Microarray synthesis and assembly of gene-length polynucleotides

• US 7,323,320 B2
• Filed: 08/10/2005
• Issued: 01/29/2008
• Est. Priority Date: 09/12/2002
• Status: Expired due to Term

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: CombiMatrix Corporation
• Inventors: Oleinikov, Andrew V.
• Primary Examiner(s): SHIBUYA, MARK LANCE
• Application Number:
  US11/201,629
• Publication Number:
  US 20050287585A1
• Time in Patent Office: 902 Days
• Field of Search: 435/91.5, 435/6, 435/41, 435/91.1, 435/91.2
• US Class Current: 435/91.5
• 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 assembling a polynucleotide from a plurality of different oligonucleotides comprising:

(a) providing the plurality of different oligonucleotides that together comprise the polynucleotide sequence, wherein each of said oligonucleotides has an overlapping sequence region corresponding to a sequence region in a next oligonucleotide, and wherein a plurality of the different oligonucleotides further comprises flanking sequence regions at the 3′ and

5′

ends thereof, wherein said flanking sequence regions comprise primer binding sites that are the same for each oligonucleotide and a sequence segment having a restriction enzyme recognition sequence for removing the primer binding sites;

(b) amplifying each oligonucleotide using a pair of primers complementary to the primer binding sites of the flanking sequence regions;

(c) cleaving the oligonucleotides to remove the primer binding sites; and

(d) assembling the cleaved oligonucleotides through the overlapping sequence regions to form a full length polynucleotide.

2. The process of claim 1 further comprising, prior to step (a), synthesizing in situ or spotting the plurality of different oligonucleotides on a microarray device or bead device each having a solid or porous surface, wherein the plurality of different oligonucleotides are attached to the solid or porous surface.

3. The process of claim 2 wherein each oligonucleotide is attached to the solid or porous surface through a cleavable linker moiety.

4. The process of claim 3 further comprising cleaving the cleavable linker moiety to cleave each oligonucleotide from the microarray or bead solid surface to form a soluble mixture of oligonucleotides.

5. The process of claim 4 wherein the cleavable linker 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 5 wherein the cleavable linker 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-deoxyguanosone-3′

-succinate, and combinations thereof.

7. The process of claim 2 wherein the flanking sequence region at an end attached to the solid or porous surface comprises a sequence segment having a restriction enzyme recognition sequence capable of being cleaved by a corresponding restriction enzyme.

8. The process of claim 7 further comprising hybridizing an oligonucleotide complementary to the flanking sequence region at an end attached to the solid or porous surface to form a double stranded sequence capable of interacting with the corresponding restriction enzyme and digesting the oligonucleotides to cleave them from the microarray device or beads to form a soluble mixture of oligonucleotides.

9. The process of claim 1 wherein the flanking sequence regions are from about 7 to about 50 bases in length.

10. The process of claim 1 wherein the restriction enzyme recognition sequence is a class II endonuclease restriction site sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme.

11. The process of claim 10 wherein the restriction endonuclease class II site corresponds to restriction sites for a restriction endonuclease class II enzyme selected from the group consisting of Mly I, BspM I, BaeI, BsaX I, BsrI, Bmr I, Btr I, BtsI, FokI, and combinations thereof.

12. The process of claim 1 wherein the flanking sequence regions further comprise a binding moiety used to purify cleaved oligonucleotides from flanking sequences.

13. The process of claim 12 wherein the process further comprises the step of labeling the flanking sequence regions during the amplification step (b) using primer sequences labeled with binding moieties.

14. The process of claim 13 wherein the binding moiety is biotin or fluorescein.

15. The process of claim 1 wherein the flanking sequence regions for each oligonucleotide are the same.

16. The process of claim 1 wherein the primer binding sites for each oligonucleotide are the same.

17. The process of claim 1 wherein the process further comprises amplifying the full length polynucleotide sequence using primers complementary to the ends thereof.

18. The process of claim 1 wherein the flanking sequence regions are designed to have minimal homology to the oligonucleotide sequences.

19. The process of claim 1 wherein assembly comprises repeated cycles of melting, self-annealing and polymerase extension.

20. The process of claim 1 wherein cleaved oligonucleotides are purified from flanking sequence regions based on size.

21. The method of claim 1 wherein each oligonucleotide has a sequence region complementary to a sequence region in a next oligonucleotide that permits formation of an overlap suitable for primer extension by polymerase.

22. The method of claim 1 wherein each oligonucleotide has a sequence region of five or more bases that is complementary to a sequence region in a next oligonucleotide.

23. The method of claim 1 wherein each oligonucleotide has a sequence region of 15-25 bases that is complementary to a sequence region in a next oligonucleotide.