Compositions and Methods for High Fidelity Assembly of Nucleic Acids

• US 20150203839A1
• Filed: 08/23/2012
• Published: 07/23/2015
• Est. Priority Date: 08/26/2011
• Status: Abandoned Application

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Schindler, Daniel, Lawton, Scott S.
• Application Number:
  US14/241,242
• Publication Number:
  US 20150203839A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1089 Design, preparation, screening or analysis of libraries using computer algorithms
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase

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.

Claims

1. A method of producing a target nucleic acid having a predefined sequence, the method comprising:

releasing a plurality of oligonucleotides synthesized on a solid support, wherein the plurality of oligonucleotides each are engineered to comprise, at both ends, a universal primer binding site and a restriction enzyme recognition sequence;

synthesizing complementary strands of the plurality of oligonucleotides using a universal primer complementary to the universal primer binding site in a polymerase based reaction, thereby providing a plurality of blunt-end double-stranded nucleic acid fragments;

enzymatically digesting the plurality of blunt-end double-stranded nucleic acid fragments to produce a plurality of cohesive-end double-stranded nucleic acid fragments that are designed to together comprise the target nucleic acid sequence, wherein the plurality of cohesive-end double-stranded nucleic acid fragments each have two different and non-complementary overhangs; and

ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase, wherein a first overhang of a first cohesive-end double-stranded nucleic acid fragment is uniquely complementary to a second overhang of a second, preselected cohesive-end double-stranded nucleic acid fragment;

thereby forming a linear and predetermined arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the linear and predetermined arrangement comprises the target nucleic acid having the predefined sequence.

2. The method of claim 1, wherein the plurality of oligonucleotides are immobilized on a solid support.

6. The method of claim 1, wherein said the restriction enzyme recognition sequence is part of the universal primer binding site and is located at the 5′

or 3′

end of the universal primer binding site.

7. The method of claim 1, wherein the universal primer has an affinity tag to facilitate affinity removal of undesirable enzymatic digestion products.

8. The method of claim 7, wherein the affinity tag is biotin.

9. The method of claim 1, wherein the plurality of blunt-end double-stranded nucleic acids comprises at least 3, 4, 5, 6, 7, 8, 10, 15 or 20 different blunt-end double-stranded nucleic acid fragments.

10. The method of claim 1, wherein each of the plurality of blunt-end double-stranded nucleic acid fragments is at least 50, 100, 200, or 300 bases long.

11. The method of claim 1, wherein the restriction enzyme recognition sequence is the same for all blunt-end double-stranded nucleic acid fragments.

12. The method of claim 1, wherein the plurality of blunt-end double-stranded nucleic acid fragments comprise at least two different restriction enzyme recognition sequences recognizable by two different restriction enzymes that are selected to produce overhangs having the same number of bases.

13. The method of claim 1, wherein the restriction enzyme recognition sequence is capable of being recognized by a type IIs restriction enzyme.

14. The method of claim 13, wherein the type IIs restriction enzyme is BsaI, BsmBI, BspQI, BtgZI, BsmFI, FokI, BbvI, any variant thereof, or any combination thereof.

15. The method of claim 1, wherein the plurality of cohesive-end double-stranded nucleic acid fragments are designed such that a cohesive end in a cohesive-end double-stranded nucleic acid fragment is uniquely complementary to a next cohesive end in an adjacent cohesive-end double-stranded nucleic acid fragment.

16. The method of claim 1, wherein the first and second overhangs are at least 3, 4, 5, 6, 7, or 8 bases long.

17. The method of claim 1, wherein the first and second overhangs differ from one another by at least 1, 2, 3 or 4 bases.

18. The method of claim 1, wherein the first and second overhangs are 5′

or 3′

overhangs.

19. The method of claim 1, further comprising, before the ligating step, purifying the plurality of cohesive-end double-stranded nucleic acid fragments to remove undesirable enzymatic digestion products.

20. The method of claim 19, wherein the undesirable enzymatic digestion products include fragments less than about 40, about 35, about 30, about 25, about 20, or about 15 bases long.

21. The method of claim 19, wherein said purifying includes differential affinity to silica, size filtration, differential precipitation with polyethylene glycol or cetyltrimethlyammonium bromide, or any combination thereof.

22. The method of claim 1, wherein the ligase is T3 DNA ligase, T4 DNA ligase, T7 DNA ligase, E. coli DNA ligase, any variant thereof, or any combination thereof.

23. The method of claim 1, wherein the target nucleic acid is a non-naturally occurring nucleic acid.

24. The method of claim 1, wherein the target nucleic acid is at least 500, 800, 1000, 1500, 2000, or 3000 bases long.

25. The method of claim 1, further comprising amplifying the target nucleic acid using a pair of primers specific to the target nucleic acid and a polymerase.

26. The method of claim 1, further comprising confirming the sequence of the target nucleic acid.

27. The method of claim 1, wherein the plurality of blunt-end double-stranded nucleic acid fragments are hierarchically assembled from synthetic oligonucleotides.

28. The method of claim 1 wherein the plurality of cohesive-end double-stranded nucleic acid fragments are ligated in a single pool.

29. The method of claim 1 wherein the plurality of cohesive-end double-stranded nucleic acid fragments are in at least two pools, each nucleic acid fragment of the first pool having a terminal end complementary to a nucleic acid fragment of the second pool.

3-5. -5. (canceled)

30-44. -44. (canceled)