Assembly of Nucleic Acid Sequences in Emulsions
First Claim
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1. A method of synthesizing a target nucleic acid sequence comprising the steps of:
- localizing a plurality of oligonucleotide subsequences defining an oligonucleotide set corresponding to a particular target nucleic acid sequence by hybridization to a predesigned sequence that is unique to each oligonucleotide set;
attaching the plurality of oligonucleotide subsequences to a bead;
placing the bead within an emulsion droplet;
separating the plurality of oligonucleotide subsequences from the bead such that the plurality of oligonucleotide subsequences remain within the emulsion droplet; and
assembling within the emulsion droplet the plurality of oligonucleotide subsequences to form the target nucleic acid sequence.
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Abstract
Methods and compositions for synthesizing nucleic acid sequences in an emulsion are provided.
136 Citations
42 Claims
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1. A method of synthesizing a target nucleic acid sequence comprising the steps of:
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localizing a plurality of oligonucleotide subsequences defining an oligonucleotide set corresponding to a particular target nucleic acid sequence by hybridization to a predesigned sequence that is unique to each oligonucleotide set; attaching the plurality of oligonucleotide subsequences to a bead; placing the bead within an emulsion droplet; separating the plurality of oligonucleotide subsequences from the bead such that the plurality of oligonucleotide subsequences remain within the emulsion droplet; and assembling within the emulsion droplet the plurality of oligonucleotide subsequences to form the target nucleic acid sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method of synthesizing a target nucleic acid sequence comprising the steps of:
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amplifying a plurality of oligonucleotide subsequences defining an oligonucleotide set corresponding to a particular target nucleic acid sequence by using orthogonal primers that hybridize to a pair of orthogonal primer binding sites that are unique to the oligonucleotide set; removing the orthogonal primer binding sites from the amplified plurality of oligonucleotide subsequences; attaching the amplified plurality of oligonucleotide subsequences to a bead; synthesizing a complementary strand to each of the amplified plurality of oligonucleotide subsequences to produce a plurality of double stranded nucleic acids; placing the bead within an emulsion droplet; separating the plurality of double stranded nucleic acids from the bead such that the plurality of double stranded nucleic acids remain within the emulsion droplet; and assembling within the emulsion droplet the plurality of double stranded nucleic acids to form the target nucleic acid sequence. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16)
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17. A method of synthesizing a plurality of target nucleic acid sequences comprising the steps of:
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for each target nucleic acid sequence within the plurality, localizing a plurality of oligonucleotide subsequences defining an oligonucleotide set corresponding to a particular target nucleic acid sequence by hybridization to a predesigned sequence that is unique to the oligonucleotide set thereby resulting in a plurality of oligonucleotide sets corresponding to the plurality of target nucleic acid sequences; attaching the plurality of oligonucleotide sets to a plurality of beads; placing the plurality of beads within a plurality of emulsion droplets; separating the plurality of double stranded nucleic acids from the bead such that the plurality of double stranded nucleic acids remain within the emulsion droplet; assembling within the emulsion droplet the plurality of double stranded nucleic acids to form one of the plurality of target nucleic acid sequences. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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30. A method of synthesizing a plurality of target nucleic acid sequences comprising the steps of:
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amplifying a plurality of oligonucleotide subsequences defining a plurality of oligonucleotide sets with each oligonucleotide set corresponding to a particular target nucleic acid sequence by using orthogonal primers that hybridize to a pair of orthogonal primer binding site sequences that are unique to each oligonucleotide set; removing the orthogonal primer binding sites from the amplified plurality of oligonucleotide subsequences; for each oligonucleotide set, attaching corresponding amplified oligonucleotide subsequences defining the set to a bead; synthesizing a complementary strand to each of the amplified oligonucleotide subsequences to produce a plurality of double stranded nucleic acids; placing the bead within an emulsion droplet; separating the plurality of double stranded nucleic acids from the bead such that the plurality of double stranded nucleic acids remain within the emulsion droplet; assembling within the emulsion droplet the plurality of double stranded nucleic acids to form one of the plurality of target nucleic acid sequences. - View Dependent Claims (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42)
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Specification