Bead emulsion nucleic acid amplification

Abstract

Disclosed are methods for nucleic acid amplification wherein nucleic acid templates, beads, and amplification reaction solution are emulsified and the nucleic acid templates are amplified to provide clonal copies of the nucleic acid templates attached to the beads. Also disclosed are kits and apparatuses for performing the methods of the invention.

1 - 44 . (canceled) 
     
     
         45 . A method of enriching for sequestered populations of amplified nucleic acid molecules, comprising:
 distributing a solution comprising a plurality of beads and a plurality of species of template nucleic acid molecules into a plurality of aqueous emulsion droplets in a continuous oil phase, wherein a first subset of the droplets comprise one or more of the beads and one or more of the species of template nucleic acid molecule compartmentalized therein, and a second subset of the droplets comprise one or more of the beads without any of the species of template nucleic acid molecules compartmentalized therein;   amplifying the species of template nucleic acid molecules within the first subset of the droplets, wherein a plurality of nucleic acid molecules complementary to the species of template nucleic acid molecules are immobilized to the one or more beads within the first subset of the droplets;   breaking the aqueous emulsion droplets to release the beads of the first and second subsets;   attaching one or more of the beads of the first subset comprising the immobilized complementary copies to one or more enrichment beads, wherein the beads of the first subset are attached to the one or more enrichment beads by hybridizing a capture primer to a portion of one or more of the immobilized complementary nucleic acid molecules and the capture primer is thereafter coupled to the enrichment beads;   isolating the one or more enrichment beads away from the beads of the second subset; and   separating the enrichment beads from the beads of the first subset by separating the capture primers from the immobilized complementary nucleic acid molecules.   
     
     
         46 . The method of  claim 45 , wherein: the solution comprises a ratio of a number of the template nucleic acid molecules to a number of the beads selected from a group consisting of a ratio that is equal to about 1, a ratio that is less than 1, and a ratio that is about 0.1. 
     
     
         47 . The method of  claim 45 , wherein: the solution further comprises a sufficient mixture of reagents necessary for performing an amplification reaction of the species of template nucleic acid molecule. 
     
     
         48 . The method of  claim 45 , wherein: the step of amplifying employs a polymerase chain reaction amplification process. 
     
     
         49 . The method of  claim 48 , wherein: the complementary nucleic acid molecules are immobilized to the beads of the first subset during the polymerase chain reaction amplification process by hybridizing a plurality of copies of the species of template nucleic acid molecules to a plurality of primers immobilized on the beads and extending the complementary nucleic acid molecules from the immobilized primers. 
     
     
         50 . The method of  claim 45 , wherein: the capture primer is complementary to a 3′ portion common to the immobilized complementary nucleic acid molecules wherein the 3′ portion comprises a sequence length selected from the group consisting of at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, and at least 40 bases. 
     
     
         51 . The method of  claim 45 , wherein the step of isolating the one or more enrichment beads away from the beads of the second subset comprises using a magnetic field enabled to isolate the one or more enrichment beads from the beads of the second subset. 
     
     
         52 . The method of  claim 45 , further comprising: determining a nucleic acid composition for the one or more species of nucleic acid molecules from the immobilized complementary nucleic acid molecules. 
     
     
         53 . The method of  claim 45 , further comprising: reusing the beads of the second subset in a subsequent amplification and enrichment process. 
     
     
         54 . A method of enriching for sequestered populations of amplified nucleic acid molecules, comprising:
 distributing a solution comprising a plurality of beads and a plurality of species of template nucleic acid molecules into a plurality of aqueous emulsion droplets in a continuous oil phase, wherein a first subset of the droplets comprise one or more of the beads and one or more of the species of template nucleic acid molecule compartmentalized therein, and a second subset of the droplets comprise one or more of the beads without any of the species of template nucleic acid molecules compartmentalized therein;   amplifying the species of template nucleic acid molecules within the first subset of the droplets, wherein a plurality of nucleic acid molecules complementary to the species of template nucleic acid molecules are immobilized to the one or more beads within the first subset of the droplets;   breaking the aqueous emulsion droplets to release the beads of the first and second subsets;   attaching one or more of the beads of the first subset comprising the immobilized complementary copies to one or more enrichment beads, wherein the beads of the first subset are attached to the one or more enrichment beads by hybridizing a capture primer to a portion of one or more of the immobilized complementary nucleic acid molecules, said capture primer comprising a binding moiety that binds to a capture moiety immobilized on the enrichment beads such that after said hybridizing, the capture primer is thereafter coupled to the enrichment beads;   isolating the one or more enrichment beads away from the beads of the second subset; and   separating the enrichment beads from the beads of the first subset by separating the capture primers from the immobilized complementary nucleic acid molecules.   
     
