Microarray Synthesis and Assembly of Gene-Length Polynucleotides

• US 20100124767A1
• Filed: 06/22/2009
• Published: 05/20/2010
• Est. Priority Date: 09/12/2002
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: CombiMatrix Corporation
• Inventors: Oleinikov, Andrew V.
• Granted Patent: US 8,058,004 B2
• Time in Patent Office: Days
• US Class Current: 435/91.500
• 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 oligonucleotides comprising:

(a) synthesizing or spotting a plurality of oligonucleotide sequences on a microarray device or bead device having a solid or porous surface, wherein a first oligonucleotide is oligo 1 and a second oligonucleotide is oligo 2 and so on, wherein the plurality of oligonucleotide sequences are attached to the solid or porous surface, and wherein the first oligonucleotide sequence has an overlapping sequence region of from about 10 to about 50 bases that is the same or substantially the same as a region of a second oligonucleotide sequence, and wherein the second oligonucleotide sequence has an overlapping region with a third oligonucleotide sequence and so on;

(b) forming complimentary oligo 1 by extending primer 1, wherein primer 1 is complimentary to oligo 1;

(c) disassociating complimentary oligo 1 from oligo 1 and annealing complimentary oligo 1 to both oligo 1 and to the overlapping region of oligo 2, wherein the annealing of complimentary oligo 1 to oligo 2 serves as a primer for extension for forming complimentary oligo 1+2;

(d) repeating the primer extension cycles of step (c) until a full-length polynucleotide is produced; and

(e) amplifying the assembled complementary full length polynucleotide to produce a full length polynucleotide in desired quantities.

2. The process for assembling a polynucleotide from a plurality of oligonucleotides of claim 1 wherein the solid or porous surface is in the form of a microarray device.

3. The process for assembling a polynucleotide from a plurality of oligonucleotides of claim 2 wherein the microarray device is a semiconductor device having a plurality of electrodes for synthesizing oligonucleotides in situ using electrochemical means to couple and decouple nucleotide bases.

4. The process for assembling a polynucleotide from a plurality of oligonucleotides of claim 1 wherein the primer extension reaction is conducted through a sequential process of melting, annealing and then extension.

5. The process for assembling a polynucleotide from a plurality of oligonucleotides of claim 4 wherein the primer extension reaction is conducted in a thermocycler PCR amplification device using the microarray having the plurality of oligonucleotides bound thereto.

6. (canceled)

7. (canceled)

8. (canceled)

9. (canceled)

10. (canceled)

11. (canceled)

12. (canceled)

13. (canceled)

14. (canceled)

15. (canceled)

16. (canceled)

17. (canceled)

18. (canceled)

19. (canceled)

20. (canceled)

21. A process for creating a mixture of oligonucleotide sequences in solution comprising:

(a) synthesizing in situ or spotting a plurality of oligonucleotide sequences on a microarray device or bead device each having a solid or porous surface, wherein the plurality of oligonucleotide sequences are attached to the solid or porous surface, and wherein each oligonucleotide sequence further comprises two flanking sequences, one at the 3′

end and the other at the 5′

end of each oligonucleotide, wherein each flanking sequence is from about 7 to about 50 bases and comprising a primer region and a sequence segment having a restriction enzyme cleavable site;

(b) amplifying each oligonucleotide using the primer regions of the flanking sequence to form a double stranded (ds) oligonucleotides; and

(c) cleaving the double stranded oligonucleotide sequences at the restriction enzyme cleavable site.

22. The process for creating a mixture of oligonucleotide sequences in solution of claim 21 wherein the flanking sequence is from about 10 to about 20 bases in length.

23. The process for creating a mixture of oligonucleotide sequences in solution of claim 21 wherein the restriction enzyme cleavable site is a class II endonuclease restriction site sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme.

24. The process for creating a mixture of oligonucleotide sequences in solution of claim 23 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, Bae I, BsaX I, Bsr I, Bmr I, Btr I, Bts I, Fok I, and combinations thereof.

25. The process for creating a mixture of oligonucleotide sequences in solution of claim 21 wherein the flanking sequence further comprises a binding moiety used to purify cleaved oligonucleotides from flanking sequences.

26. The process for creating a mixture of oligonucleotide sequences in solution of claim 21 wherein the process further comprises the step of labeling the flanking sequence during the amplification step (b) using primer sequences labeled with binding moieties.

27. The process for creating a mixture of oligonucleotide sequences in solution of claim 26 wherein a binding moiety is a small molecule able to be captured, such as biotin captured by avidin or streptavidin, or fluorescein able to be captured by an anti-fluorescein antibody.

28. A process for creating a mixture of oligonucleotide sequences in solution comprising:

(a) synthesizing in situ or spotting a plurality of oligonucleotide sequences on a microarray device or bead device each having a solid or porous surface, wherein the plurality of oligonucleotide sequences are attached to the solid or porous surface, and wherein each oligonucleotide sequence has a sequence segment having a cleavable linker moiety;

(b) cleaving the oligonucleotide sequences at the cleavable linker site to cleave each oligonucleotide sequence from the microarray or bead solid surface to form a soluble mixture of oligonucleotides.

29. The process for creating a mixture of oligonucleotide sequences in solution of claim 28 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.

30. The process for creating a mixture of oligonucleotide sequences in solution of claim 29 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.

31. (canceled)

32. (canceled)

33. (canceled)

34. (canceled)