Methods of Primer Extension Using Porous Particle Supports

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 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 primer including a first nucleic acid sequence;   (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.   
     
     
         25 . The method of  claim 24 , further including forming an emulsion comprising a dispersed aqueous phase of micelles such that at least one micelle contains a single particle and one or more adapter oligonucleotides. 
     
     
         26 . The method of  claim 24 , further comprising enriching the particles including the sequence-specific primers. 
     
     
         27 . The method of  claim 26 , 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. 
     
     
         28 . The method of  claim 26 , 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. 
     
     
         29 . The method of  claim 24 , wherein at least one particle in the population of particles includes a polyacrylamide gel. 
     
     
         30 . The method of  claim 29 , wherein the polyacrylamide gel has a T value from about 5% to about 10%. 
     
     
         31 . The method of  claim 24 , wherein the primer is covalently attached to the particle via an acrydite group. 
     
     
         32 . The method of  claim 24 , wherein at least one particle in the population of particles has an average pore size of from about 20 to about 150 nm. 
     
     
         33 . The method of  claim 24 , wherein at least one particle in the population of particles is substantially spherical or spheroidal in shape. 
     
     
         34 . The method of  claim 33 , wherein the at least one particle has an average diameter of from about 0.5 μm to about 30 μm. 
     
     
         35 . The method of  claim 24 , wherein at least one particle in the population of particles is non-spherical.