Compositions and methods for multiplex nucleic acids synthesis

Patent metadata

Field	Value
Publication number	US-10273471-B2
Application number	US-201414766195-A
Country	US
Kind code	B2
Filing date	Mar 13, 2014
Priority date	Mar 15, 2013
Publication date	Apr 30, 2019
Grant date	Apr 30, 2019

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.

First claim

The invention claimed is:

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, wherein the anchor oligonucleotides are attached to a support;
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.

2. The method of claim 1 wherein the first plurality of overhangs are designed to be relatively mutually orthogonal to each other.

3. The method of claim 1 wherein the second plurality of overhangs are designed to be relatively mutually orthogonal to each other.

4. The method of claim 1 wherein the support is solid.

5. The method of claim 4 wherein the support is a bead.

6. The method of claim 1 wherein the support is a stem loop polynucleotide.

7. The method of claim 1 wherein each of the plurality of the double-stranded anchor oligonucleotides is immobilized to the same support.

8. The method of claim 1 wherein each of the plurality of the double-stranded anchor oligonucleotides is immobilized to a different support.

9. The method of claim 1 wherein steps a)-d) are performed in a single reaction volume.

10. A method of producing at least one target nucleic acid having a predefined sequence, the method comprising:
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; wherein the first plurality of overhangs are each designed to be relatively mutually orthogonal to each other; and wherein the second plurality of overhangs are each designed to be relatively mutually orthogonal to each other;
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; wherein the third plurality of overhangs are each designed to be relatively mutually orthogonal to each other; and wherein the fourth plurality of overhangs are each designed to be relatively mutually orthogonal to each other; 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.

11. The method of claim 10 further comprising, repeating steps a)-c).

12. A method of producing at least one target nucleic acid having a predefined sequence, the method comprising:
ligating a first plurality of double-stranded oligonucleotides with a second plurality of double-stranded oligonucleotides at a 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 a 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;
wherein the first plurality of 3′ overhangs are each designed to be relatively mutually orthogonal to each other; wherein the first plurality of 5′ overhangs are each designed to be relatively mutually orthogonal to each other; wherein the second plurality of 3′ overhangs are each designed to be relatively mutually orthogonal to each other; and wherein the second plurality of 5′ overhangs are each designed to be relatively mutually orthogonal to each other;
thereby forming at least one target nucleic acid.

13. The method of claim 12 further comprising, repeating the ligating step.

14. The method of claim 2 wherein the second plurality of overhangs are designed to be relatively mutually orthogonal to each other.

Assignees

• Gen9 Inc
• Gen 9 Inc

Inventors

• Jacobson Joseph
• Schindler Daniel
• Saaem Ishtiaq
• Lawton Scott
• Goldberg Martin J
• Hudson Michael E
• Kung Li-Yun A

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/1031 Primary

  mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR · CPC title

• B01J2219/00722

  Nucleotides · CPC title

• B01J2219/005

  Beads · CPC title