Compositions and Methods For High Fidelity Assembly of Nucleic Acids
First Claim
31. A method for designing a plurality of starting nucleic acids to be assembled into a target nucleic acid, the method comprising:
- (a) obtaining an input target sequence of a target nucleic acid;
(b) selecting a plurality of subsequences therein such that every two adjacent subsequences overlap with each other by N bases;
(c) storing the resulting overlapping N-base sequences in a memory;
(d) comparing the overlapping N-base sequences to one another to ensure that they differ from one another by at least one base; and
(e) repeating steps (b) to (d) until a plurality of satisfactory nucleic acid fragments are obtained wherein any two adjacent starting nucleic acid fragments uniquely overlap with each other by N bases.
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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
43 Claims
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31. A method for designing a plurality of starting nucleic acids to be assembled into a target nucleic acid, the method comprising:
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(a) obtaining an input target sequence of a target nucleic acid; (b) selecting a plurality of subsequences therein such that every two adjacent subsequences overlap with each other by N bases; (c) storing the resulting overlapping N-base sequences in a memory; (d) comparing the overlapping N-base sequences to one another to ensure that they differ from one another by at least one base; and (e) repeating steps (b) to (d) until a plurality of satisfactory nucleic acid fragments are obtained wherein any two adjacent starting nucleic acid fragments uniquely overlap with each other by N bases. - View Dependent Claims (35, 36, 37, 38, 39, 42)
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32-1. The method of claim 32, wherein the restriction enzyme recognition site is a type IIS recognition site.
- 40. The plurality of starting nucleic acids of claim 40, each further comprising an engineered universal primer binding site for amplifying the plurality of starting nucleic acids therefrom.
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43. A computer program product for designing a plurality of starting nucleic acids to be assembled into a target nucleic acid, said program residing on a hardware computer readable storage medium and having a plurality of instructions which, when executed by a processor, cause the processor to perform operations comprising:
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(a) obtaining a target sequence of a target nucleic acid; (b) selecting a plurality of subsequences therein such that every two adjacent subsequences overlap with each other by N bases; (c) storing the resulting overlapping N-base sequences in a memory; (d) comparing the overlapping N-base sequences to one another to ensure that they differ from one another by at least one base; and (e) repeating steps (b) to (d) until a plurality of satisfactory starting nucleic acids are obtained wherein any two adjacent starting nucleic acids uniquely overlap with each other by N bases.
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Specification