Compositions, Methods and Apparatus for Oligonucleotides Synthesis

• US 20160122755A1
• Filed: 03/13/2014
• Published: 05/05/2016
• 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: Lawton, Scott S., Hall, Giles F.
• Granted Patent: US 10,280,417 B2
• 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 15/1031 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, 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, the 3′

flanking sequence comprising a primer recognition site and a restriction enzyme recognition site and the 5′

flanking sequence comprising a primer recognition site, a restriction enzyme recognition site and an additional padding nucleotide sequence, wherein each padding nucleotide sequence has a different length;

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

c. isolating the oligonucleotide having the correct predefined internal sequence.

3. The method of claim 1 further comprising amplifying the plurality of oligonucleotides.

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

5. The method of claim 3 further comprising subjecting the plurality of amplified oligonucleotides to error removal prior or after exposing the amplified oligonucleotides to a restriction enzyme under conditions suitable to promote digestion.

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 endonuclease is a type IIS restriction endonuclease.

8. The method of claim 1 wherein in the step of designing the padding nucleotide sequence is between the primer recognition site and the restriction enzyme recognition site.

9. The method of claim 1 wherein in the step of designing the padding nucleotide sequence is upstream the primer recognition site.

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

11. The method of claim 1 wherein in the step of synthesizing, the plurality of oligonucleotides comprise error containing oligonucleotides and error-free oligonucleotides.

12. The method of claim 1 wherein the step of isolating comprises sequencing the oligonucleotides.

2. (canceled)

13. A redundant array comprising at least one plurality of oligonucleotide sequences, each oligonucleotide of each plurality of oligonucleotides having a different length, each oligonucleotide having an identical predefined internal sequence and 5′

and 3′

flanking sequences flanking the 5′ and

the 3′

end of the internal sequence,

the 3′

flanking region comprising, from 5′

to 3′

, a restriction enzyme recognition site and primer recognition site,

the 5′

flanking region comprising from 5′

to 3′

, a primer recognition site and a restriction enzyme recognition site, and further comprising an additional padding nucleotide sequence, wherein each padding nucleotide sequence has a different length,

the redundant array comprising redundant internal sequences.

14. The redundant array of claim 13 wherein the padding nucleotide sequence is between the primer recognition site and the restriction enzyme recognition site.

15. The redundant array of claim 13 wherein the padding nucleotide sequence is 5′

of the primer recognition site.

16. The redundant array of claim 13 wherein each internal sequence is represented by two sets of oligonucleotides, the second set of oligonucleotides being a reverse-complement of the first set of oligonucleotides.

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

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

end of the target nucleic acid,

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

end of the target nucleic acid; and

c. optionally one or more plurality of oligonucleotides comprising an internal sequence identical to a different portion of a sequence of a target nucleic acid, each of the plurality of oligonucleotides having an overlapping sequence region corresponding to a sequence region in a next oligonucleotide to together comprise the target nucleic acid;

wherein each plurality of oligonucleotides has 5′ and

3′

flanking sequences at the 5′ and

the 3′

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

wherein the 5′

flanking sequence has an additional padding nucleotide sequence.

18. The composition of claim 17 wherein the padding nucleotide sequence is between the primer recognition site and the restriction enzyme recognition site.

19. The composition of claim 17 wherein the padding nucleotide sequence is 5′

of the primer recognition site.

20. The composition of claim 17 wherein the padding nucleotide sequence is from 4 bps long to 10 bps long.

21. The composition of claim 17 wherein the restriction enzyme recognition site is a type IIS restriction enzyme recognition site.