METHODS AND COMPOSITIONS FOR LOADING OF POLYMERASE COMPLEXES

• US 20170159119A1
• Filed: 11/17/2016
• Published: 06/08/2017
• 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
• Granted Patent: US 10,731,211 B2
• Time in Patent Office: Days
• 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 buffer;

(b) exposing the surface to a spike solution comprising polymerase-template complexes, wherein the spike solution has a higher density than the buffer, such that the spike solution sinks to the bottom of the array regions as a thin layer,

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 spike solution with a higher density than the buffer.

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

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

4. The method of claim 1, wherein the spike solution comprises a volume excluding buffer.

5. The method of claim 1, wherein the spike 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 spike 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 spike 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 spike 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 spike solution.

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

11. The method of claim 1, wherein the distributing occurs at least 10 times faster as compared to distributing without the spike solution having a higher density than the buffer.

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

13. The method of claim 1, wherein the spike solution has a volume that is about 1% to about 20% of the volume of the buffer.

14. The method of claim 13, wherein the spike solution has a volume that is about 5% to about 15% of the volume of the buffer.

15. The method of claim 13, wherein the spike solution has a volume that is about 10% of the volume of the buffer.

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

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

18. The method of claim 17, wherein the paramagnetic particle comprises a plurality of oligonucleotides, wherein the oligonucleotides comprise sequences complementary to a primer binding site on the templates.

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

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

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

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

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

24. 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.

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

26. The method of claim 24, wherein the first hairpin oligonucleotide comprises a primer binding site, and the second hairpin oligonucleotide comprises a capture adapter.

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

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

29. The method of claim 1, wherein the spike solution has greater viscosity than the buffer.

30. The method of claim 1, wherein the volume of the spike solution is at a ratio of about 1:

10 to that of the buffer.

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

32-109. -109. (canceled)