Compositions and Methods for Synthesis of High Fidelity Oligonucleotides

• US 20150368687A1
• Filed: 03/13/2014
• Published: 12/24/2015
• 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: Hudson, Michael E., Jacobson, Joseph, Saaem, Ishtiaq E., Archer, Stephen F., Sann-Rorth, Sophea, Fanti, Ellen J.
• Granted Patent: US 10,053,719 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 15/66 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

Abstract

Aspects of the invention relate to methods, compositions for synthesizing high fidelity oligonucleotides.

Claims

1. A method for synthesizing high fidelity target oligonucleotides, the method comprising:

a. providing an array comprising a plurality of nucleic acids, each nucleic acid having a sequence comprising a template oligonucleotide sequence and a primer binding sequence, wherein the template oligonucleotide sequence is a reverse complement of the target oligonucleotide;

b. releasing the plurality of nucleic acids from the array;

c. circularizing the plurality of nucleic acids;

d. providing a primer, wherein the primer comprises a sequence complementary to the primer binding sequence, a restriction enzyme recognition site and self-complementary termini, under conditions promoting annealing of the primer to the primer binding sites;

e. amplifying the circularized nucleic acids in rolling-circle amplification reaction using the primer thereby forming a single-stranded tandem nucleic acid sequences;

f. removing the primer sequence of the single-stranded tandem nucleic acid sequences under conditions promoting hairpin formation of the primer; and

g. isolating the target oligonucleotides.

2. The method of claim 1 wherein in the step of releasing the plurality of nucleic acids is released in a pool.

3. The method according to claim 1 wherein the plurality of nucleic acids is immobilized on the array by their 3′

terminus.

4. The method according to claim 1 wherein the plurality of nucleic acids is synthesized on the array in a 3′

-5′

direction.

5. The method according to claim 1 wherein the plurality of nucleic acids is synthesized on the array in a 5′

-3′

direction.

6. The method according to claim 1 wherein the primer binding sequence is at least 100 bps long.

7. The method according to claim 1 wherein in the step of providing the array, the array comprises a plurality of template oligonucleotides and the primer binding sequence is ligated to each of the plurality of the template oligonucleotides.

8. The method according to claim 1 wherein, in the step of providing the array, the array comprises a porous reaction layer coupled to a phosphorylation reagent.

10. The method according to claim 1 wherein the primer binding site is 3′

or 5′

of the template oligonucleotide sequence.

11. The method according to claim 1 wherein the plurality of nucleic acids is phosphorylated at the 3′

end and wherein the step of releasing further comprises dephosphorylating the 3′

end of the nucleic acids and phosphorylating the 5′

end of the nucleic acids.

13. The method according to claim 1 wherein the step of circularizing is in presence of a ligase.

14. The method according to claim 1 further comprising purifying the target oligonucleotides.

15. The method according to claim 1 wherein the primers contain dT-biotin and wherein, in the step of isolating, the oligonucleotides are purified using streptavidin beads.

26. The method of claim 1 wherein in step (a) the plurality of nucleic acids is synthesized on an array.

9. (canceled)

12. (canceled)

16. (canceled)

17. (canceled)

18. A method of synthesizing high fidelity oligonucleotides the method comprising:

a. providing a plurality of nucleic acids, each nucleic acid comprising a template sequence, a 5′

end flanking region and a 3′

end flanking region, wherein the 5′

end flanking region comprises a first primer binding sequence and the 3′

end flanking region comprises a second primer binding sequence and a restriction enzyme recognition site;

b. providing a first primer capable of annealing the first binding sequence and a second primer capable of annealing to the second primer binding sequence, the first primer comprising biotin at the 5′

end and uracil at the 3′

end;

c. amplifying the nucleic acids to form a plurality of double-stranded nucleic acids;

d. removing the second primer binding site by enzymatic cleavage using a suitable restriction enzyme, thereby forming a plurality of enzymatic cleaved double-stranded nucleic acids;

e. isolating the plurality of enzymatic cleaved double-stranded nucleic acids using a solid support comprising streptavidin;

f. denaturing the plurality of enzymatic cleaved double-stranded nucleic acids to form a plurality of single-stranded nucleic acids;

g. removing the uracil at the 5′

end of the plurality of single-stranded nucleic acids; and

h. releasing the plurality of single-stranded nucleic acids from the solid support.

19. The method of claim 18 wherein the plurality of nucleic acids is provided on an array.

20. The method according to claim 18 wherein the primers are universal or semi-universal primers.

21. The method according to claim 18 further comprising subjecting the double-stranded nucleic acids to error removal.

24. The method according to claim 18 wherein the uracil is removed using a DNA-glycosylase and endonuclease.

25. The method according to claim 18 wherein the step of releasing comprises heating the single-stranded nucleic acids to break abasic bonds.

22. (canceled)

23. (canceled)