Methods and Devices for In Situ Nucleic Acid Synthesis

• US 20130309725A1
• Filed: 05/26/2011
• Published: 11/21/2013
• Est. Priority Date: 05/28/2010
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Schindler, Daniel, Ramu, Senthil
• Granted Patent: US 9,187,777 B2
• Time in Patent Office: Days
• US Class Current: 435/91.5
• 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 15/1093 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

Abstract

Disclosed are compositions, methods and devices for the in situ synthesis of nucleic acids. In an exemplary embodiment, a support-bound oligonucleotide is elongated by addition of one or more nucleotides by hybridization of a partially double-stranded oligonucleotide, ligation and removal of unwanted nucleotides.

Claims

1. A method for synthesizing a nucleic acid having a predefined sequence, the method comprising:

a. providing a support comprising an anchor oligonucleotide at a first feature;

b. hybridizing a partially double-stranded first oligonucleotide to the anchor oligonucleotide wherein the first oligonucleotide comprises a 5′

overhang and a ligatable predetermined addition nucleotide at a 3′

end of the double-stranded portion;

c. ligating the partially double-stranded first oligonucleotide to the anchor oligonucleotide thereby generating a first ligation product; and

d. removing unwanted nucleotides from the first ligation product thereby generating a first elongated product comprising the predetermined addition nucleotide.

2. The method of claim 1 wherein in the step of providing the anchor oligonucleotide is support-bound.

3. The method of claim 1 wherein in the step of providing the anchor oligonucleotide is in solution within a droplet.

4. The method of claim 1 wherein in the step of providing the support comprises a plurality of features, each feature comprising a plurality of single-stranded anchor oligonucleotides.

5. The method of claim 1 further comprising repeating steps b), c) and d) thereby providing the nucleic acid having the predefined sequence.

6. The method of claim 1 wherein in the step of hybridizing the partially double-stranded oligonucleotides comprises a double-stranded portion and a single-stranded 5′

overhang, wherein the single-stranded overhang comprises degenerate bases.

7. The method of claim 1 wherein the partially double-stranded oligonucleotide is generated by hybridizing a first construction oligonucleotide comprising at its 5′

end a predefined sequence and at its 3′

end the desired ligatable addition nucleotide to a longer oligonucleotide comprising at its 5′

end a single-stranded overhang a nucleotide complementary to the predetermined addition nucleotide, and at its 3′

end a sequence complementary to the 5′

end predefined sequence of the first construction oligonucleotide.

8. The method of claim 1 wherein the single-stranded overhang is complementary to the 5′

end of the anchor oligonucleotide.

9. The method of claim 7 wherein the nucleotide upstream the predetermined addition nucleotide on the first construction oligonucleotide is a RNA base.

10. The method of claim 1 wherein the step of removing comprises cleaving the first ligated product using a RNase.

11. The method of claim 1 wherein in the step of hybridizing the double-stranded portion of the partially double-stranded oligonucleotide comprises a restriction endonuclease binding site.

12. The method of claim 11 wherein the restriction endonuclease binding site is a type IIS endonuclease binding site.

13. The method of claim 1 wherein the step of removing nucleotides from the ligation product comprises cleaving the ligation product with a restriction enzyme.

14. The method of claim 13 wherein cleavage of the ligation product provides an elongated product comprising the predetermined addition nucleotide.

15. The method of claim 1 further comprising melting off nucleotides from the elongated product.

16. The method of claim 1 wherein in the step of hybridizing the partially double-stranded oligonucleotides comprises a detectable label.

17. The method of claim 16 wherein the label is a fluorescent label.

18. The method of claim 16 further comprising analyzing the hybridization step and/or the ligation step wherein the presence of the detectable label is indicative of the completion of the steps.

19. The method of claim 16 further comprising analyzing the removing step wherein the absence of detectable label is indicative of the completion of the step.

20. The method of claim 1 wherein in the step of hybridizing the partially double-stranded first oligonucleotide is deposited at the first feature using an ink jet device.

21. The method of claim 18 wherein the steps of analyzing are performed using an imaging system.

22. The method of claim 21 wherein the imaging system is a CCD.

23. The method of claim 1 wherein in the step of hybridizing a plurality of partially double-stranded oligonucleotides are provided at a plurality of different features of the support.

24. The method of claim 23 wherein the plurality of partially double-stranded oligonucleotides comprises a 5′

single-stranded overhang, a predetermined addition nucleotide and a double-stranded sequence and wherein the double-stranded sequence is identical within the plurality of partially double-stranded oligonucleotides.

25. The method of claim 24 wherein the oligonucleotides of plurality of oligonucleotides differ only with at least one predetermined nucleotide addition.

26. The method of claim 25 wherein the predetermined nucleotide addition is A, T, G or C.

27. The method of claim 1 wherein the first elongated product comprises one or more predetermined addition nucleotides.

32. The method of claim 1 wherein the predetermined nucleotide addition is A, T, G or C.

28. A composition for synthesizing a plurality of nucleic acids on a surface of a solid support, the composition comprising a plurality of partially double-stranded oligonucleotides, wherein the plurality of partially double-stranded oligonucleotides comprises a 5′

single-stranded sequence, a double-stranded sequence comprising a predefined sequence, and a predetermined ligatable addition nucleotide at a 3′

end of the shorter strand of the double-stranded sequence, wherein the double-stranded sequence is identical within the plurality of partially double-stranded oligonucleotides.

29. The composition of claim 28 wherein the double-stranded portion comprises a restriction enzyme binding site.

30. The composition of claim 28 wherein the shorter strand comprises at least one RNA base upstream of the predetermined addition nucleotide.

31. The composition of claim 28 wherein the 5′

single-stranded sequence is a degenerate sequence.