Compositions and Methods for Multiplex Nucleic Acids Synthesis

• US 20150376602A1
• Filed: 03/13/2014
• Published: 12/31/2015
• Est. Priority Date: 03/15/2013
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Schindler, Daniel, Goldberg, Martin J., Hudson, Michael E., Kung, Li-yun A., Saaem, Ishtiaq, Lawton, Scott
• Granted Patent: US 10,273,471 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

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.

Claims

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.

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 anchor oligonucleotides are attached to a support.

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

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

7. The method of claim 4 wherein the support is a stem loop polynucleotide.

8. The method of claim 4 wherein each of the plurality of the double-stranded anchor oligonucleotides is immobilized to the same support.

9. The method of claim 4 wherein each of the plurality of the double-stranded anchor oligonucleotides is immobilized to a different support.

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

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

11. 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;

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.

12. The method of claim 11 wherein the first, second, third and fourth pluralities of overhangs are each designed to be relatively mutually orthogonal to each other.

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

19. The method of claim 12 further comprising, optionally, repeating steps a)-c).

14. 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 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.

15. The method of claim 14 wherein the first and second pluralities of 5′

and 3′

overhangs are each designed to be relatively mutually orthogonal to each other.

16. The method of claim 14 or claim 15 further comprising, optionally, repeating the ligating step.

17. 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.

18. The method of claim 17, wherein the first and second pluralities of blunt-ended double-stranded oligonucleotides are error corrected or error reduced prior to melting.