Microarray synthesis and assembly of gene-length polynucleotides

• US 10,450,560 B2
• Filed: 05/01/2015
• Issued: 10/22/2019
• Est. Priority Date: 09/12/2002
• Status: Active Grant

• 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:
  US14/701,957
• Publication Number:
  US 20160001247A1
• Time in Patent Office: 1,635 Days
• Field of Search: None
• 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 process for creating a mixture of double-stranded fragments in solution, the process comprising:

a) predetermining a target polynucleotide sequence;

b) providing a plurality of single-stranded oligonucleotides bound on a solid or porous surface, wherein each single-stranded oligonucleotide comprise a fragment of the predetermined target polynucleotide sequence, and a flanking sequence, wherein the flanking sequence comprises a primer binding site and a restriction enzyme site permitting removal of the primer binding site, and

wherein each single-stranded oligonucleotide is bound to the surface via a cleavable linker moiety;

c) providing a plurality of complementary oligonucleotides, wherein each oligonucleotide of the plurality of complementary oligonucleotide is complementary to the flanking sequence;

d) hybridizing the plurality of said single-stranded oligonucleotides to the plurality of complementary oligonucleotides;

e) extending the hybridized plurality of complementary oligonucleotides to form double-stranded fragments; and

f) cleaving each oligonucleotides at the cleavable linker moiety to form a mixture of double-stranded fragments,

wherein the mixture of double-stranded fragments together comprise the predetermined target polynucleotide sequence.

2. The process of claim 1 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.

3. The process of claim 1, wherein the flanking sequence is located at the 3′

end, or the 5′

end, or the 3′

end and the 5′

end of each oligonucleotide of the plurality of single-stranded oligonucleotides.

4. The process of claim 3 wherein each oligonucleotide of the plurality of single-stranded oligonucleotides has a sequence region that is the same as or complementary to another sequence region of a next oligonucleotide.

5. The process of claim 1, wherein the cleavable linker moiety is a chemical composition having a succinate moiety bound to a nucleotide moiety such that cleavage produces a 3′

hydroxy nucleotide.

6. The process of claim 1, wherein the cleavable linker moiety is selected from the group consisting of 5′

-dimethoxytrityl-thymidine-3′

succinate, 4-N-benzoyl-5′

-dimethoxytrityl-deoxycytidine-3′

-succinate, 1-N-benzoyl-5′

-dimethoxytrityl-deoxyadenosine-3′

-succinate, 2-N-isobutyryl-5′

-dimethoxytrityl-deoxyguanosine-3′

-succinate, and combinations thereof.