Methods for seamless nucleic acid assembly

• US 10,696,965 B2
• Filed: 06/12/2018
• Issued: 06/30/2020
• Est. Priority Date: 06/12/2017
• Status: Active Grant

• Current Assignee: Twist Bioscience Corporation
• Original Assignee: Twist Bioscience Corporation
• Inventors: Nugent, Rebecca, Chen, Siyuan, Lee, Elian, Raynard, Nathan
• Primary Examiner(s): Bhat, Narayan K
• Application Number:
  US16/006,581
• Publication Number:
  US 20180355351A1
• Time in Patent Office: 749 Days
• 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 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/93 Ligases (6)
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/101 Linear amplification, i.e. non exponential
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Y 301/11002 Exodeoxyribonuclease III (3.1.11.2)

Abstract

Provided herein are methods, systems, and compositions for seamless nucleic acid assembly. Methods, systems, and compositions as provided herein provide for efficient assembly of nucleic acids without primer removal. Methods, systems, and compositions for seamless nucleic acid assembly comprise use of an endonuclease or exonuclease, optionally in conjunction with additional enzymes to assemble nucleic acids or polynucleotides.

Claims

1. A method for nucleic acid assembly, comprising:

(a) providing a plurality of polynucleotides, wherein each of the polynucleotides do not comprise a terminal region of sequence homology to another polynucleotide of the plurality of polynucleotides; and

(b) mixing the plurality of polynucleotides with an exonuclease, a flap endonuclease, a polymerase, and a ligase, wherein the plurality of polynucleotides are annealed in a processive predetermined order based on a complementary sequence between adjacent polynucleotides.

2. The method of claim 1, wherein the exonuclease is exonuclease III.

3. The method of claim 2, wherein a concentration of the exonuclease III is in a range of about 0.1 U to about 10 U.

4. The method of claim 1, wherein the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN1.

5. The method of claim 4, wherein a concentration of the flap endonuclease 1 is in a range of about 0.32 U to about 4.8 U.

6. The method of claim 1, wherein the polymerase comprises 5′

to 3′

polymerase activity.

7. The method of claim 6, wherein a concentration of the polymerase is in a range of about 0.01 U to about 2 U.

8. The method of claim 1, wherein the polymerase is a DNA polymerase.

9. The method of claim 1, wherein the ligase catalyzes joining of at least two nucleic acids.

10. The method of claim 9, wherein a concentration of the ligase is up to about 2.0 U.

11. The method of claim 9, wherein a concentration of the ligase is in a range of about 4.0 U to about 8.0 U.

12. The method of claim 1, wherein step (b) occurs at a temperature of about 30°

C. to about 60°

C.

13. The method of claim 1, wherein mixing the plurality of polynucleotides with the flap endonuclease results in a 5′

overhang.

14. The method of claim 1, wherein the flap endonuclease is flap endonuclease 1.

15. The method of claim 1, wherein the exonuclease is exonuclease III;

the flap endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN1; and

the polymerase is a DNA polymerase.

16. The method of claim 1, wherein a concentration of the exonuclease is in a range of about 0.1 U to about 10 U;

a concentration of the flap endonuclease is in a range of about 0.32 U to about 4.8 U;

a concentration of the polymerase is in a range of about 0.01 U to about 2 U; and

a concentration of the ligase is in a range of about 4.0 U to about 8.0 U.

17. The method of claim 1, wherein a concentration of the exonuclease is in a range of about 0.1 U to about 10 U;

a concentration of the flap endonuclease is in a range of about 0.32 U to about 4.8 U;

a concentration of the polymerase is in a range of about 0.01 U to about 2 U; and

a concentration of the ligase is up to about 2.0 U.

18. A method for nucleic acid assembly, comprising:

(a) providing a first double stranded nucleic acid;

(b) providing a second double stranded nucleic acid;

(c) providing a third double stranded nucleic acid comprising in 5′

to 3′

order;

a 5′

flanking adapter sequence, a first homology sequence to the first double stranded nucleic acid, a second homology sequence to the second double stranded nucleic acid, and a 3′

flanking adapter sequence, wherein the first double stranded nucleic acid, the second double stranded nucleic acid, and the third double stranded nucleic acid comprise non-homologous sequences at terminal regions; and

(d) mixing the first double stranded nucleic acid, the second double stranded nucleic acid, and the third double stranded nucleic acid with a reaction mixture comprising an exonuclease, an endonuclease, a polymerase, and a ligase.

19. The method of claim 18, wherein the exonuclease is exonuclease III.

20. The method of claim 18, wherein the endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN1.

21. The method of claim 18, wherein the polymerase is a DNA polymerase.

22. The method of claim 18, wherein a concentration of the exonuclease is in a range of about 0.1 U to about 10 U;

a concentration of the endonuclease is in a range of about 0.32 U to about 4.8 U;

a concentration of the polymerase is in a range of about 0.01 U to about 2 U; and

a concentration of the ligase is in a range of about 4.0 U to about 8.0 U.

23. The method of claim 18, wherein a concentration of the exonuclease is in a range of about 0.1 U to about 10 U;

a concentration of the endonuclease is in a range of about 0.32 U to about 4.8 U;

a concentration of the polymerase is in a range of about 0.01 U to about 2 U; and

a concentration of the ligase is up to about 2.0 U.

24. A method for nucleic acid assembly, comprising:

a. providing a first double stranded nucleic acid comprising in 5′

to 3′

order;

a 5′

flanking adapter sequence, a homology sequence, an insert sequence, and a 3′

flanking adapter sequence;

b. providing a second double stranded nucleic acid comprising in 5′

to 3′

order;

a 5′

flanking adapter sequence, a homology sequence, an insert sequence, and a 3′

flanking adapter sequence; and

c. mixing the first double stranded nucleic acid and the second double stranded nucleic acid with a reaction mixture comprising an exonuclease, an endonuclease, a polymerase, and a ligase.

25. The method of claim 24, wherein the exonuclease is exonuclease III.

26. The method of claim 24, wherein the endonuclease is flap endonuclease 1, exonuclease 1, XPG, Dna2, or GEN1.

27. The method of claim 24, wherein the polymerase is a DNA polymerase.

28. The method of claim 24, wherein a concentration of the exonuclease is in a range of about 0.1 U to about 10 U;

a concentration of the endonuclease is in a range of about 0.32 U to about 4.8 U;

a concentration of the polymerase is in a range of about 0.01 U to about 2 U; and

a concentration of the ligase is in a range of about 4.0 U to about 8.0 U.

29. The method of claim 24, wherein a concentration of the exonuclease is in a range of about 0.1 U to about 10 U;

a concentration of the endonuclease is in a range of about 0.32 U to about 4.8 U;

a concentration of the polymerase is in a range of about 0.01 U to about 2 U; and

a concentration of the ligase is up to about 2.0 U.