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US 12,416,048 B2Granted
Nucleic acid synthesis techniques
AssigneeIllumina, Inc.
InventorsMostafa Ronaghi, Molly He, Cheng-yao Chen, Michael Previte, M. Shane Bowen
Life of this patent
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Abstract
A method for synthesizing a nucleic acid includes synthesizing one or more nucleic acid fragments on a substrate. The synthesized one or more nucleic acid fragments may be amplified on the substrate. The method also includes sequencing the synthesized or amplified one or more nucleic acid fragments on the substrate. The sequencing may provide feedback to designs of the one or more nucleic acid fragments. The method further includes harvesting the synthesized or amplified one or more nucleic acid fragments based on sequencing. The synthesized or amplified one or more nucleic acid fragments may be assembled to generate a target nucleic acid.
Claims20
A system for synthesizing a nucleic acid, comprising:
- (a) a synthesis substrate comprising a plurality of flow channels, each of the plurality of flow channels (i) comprising a plurality of immobilized primer oligonucleotides and (ii) lacking a template nucleic acid complementary to the plurality of immobilized primer oligonucleotides;
- (b) a processor-based device storing executable instructions and coupled to the synthesis substrate, wherein the executable instructions are configured to:
- (i) receive one or more synthesis signals for each respective flow channel indicative of a presence of a first polymerase extending de novo the plurality of immobilized primer oligonucleotides in the absence of the template nucleic acid to generate a respective plurality of single-stranded fragment polynucleotides based on one of a plurality of target sequences;
- (ii) control entry of a first sequencing reagent, a second sequencing reagent, and a second polymerase into the flow channels based on the one or more synthesis signals, wherein the first sequencing reagent comprises one or more nucleotide monomers, wherein the one or more nucleotide monomers form a plurality of polynucleotides complementary to at least a portion of the plurality of fragment polynucleotides, wherein the second sequencing reagent comprises at least one nucleotide monomer, and wherein the at least one nucleotide monomer of the second sequencing reagent comprises a reversibly terminating moiety;
- iii) receive one or more sequencing signals for each respective flow channel indicative of a presence of the first sequencing reagent and the second sequencing reagent in the presence of the second polymerase;
- (iv) determine a sequence of each of the plurality of fragment polynucleotides based on the one or more sequencing signals;
- and
- (v) provide an indication related to a comparison of the sequence of each of the plurality of fragment polynucleotides with the respective target sequence;
- (c) an assembly cell comprising an assembly substrate comprising an immobilized anchor oligonucleotide, wherein the assembly cell is configured to gather each of the plurality of fragment polynucleotides from each of the plurality of flow channels and assemble each of the plurality of fragment polynucleotides to generate an assembled polynucleotide;
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- (d) the assembled polynucleotide, wherein the assembled polynucleotide comprises double-stranded regions and single-stranded gaps regions, wherein the single-stranded regions are in both strands, and wherein the assembled polynucleotide is immobilized on the assembly substrate via the immobilized anchor oligonucleotide.
8 more elements
A system for synthesizing a nucleic acid, comprising:
- (a) a synthesis substrate comprising a plurality of flow channels, each of the plurality of flow channels (i) comprising a plurality of immobilized primer oligonucleotides and (ii) lacking a template nucleic acid complementary to the plurality of immobilized primer oligonucleotides;
- (b) a processor-based device storing executable instructions and coupled to the synthesis substrate, wherein the executable instructions are configured to:
- (i) receive one or more synthesis signals for each respective flow channel indicative of a presence of a first polymerase extending de novo the plurality of immobilized primer oligonucleotides in the absence of the template nucleic acid to generate a respective plurality of single-stranded fragment polynucleotides based on one of a plurality of target sequences, wherein the first polymerase comprises terminal deoxynucleotidyl transferase (TdT);
- (ii) control entry of a first sequencing reagent, a second sequencing reagent, and a second polymerase into the flow channels based on the one or more synthesis signals, wherein the first sequencing reagent comprises one or more nucleotide monomers, wherein the one or more nucleotide monomers form a plurality of polynucleotides complementary to at least a portion of the plurality of fragment polynucleotides, wherein the second sequencing reagent comprises at least one nucleotide monomer, and wherein the at least one nucleotide monomer of the second sequencing reagent comprises a reversibly terminating moiety;
- (iii) receive one or more sequencing signals for each respective flow channel indicative of a presence of the first sequencing reagent and the second sequencing reagent in the presence of the second polymerase;
- (iv) determine a sequence of each of the plurality of fragment polynucleotides based on the one or more sequencing signals;
- and
- (v) provide an indication related to a comparison of the sequence of each of the plurality of fragment polynucleotides with the respective target sequence;
- (c) a processor configured to generate a sequence accuracy score or metric for each of the plurality of fragment polynucleotides;
- (d) an assembly cell comprising an assembly substrate comprising an immobilized anchor oligonucleotide, wherein the assembly cell is configured to gather each of the plurality of fragment polynucleotides from each of the plurality of flow channels and assemble each of the plurality of fragment polynucleotides to generate an assembled polynucleotide;
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- (e) the assembled polynucleotide, wherein the assembled polynucleotide comprises double-stranded regions and single-stranded regions, wherein the single-stranded regions are in both strands, and wherein the assembled polynucleotide is immobilized on the assembly substrate via the immobilized anchor oligonucleotide.
9 more elements
Dependencies are inferred from the claim text.