SCAFFOLDED NUCLEIC ACID POLYMER PARTICLES AND METHODS OF MAKING AND USING

• US 20100304982A1
• Filed: 05/24/2010
• Published: 12/02/2010
• Est. Priority Date: 05/29/2009
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: ION Torrent Systems Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Leamon, John, Light, David, Rothberg, Jonathan M., Hinz, Wolfgang
• Granted Patent: US 8,574,835 B2
• Time in Patent Office: Days
• US Class Current: 506/7
• 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

3-1. The amplicon library of claim 1 wherein said volumes of said polymer networks are spheroidal.

8. A method of making an amplicon library comprising the steps of:

combining in an amplification reaction mixture a library of polynucleotide fragments each having at least one primer binding site and a population of non-nucleosidic polymer networks, each such polymer network having a volume of less than 1.4×

10⁴ μ

m³ and having primers attached thereto, and the volumes of the non-nucleosidic polymer networks having a coefficient of variation of fifteen percent or less;

forming an emulsion having a dispersed aqueous phase of micelles such that at least one micelle contains at most one polymer network and one polynucleotide fragment; and

performing an amplification reaction in the micelles so that primers of the polymer networks are each extended along a polynucleotide fragment annealed thereto so that clonal populations of complements of such polynucleotide fragments are formed on the polymer networks, thereby forming an amplicon library.

9. The method of claim 8 wherein said amplification reaction is a polymerase chain reaction.

10. The method of claim 8 wherein said amplification reaction is an isothermal amplification reaction.

11. The method of claim 10 wherein said polynucleotide fragments have a size in the range of from 150 to 500 nucleotides.

12. A method of making an amplicon library comprising the steps of:

combining in a polymerase chain reaction mixture a library of polynucleotide fragments each having a first primer binding site at one end and a second primer binding site at the other end, and a population of nucleic acid polymer particles each comprising a non-nucleosidic polymer network having attached thereto a first primer and a second primer such that each polynucleotide fragment is capable of annealing to a first primer by its first primer binding site and to a second primer by a complement of its second primer binding site;

performing a polymerase chain reaction so that primers of the polymer networks are extended along polynucleotide fragments annealed thereto so that clonal populations of complements of such polynucleotide fragments are formed on the polymer networks, thereby forming an amplicon library.

13. The method of claim 12 wherein each of said polymer networks has a volume and wherein a concentration of said polynucleotide fragments and a concentration of said polymer networks are selected so that in said step of combining at least 10 percent of said polymer networks have at a single said polynucleotide fragment within its volume.

14. The method of making an amplicon library of claim 12 wherein said non-nucleosidic polymer network has a volume of less than 1.4×

10⁴ μ

m³ and the volumes of said non-nucleosidic polymer networks have a coefficient of variation of fifteen percent or less.

15. A method of determining a sequence of a nucleic acid, the method comprising the steps of:

disposing an amplicon library in a planar array, the amplicon library comprising a plurality of amplicons, each amplicon comprising a clonal population of a single polynucleotide from a nucleic acid library, each polynucleotide of the clonal population being attached to a non-nucleosidic polymer network, each such polymer network having a volume and the polynucleotides of the clonal population being attached to the polymer network throughout its volume, wherein the number of attached polynucleotides is at least 6.9×

10⁴ per μ

m³ and the volumes of the polymer networks have a coefficient of variation of fifteen percent or less.

performing a sequencing reaction simultaneously on the fragments of the amplicons of the library.

16. The method of claim 15 wherein said planar array is an array of sample retaining regions such that substantially each sample retaining region has at most one amplicon.

17. The method of claim 16 wherein said array of sample retaining regions is a microwell array of regularly spaced microwells having a pitch therebetween of 10 μ

m or less.

18. The method of claim 17 wherein said microwell array is disposed on an integrated array of chemical field effect transistors in a circuit-supporting substrate, wherein the integrated array has a pitch of 10 μ

m or less and each microwell is positioned on at least one chemical field effect transistor that is configured to generate at least one output signal related to an amount or concentration of a reaction byproduct generated in a sequencing reaction on an amplicon in such microwell.

19. A composition comprising:

a field effect transistor array in a circuit-supporting substrate, such transistor array having disposed on its surface an array of sample retaining regions such that each sample retaining region is positioned on at least one field effect transistor; and

a population of nucleic acid polymer particles randomly distributed in the sample retaining regions, the population comprising a plurality of different nucleic acids and the nucleic acid polymer particles each having a volume and the volumes having a coefficient of variation of fifteen percent or less.

20. The composition of claim 19 wherein there is at most one said nucleic acid polymer particle per said sample retaining region.

21. A composition of nucleic acid polymer particles each comprising oligonucleotides attached to a non-nucleosidic polymer network, each such polymer network having a volume and the oligonucleotides being attached to the polymer network throughout its volume, wherein the oligonucleotides have an average nearest neighbor distance in the range of from 10 to 20 nm and the volumes of the non-nucleosidic polymer networks have a coefficient of variation of fifteen percent or less.

22. The composition of claim 21 wherein said oligonucleotides are primers each attached to said polymer network by its 5′

end.

23. A method of amplifying a target polynucleotide in a sample, the method comprising the steps of:

combining a sample and nucleic acid polymer particles in a polymerase chain reaction mixture, each such particle comprising a non-nucleosidic polymer network having attached throughout its volume a first primer and a second primer each at a concentration of at least 1×

10⁵ primers per mm³, such that the target polynucleotide is capable of annealing to the first primer by a first region and to a second primer by a complement of a second region; and

performing a polymerase chain reaction so that primers on a nucleic acid polymer particle are repeatedly extended along a target polynucleotide annealed thereto so that a clonal population of complements of such target polynucleotide is formed on the polymer networks.