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US 8,012,690 B2Granted
Bead emulsion nucleic acid amplification
Assignee454 Life Sciences Corporation
InventorsJan Berka, Yi-Ju Chen, John H. Leamon, Steve Lefkowitz, Kenton L. Lohman, Vinod B. Makhijani, Jonathan M. Rothberg, Gary J. Sarkis, Maithreyan Srinivasan, Michael P. Weiner
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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.
Claims19
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;
- and
- 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 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.
5 more elements
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 at least one of the beads and one or more species of the template nucleic acid molecules compartmentalized therein, and a second subset of the droplets comprise at least one 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 during the amplification a plurality of copies of the species of template nucleic acid molecule hybridize to a plurality of first primers immobilized on the one or more beads and a plurality of complementary nucleic acid molecules are extended therefrom;
- breaking the aqueous emulsion droplets to release the beads of the first and second subsets;
- disassociating the hybridized copies of the species of the template nucleic acid molecules from the first primers and extended complementary nucleic acid molecules immobilized on the beads of the first subset;
- hybridizing a 3′ end of one or more of the complementary nucleic acid molecules immobilized on the beads of the first subset to one or more second primers disposed on one or more enrichment beads enabled for isolation under a selective condition thereby linking the enrichment beads to one or more of the beads of the first subset;
- applying the selective condition to isolate the one or more enrichment beads from the beads of the second subset;
- and
- separating the enrichment beads from the beads of the first subset by separating the second primers from the immobilized complementary nucleic acid molecules.
5 more elements
Dependencies are inferred from the claim text.