Compositions and methods for synthetic gene assembly

• US 9,677,067 B2
• Filed: 05/13/2016
• Issued: 06/13/2017
• Est. Priority Date: 02/04/2015
• Status: Active Grant

• Current Assignee: Twist Bioscience Corporation
• Original Assignee: Twist Bioscience Corporation
• Inventors: Toro, Esteban, Treusch, Sebastian, Chen, Siyuan, Wu, Cheng-Hsien
• Primary Examiner(s): Zhang, Kaijiang
• Application Number:
  US15/154,879
• Publication Number:
  US 20160264958A1
• Time in Patent Office: 396 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides

Abstract

Methods and compositions are provided for assembly of large nucleic acids where the assembled large nucleic acids lack internal sequence modifications made during the assembly process.

Claims

1. A method for nucleic acid assembly, the method comprising:

a) providing a predetermined nucleic acid sequence;

b) providing a plurality of precursor double-stranded nucleic acid fragments, each precursor double-stranded nucleic acid fragment having two strands, wherein each of the two strands comprises a sticky end sequence of 5′

-A (N^x) T-3′

(SEQ ID NO.;

     1) or 5′

-G (N′

)C-3′

(SEQ ID NO.;

     16), wherein N is a nucleotide, wherein x is the number of nucleotides between nucleotides A and T or between G and C, and wherein x is 1 to 10, and wherein no more than two precursor double-stranded nucleic acid fragments comprise the same sticky end sequence;

c) providing primers comprising a nicking endonuclease recognition site and a sequence comprising (i) 5′

-A (N^x) U-3′

(SEQ ID NO.;

     80) corresponding to each of the different sticky end sequences of 5′

-A (N^x) T-3′

(SEQ ID NO.;

     1) or (ii) 5′

-G (N^x) U-3′

(SEQ ID NO.;

     81) corresponding to each of the different sticky end sequences of 5′

-G (N^x)C-3′

(SEQ ID NO.;

     16);

d) performing a polynucleotide extension reaction to form a polynucleotide extension reaction product that is double-stranded nucleic acid fragments;

e) subjecting the polynucleotide extension reaction product to nicking and cleavage reactions to form double-stranded nucleic acid fragments with 3′

overhangs; and

f) annealing the double-stranded nucleic acid fragments with 3′

overhangs to form a nucleic acid encoding for the predetermined nucleic acid sequence that does not include the nicking endonuclease recognition site.

2. The method of claim 1, wherein x is 4, 5, or 6.

3. The method of claim 1, wherein the predetermined nucleic acid sequence is 1 kb to 100 kb in length.

4. The method of claim 1, wherein the plurality of precursor double-stranded nucleic acid fragments are each at least 100 bases in length.

5. The method of claim 1, wherein the sticky end sequences are each at least 4 bases long.

6. The method of claim 1, wherein step “

c”

further comprises providing (i) a forward primer comprising, in order 5′

to 3′

;

a first outer adaptor region and a nucleic acid sequence from a first terminal portion of the predetermined nucleic acid sequence; and

(ii) a reverse primer, comprising, in order 5′

to 3′

;

a second outer adaptor region and a nucleic acid sequence from a second terminal portion of the predetermined nucleic acid sequence.

7. The method of claim 6, wherein the annealed double-stranded nucleic acid fragments comprise the first outer adaptor region and the second outer adapter region.

8. The method of claim 1, wherein the nicking and cleavage reactions comprise adding an enzyme that is a nicking endonuclease, N-glycosylase or AP-lyase.

9. The method of claim 8, wherein the enzyme is endonuclease VIII.

10. The method of claim 8, wherein the nicking endonuclease is Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.BsmAI, Nt.BspQI, Nt.BstNBI, or Nt.CviPII.

11. The method of claim 1, further comprising ligating the annealed double-stranded nucleic acid fragments.

12. The method of claim 1, wherein annealing comprises thermocycling between a maximum and a minimum temperature, thereby generating a first overhang from a first double-stranded DNA fragment and a second overhang from a second double-stranded DNA fragment, wherein the first and the second overhangs are complimentary, hybridizing the first and second overhangs to each other;

and ligating.

