Methods for Nucleic Acid Assembly and High Throughput Sequencing

• US 20150191719A1
• Filed: 06/24/2013
• Published: 07/09/2015
• Est. Priority Date: 06/25/2012
• Status: Abandoned Application

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Hudson, Michael E., Kung, Li-yun A., Schindler, Daniel, Archer, Stephen, Saaem, Ishtiaq
• Application Number:
  US14/408,103
• Publication Number:
  US 20150191719A1
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• 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: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/501 Ligase

Abstract

Methods and apparatus of some aspects of the invention relate to the synthesis of high fidelity polynucleotides. In particular, aspects of the invention relate to concurrent enzymatic removal of amplification sequences and ligation of processed oligonucleotides into nucleic acid assemblies. According to some embodiments, the invention provides a method for producing a target nucleic acid having a predefined sequence. In some embodiments, the method comprises the step of providing a plurality of oligonucleotides, wherein each oligonucleotides comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) a 5′ sequence flanking the 5′ end of the internal sequence and a 3′ flanking sequence flanking the 3′ end of the internal sequence, each of the flanking sequence comprising a primer recognition site for a primer pair and a restriction enzyme recognition site.

Claims

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

a) providing a first mixture comprising;

(i) a first pool of oligonucleotides comprising a first plurality of oligonucleotides comprising a sequence identical to the 5′

end of the target nucleic acid, a second plurality of oligonucleotides comprising a sequence identical to the 3′

end of the target nucleic acid, and a plurality of oligonucleotides comprising a sequence identical to a different portion of a sequence of a target nucleic acid, each of the oligonucleotides having an overlapping sequence region corresponding to a sequence region in a next oligonucleotide, the oligonucleotides in the first pool together comprising the target nucleic acid sequence;

(ii) a restriction enzyme, and

b) exposing the first mixture to a ligase and subjecting the first mixture comprising the ligase to conditions suitable to promote restriction enzyme digestion and ligation, thereby generating the target nucleic acid.

3. The method of claim 1 further comprising, prior to step (a), providing a plurality of construction oligonucleotides, wherein each construction oligonucleotide comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) 5′

and 3′

flanking sequences flanking the 5′

end and the 3′

end of the internal sequence, each of the flanking sequence comprising a primer recognition site for a primer pair and a restriction enzyme recognition site.

4. The method of claim 3 further comprising amplifying the plurality of construction oligonucleotides.

5. The method of claim 4 further comprising subjecting the plurality of amplified oligonucleotides to error removal.

6. The method of claim 5 wherein the plurality of amplified oligonucleotides are contacted with a mismatch binding agent, wherein the mismatch binding agent selectively binds and cleaves the double-stranded oligonucleotides comprising a mismatch.

7. The method of claim 4 wherein the restriction enzyme and the ligase are added to a single pool of amplified oligonucleotides under conditions suitable to promote digestion and ligation, thereby generating a mixture comprising the assembled target nucleic acid sequences, and the flanking sequences.

9. The method of claim 1 wherein the restriction enzyme is a Type IIS restriction enzyme.

10. The method of claim 7 wherein the restriction enzyme is a Type IIS restriction enzyme, wherein digestion with the Type IIS restriction enzyme produces a plurality of cohesive end double-stranded oligonucleotides and wherein the plurality of cohesive end double-stranded oligonucleotides are ligated in a unique linear arrangement.

11. The method of claim 1 further comprising amplifying the target nucleic acid using a primer pair capable of recognizing a primer recognition site at the 5′

end of the first oligonucleotide and 3′

end of second oligonucleotide.

14. The method of claim 1 further comprising sequencing at least one target nucleic acid to confirm its sequence accuracy and isolating at least one target nucleic acid having the predefined sequence from a pool of nucleic acid sequences.

16. The method of claim 1 further comprising

c) providing a second mixture comprising

(i) a second pool of oligonucleotides comprising a first plurality of oligonucleotides comprising a sequence identical to the 5′

end of the target nucleic acid, a second plurality of oligonucleotides comprising a sequence identical to the 3′

end of the target nucleic acid, and a plurality of oligonucleotides comprising a sequence identical to a different portion of a sequence of a target nucleic acid, each of the oligonucleotides having an overlapping sequence region corresponding to a sequence region in a next oligonucleotide, the oligonucleotides in the second pool together comprising the second target nucleic acid;

(ii) a restriction enzyme, and

d) exposing the second mixture to a ligase and subjecting the second mixture comprising the ligase to conditions suitable to promote restriction enzyme digestion and ligation, thereby generating a second target nucleic acid.

17. The method of claim 16 further comprising assembling at least two target nucleic acids.

19. The method of claim 17 wherein the second plurality of oligonucleotides of the first pool of oligonucleotides comprises a restriction endonuclease recognition site for a restriction endonuclease and the first plurality of oligonucleotides of the second pool of oligonucleotides comprises a restriction endonuclease recognition site for the restriction endonuclease.

20. The method of claim 19 wherein the at least two target nucleic acids are subjected to restriction endonuclease digestion and ligation thereby forming a long target nucleic acid construct.

21. The method of claim 20, wherein the long target nucleic acid construct is at least about 10 kilobases in length.

2. (canceled)

8. (canceled)

12. (canceled)

13. (canceled)

15. (canceled)

18. (canceled)

22. (canceled)

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

a) providing a plurality of oligonucleotides, wherein each oligonucleotide comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) 5′

flanking sequence flanking the 5′

end of the internal sequence and 3′

flanking sequences flanking the 3′

end of the internal sequence, each of the flanking sequences comprising a primer recognition site for a primer pair and a restriction enzyme recognition site for a restriction endonuclease;

b) amplifying at least a subset of the oligonucleotides using the primer pair thereby generating a plurality of amplified oligonucleotides;

c) optionally subjecting the plurality of amplified oligonucleotides to error removal;

d) providing a circular vector having a restriction enzyme recognition site for the restriction endonuclease; and

c) exposing the plurality of amplified oligonucleotides and circular vector to the restriction enzyme and ligase in a single pool, wherein the restriction enzyme is capable of recognizing the restriction enzyme recognition sites, thereby assembling the target nucleic acid in the vector.

24. The method of claim 23 further comprising transforming the vector into a host cell.

25. A composition for the assembly of a target nucleic acid having a predefined sequence comprising:

a) a pool of oligonucleotides comprising a first plurality of oligonucleotides comprising a sequence identical to the 5′

end of the target nucleic acid, a second plurality of oligonucleotides comprising a sequence identical to the 3′

end of the target nucleic acid, and one or more plurality of oligonucleotides comprising a sequence identical to a different portion of a sequence of a target nucleic acid, each of the oligonucleotides having an overlapping sequence region corresponding to a sequence region in a next oligonucleotide, the oligonucleotides in the pool together comprising the target nucleic acid;

b) a plurality of common sequences comprising a primer recognition site for a primer pair and a restriction endonuclease recognition site;

c) a restriction endonuclease;

d) a ligase; and

e) a linearized vector having a 5′

end compatible with the first plurality of oligonucleotides and a 3′

end compatible with the second plurality of oligonucleotides.

26. The composition of claim 25 wherein the oligonucleotides are amplified, error corrected, or amplified and error corrected.

29. The composition of claim 25 wherein the restriction endonuclease is a Type IIS restriction endonuclease.

27. (canceled)

28. (canceled)