Methods for seamless nucleic acid assembly
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.
1. A method for nucleic acid assembly comprising:
(a) providing at least 10 different nucleic acid fragments, wherein each of the at least 10 different nucleic acid fragments does not comprise a terminal region of sequence homology to another fragment of the at least 10 different nucleic acid fragments; and
(b) mixing the at least 10 different nucleic acid fragments with a plurality of enzymes, wherein the plurality of enzymes is selected from an endonuclease, exonuclease, a polymerase, and a ligase to form a nucleic acid; and
wherein the endonuclease is a flap endonuclease.
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 provided is about 0.32 U to about 4.8 U.
6. The method of claim 4 , wherein a concentration of the flap endonuclease 1 provided is less than about 5.0 U.
7. The method of claim 1 , wherein the polymerase comprises 5′ to 3′ polymerase activity.
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 1 , wherein a concentration of the polymerase is in a range of about 0.01 U to about 2 U.
11. The method of claim 1 , wherein a concentration of the ligase is up to about 2.0 U.
12. The method of claim 1 , wherein a concentration of the ligase is in a range of about 4.0 U to about 8.0 U.
13. The method of claim 1 , wherein step (b) occurs at a temperature of about 30° C. to about 60° C.
14. The method of claim 1 , wherein mixing the at least 10 different fragments with the endonuclease results in a 5′ overhang.
15. The method of claim 1 , wherein the exonuclease is exonuclease III; the 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 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 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.