13. The method of claim 1, wherein a polymerase lacking 3′

to 5′

proofreading activity is added during the polynucleotide extension reaction.

14. The method of claim 13, wherein the polymerase is a Family A polymerase or a Family B high fidelity polymerase engineered to tolerate base pairs comprising uracil.

15. The method of claim 1, wherein the plurality of precursor double-stranded nucleic acid fragments comprise an adaptor sequence comprising the nicking endonuclease recognition site.

16. The method of claim 1, wherein one or more of the plurality of precursor double-stranded nucleic acid fragments is a linear vector sequence.

17. A method for nucleic acid assembly, the method comprising:

a) providing a predetermined nucleic acid sequence;

b) synthesizing a plurality of precursor double-stranded nucleic acid fragments, each precursor double-stranded nucleic acid fragment having two strands, wherein each of the two strands comprises a sticky end sequence of 5′

-A (Nx) T-3′

(SEQ ID NO.;

     1) or 5′

-G (Nx)C-3′

(SEQ ID NO.;

     16), wherein N is a nucleotide, wherein x is the number of nucleotides between nucleotides A and T or between G and C, and wherein x is 1 to 10, and wherein no more than two precursor double-stranded nucleic acid fragments comprise the same sticky end sequence;

c) providing primers comprising a nicking endonuclease recognition site and a sequence comprising (i) 5′

-A (Nx) M-3′

(SEQ ID NO.;

     82) corresponding to each of the different sticky end sequences of 5′

-A (Nx) T-3′

(SEQ ID NO.;

     1) or (ii) 5′

-G (Nx) M-3′

(SEQ ID NO.;

     83) corresponding to each of the different sticky end sequences of 5′

-G (Nx)C-3′

(SEQ ID NO.;

     16), wherein M is a non-canonical base, wherein the primers are each 7 to 70 bases in length;

d) performing a polynucleotide extension reaction to form a polynucleotide extension reaction product that is double-stranded nucleic acid fragments;

e) subjecting the polynucleotide extension reaction product to nicking and cleavage reactions to form double-stranded nucleic acid fragments with 3′

overhangs; and

f) annealing the double-stranded nucleic acid fragments with 3′

overhangs to form a nucleic acid encoding for the predetermined nucleic acid sequence that does not include the nicking endonuclease recognition site.

18. The method of claim 17, wherein x is 4, 5, or 6.

19. The method of claim 17, wherein the non-canonical base is uracil, inosine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, acetylcytosine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N-6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 1-methyladenine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, 5-ethylcytosine, N6-adenine, N6-methyladenine, N,N-dimethyladenine, 8-bromoadenine, 7-methylguanine, 8-bromoguanine, 8-chloroguanine, 8-aminoguanine, 8-methylguanine, 8-thioguanine, 5-ethyluracil, 5-propyluracil, 5-methylaminomethyluracil, methoxyarninomethyl-2-thiouracil, beta-D-mannosylqueosine, 5′

-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid, pseudouracil, 1-methylpseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-hydroxymethyluracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-S-oxyacetic acid, 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, 5-(2-bromovinyl)uracil, 2-aminopurine, 6-hydroxyaminopurine, 6-thiopurine, or 2,6-diaminopurine.

20. The method of claim 17, wherein the non-canonical base is uracil.

21. The method of claim 17, wherein one of the plurality of precursor double-stranded nucleic acid fragments comprises a portion of linear vector.

22. The method of claim 17, wherein no more than two nucleotides from the Nx portion of the sticky end sequence have the same identity.

23. The method of claim 17, wherein the plurality of precursor double-stranded nucleic acid fragments comprise an adaptor sequence comprising the nicking endonuclease recognition site.

24. The method of claim 17, wherein the predetermined nucleic acid sequence is 1 kb to 100 kb in length.

25. The method of claim 17, wherein the sticky end sequences are each at least 4 bases long in each precursor nucleic acid.