Microarray synthesis and assembly of gene-length polynucleotides

• US 10,640,764 B2
• Filed: 02/23/2017
• Issued: 05/05/2020
• 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:
  US15/440,293
• Publication Number:
  US 20170240886A1
• Time in Patent Office: 1,167 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 assembling a target polynucleotide, comprising:

(a) providing a plurality of single-stranded nucleic acid sequences attached to a solid or porous surface, wherein each single-stranded nucleic acid sequence comprises a different fragment of a single strand of a target polynucleotide sequence and has an overlapping sequence with another single-stranded nucleic acid sequence, wherein the plurality of single-stranded nucleic acid sequences together comprise the single strand of the target polynucleotide sequence;

(b) amplifying each single-stranded nucleic acid sequence to form a plurality of double-stranded nucleic acid sequences using a primer complementary to a primer region of at least one flanking sequence located at a 3′

end, a 5′

end, or a 3′

end and a 5′

end of each single-stranded nucleic acid sequence;

(c) releasing the plurality of double-stranded nucleic acid sequences into a solution; and

(d) subjecting the released double-stranded nucleic acid sequences to assembly conditions comprising one or more of ligation, primer extension and polymerase chain reaction.

2. The process of claim 1, wherein the solid or porous surface is a microarray device or bead device.

3. The process of claim 1, wherein the at least one 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.

4. The process of claim 3, wherein the at least one flanking sequence is from about 10 to about 20 bases in length.

5. The process of claim 3, wherein the restriction enzyme cleavable site is a class II endonuclease restriction sequence.

6. The process of claim 5, wherein the class II endonuclease restriction sequence is cleavable by 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.

7. The process of claim 1, further comprising cleaving the primer region from the double-stranded nucleic acid sequences at the restriction enzyme cleavable site, thereby releasing the plurality of double-stranded nucleic acid sequences into solution.

8. The process of claim 7, wherein the at least one flanking sequence further comprises a binding moiety for purifying the cleaved nucleic acids.

9. The process of claim 1, further comprising labeling the at least one flanking sequence during the amplifying step using a primer labeled with a binding moiety.

10. The process of claim 9, wherein the binding moiety is biotin or fluorescein.

11. A process for creating a plurality of double-stranded fragments, comprising:

(a) providing a plurality of single-stranded nucleic acid sequences bound on a solid or porous surface, wherein each single-stranded nucleic acid sequence comprises (i) a fragment of a single strand of a target polynucleotide sequence and (ii) an overlapping sequence with another single-stranded nucleic acid sequence, wherein the plurality of single-stranded nucleic acid sequences together comprise the single strand of the target polynucleotide sequence;

(b) amplifying each single-stranded nucleic acid sequence to form a plurality of double-stranded nucleic acid sequences using a primer complementary to a primer region of at least one flanking sequence located at a 3′

end, a 5′

end, or the 3′

end and the 5′

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

(c) cleaving the primer region from the plurality of double-stranded nucleic acid sequences at the restriction enzyme cleavable sites, thereby producing the plurality of double-stranded fragments that together comprise the target polynucleotide sequence, wherein the plurality of double-stranded fragments are assembled to form the target polynucleotide sequence.

12. The process of claim 11, wherein each double-stranded nucleic acid sequence has an overlapping region corresponding to another double-stranded nucleic acid sequence.

13. The process of claim 11, wherein the at least one flanking sequence of each fragment is the same.

14. The process of claim 11, wherein the at least one flanking sequence is designed to have minimal homology to the target polynucleotide sequence.

15. The process of claim 11, wherein restriction enzyme cleavable site is a class II endonuclease restriction sequence.

16. The process of claim 15, wherein the class II endonuclease restriction sequence is cleavable by 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.

17. The process of claim 11, further comprising purifying the plurality of double-stranded fragments by size fractionation.

18. A process for creating a mixture of double-stranded fragments, comprising:

(a) providing a plurality of single-stranded nucleic acid sequences,

wherein each single-stranded nucleic acid sequence comprises (i) a fragment of a single strand of a target polynucleotide sequence, (ii) an overlapping sequence with another single-stranded nucleic acid sequence, and (iii) at least one flanking sequence at a 3′

end, a 5′

end, or the 3′

end and the 5′

end of each fragment,

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

wherein the plurality of single-stranded nucleic acid sequences together comprise the single strand of the target polynucleotide sequence;

(b) amplifying each single-stranded nucleic acid sequence using a primer complementary to the primer region of the at least one flanking sequence to form a plurality of double-stranded nucleic acid sequences; and

(c) cleaving the primer region from the plurality of double-stranded nucleic acid sequences at the restriction enzyme cleavable sites, thereby producing a plurality of double-stranded fragments that together comprise the target polynucleotide sequence, wherein the plurality of double-stranded fragments are assembled to form the target polynucleotide sequence.

19. A process for creating a mixture of double-stranded nucleic acid sequences, comprising:

(a) providing a plurality of single-stranded nucleic acid sequences,

wherein each single-stranded nucleic acid sequence comprises (i) a fragment of a single strand of a target polynucleotide sequence, (ii) an overlapping sequence with another single-stranded nucleic acid sequence, and (iii) at least one flanking sequence at a 3′

end, a 5′

end, or at the 3′

end and the 5′

end of each fragment,

wherein the at least one flanking sequence comprises a primer binding site, each primer binding site being complementary to a primer permitting amplification of the plurality of single-stranded nucleic acid sequences, and

wherein the plurality of single-stranded nucleic acid sequences together comprise the single strand of the target polynucleotide sequence; and

(b) subjecting the plurality of single-stranded nucleic acid sequences to amplification to form double-stranded nucleic acids comprising double-stranded fragments, the double-stranded fragments together comprising the target polynucleotide sequence, wherein the double-stranded fragments are assembled to form the target polynucleotide sequence.

20. The process of claim 19, wherein the at least one flanking sequence further comprises a sequence segment permitting removal of the primer binding site.

21. The process of claim 20, wherein the sequence segment permitting removal of the primer binding site is a class II endonuclease restriction sequence.

22. The process of claim 20, wherein each single-stranded nucleic acid sequence further comprises a cleavable linker moiety.

23. The process of claim 20, wherein the at least one flanking sequence is designed to have minimal homology to the target polynucleotide sequence.

24. The process of claim 20, wherein each single-stranded nucleic acid sequence has a sequence region that is the same as or complementary to a sequence region of another single-stranded nucleic acid sequence.

25. The process of claim 20, wherein the plurality of single-stranded nucleic acid sequences are provided on a solid or porous surface.