     
         55 . The method of  claim 54 , wherein: the solution comprises a ratio of a number of the template nucleic acid molecules to a number of the beads selected from a group consisting of a ratio that is equal to about 1, a ratio that is less than 1, and a ratio that is about 0.1. 
     
     
         56 . The method of  claim 54 , wherein: the solution further comprises a sufficient mixture of reagents necessary for performing an amplification reaction of the species of template nucleic acid molecule. 
     
     
         57 . The method of  claim 54 , wherein: the step of amplifying employs a polymerase chain reaction amplification process. 
     
     
         58 . The method of  claim 57 , wherein: the complementary nucleic acid molecules are immobilized to the beads of the first subset during the polymerase chain reaction amplification process by hybridizing a plurality of copies of the species of template nucleic acid molecules to a plurality of primers immobilized on the beads and extending the complementary nucleic acid molecules from the immobilized primers. 
     
     
         59 . The method of  claim 54 , wherein: the capture primer is complementary to a 3′ portion common to the immobilized complementary nucleic acid molecules wherein the 3′ portion comprises a sequence length selected from the group consisting of at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, and at least 40 bases. 
     
     
         60 . The method of  claim 54 , wherein the step of isolating the one or more enrichment beads away from the beads of the second subset comprises using a magnetic field enabled to isolate the one or more enrichment beads from the beads of the second subset. 
     
     
         61 . The method of  claim 54 , further comprising: determining a nucleic acid composition for the one or more species of nucleic acid molecules from the immobilized complementary nucleic acid molecules. 
     
     
         62 . The method of  claim 54 , further comprising: reusing the beads of the second subset in a subsequent amplification and enrichment process. 
     
     
         63 . A method of enriching for sequestered populations of amplified nucleic acid molecules, comprising:
 distributing a solution comprising a plurality of beads and a plurality of species of template nucleic acid molecules into a plurality of aqueous emulsion droplets in a continuous oil phase, wherein a first subset of the droplets comprise one or more of the beads and one or more of the species of template nucleic acid molecule compartmentalized therein, and a second subset of the droplets comprise one or more of the beads without any of the species of template nucleic acid molecules compartmentalized therein;   amplifying the species of template nucleic acid molecules within the first subset of the droplets, wherein a plurality of nucleic acid molecules complementary to the species of template nucleic acid molecules are immobilized to the one or more beads within the first subset of the droplets;   breaking the aqueous emulsion droplets to release the beads of the first and second subsets;   attaching one or more of the beads of the first subset comprising the immobilized complementary copies to one or more enrichment beads, wherein the beads of the first subset are attached to the one or more enrichment beads by hybridizing a capture primer to a portion of one or more of the immobilized complementary nucleic acid molecules, said capture primer comprising a binding moiety that binds to a capture moiety immobilized on the enrichment beads such that after said hybridizing, the capture primer is thereafter coupled to the enrichment beads, wherein the binding moiety and the capture moiety comprise a biotin streptavidin binding pair;   isolating the one or more enrichment beads away from the beads of the second subset; and   separating the enrichment beads from the beads of the first subset by separating the capture primers from the immobilized complementary nucleic acid molecules.   
     
     
         64 . The method of  claim 63 , wherein: the solution comprises a ratio of a number of the template nucleic acid molecules to a number of the beads selected from a group consisting of a ratio that is equal to about 1, a ratio that is less than 1, and a ratio that is about 0.1. 
     
     
         65 . The method of  claim 63 , wherein: the solution further comprises a sufficient mixture of reagents necessary for performing an amplification reaction of the species of template nucleic acid molecule. 
     
     
         66 . The method of  claim 63 , wherein: the step of amplifying employs a polymerase chain reaction amplification process. 
     
     
         67 . The method of  claim 66 , wherein: the complementary nucleic acid molecules are immobilized to the beads of the first subset during the polymerase chain reaction amplification process by hybridizing a plurality of copies of the species of template nucleic acid molecules to a plurality of primers immobilized on the beads and extending the complementary nucleic acid molecules from the immobilized primers. 
     
     
         68 . The method of  claim 63 , wherein: the capture primer is complementary to a 3′ portion common to the immobilized complementary nucleic acid molecules wherein the 3′ portion comprises a sequence length selected from the group consisting of at least 15 bases, at least 20 bases, at least 25 bases, at least 30 bases, at least 35 bases, and at least 40 bases. 
     
     
         69 . The method of  claim 63 , wherein the step of isolating the one or more enrichment beads away from the beads of the second subset comprises using a magnetic field enabled to isolate the one or more enrichment beads from the beads of the second subset. 
     
     
         70 . The method of  claim 63 , further comprising: determining a nucleic acid composition for the one or more species of nucleic acid molecules from the immobilized complementary nucleic acid molecules. 
     
     
         71 . The method of  claim 63 , further comprising: reusing the beads of the second subset in a subsequent amplification and enrichment process.