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US 12,668,791 B2Granted
Compositions and methods for multiplex nucleic acids synthesis
AssigneeTwist Bioscience Corporation
InventorsJoseph Jacobson, Daniel Schindler, Ishtiaq Saaem, Scott S. Lawton, Martin J. Goldberg, Michael E. Hudson, Li-Yun A. Kung
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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.
Claims14
A method of producing at least one target nucleic acid having a predefined sequence, the method comprising:
- (a) providing a first plurality of double-stranded anchor oligonucleotides having a first plurality of overhangs, wherein the anchor oligonucleotides are attached to a support, and wherein the first plurality of overhangs are designed to be relatively mutually orthogonal to each other;
- (b) providing a first plurality of double-stranded construction oligonucleotides having a second plurality of overhangs and a third plurality of overhangs, wherein the second plurality of overhangs are designed to be complementary to the first plurality of overhangs, wherein the second plurality of overhangs are designed to be relatively mutually orthogonal to each other, and wherein the third plurality of overhangs are designed to be relatively mutually orthogonal to each other;
- (c) hybridizing the first plurality of overhangs with the second plurality of overhangs and ligating the double-stranded anchor oligonucleotides with the double-stranded construction oligonucleotides to form a second plurality of double-stranded anchor oligonucleotides with the third plurality of overhangs;
- and
- (d) repeating steps (a) through (c), using the second plurality of double-stranded anchor oligonucleotides and a second plurality of double-stranded construction oligonucleotides having a fourth and fifth plurality of overhangs, wherein the fourth plurality of overhangs are designed to be complementary to the third plurality of overhangs, wherein the fourth plurality of overhangs are designed to be relatively mutually orthogonal to each other, and wherein the fifth plurality of overhangs are designed to be relatively mutually orthogonal to each other thereby generating the at least one target nucleic acid.
2 more elements
A method of producing a plurality of double-stranded oligonucleotides having a plurality of overhangs, comprising:
- (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.
Dependencies are inferred from the claim text.