# US 12,595,500 B2 · High efficiency, small volume nucleic acid synthesis

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- Number: US 12,595,500 B2
- Document ID: US12595500B2
- Status: Granted
- Kind: B2
- Application number: 17/405,548
- Filed: 2021-08-18
- Published: 2022-06-09
- Granted: 2026-04-07
- Earliest claimed: 2011-09-26
- Assignees: Life Technologies Corporation
- Applicants: Life Technologies Corporation; Thermo Fisher Scientific Geneart GmbH
- Inventors: Todd Peterson; Axel Trefzer; Thomas Poehmerer
- Web page: https://patentgrep.com/patent/US12595500B2

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 15/1031: {mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR}
- C07H 21/04: with deoxyribosyl as saccharide radical
- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07H: SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS (derivatives of aldonic or saccharic acids C07C, C07D; aldonic acids, saccharic acids C07C59/105, C07C59/285; cyanohydrins C07C255/16; glycals C07D; compounds of unknown constitution C07G; polysaccharides, derivatives thereof C08B; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; sugar industry C13)

## Abstract

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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.

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 1)

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

### Claim 5 (depends on 1)

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

### Claim 6 (depends on 1)

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

### Claim 7 (depends on 6)

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

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 8)

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.

### Claim 10 (depends on 8)

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.

### Claim 11 (depends on 1)

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.

### Claim 12 (depends on 1)

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.

### Claim 13 (depends on 1)

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

### Claim 14 (depends on 1)

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