# US 2019/0376103 A1 · Methods for in vitro joining and combinatorial assembly of nucleic acid molecules

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- Number: US 2019/0376103 A1
- Document ID: US20190376103A1
- Status: Published application
- Kind: A1
- Application number: 16/528,312
- Filed: 2019-07-31
- Published: 2019-12-12
- Earliest claimed: 2008-02-15
- Assignees: SGI-DNA, Inc.
- Applicants: SGI-DNA, Inc.
- Inventors: Daniel G. Gibson; Hamilton O. Smith; Clyde A. Hutchison; Lei Young; J. Craig Venter
- Web page: https://patentgrep.com/patent/US20190376103A1

## CPC

- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/93: {Ligases (6)}
- C12N 15/10: Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34)
- C12N 15/1027: {by DNA shuffling, e.g. RSR, STEP, RPR}
- C12N 15/64: General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
- C12N 15/66: General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
- 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)

## Abstract

The present invention relates to methods of joining two or more double-stranded (ds) or single-stranded (ss) DNA molecules of interest in vitro, wherein the distal region of the first DNA molecule and the proximal region of the second DNA molecule of each pair share a region of sequence identity. The method allows the joining of a large number of DNA fragments, in a predetermined order and orientation, without the use of restriction enzymes. It can be used, e.g., to join synthetically produced sub-fragments of a gene or genome of interest. Kits for performing the method are also disclosed. The methods of joining DNA molecules may be used to generate combinatorial libraries useful to generate, for example, optimal protein expression through codon optimization, gene optimization, and pathway optimization.

## Claims

### Claim 1

An in vitro method of joining a set of two or more double-stranded (ds) or single-stranded (ss) DNA molecules, wherein adjacent DNA molecules to be joined contain overlapping sequences at their termini, said method comprising:
contacting in vitro the two or more DNA molecules in a single vessel with a mixture of components comprising;
(a) an isolated non-thermostable 5′ to 3′ exonuclease that lacks 3′ exonuclease activity,
(b) a crowding agent selected from the group consisting of: polyethylene glycol and dextran;
(c) an isolated thermostable DNA polymerase,
(d) an isolated thermostable ligase,
(e) a mixture of dNTPs, and
(f) a suitable buffer,
under isothermal conditions and in amounts effective for joining the two or more DNA molecules to form a first assembled dsDNA molecule in a concerted reaction in said single vessel.

### Claim 2 (depends on 1)

The method of claim 1, wherein the exonuclease of (a) is a T5 exonuclease.

### Claim 3 (depends on 1)

The method of claim 1, wherein
the crowding agent of (b) is PEG, and/or
the ligase of (d) is Taq ligase.

### Claim 4 (depends on 3)

The method of claim 3 wherein the crowding agent of (b) is PEG and the ligase of (d) is Taq ligase.

### Claim 5 (depends on 3)

The method of claim 3, wherein the exonuclease of (a) is a T5 exonuclease.

### Claim 6 (depends on 4)

The method of claim 4, wherein the exonuclease of (a) is a T5 exonuclease.

### Claim 7 (depends on 1)

The method of claim 1, wherein the conditions are also suitable for digesting any unpaired, non-homologous, single stranded DNAs following the joining reaction.

### Claim 8 (depends on 8)

The method of claim 8, wherein at least some of the DNA molecules to be joined comprise, at one terminus, a sequence that is non-homologous to any of the DNA molecules of interest.

### Claim 9 (depends on 8)

The method of claim 8, wherein the non-homologous sequences comprise one or more binding regions for PCR primers, and/or regions of homology to vector sequences, and/or recognition sites for one or more restriction enzymes.

### Claim 10 (depends on 1)

The method of claim 1, further comprising repeating the method to join a second set of two or more DNA molecules to one another to obtain a second assembled DNA molecule, and then joining the first and the second assembled DNA molecules to obtain a third assembled ds DNA molecule.