Screening of nucleic acid sequence variations via random loading of optically-encoded particles

Abstract

Embodiments disclosed herein provide reagents and methods for high-throughput screening of nucleic acid sequence variations in nucleic acid containing specimens. Nucleic acid specimens to be screened are loaded into separate discrete volumes. Optically encoded particles are used to deliver primers to amplify one or more sequences comprising the nucleic acid sequence variation. The optically encoded particles may be delivered to the discrete volumes randomly resulting in a random combination of optically encoded particles in each well, or a unique combination of optically encoded particles may be specifically assigned to each discrete volume. The observable combination of optically encoded particles may then be used to identify each discrete volume.

1 .- 10 . (canceled) 
     
     
         11 . A method for optically encoding individual discrete volumes comprising:
 assigning optical particles with individual discrete volumes, the observable pattern of discrete optically encoded particles in each discrete volume thereby allowing each discrete volume to be independently identified.   
     
     
         12 . The method of  claim 11 , wherein the optical particles are a colloidal metal particle, a microsphere, a nanotube, a nanorod, a nanoshell, a dendrimer, a quantum dot, or a hydrogel particle. 
     
     
         13 . The method of  claim 11 , wherein the optically encoded particles comprise distinct types of particles within the set of encoded particles that may further comprise one or more unique nucleic acid particle identifier that are associated only with that distinct particle type within the set. 
     
     
         14 . The method of  claim 11 , further comprising optically detecting a combination of optically encoded particles delivered to each discrete volume. 
     
     
         15 . The method of  claim 14 , wherein the detecting comprises capturing one or more images in one or more channels corresponding to the optically encoded particles. 
     
     
         17 . The method of  claim 11 , further comprising distributing a nucleic acid containing species to the individual discrete volumes, and hybridizing one or more primers to one or more target sequences in the nucleic acid containing species. 
     
     
         18 . A method for optically encoding individual discrete volumes comprising:
 associating optical particles with nucleic acids comprising a barcode sequence, thereby generating optically encoded particles; and   distributing the optically encoded particles to individual discrete volumes, optionally wherein the nucleic acid is a primer.   
     
     
         19 . The method of  claim 18 , wherein the optical particles are a colloidal metal particle, a microsphere, a nanotube, a nanorod, a nanoshell, a dendrimer, a quantum dot, or a hydrogel particle. 
     
     
         20 . The method of  claim 18 , wherein the optically encoded particles comprise distinct types of particles within the set of encoded particles that may further comprise one or more unique nucleic acid particle identifier that are associated only with that distinct particle type within the set. 
     
     
         21 . The method of  claim 18 , wherein associating the optically encoded particles with primers comprises:
 mixing oligonucleotide functionalized particles with the primers and optically-labeled oligonucleotides;   annealing the mixture thereby generating optically encoded particles; and   distributing the optically encoded particles to individual discrete volumes.   
     
     
         22 . The method of  claim 21 , wherein the optically-labeled oligonucleotides are labeled with distinct ratios of one or more optical labels, optionally wherein the optical labels are dyes comprising FMA, Cy3 and/or Cy5. 
     
     
         23 . The method of  claim 18 , further comprising optically detecting a combination of optically encoded particles delivered to each discrete volume. 
     
     
         24 . The method of  claim 23 , wherein the detecting comprises capturing one or more images in one or more channels corresponding to the optically encoded particles. 
     
     
         25 . The method of  claim 21 , wherein the oligonucleotide functionalized particle is an oligonucleotide functionalized hydrogel, optionally a PEG hydrogel or agarose. 
     
     
         26 . The method of  claim 18 , further comprising distributing a nucleic acid containing species to the individual discrete volumes, and hybridizing the primers to one or more target sequences in the nucleic acid containing species.