Methods of Making Libraries of Nucleic Acids Using Porous Particles

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 .- 23 . (canceled) 
     
     
         24 . A method of making an amplicon, comprising:
 (a) combining a library of polynucleotides and a population of particles in an amplification reaction mixture, wherein each polynucleotide includes a primer binding site and each particle includes a non-nucleosidic polymer network and a plurality of primers that are covalently attached to the network throughout the volume of the particle at an average density of at least 1×10 5  per μm 3 ; and   (b) performing an amplification reaction so at least one primer of a plurality of particles is extended along a polynucleotide fragment annealed thereto, thereby forming an amplicon library including a plurality of particles, each particle of the library including a clonal population of polynucleotides.   
     
     
         25 . The method of  claim 24 , further including forming an emulsion of micelles prior to performing the amplification reaction, wherein the emulsion includes a plurality of micelles containing a single polynucleotide and a particle. 
     
     
         26 . The method of  claim 25 , further comprising breaking the emulsion. 
     
     
         27 . The method of  claim 26 , wherein the breaking further includes adding n-butanol, n-popanol, or i-propanol to the emulsion. 
     
     
         28 . The method of  claim 24 , further including enriching the particles including amplicons from the particles that do not include amplicons. 
     
     
         29 . The method of  claim 28 , wherein the enriching includes separating particles including amplicons from particles that do not include amplicons using affinity-based separation. 
     
     
         30 . The method of  claim 28 , wherein the enriching includes electrophoretically separating particles including amplicons from particles that do not include amplicons. 
     
     
         31 . The method of  claim 24 , wherein the amplification reaction is a polymerase chain reaction. 
     
     
         32 . The method of  claim 24 , wherein the amplification reaction is an isothermal reaction. 
     
     
         33 . The method of  claim 24 , wherein a plurality of polynucleotides of the library are from 150 to 500 nucleotides in length. 
     
     
         34 . The method of  claim 25 , wherein at least about 10 percent of the micelles contain a single polynucleotide and a single particle. 
     
     
         35 . The method of  claim 24 , wherein at least one particle includes a polyacrylamide gel. 
     
     
         36 . The method of  claim 35 , wherein the polyacrylamide gel has a T value from 5% to 10%.