Scaffolded nucleic acid polymer particles and methods of making and using

• US 8,574,835 B2
• Filed: 05/24/2010
• Issued: 11/05/2013
• 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
• Primary Examiner(s): CROW, ROBERT THOMAS
• Application Number:
  US12/785,685
• Publication Number:
  US 20100304982A1
• Time in Patent Office: 1,261 Days
• Field of Search: 435/6, 435/6.1, 435/283.1, 435/287.1, 435/287.2, 257/253
• US Class Current: 435/6.1
• 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 of detecting a nucleotide incorporation, comprising:

(a) disposing a particle in a reaction chamber including, or capacitively coupled to, a field effect transistor, wherein the particle includes a hydrophillic non-nucleosidic polymer network and a plurality of nucleic acids attached to the non-nucleosidic polymer network, the particle having a coefficient of variance of volume of not greater than 15%, the non-nucleosidic polymer network includes a polyacrylamide gel having a total monomer percentage in a range of 3% to 20% and being permeable to proteins having a size in the range of 50 kilodaltons to 200 kilodaltons;

(b) performing a nucleotide incorporation whereby a nucleotide is incorporated into one or more of the plurality of nucleic acids attached to the polymer network of the particle, and wherein the particle includes a sufficiently high concentration of attached nucleic acids to emit, upon such performing, a pH signal that can be detected by the field effect transistor; and

(c) detecting the pH signal using the field effect transistor, thereby detecting the nucleotide incorporation.

2. The method of claim 1, further including disposing a plurality of particles into a plurality of reaction chambers in an array of reaction chambers, wherein at least one of the reaction chambers of the plurality of reaction chambers includes or is operatively coupled to a field effect transistor.

3. The method of claim 2, wherein at least one reaction chamber of the plurality of reaction chambers has a volume that is no greater than 10 μ

m³.

4. The method of claim 2, wherein the array of reaction chambers includes at least 10⁵ microwells.

5. The method of claim 2, wherein at least one particle in the plurality of particles includes a clonal population of nucleic acids.

6. The method of claim 2, wherein at least one particle in the plurality of particles includes a template nucleic acid having a sequence that differs from the sequence of a template nucleic acid of at least one other particle in the plurality of particles.

7. The method of claim 2, wherein at least one of the plurality of reaction chambers includes no more than one particle.

8. The method of claim 1, wherein the field effect transistor is an ion-sensitive field effect transistor.

9. The method of claim 1, wherein at least one of the plurality of nucleic acids attached to the polymer network of the particle is covalently attached to the polymer network.

10. The method of claim 1, wherein at least one of the plurality of nucleic acids attached to the polymer network of the particle is hybridized to a nucleic acid that is covalently attached to the polymer network.

11. The method of claim 10, wherein at least one of the plurality of nucleic acids attached to the polymer network of the particle includes an oligonucleotide primer.

12. The method of claim 1, further including performing a second nucleotide incorporation to incorporate a second nucleotide into one or more of the plurality of nucleic acids attached to the polymer network of the particle.

13. The method of claim 1, further including identifying the incorporated nucleotide.

14. The method of claim 1, wherein the particle in the reaction chamber is formed by:

combining a library of polynucleotides and a population of particles in an amplification reaction mixture, wherein at least one polynucleotide includes a primer binding site and particles of the population of particles include the hydrophilic non-nucleosidic polymer network and a plurality of primers that are attached to the non-nucleosidic polymer network throughout the volume of the particle; and

performing an amplification reaction, thereby forming an amplicon library including a plurality of particles having a clonal population of polynucleotides; and

introducing at least one particle having a clonal population of polynucleotides into a reaction chamber coupled to a field effect transistor.

15. The method of claim 14, further including separating particles including amplicons from particles that do not include amplicons.

16. The method of claim 15, wherein the separating includes separating particles including amplicons from particles that do not include amplicons using affinity-based separation.

17. The method of claim 16, wherein the separating includes annealing an oligonucleotide with a capture moiety to particles including amplicons and capturing the particles including amplicons with a streptavidinated magnetic bead based on the annealed oligonucleotide with the capture moiety.

18. The method of claim 14, wherein the amplification reaction is a polymerase chain reaction.

19. The method of claim 14, wherein the amplification reaction is an isothermal reaction.