Scaffolded nucleic acid polymer particles and methods of making and using

• US 10,612,017 B2
• Filed: 09/04/2018
• Issued: 04/07/2020
• Est. Priority Date: 05/29/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Hinz, Wolfgang, Leamon, John, Light, David, Rothberg, Jonathan M.
• Primary Examiner(s): Crow, Robert T.
• Application Number:
  US16/121,615
• Publication Number:
  US 20190071667A1
• Time in Patent Office: 581 Days
• Field of Search: None
• 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 2219/00466 in a slurry
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C40B 40/08 Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 50/06 Biochemical methods, e.g. using enzymes or whole viable microorganisms
• CPC Class Codes: Y10T 428/2982 Particulate matter [e.g., sphere, flake, etc.]

Abstract

The invention provides particle compositions having applications in nucleic acid analysis. Nucleic acid polymer particles of the invention allow polynucleotides to be attached throughout their volumes for higher loading capacities than those achievable solely with surface attachment. In one aspect, nucleic acid polymer particles of the invention comprise polyacrylamide particles with uniform size distributions having low coefficients of variations, which result in reduced particle-to-particle variation in analytical assays. Such particle compositions are used in various amplification reactions to make amplicon libraries from nucleic acid fragment libraries.

Claims

1. A method for forming a particle, the method comprising:

suspending an emulsion in an initiator-saturated oil-based continuous phase, the emulsion comprising the continuous phase and a disperse phase, the disperse phase including an acrylamide monomer, a bis-acrylamide crosslinker, a surfactant, and an acrydite oligonucleotide in an aqueous solution;

heating the emulsion to polymerize the acrylamide monomer, bis-acrylamide crosslinker and acrydite oligonucleotide to form acrylamide gel particles having a plurality of oligonucleotides attached through a polymer network of the acrylamide gel; and

removing the initiator-saturated oil-based continuous phase; and

resuspending the acrylamide gel particles in a buffered aqueous solution,

wherein the acylamide gel particles have a coefficient of variance of volume of not greater than 15%, a total monomer percentage of the acrylamide gel particles is in a range of 3% to 20%, and the acrylamide gel particles are permeable to proteins having a size in the range of 50 kilodaltons to 200 kilodaltons.

2. The method of claim 1, further comprising cooling the emulsion following polymerization and prior to removing the continuous phase.

3. The method of claim 1, wherein removing the continuous phase includes centrifuging to form a pellet of the acrylamide gel particles.

4. The method of claim 1, further comprising resuspending in butanol, centrifuging, and removing the butanol, following removing the initiator-saturated oil-based continuous phase and prior to resuspending the acrylamide gel particles in the buffered aqueous solution.

5. The method of claim 1, wherein the acrylamide gel particles include the oligonucleotide through their volume at an average density of at least 6.9×

10⁴ per μ

m³.

6. The method of claim 1, further comprising forming the emulsion with an oil-based continuous phase and the disperse phase, and removing the oil-based continuous phase.

7. The method of claim 6, wherein removing the oil-based continuous phase includes centrifuging the emulsion.

8. The method of claim 6, wherein forming the emulsion includes extruding the disperse phase through a membrane into the oil-based continuous phase.

9. The method of claim 8, wherein the membrane has orifices having a diameter of 25% to 35% of the diameter of the acrylamide gel particles.

10. The method of claim 1, wherein heating includes heating in a heated tube.

11. The method of claim 1, wherein heating includes heating in an oven.

12. The method of claim 1, wherein the acrylamide gel particles have an average diameter of from about 0.5 μ

m to about 10 μ

m.

13. The method of claim 1, wherein the total monomer percentage of the acrylamide gel particles is in a range of 5% to 10%.

14. The method of claim 1, wherein the acrylamide gel particles have an average pore size in a range of 20 nm to 150 nm.

15. The method of claim 1, further comprising:

combining a library of polynucleotides and the acrylamide gel particles in an amplification reaction mixture, wherein at least one polynucleotide includes a primer binding site complementary to the oligonucleotide; and

performing an amplification reaction in the amplification reaction mixture, thereby forming an amplicon library including a plurality of particles having a clonal population of polynucleotides from the acrylamide gel particles.

16. The method of claim 15, wherein the amplification reaction is a polymerase chain reaction.

17. The method of claim 15, wherein the amplification reaction is an isothermal reaction.

18. The method of claim 15, introducing at least one acrylamide gel particle having a clonal population of polynucleotides into a reaction chamber coupled to a field effect transistor.

19. The method of claim 18, wherein the field effect transistor is an ion-sensitive field effect transistor.

20. The method of claim 1, wherein the acrylamide gel particles are substantially spherical or spheroidal in shape.