Granted Patent B2

US 12,595,500 · App. 17/405,548 · Granted Apr 7, 2026

Inventors: Todd Peterson (Coronado, CA); Axel Trefzer (Tegernheim, DE); Thomas Poehmerer (Regensburg, DE)

Assignee: Life Technologies Corporation

C12P19/34C12N15/1031C12N15/1093

Abstract

Claims (22)

1 . A method for the generation of an assembled nucleic acid molecule, the method comprising:

(a) synthesizing a plurality of nucleic acid molecules, wherein each nucleic acid molecule is prepared in a well of a plate in an average amount of from 0.001 nanomoles to 1,000 nanomoles;

(b) combining the nucleic acid molecules generated in (a) to produce a pool of single-stranded nucleic acid molecules;

(c) generating double-stranded nucleic acid molecules from nucleic acid molecules present in the pool of single-stranded nucleic acid molecules formed in step (b) to generate a pool of double-stranded nucleic acid molecules;

(d) eliminating nucleic acid molecules which contain sequence errors from the pool of double-stranded nucleic acid molecules formed in step (c) by mismatch endonuclease correction to produce an error corrected pool of double-stranded nucleic acid molecules; and

(e) simultaneously assembling the nucleic acid molecules in the error corrected pool of double-stranded nucleic acid molecules and an acceptor nucleic acid molecule to form the assembled nucleic acid molecule.

2 . The method of claim 1 , wherein synthesis of the nucleic acid molecules on beads present in the wells of the plate involves deblocking in step (a) by an acid generated in a redox reaction mixture in the wells of the plate.

3 . The method of claim 2 , wherein the redox reaction mixture in the wells of the plate contains hydroquinone.

4 . The method of claim 1 , wherein the assembling in step (e) is mediated by polymerase chain reaction.

5 . The method of claim 1 , wherein the pool of double-stranded nucleic acid molecules have blunt termini.

6 . The method of claim 1 , wherein the assembled nucleic acid molecule is closed, circular.

7 . The method of claim 6 , wherein the assembled nucleic acid molecule is a plasmid.

8 . The method of claim 1 , wherein the mismatch endonuclease correction comprises:

(i) denaturing the double-stranded nucleic acid molecules in the pool formed in step (c);

(ii) re-annealing the denatured nucleic acid molecules to form double-stranded nucleic acid molecules comprising mismatched base pairs at nucleotide positions corresponding to sequence errors; and

(iii) contacting the re-annealed nucleic acid molecules with at least one mismatch endonuclease under conditions suitable for cleaving the nucleic acid molecules at the mismatched base pairs.

9 . The method of claim 8 , wherein the at least one mismatch endonuclease is selected from T7 Endonuclease 1, endonuclease VII, RES I endonuclease, CEL I endonuclease, and/or SP endonuclease.

10 . The method of claim 8 , wherein the at least one mismatch endonuclease is T7 Endonuclease 1 and wherein step (d) further comprises adding a DNA ligase to the error corrected pool of double-stranded nucleic acid molecules.

11 . The method of claim 1 , wherein the generating double-stranded nucleic acid molecules in step (c) comprises joining some or all of the nucleic acid molecules present in the pool formed in (b) to form a plurality of larger nucleic acid molecules; wherein the joining in step (c) is mediated by polymerase chain reaction and/or ligases.

12 . The method of claim 1 , wherein step (b) further comprises combining nucleic acid molecules generated in (a) with nucleic acid molecules obtained by other means to form a pool, wherein said other means include PCR, restriction enzyme digest, or exonuclease treatment.

13 . The method of claim 1 , wherein two or more nucleic acid molecules are simultaneously formed.

14 . The method of claim 1 , wherein the plate is a microfluidic chip.

Assignments (2)

Continuity (10)

References Cited (192)