Methods and Devices for In Situ Nucleic Acid Synthesis

• US 20160097051A1
• Filed: 10/16/2015
• Published: 04/07/2016
• Est. Priority Date: 05/28/2010
• Status: Abandoned Application

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Ramu, Senthil, Schindler, Daniel
• Application Number:
  US14/884,883
• Publication Number:
  US 20160097051A1
• Time in Patent Office: 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 composition for synthesizing a plurality of target nucleic acids on a surface of a solid support, the composition comprising a plurality of partially double-stranded oligonucleotides, each having a first strand and a second strand such that each partially double-stranded oligonucleotide comprises a double-stranded portion and a 5′

overhang on the second strand, wherein the double-stranded portion comprises at least one predetermined addition nucleotide at the 3′

end of the first strand, and wherein the double-stranded portion has a predefined sequence that is identical across the plurality of partially double-stranded oligonucleotides.

2. The composition of claim 1 wherein the double-stranded portion comprises a restriction enzyme binding site.

3. The composition of claim 1 wherein the shorter strand comprises at least one ribonucleotide upstream of the at least one predetermined addition nucleotide.

4. The composition of claim 1 wherein the 5′

overhang comprises a degenerate sequence.

5. The composition of claim 1, further comprising a support having a plurality of features, each feature comprising a plurality of single-stranded support-bound oligonucleotides that can be ligated with the partially double-stranded oligonucleotides at the 3′

end of the predetermined addition nucleotide.

6. The composition of claim 5, wherein the plurality of single-stranded support-bound oligonucleotides each have sequence complementarity to the 5′

overhang.

7. The composition of claim 5, further comprising an ink jet programmed to provide the partially double-stranded oligonucleotides at a select feature.

8. A composition for synthesizing a plurality of target nucleic acids on a surface of a solid support, the composition comprising:

a support having a plurality of features, each feature comprising a plurality of single-stranded support-bound oligonucleotides; and

a first partially double-stranded oligonucleotide comprising a first strand and a second strand forming a double-stranded portion and a 5′

overhang on the second strand, wherein the first strand comprises a first predetermined ligatable addition nucleotide at the 3′

end;

wherein the first partially double-stranded oligonucleotide can be ligated to a first support-bound oligonucleotide at the first predetermined ligatable addition nucleotide, thereby generating a first ligation product comprising the first predetermined addition nucleotide; and

wherein the first ligation product is cleavable thereby generating a first elongated support-bound oligonucleotide comprising the first predetermined addition nucleotide.

9. The composition of claim 8 further comprising a second partially double-stranded oligonucleotide comprising a second predetermined ligatable addition nucleotide.

10. The composition of claim 9 wherein the first and second partially double-stranded oligonucleotides comprise in the double-stranded portion an identical predefined sequence.

11. The composition of claim 8 wherein the double-stranded portion comprises a restriction enzyme binding site.

12. The composition of claim 8 wherein the first ligation product is cleavable by a restriction enzyme.

13. The composition of claim 8 wherein the shorter strand comprises at least one ribonucleotide upstream of the first predetermined ligatable addition nucleotide.

14. The composition of claim 13 wherein the first ligation product is cleavable by an RNase.

15. The composition of claim 8 wherein the 5′

overhang comprises a degenerate sequence.

16. The composition of claim 8, wherein the plurality of single-stranded support-bound oligonucleotides each have sequence complementarity to the 5′

overhang.

17. The composition of claim 8, further comprising an ink jet programmed to provide the first partially double-stranded oligonucleotide at a select feature.

18. The composition of claim 8 wherein the first partially double-stranded oligonucleotide is prepared by hybridizing the first strand to the second strand, wherein the first strand comprises at its 5′

end a predefined sequence and at its 3′

end the first predetermined ligatable addition nucleotide, and wherein the second strand comprises at its 3′

end a sequence complementary to the predefined sequence and a nucleotide complementary to the first predetermined ligatable addition nucleotide.

19. The composition of claim 8 wherein the first partially double-stranded oligonucleotide comprises a detectable label.

20. The composition of claim 8 further comprising an imaging system.