Compositions and Methods for High Fidelity Assembly of Nucleic Acids
First Claim
1. A method of producing a target nucleic acid having a predefined sequence, the method comprising:
- releasing a plurality of oligonucleotides synthesized on a solid support, wherein the plurality of oligonucleotides each are engineered to comprise, at both ends, a universal primer binding site and a restriction enzyme recognition sequence;
synthesizing complementary strands of the plurality of oligonucleotides using a universal primer complementary to the universal primer binding site in a polymerase based reaction, thereby providing a plurality of blunt-end double-stranded nucleic acid fragments;
enzymatically digesting the plurality of blunt-end double-stranded nucleic acid fragments to produce a plurality of cohesive-end double-stranded nucleic acid fragments that are designed to together comprise the target nucleic acid sequence, wherein the plurality of cohesive-end double-stranded nucleic acid fragments each have two different and non-complementary overhangs; and
ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase, wherein a first overhang of a first cohesive-end double-stranded nucleic acid fragment is uniquely complementary to a second overhang of a second, preselected cohesive-end double-stranded nucleic acid fragment;
thereby forming a linear and predetermined arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the linear and predetermined arrangement comprises the target nucleic acid having the predefined sequence.
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Abstract
Aspects of the invention relate to methods, compositions and algorithms for designing and producing a target nucleic acid. The method can include: (1) providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends thereof; (2) producing via enzymatic digestion a plurality of cohesive-end double-stranded nucleic acid fragments each having two different and non-complementary overhangs; (3) ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase; and (4) forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid. In certain embodiments, the plurality of blunt-end double-stranded nucleic acid fragments can be provided by: releasing a plurality of oligonucleotides synthesized on a solid support; and synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.
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Citations
30 Claims
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1. A method of producing a target nucleic acid having a predefined sequence, the method comprising:
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releasing a plurality of oligonucleotides synthesized on a solid support, wherein the plurality of oligonucleotides each are engineered to comprise, at both ends, a universal primer binding site and a restriction enzyme recognition sequence; synthesizing complementary strands of the plurality of oligonucleotides using a universal primer complementary to the universal primer binding site in a polymerase based reaction, thereby providing a plurality of blunt-end double-stranded nucleic acid fragments; enzymatically digesting the plurality of blunt-end double-stranded nucleic acid fragments to produce a plurality of cohesive-end double-stranded nucleic acid fragments that are designed to together comprise the target nucleic acid sequence, wherein the plurality of cohesive-end double-stranded nucleic acid fragments each have two different and non-complementary overhangs; and ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase, wherein a first overhang of a first cohesive-end double-stranded nucleic acid fragment is uniquely complementary to a second overhang of a second, preselected cohesive-end double-stranded nucleic acid fragment; thereby forming a linear and predetermined arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the linear and predetermined arrangement comprises the target nucleic acid having the predefined sequence. - View Dependent Claims (2, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)
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3-5. -5. (canceled)
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30-44. -44. (canceled)
Specification