Microarray synthesis and assembly of gene-length polynucleotides

• US 9,023,601 B2
• Filed: 09/30/2011
• Issued: 05/05/2015
• Est. Priority Date: 09/12/2002
• Status: Expired due to Term

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Oleinikov, Andrew V.
• Primary Examiner(s): Kim, Young J
• Application Number:
  US13/250,207
• Publication Number:
  US 20120270750A1
• Time in Patent Office: 1,313 Days
• US Class Current: 435/6.11
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00385 Printing
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: B01J 2219/00454 by chemical cleavage from the solid support
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00639 the compounds being trapped in or bound to a porous medium
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00675 In-situ synthesis on the substrate
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00713 Electrochemical synthesis
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• 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: C12Q 1/6837 : using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 50/14 : Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 80/00 : Linkers or spacers specially adapted for combinatorial chemistry or libraries, e.g. traceless linkers or safety-catch linkers

Abstract

There is disclosed a process for in vitro synthesis and assembly of long, gene-length polynucleotides based upon assembly of multiple shorter oligonucleotides synthesized in situ on a microarray platform. Specifically, there is disclosed a process for in situ synthesis of oligonucleotide fragments on a solid phase microarray platform and subsequent, “on device” assembly of larger polynucleotides composed of a plurality of shorter oligonucleotide fragments.

Claims

1. A composition for assembly of a target polynucleotide sequence, the composition comprising a plurality of different synthesized oligonucleotide sequences, wherein the plurality of different synthesized oligonucleotide sequences are immobilized on a solid or porous surface, each oligonucleotide sequence comprising a fragment of a target polynucleotide sequence and at least one flanking sequence, wherein the at least one flanking sequence is at the 3′

end, or the 5′

end, or the 3′

end and the 5′

end of each fragment, and wherein each flanking sequence comprises a primer binding site and a sequence segment designed to permit removal of the primer binding site such that the fragments together comprise the target polynucleotide sequence.

2. The composition of claim 1 wherein said sequence segment permitting removal of the primer binding site is a class II endonuclease restriction site sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme.

3. The composition of claim 1 wherein each oligonucleotide comprises a cleavable linker moiety.

4. The composition of claim 1 wherein each oligonucleotide comprises a binding moiety.

5. The composition of claim 1 wherein the flanking sequences are the same.

6. The composition of claim 1 wherein the flanking sequences are designed to have minimal homology to the oligonucleotide fragment sequences.

7. The composition of claim 1 wherein each fragment sequence further comprises an overlapping sequence corresponding to a next oligonucleotide of the composition, and wherein the overlapping sequence is the same as a sequence of a next oligonucleotide, or is complementary to a sequence of the next oligonucleotide.

8. A device comprising a solid or porous surface having a plurality of cleavable oligonucleotide sequences immobilized thereon, wherein each oligonucleotide sequence has an overlapping sequence and at least one flanking sequence,

wherein the overlapping sequence correspond to a sequence of a next oligonucleotide of the plurality of oligonucleotides, and

wherein the at least one flanking sequence is at the 3′

end, the 5′

end, or at the 3′

end and 5′

end of each overlapping sequence, each flanking sequence comprising a primer binding site and a sequence segment permitting removal of the primer binding site, such that the overlapping sequences together comprise the target polynucleotide sequence.

9. The device of claim 8 wherein each oligonucleotide comprises a cleavable linker moiety.

10. The device of claim 8 wherein the sequence segment comprises a restriction enzyme cleavable site capable of being cleaved by a type II restriction endonuclease.

11. The device of claim 8 wherein the flanking sequence are the same.

12. The device of claim 8 wherein the flanking sequences are designed to have minimal homology to the overlapping sequences.

13. The device of claim 8 wherein each oligonucleotide has an overlapping-sequence that is the same as or complementary to a sequence of the next oligonucleotide.

14. A process for creating a mixture of oligonucleotide sequences in solution, the process comprising:

(a) providing a plurality of oligonucleotide sequences bound on a solid or porous surface, wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence and wherein each oligonucleotide sequence further comprises at least one flanking sequence at the 3′

end, the 5′

end, or at the 3′

end and the 5′

end of each fragment, wherein each flanking sequence comprises a primer binding site and a sequence segment having a restriction enzyme site permitting removal of the primer binding site such that the fragments together comprise the target polynucleotide sequence and wherein each oligonucleotide sequence has a cleavable site; and

(b) cleaving the oligonucleotide sequences at the cleavable site to form a soluble mixture of oligonucleotides.

15. The process of claim 14 wherein the restriction enzyme site is a class II endonuclease restriction site sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme.

16. The process of claim 14 wherein each oligonucleotide comprises a cleavable linker moiety.

17. The process of claim 14 wherein the flanking sequence regions are designed to have minimal homology to the oligonucleotide sequences.

18. The process of claim 14 wherein each oligonucleotide has a sequence region that is the same as or complementary to the sequence region of the next oligonucleotide.