Methods of 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 sequencing a template nucleic acid comprising:
 (a) disposing a particle in a reaction chamber comprising or capacitively coupled to a field effect transistor (FET), wherein the particle comprises a non-nucleosidic polymer network and template nucleic acids attached to the non-nucleosidic polymer network throughout its volume at a density of at least about 6.9×10 4  per μm 3 ;   (b) annealing a primer to one or more of the template nucleic acids attached to the non-nucleosidic polymer network;   (c) performing a polymerase extension reaction on the primer annealed to the one or more template nucleic acids by incorporating a first nucleotide into the annealed primer using a polymerase; and   (d) detecting a signal output in the FET in response to the incorporation of the nucleotide into the primer.   
     
     
         25 . The method of  claim 24 , further including disposing a plurality of particles into a plurality of reaction chambers in an array of reaction chambers, wherein the each reaction chamber of the plurality of reaction chambers comprises or is capacitively coupled to a field effect transistor (FET). 
     
     
         26 . The method of  claim 25 , wherein each particle in the plurality of particles has a volume, and the coefficient of variation of the volumes of the plurality of particles is less than about 15 percent. 
     
     
         27 . The method of  claim 25 , wherein each reaction chamber of the plurality of reaction chambers has a volume that is is no greater than about 10 μm 3 . 
     
     
         28 . The method of  claim 25 , wherein the array of reaction chambers includes at least about 10 5  microwells. 
     
     
         29 . The method of  claim 25 , wherein each particle in the plurality of particles includes a clonal population of nucleic acids. 
     
     
         30 . The method of  claim 25 , wherein each 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 any other particle in the plurality of particles. 
     
     
         31 . The method of  claim 25 , wherein at least one of the plurality of reaction chambers includes no more than one particle. 
     
     
         32 . The method of  claim 24 , wherein the non-nucleosidic polymer network includes polyacrylamide. 
     
     
         33 . The method of  claim 25 , wherein at least one of the plurality of particles is substantially spherical or spheroidal in shape. 
     
     
         34 . The method of  claim 33 , wherein the plurality of particles have diameters of from about 0.5 μm to about 10 μm. 
     
     
         35 . The method of  claim 24 , wherein the FET is an ion-sensitive field effect transistor (ISFET). 
     
     
         36 . The method of  claim 24 , further comprising washing the reaction chamber and incorporating a second nucleotide into the primer, where the second nucleotide is of the same or different type from the first nucleotide.