Compositions, methods and apparatus for oligonucleotides synthesis

• US 10,280,417 B2
• Filed: 03/13/2014
• Issued: 05/07/2019
• Est. Priority Date: 03/13/2013
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Hall, Giles F., Lawton, Scott S.
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US14/765,961
• Publication Number:
  US 20160122755A1
• Time in Patent Office: 1,881 Days
• Field of Search: None
• 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 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1068 Template (nucleic acid) mediated chemical library synthesis, e.g. chemical and enzymatical DNA-templated organic molecule synthesis, libraries prepared by non ribosomal polypeptide synthesis [NRPS], DNA/RNA-polymerase mediated polypeptide synthesis
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

Aspects of the invention relate to methods, compositions for synthesizing oligonucleotides having a predefined sequence.

Claims

1. A method for generating an oligonucleotide having a predefined sequence, the method comprising:

a. designing at least one plurality of oligonucleotides, each oligonucleotide of each plurality of oligonucleotides having a different length and each oligonucleotide having an identical predefined internal sequence, a 3′

flanking sequence at the 3′

end of the internal sequence and a 5′

flanking sequence at the 5′

end of the internal sequence,

wherein the 3′

flanking sequence comprises a primer recognition site and a restriction enzyme recognition site;

wherein the 5′

flanking sequence comprises a primer recognition site, a restriction enzyme recognition site and a padding nucleotide sequence, wherein each oligonucleotide of each plurality of oligonucleotides comprises a padding nucleotide sequence of a different length;

b. synthesizing the at least one plurality of oligonucleotides on a solid support; and

c. isolating the oligonucleotide having the predefined sequence.

2. The method of claim 1 further comprising amplifying the at least one plurality of oligonucleotides to produce a plurality of amplified oligonucleotides.

3. The method of claim 2 further comprising exposing the plurality of amplified oligonucleotides to a restriction enzyme under conditions suitable to promote digestion.

4. The method of claim 2 further comprising subjecting the plurality of amplified oligonucleotides to error removal prior to or after exposing the plurality of amplified oligonucleotides to a restriction enzyme under conditions suitable to promote digestion.

5. The method of claim 4 wherein the plurality of amplified oligonucleotides are contacted with a mismatch binding agent, wherein the mismatch binding agent selectively binds and cleaves double-stranded oligonucleotides comprising a mismatch.

6. The method of claim 3 wherein the restriction enzyme is a type IIS restriction endonuclease.

7. The method of claim 1 wherein the padding nucleotide sequence is positioned between the primer recognition site and the restriction enzyme recognition site within the 5′

flanking sequence.

8. The method of claim 1 wherein the padding nucleotide sequence is positioned upstream of the primer recognition site within the 5′

flanking sequence.

9. The method of claim 1, wherein the padding nucleotide sequence is from 4 bps long to 10 bps long.

10. The method of claim 1 wherein the plurality of oligonucleotides comprise error containing oligonucleotides and error-free oligonucleotides.

11. The method of claim 1 wherein the method further comprises sequencing the oligonucleotides isolated from step c.

12. A redundant array comprising at least one plurality of oligonucleotide sequences, each oligonucleotide of each plurality of oligonucleotides having a different length and each oligonucleotide of each plurality of oligonucleotides having an identical predefined internal sequence, a 3′

flanking sequence at the 3′

end of the internal sequence and a 5′

flanking sequence at the 5′

end of the internal sequence,

wherein the 3′

flanking sequence comprises from 5′

to 3′

, a restriction enzyme recognition site and a primer recognition site;

wherein the 5′

flanking sequence comprises from 5′

to 3′

, a primer recognition site and a restriction enzyme recognition site, and further comprises a padding nucleotide sequence, wherein each padding nucleotide sequence has a different length; and

wherein the redundant array comprises redundant internal sequences.

13. The redundant array of claim 12 wherein the padding nucleotide sequence is positioned between the primer recognition site and the restriction enzyme recognition site within the 5′

flanking sequence.

14. The redundant array of claim 12 wherein the padding nucleotide sequence is positioned upstream of the primer recognition site sequence is positioned upstream of the primer recognition site within the 5′

flanking sequence.

15. The redundant array of claim 12 wherein each internal sequence is represented by two sets of oligonucleotides, a second set of oligonucleotides being a reverse-complement of a first set of oligonucleotides.

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

a. a first plurality of oligonucleotides comprising a first internal sequence identical to the 5′

end of the target nucleic acid;

b. a second plurality of oligonucleotides comprising a second internal sequence identical to the 3′

end of the target nucleic acid; and

c. optionally one or more pluralities of oligonucleotides each comprising an internal sequence identical to a different portion of the sequence of the target nucleic acid, wherein each oligonucleotide of each plurality of oligonucleotides has a sequence region overlapping with a sequence region in each oligonucleotide of another plurality of oligonucleotides, wherein each internal sequence of the one or more pluralities of oligonucleotides together with the first and second internal sequences comprise the target nucleic acid;

wherein each oligonucleotide has 5′ and

3′

flanking sequences at the 5′

end and the 3′

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

wherein each 5′

flanking sequence further comprises a padding nucleotide sequence that has a different length than other padding sequences within each plurality of oligonucleotides.

17. The composition of claim 16 wherein the padding nucleotide sequence is positioned between the primer recognition site and the restriction enzyme recognition site within the 5′

flanking sequence.

18. The composition of claim 16 wherein the padding nucleotide sequence is positioned upstream of the primer recognition site within the 5′

flanking sequence.

19. The composition of claim 16 wherein the padding nucleotide sequence is from 4 bps long to 10 bps long.

20. The composition of claim 16 wherein the restriction enzyme recognition site is a type IIS restriction enzyme recognition site.

21. The composition of claim 16, wherein the one or more pluralities of oligonucleotides of part c are present in the composition.