Combined extension and ligation for nucleic acid assembly
First Claim
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1. A method of synthesizing a target nucleic acid, the method comprising:
- analyzing a sequence of a target nucleic acid for the presence of one or more predetermined interfering sequence features that are predicted to disrupt a multiplex oligonucleotide assembly reaction,assembling one or more first fragments of the target nucleic acid using a polymerase-mediated oligonucleotide assembly reaction, wherein no predetermined interfering sequence features are present in any of the one or more first fragments,if one or more predetermined interfering sequence features are present in the target nucleic acid, assembling one or more second fragments of the target nucleic acid using a ligase-mediated oligonucleotide assembly reaction, wherein the one or more second fragments comprise the one or more predetermined interfering sequence features, andassembling the target nucleic acid from the one or more first fragments and the one or more second fragments if the target nucleic acid contained one or more predetermined interfering sequence features.
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Abstract
Certain aspects of the present invention provide methods for assembling nucleic acid molecules. Some embodiments involve analyzing nucleic acid sequences and determining appropriate assembly strategies based on the presence or absence of sequence features that are known or predicted to interfere with extension-based and/or ligation-based assembly techniques. Aspects of the invention also provide kits, compositions, devices, and systems for assembling synthetic nucleic acids using polymerase-based techniques, ligase-based techniques, or combinations thereof.
181 Citations
57 Claims
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1. A method of synthesizing a target nucleic acid, the method comprising:
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analyzing a sequence of a target nucleic acid for the presence of one or more predetermined interfering sequence features that are predicted to disrupt a multiplex oligonucleotide assembly reaction, assembling one or more first fragments of the target nucleic acid using a polymerase-mediated oligonucleotide assembly reaction, wherein no predetermined interfering sequence features are present in any of the one or more first fragments, if one or more predetermined interfering sequence features are present in the target nucleic acid, assembling one or more second fragments of the target nucleic acid using a ligase-mediated oligonucleotide assembly reaction, wherein the one or more second fragments comprise the one or more predetermined interfering sequence features, and assembling the target nucleic acid from the one or more first fragments and the one or more second fragments if the target nucleic acid contained one or more predetermined interfering sequence features. - View Dependent Claims (6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19)
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2. A method of designing a target nucleic acid assembly, the method comprising:
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analyzing a sequence of a target nucleic acid for the presence of one or more predetermined interfering sequence features that are predicted to disrupt a multiplex oligonucleotide assembly reaction, selecting one or more first fragments of the target nucleic acid for assembly in a polymerase-mediated oligonucleotide assembly reaction, wherein no predetermined interfering sequence features are present in any of the one or more first fragments, and if one or more predetermined interfering sequence features are present in the target nucleic acid, selecting one or more second fragments of the target nucleic acid for assembly in a ligase-mediated oligonucleotide assembly reaction, wherein the one or more second fragments comprise the one or more predetermined interfering sequence features, wherein the target nucleic acid can be assembled from the one or more first fragments and the one or more second fragments if they are selected. - View Dependent Claims (3, 4, 5)
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20-55. -55. (canceled)
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56. A system for assembling a target nucleic acid, the system comprising:
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a means for analyzing a sequence of a target nucleic acid for the presence of one or more predetermined interfering sequence features that are predicted to disrupt a multiplex oligonucleotide assembly reaction, a means for assembling one or more first fragments of the target nucleic acid using a polymerase-mediated oligonucleotide assembly reaction, wherein no predetermined interfering sequence features are present in any of the one or more first fragments, a means for assembling one or more second fragments of the target nucleic acid using a ligase-mediated oligonucleotide assembly reaction if one or more predetermined interfering sequence features are present in the target nucleic acid, wherein the one or more second fragments comprise the one or more predetermined interfering sequence features, and a means for assembling the target nucleic acid from the one or more first and second fragments.
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57-61. -61. (canceled)
Specification