METHODS AND COMPOSITIONS FOR RAPID SEAMLESS DNA ASSEMBLY

• US 20160230175A1
• Filed: 02/11/2015
• Published: 08/11/2016
• Est. Priority Date: 02/11/2015
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Carstens, Carsten
• Granted Patent: US 9,834,774 B2
• Time in Patent Office: Days
• US Class Current: 1/1
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host

Abstract

Provided herein are methods for assembling DNA fragments employing at least three enzymatic activities: DNA polymerase, flap endonuclease, and DNA ligase. Certain aspects include methods for generating closed circular DNA products, e.g., plasmid vectors, by assembling various DNA fragments having complementary ends that hybridize to one another. The resulting circular products can be introduced into host cells and selected for desired properties. Kits for performing the method are also provided.

Claims

1. A DNA assembly method comprising:

(a) obtaining at least two DNA fragments, wherein the at least two DNA fragments comprise ends that can selectively hybridize with one another; and

(b) contacting the at least two DNA fragments with;

(i) a DNA polymerase;

(ii) a flap endonuclease; and

(iii) a DNA ligase;

under reaction conditions that promote hybridization of the at least two DNA fragments and support the activities of components (i), (ii) and (iii), to produce a product comprising an assembled circular DNA comprising at least a portion of each of the at least two DNA fragments.

2. The method of claim 1, further comprising performing at least one thermal cycling incubation step after the contacting step (b).

3. The method of claim 2, wherein multiple thermal cycling incubations steps are performed.

4. The method of claim 1, wherein the at least two DNA fragments comprise one or more cassettes selected from the group consisting of:

one or more selectable marker cassettes, one or more origin of replication cassette, one or more additional functional cassettes, one or more target cassettes, and combinations thereof.

5. The method of claim 4, wherein the one or more functional cassettes are selected from the group consisting of:

promoter cassettes, N-terminal purification tag cassettes, C-terminal purification tag cassettes, shuttle origin of replication cassettes, terminator cassettes, protein expression enhancer cassettes, and shuttle selectable marker cassettes.

6. The method of claim 4, wherein the one or more target cassettes comprises a polynucleotide sequence encoding a polypeptide or a regulatory RNA.

7. The method of claim 1, wherein at least three DNA fragments comprising ends that can selectively hybridize with one another are obtained in step (a), and wherein the product produced comprises an assembled circular DNA comprising at least a portion of each of the at least three DNA fragments.

8. The method of claim 1, wherein at least five DNA fragments comprising ends that can selectively hybridize with one another are obtained in step (a), and wherein the product produced comprises an assembled circular DNA comprising at least a portion of each of the at least five DNA fragments.

9. The method of claim 1, wherein the assembled circular DNA is a plasmid vector comprises an origin of replication for a bacterial host cell, a selectable marker cassette, and an expression cassette for a gene of interest.

10. The method of claim 1, wherein the ends of the at least two DNA fragments that can selectively hybridize are at least 20 nucleotides in length.

11. The method of claim 10, wherein the ends of the at least two DNA fragments that can selectively hybridize have a T_m of at least 45°

C.

12. The method of claim 1, wherein the DNA polymerase is thermostable.

13. The method of claim 12, wherein the DNA polymerase is selected from the group consisting of:

Pfu DNA polymerase SSO7 fusion, Pfu DNA polymerase, Tma DNA polymerase, Tli DNA polymerase, KOD DNA polymerase, JDF-3 DNA polymerase, PGB-D DNA polymerase, Tgo DNA polymerase, and Pyrolobus furmarius DNA polymerase.

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

15. The method of claim 14, wherein the flap endonuclease is selected from the group consisting of:

Pfu FEN-1 and Dna2.

16. The method of claim 15, wherein the DNA ligase is thermostable.

17. The method of claim 16, wherein the DNA ligase is selected from the group consisting of:

Pfu DNA ligase, Tth DNA ligase, Taq DNA ligase, Thermus filiformis ligase, Rhodothermus marinus DNA ligase, Thermus scotoductus DNA ligase, and Bacillus stearothermophilus DNA ligase.

18. The method of claim 1, wherein the DNA polymerase is a non strand-displacing DNA polymerase.

19. The method of claim 1, further comprising:

introducing the DNA assembly product of step (b) into a host cell, and

identifying a host cell that harbors the assembled circular DNA.