Methods and devices for in situ nucleic acid synthesis

• US 9,187,777 B2
• Filed: 05/26/2011
• Issued: 11/17/2015
• 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, Ramu, Senthil, Schindler, Daniel
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/700,291
• Publication Number:
  US 20130309725A1
• Time in Patent Office: 1,636 Days
• US Class Current: 1/1
• 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 a plurality of features, each feature comprising a plurality of single-stranded support-bound oligonucleotides;

b) hybridizing a partially double-stranded first oligonucleotide to at least a first support-bound oligonucleotide at a first feature, wherein the first partially double-stranded 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 first support-bound oligonucleotide thereby generating a first ligation product comprising the predetermined addition nucleotide; and

d) cleaving the first ligation product thereby generating a first elongated support-bound oligonucleotide comprising the predetermined addition nucleotide.

2. The method of claim 1 wherein in the step of providing the at least first oligonucleotide is in solution within a droplet and is deposited at the first feature.

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

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

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

5. The method of claim 1 wherein the partially double-stranded oligonucleotide having a 5′

overhang is generated by hybridizing a first single-stranded construction oligonucleotide comprising at its 5′

end a predefined sequence and at its 3′

end the predetermined ligatable addition nucleotide to a longer single-stranded oligonucleotide comprising at its 3′

end a sequence complementary to the 5′

end predefined sequence of the first construction oligonucleotide and comprising a nucleotide complementary to the predetermined ligatable addition nucleotide upstream to the 3′

end sequence.

6. The method of claim 5 wherein the first construction oligonucleotide further comprises RNA base upstream of the predetermined addition nucleotide.

7. The method of claim 1 wherein the 5′

overhang of the partially double-stranded oligonucleotide is complementary to the 5′

end of the support-bound oligonucleotide.

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

9. 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.

10. The method of claim 9 wherein the restriction endonuclease binding site is a type IIS endonuclease binding site.

11. The method of claim 1 wherein in the step of cleaving the ligation product is cleaved with a restriction enzyme.

12. The method of claim 11 wherein cleavage of the ligation product provides an elongated support-bound oligonucleotide comprising one or more predetermined addition nucleotides.

13. The method of claim 1 further comprising melting off nucleotides from the elongated support-bound oligonucleotide.

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

15. The method of claim 14 wherein the label is a fluorescent label.

16. The method of claim 14 further comprising analyzing the hybridization step and/or the ligation step and wherein the presence of the detectable label is indicative of the completion of the steps.

17. The method of claim 16 wherein the steps of analyzing are performed using an imaging system.

18. The method of claim 17 wherein the imaging system is a CCD.

19. The method of claim 14 further comprising analyzing the cleaving step and wherein the absence of detectable label is indicative of the completion of the step.

20. The method of claim 1 further comprising depositing the partially double-stranded first oligonucleotide at the first feature using an ink jet device.

21. 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.

22. The method of claim 21 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.

23. The method of claim 22 wherein the oligonucleotides of the plurality of partially double-stranded oligonucleotides differ only with at least one predetermined nucleotide addition.

24. The method of claim 23 wherein the predetermined nucleotide addition is A, T, G or C.

25. The method of claim 1 wherein the first elongated support-bound oligonucleotide comprises one or more predetermined addition nucleotides.

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