Compositions and Methods for Multiplex Nucleic Acids Synthesis
First Claim
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1. A method of producing at least one target nucleic acid having a predefined sequence, the method comprising:
- a. providing a plurality of double-stranded anchor oligonucleotides having a first plurality of overhangs;
b. providing a plurality of double-stranded construction oligonucleotides having a second plurality of overhangs, wherein the second plurality of overhangs are designed to be complementary to the first plurality of overhangs;
c. hybridizing and ligating the double-stranded anchor oligonucleotides with the double-stranded construction oligonucleotides; and
d. optionally, repeating steps a)-c), thereby generating the at least one target nucleic acid.
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Abstract
Aspects of the invention relate to methods, compositions for designing and producing a target nucleic acid. In particular, aspects of the invention relate to the multiplex synthesis of target polynucleotides.
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Citations
20 Claims
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1. A method of producing at least one target nucleic acid having a predefined sequence, the method comprising:
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a. providing a plurality of double-stranded anchor oligonucleotides having a first plurality of overhangs; b. providing a plurality of double-stranded construction oligonucleotides having a second plurality of overhangs, wherein the second plurality of overhangs are designed to be complementary to the first plurality of overhangs; c. hybridizing and ligating the double-stranded anchor oligonucleotides with the double-stranded construction oligonucleotides; and d. optionally, repeating steps a)-c), thereby generating the at least one target nucleic acid. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 20)
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11. A method of producing at least one target nucleic acid having a predefined sequence, the method comprising:
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a. ligating a first plurality of double-stranded oligonucleotides having a first plurality of overhangs with a second plurality of double-stranded oligonucleotides having a second plurality of overhangs, wherein the first plurality of overhangs are designed to be complementary to the second plurality of overhangs, thereby producing a first set of polynucleotides; b. ligating a third plurality of double-stranded oligonucleotides having a third plurality of overhangs with a fourth plurality of double-stranded oligonucleotides having a fourth plurality of overhangs, wherein the third plurality of overhangs are designed to be complementary to the fourth plurality of overhangs, thereby producing a second set of polynucleotides; and c. ligating the first set of polynucleotides with the second set of polynucleotides, wherein the first set of polynucleotides and the second set of polynucleotides are designed to have complementary overhangs, thereby forming at least one target nucleic acid. - View Dependent Claims (12, 13, 19)
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14. A method of producing at least one target nucleic acid having a predefined sequence, the method comprising:
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ligating a first plurality of double-stranded oligonucleotides with a second plurality of double-stranded oligonucleotides at 5 ‘
end of the second plurality of double-stranded oligonucleotides, and ligating the second plurality of double-stranded oligonucleotides with a third plurality of double-stranded oligonucleotides at 3’
end of the second plurality of double-stranded oligonucleotides, wherein the first plurality of double-stranded oligonucleotides have a first plurality of 3′
overhangs, the second plurality of double-stranded oligonucleotides have a first plurality of 5′
overhangs and a second plurality of 3′
overhangs, and the third plurality of double-stranded oligonucleotides have a second plurality of 5′
overhangs, wherein the first plurality of 3′
overhangs are designed to be complementary to the first plurality of 5′
overhangs, and the second plurality of 3′
overhangs are designed to be complementary to the second plurality of 5′
overhangs;thereby forming at least one target nucleic acid. - View Dependent Claims (15, 16)
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17. A method of producing a plurality of double-stranded oligonucleotides having a plurality of overhangs, comprising:
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a) melting a first plurality of blunt-ended double-stranded oligonucleotides and a second plurality of blunt-ended double-stranded oligonucleotides to form a plurality of single-stranded oligonucleotides; and b) re-annealing the plurality of single-stranded oligonucleotides to form a plurality of double-stranded oligonucleotides having a plurality of overhangs, wherein the plurality of overhangs are designed to be substantially mutually orthogonal to each other. - View Dependent Claims (18)
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Specification