Scaffolded nucleic acid polymer particles and methods of making and using

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.

1 .- 20 . (canceled) 
     
     
         21 . A method of making a set of particles including a plurality of sequence-specific primers, comprising:
 (a) obtaining a population of particles, wherein a plurality of particles in the population each includes a non-nucleosidic polymer network and includes primers having a first nucleic acid sequence attached to the non-nucleosidic polymer network through its volume, the population having a coefficient of variance of volume of not greater than 15%, the non-nucleosidic polymer network including 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) annealing an adapter oligonucleotide to a primer of a particle in the population, thereby forming an annealed primer, wherein the adapter oligonucleotide includes a first region that is complementary to the first nucleic acid sequence of the primer and a second region that has a sequence identical to a sequence of a target polynucleotide; and   (c) extending the annealed primer along the second region of the adapter oligonucleotide, thereby forming a sequence-specific primer.   
     
     
         22 . The method of  claim 21 , further including forming an emulsion comprising a dispersed aqueous phase of droplets such that at least one droplet contains one or more particles and a single adapter oligonucleotides. 
     
     
         23 . The method of  claim 21 , further comprising enriching the particles including the sequence-specific primers 
     
     
         24 . The method of  claim 23 , wherein the enriching includes separating one or more particles including a sequence-specific primer from one or more particles that do not include a sequence-specific primer using affinity-based separation. 
     
     
         25 . The method of  claim 23 , wherein the enriching comprises electrophoretically separating one or more particles including a sequence-specific primer from one or more particles that do not include a sequence-specific primer. 
     
     
         26 . The method of  claim 21 , wherein the polyacrylamide gel has a T value from about 5% to about 10%. 
     
     
         27 . The method of  claim 21 , wherein the primer is covalently attached to the particle via an acrydite group. 
     
     
         28 . The method of  claim 21 , wherein at least one particle in the population of particles has an average pore size of from about 20 to about 150 nm. 
     
     
         29 . The method of  claim 21 , wherein at least one particle in the population of particles is substantially spherical or spheroidal in shape. 
     
     
         30 . The method of  claim 29 , wherein the at least one particle has an average diameter of from about 0.5 μm to about 30 μm. 
     
     
         31 . A method of analyzing making an amplicon library, comprising:
 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 at least one particle includes a hydrophilic non-nucleosidic polymer network and a plurality of primers that are attached to the network throughout the volume of the particle and that are complementary to the primer binding site, the at least one particle having a coefficient of variance of volume of not greater than 15%, the non-nucleosidic polymer network including 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; and   performing an amplification reaction, thereby extending at least one primer of a plurality of particles along a polynucleotide fragment annealed thereto, 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 operatively coupled to a field effect transistor (FET).   
     
     
         32 . The method of  claim 31 , further including forming an emulsion of droplets prior to performing the amplification reaction, wherein the emulsion includes a plurality of droplets containing a single polynucleotide and one or more particles. 
     
     
         33 . The method of  claim 32 , further comprising breaking the emulsion. 
     
     
         34 . The method of  claim 33 , wherein the breaking further includes adding n-butanol, n-propanol or i-propanol to the emulsion. 
     
     
         35 . The method of  claim 31 , further including enriching the particles including amplicons from the particles that do not include amplicons. 
     
     
         36 . The method of  claim 35 , wherein the enriching includes separating particles including amplicons from particles that do not include amplicons using affinity-based separation. 
     
     
         37 . The method of  claim 31 , wherein the amplification reaction is a polymerase chain reaction. 
     
     
         38 . The method of  claim 31 , wherein the amplification reaction is an isothermal reaction. 
     
     
         39 . The method of  claim 31 , wherein a signal of the field effect transistor (FET) indicates a nucleotide incorporation. 
     
     
         40 . The method of  claim 39 , wherein the nucleotide incorporation includes incorporation of a nucleotide into a polynucleotide covalently attached, or hybridized, to a particle having a clonal population of polynucleotides.