Methods and compositions for loading of polymerase complexes

• US 10,731,211 B2
• Filed: 11/17/2016
• Issued: 08/04/2020
• Est. Priority Date: 11/18/2015
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Sheikholeslami, Sassan, Hunkapiller, Michael, Popovich, Natasha, Sun, Lei, Miller, Erik, Kamtekar, Satwik
• Primary Examiner(s): Horlick, Kenneth R
• Application Number:
  US15/354,803
• Publication Number:
  US 20170159119A1
• Time in Patent Office: 1,356 Days
• Field of Search: None
• CPC Class Codes: C12N 11/02 Enzymes or microbial cells immobilised on or in an organic carrier
• CPC Class Codes: C12Q 1/6846 Common amplification features
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2527/153 Viscosity
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2563/143 Magnetism, e.g. magnetic label
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode

Abstract

The present disclosure provides methods, compositions, and systems for distributing polymerase compositions into array regions. In particular, the described methods, compositions, and systems utilize density differentials and/or additives to increase efficiency in the distribution of polymerase compositions to a surface as compared to methods utilizing only diffusion control.

Claims

1. A method of distributing polymerase molecules into a plurality of array regions, the method comprising:

(a) providing a surface comprising a plurality of array regions, wherein the plurality of array regions are bathed in a first solution comprising a buffer;

(b) exposing the surface of step a) to a second solution comprising polymerase-template complexes, wherein the second solution has a higher density than the first solution, and wherein the second solution has a volume that is about 1% to about 20% of the volume of the first solution, such that the second solution sinks to the bottom of the array regions and forms a thin layer underlying the first solution,

thereby distributing the polymerase-template complexes into the plurality of the array regions, wherein the distributing occurs at a faster rate as compared to distributing without a second solution with a higher density than the first solution.

2. The method of claim 1, wherein the second solution comprises a neutral and hydrophilic polysaccharide.

3. The method of claim 1, wherein the second solution comprises a highly branched, high-mass polysaccharide.

4. The method of claim 1, wherein the second solution comprises a volume excluding reagent.

5. The method of claim 1, wherein the second solution comprises an additive selected from the group consisting of dextran, aminodextran, dextrin, cluster dextrin, Ficoll, polyetheylene glycol, sucrose, DMSO, glycerol, and pullulan.

6. The method of claim 1, wherein the second solution comprises Ficoll.

7. The method of claim 1, wherein the distributing occurs at about a 2 to about a 25-fold faster rate as compared to distributing without the second solution.

8. The method of claim 1, wherein the distributing occurs at about a 5 to about a 20-fold faster rate as compared to distributing without the second solution.

9. The method of claim 1, wherein the distributing occurs at about a 10 to about a 15-fold faster rate as compared to distributing without the second solution.

10. The method of claim 1, wherein the distributing occurs at least 5 times faster as compared to distributing without the second solution.

11. The method of claim 1, wherein the distributing occurs at least 10 times faster as compared to distributing without the second solution.

12. The method of claim 1, wherein the distributing occurs at least 15 times faster as compared to distributing without the second solution.

13. The method of claim 1, wherein the volume of the second solution is about 5% to about 15% of the volume of the first solution.

14. The method of claim 1, wherein the volume of the second solution is about 10% of the volume of the first solution.

15. The method of claim 1, wherein prior to the exposing step (b), the second solution is cleaned to remove free polymerases that are not part of polymerase-template complexes.

16. The method of claim 15, wherein the second solution is cleaned by applying a paramagnetic particle to the second solution to capture free polymerases that are not part of polymerase-template complexes.

17. The method of claim 16, wherein the paramagnetic particle comprises a plurality of oligonucleotides, wherein the oligonucleotides comprise sequences complementary to a primer.

18. The method of claim 1, wherein the array regions comprise nanowells.

19. The method of claim 18, wherein the nanowells comprise zero mode waveguides (ZMWs).

20. The method of claim 1, wherein the distributing is complete in about 1-3 hours.

21. The method of claim 1, wherein the template of the polymerase template complexes comprises at least about 10-20 kilobasepairs (kbp) in length.

22. The method of claim 1, wherein the template of the polymerase template complexes comprises at least about 10 kbp in length.

23. The method of claim 1, wherein the template comprises:

(i) a double stranded nucleic acid segment having a first and second end;

(ii) a first hairpin oligonucleotide connecting each strand of the single template nucleic acid at the first end;

(iii) a second hairpin oligonucleotide connecting each strand of the single template nucleic acid at the second end.

24. The method of claim 23, wherein the first hairpin oligonucleotide has an identical sequence to the second hairpin oligonucleotide.

25. The method of claim 23, wherein the first hairpin oligonucleotide comprises a primer binding site, and the second hairpin oligonucleotide comprises a capture adapter.

26. The method of claim 1, wherein the templates of the polymerase-template complexes are further hybridized to primers.

27. The method of claim 1, wherein subsequent to the distributing, the polymerase-template complexes are immobilized in the array regions.

28. The method of claim 1, wherein the second solution has greater viscosity than the first solution.

29. The method of claim 1, wherein the second solution has a density of about 1.1 to about 1.5 g/cm³ and a viscosity of about 1.5-5 cP.