of the present invention remove free polymerases from the compositions using one or more purification steps, including protease treatment, thus enriching the compositions for polymerases complexed with a template nucleic acid.

Claims

1. A method for enriching a loading composition for polymerase enzyme complexes, the method comprising:

(a) providing a loading composition comprising free polymerases and polymerase enzyme complexes, wherein the polymerase enzyme complexes are in an inactive form, and wherein each polymerase enzyme complex comprises a polymerase enzyme bound to a nucleic acid template;

(b) applying a protease to the loading composition, which protease is more effective against the free polymerases than against polymerases in the polymerase enzyme complexes, and performing limited proteolytic digestion to reduce the amount of free polymerase relative to the amount of polymerase enzyme complex present in the composition, thereby enriching the loading composition for polymerase enzyme complexes; and

(c) distributing the loading composition onto a substrate to provide a loaded substrate.

2. The method of claim 1, wherein the loading composition is purified to enhance the loading composition for polymerase enzyme complexes.

3. The method of claim 1, wherein applying step (b) is conducted after distributing step (c).

4. The method of claim 1, wherein the protease comprises an endopeptidase.

5. The method of claim 1, wherein the protease comprises trypsin.

6. The method of claim 1, wherein the substrate comprises an array of nanoscale wells.

7. The method of claim 1, wherein the distributing step (c) comprises immobilizing the polymerase enzyme complexes to the substrate.

8. The method of claim 7, wherein the polymerase enzyme complexes comprise a reactive element, and the immobilizing occurs through an interaction of the reactive element and a binding site on the substrate.

9. The method of claim 8, wherein the reactive element comprises streptavidin and the binding site comprises biotin.

10. The method of claim 8, wherein the reactive element is resistant to cleavage by the protease.

11. The method of claim 1, wherein the inactive form of the polymerase enzyme complexes is maintained by including a non-catalytic metal ion in the loading composition.

12. The method of claim 11, wherein the non-catalytic metal ion is a member selected from the group consisting of:

strontium, cobalt, tin, calcium, nickel, europium, barium, iron, and zinc.

13. The method of claim 1, wherein the loading composition comprises one or more nucleotides or nucleotide analogs.

14. The method of claim 1, wherein the inactive form of the polymerase enzyme complexes is maintained by including at least one non-hydrolyzable nucleotide analog in the loading composition.

15. The method of claim 1, comprising step (d) determining a nucleotide sequence of at least a portion of the template.

16. The method of claim 15, wherein determining the nucleotide sequence comprises:

(i) providing a sequencing reaction mixture to the loaded substrate, the sequencing reaction mixture comprising at least one catalytic metal ion and one or more nucleotides or nucleotide analogs;

(ii) performing a polymerization reaction in which the polymerase replicates at least a portion of the template in a template-dependent manner, whereby the one or more nucleotides or nucleotide analogs are incorporated into the resulting nucleic acid; and

(iii) identifying a time sequence of incorporation of the one or more nucleotides or nucleotide analogs into the resulting nucleic acid.

17. The method of claim 16, wherein the performing step (ii) is conducted in the presence of a protease inhibitor.

18. The method of claim 16, wherein the one or more nucleotide or nucleotide analogs are labeled nucleotides or nucleotide analogs.

19. The method of claim 1, wherein the substrate comprises nanopores.

20. A method for enriching a loading composition for enzyme complexes, the method comprising:

(a) providing a loading composition comprising free enzymes and enzyme complexes, wherein the enzyme complexes are in an inactive form;

(b) applying a protease to the loading composition, which protease is more effective against the free enzymes than against enzymes in the enzyme complexes, and performing limited proteolytic digestion to reduce the amount of free enzyme relative to the amount of enzyme complex present in the composition, thereby enriching the loading composition for enzyme complexes; and

(c) distributing the loading composition onto a substrate to provide a loaded substrate.

21. The method of claim 20, wherein the substrate comprises nanopores or an array of nanoscale wells.