Microarray Synthesis and Assembly of Gene-Length Polynucleotides

• US 20130017977A1
• Filed: 09/14/2012
• Published: 01/17/2013
• 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.
• Granted Patent: US 9,051,666 B2
• Time in Patent Office: Days
• US Class Current: 506/16
• 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 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 comprises a fragment of a target polynucleotide sequence;

(b) amplifying each oligonucleotide sequence to form a plurality of double stranded oligonucleotide sequences using primers complementary to a primer region of flanking sequences located at the 3′

end and the 5′

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

(c) cleaving the primer regions from the plurality of double stranded oligonucleotide sequences at the restriction enzyme cleavable sites, thereby to produce a plurality of fragments of the target polynucleotide sequence,

wherein the plurality of fragments together comprise the target polynucleotide sequence.

2. The process for creating a mixture of oligonucleotide sequences in solution of claim 1, wherein the flanking sequence is from about 10 to about 20 bases in length.

3. The process for creating a mixture of oligonucleotide sequences in solution of claim 1 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.

4. The process for creating a mixture of oligonucleotide sequences in solution of claim 3, wherein the restriction endonuclease class II site corresponds to the restriction site 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.

5. The process for creating a mixture of oligonucleotide sequences in solution of claim 1, wherein the flanking sequences further comprise a binding moiety used to purify cleaved oligonucleotides from flanking sequences.

6. The process for creating a mixture of oligonucleotide sequences in solution of claim 1, wherein the process further comprises the step of labeling the flanking sequences during the amplification step (b) using primer sequences labeled with binding moieties.

7. The process for creating a mixture of oligonucleotide sequences in solution of claim 6, wherein the binding moiety is biotin, or fluorescein.

10. The process for creating a mixture of oligonucleotide sequences in solution of claim 1, wherein the flanking sequence regions for each oligonucleotide are the same.

11. The process for creating a mixture of oligonucleotide sequences in solution of claim 1, wherein the plurality of oligonucleotide sequences has two or more different flanking region sequences.

12. The process for creating a mixture of oligonucleotide sequences in solution of claim 1, wherein the flanking sequences are designed to have minimal homology to the oligonucleotide sequences.

13. The process for creating a mixture of oligonucleotide sequences in solution of claim 1, wherein each flanking sequence has a plurality of restriction enzyme cleavable sites.

14. The process for creating a mixture of oligonucleotide sequences in solution of claim 13, wherein at least one restriction enzyme cleavable site is a class II endonuclease restriction sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme.

15. The process for creating a mixture of oligonucleotide sequences in solution of claim 1, further comprising purifying the cleaved double stranded oligonucleotide sequences by size fractionation.

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

(a) providing a plurality of oligonucleotides sequences bound on a solid surface, wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence;

(b) amplifying each oligonucleotide sequence to form a plurality of double stranded oligonucleotide sequences using primers complementary to a pair of primer regions of flanking sequences located at the 3′

end and at the 5′

end of each fragment, wherein each flanking sequence comprises one of the pair of primer regions and a sequence segment having a restriction enzyme cleavable site, and wherein the plurality of oligonucleotides has two or more different flanking region sequences; and

(c) cleaving the primer regions from the plurality of double stranded oligonucleotide sequences at the restriction enzyme cleavable sites, thereby to produce a plurality of fragments,

wherein the plurality of fragments together comprise the target polynucleotide sequence.

9. The process for creating a mixture of oligonucleotide sequences in solution of claim 8, wherein each double stranded oligonucleotide sequence has an overlapping region corresponding to a next oligonucleotide sequence within the polynucleotide sequence.

16. The process for creating a mixture of oligonucleotide sequences in solution of claim 8, 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.

17. The process for creating a mixture of oligonucleotide sequences in solution of claim 16, wherein the restriction endonuclease class II site corresponds to the restriction site 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.

18. The process for creating a mixture of oligonucleotide sequences in solution of claim 8 wherein each flanking sequence has a plurality of restriction enzyme cleavable sites.

19. The process for creating a mixture of oligonucleotide sequences in solution of claim 18, wherein at least one restriction enzyme cleavable site is a class II endonuclease restriction sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme.

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

providing a plurality of oligonucleotides sequences, wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence and further comprises two flanking sequences, one at the 3′

end and the other at the 5′

end of each fragment, wherein each flanking sequence comprises a primer region and a sequence segment having a restriction enzyme cleavable site;

amplifying each oligonucleotide sequence using primers complementary to a pair of primer regions of the flanking sequences to form a plurality of double stranded oligonucleotide sequences; and

cleaving the primer regions from the plurality of double stranded oligonucleotide sequences at the restriction enzyme cleavable sites, thereby to produce a plurality of fragments,

wherein the plurality of fragments together comprise the target polynucleotide sequence.

21. The process for creating a mixture of oligonucleotide sequences in solution of claim 20, wherein the plurality of oligonucleotide sequences has two or more different flanking region sequences.

22. The process for creating a mixture of oligonucleotide sequences in solution of claim 20, wherein the flanking sequence regions for each oligonucleotide are the same.

23. A process for creating a mixture of oligonucleotide sequences in solution, the process comprising providing a plurality of oligonucleotides sequences, wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence and the fragments together comprising the target polynucleotide sequence and wherein each oligonucleotide 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, each primer biding region being complementary to a primer permitting amplification of the plurality of oligonucleotides.

24. The process of claim 23, wherein each flanking sequence further comprises a sequence segment permitting removal of the primer binding sites.

25. The process of claim 24, wherein the 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.

26. The process of claim 23, wherein each oligonucleotide further comprises a cleavable linker moiety.

27. The process of claim 23, wherein the flanking sequence regions are designed to have minimal homology to the oligonucleotide sequences.

28. The process of claim 23, wherein each oligonucleotide has a sequence region that is the same as or complementary to the sequence region of the next oligonucleotide.

29. The process of claim 23, wherein the plurality of oligonucleotides are provided bound on a solid or porous surface.

30. A composition for assembly of a target polynucleotide sequence, the composition comprising a plurality of different oligonucleotides sequences,

wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence and the fragments together comprising the target polynucleotide sequence,

wherein each oligonucleotide 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, and

wherein each flanking sequence comprises a primer binding site.

32. The composition of claim 30, further comprising at least one primer complementary to the primer binding site of the at least one flanking sequence and permitting amplification of the plurality of different oligonucleotides.

33. The composition of claim 30, wherein each flanking sequence further comprises a sequence segment permitting removal of the primer binding sites.

34. The composition of claim 33, wherein the sequence segment permitting removal of the primer binding site is a restriction enzyme cleavable site.

35. The composition of claim 34, 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.

36. The composition of claim 30, wherein the flanking sequence regions for the plurality of different oligonucleotides are the same.

37. The composition of claim 30, wherein the flanking sequence regions for the plurality of different oligonucleotides are two or more different flanking sequence regions.

38. The composition of claim 30, wherein each oligonucleotide has a sequence region that is the same as or complementary to the sequence region of another oligonucleotide.

39. The composition of claim 30, wherein the plurality of different oligonucleotides are provided bound on a solid or porous surface.

40. The composition of claim 39 wherein each oligonucleotide comprises a cleavable linker moiety.

41. The composition of claim 39 wherein each oligonucleotide further comprises a sequence segment permitting removal of the primer binding sites.

42. The composition of claim 41 wherein the sequence segment has a restriction enzyme cleavable site is capable of being cleaved by a type II restriction endonuclease.

43. The composition of claim 39 wherein the flanking sequence regions are the same.

44. The composition of claim 39 wherein the flanking sequence regions are designed to have minimal homology to the oligonucleotide sequences.

45. The composition of claim 39 wherein each oligonucleotide has a sequence region that is the same as or complementary to the sequence region of the next oligonucleotide.

39-1. A solid or porous surface comprising a plurality of cleavable oligonucleotides sequences immobilized thereon, wherein each oligonucleotide sequence has an overlapping sequence corresponding to a next oligonucleotide of the composition, wherein the plurality of overlapping sequences of the composition together comprise the polynucleotide sequence and wherein each oligonucleotide further comprises at least one flanking sequence at the 3′

end, the 5′

end, or at the 3′

end and 5′

end of each overlapping sequence, wherein each flanking sequence comprises a primer binding site, each primer biding site being complementary to a primer permitting amplification of the plurality of oligonucleotides.