# US 2021/0340598 A1 · Methods for assembling dna molecules

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- Number: US 2021/0340598 A1
- Document ID: US20210340598A1
- Status: Published application
- Kind: A1
- Application number: 17/373,115
- Filed: 2021-07-12
- Published: 2021-11-04
- Earliest claimed: 2016-12-14
- Assignees: Codex DNA, Inc.
- Applicants: Codex DNA, Inc.
- Inventors: John E. Gill; Daniel G. Gibson; Lixia Fu
- Web page: https://patentgrep.com/patent/US20210340598A1

## CPC

- C12Q 1/686: Polymerase chain reaction [PCR]
- C07H 19/073: with 2-deoxyribosyl as the saccharide radical
- C07H 19/173: with 2-deoxyribosyl as the saccharide radical
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/222: {Clustered regularly interspaced short palindromic repeats [CRISPR]-associated [CAS] enzymes}
- C12N 15/09: Recombinant DNA-technology
- 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 15/1031: {mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Y 302/02027: Uracil-DNA glycosylase (3.2.2.27)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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)}
- C12Y: ENZYMES

## Abstract

The invention provides compositions and methods for assembling a DNA molecule having a desired sequence. The methods involve contacting a DNA polymerase, dNTPs, and a plurality of pairs of oligonucleotides. The oligonucleotides of a pair have a portion of the desired sequence, and an internal sequence that overlaps and is complementary to an internal sequence of the other oligonucleotide of the pair, and, when arranged in order, they have at least a portion of the desired sequence. The oligonucleotides also have a 3′ or a 5′ primer binding sequence having a binding site for a primer. The oligonucleotides that correspond to the end oligonucleotides of the desired sequence also have a universal 3′ flanking sequence and a universal 5′ flanking sequence, respectively.

## Claims

### Claim 1

A method of removing primer binding sequences from a dsDNA molecule comprising:
contacting a dsDNA molecule with an enzyme mixture comprising an enzyme that specifically cleaves primer binding sequences at a non-standard base;
wherein the dsDNA molecule comprises a 3′ and a 5′ primer binding sequence having at least one non-standard base;
wherein the dsDNA molecule further comprises a universal 3′ flanking sequence and a universal 5′ flanking sequence comprised to the inside of the 3′ and 5′ primer binding sequences, respectively;
to thereby remove the primer binding sequences from the dsDNA molecule.

### Claim 2 (depends on 1)

The method of claim 1 wherein the non-standard base is deoxyuridine.

### Claim 3 (depends on 1)

The method of claim 1 wherein the enzyme that specifically cleaves primer binding sequences at a non-standard base is uracil DNA-glycosylase (UDG).

### Claim 4 (depends on 3)

The method of claim 3 wherein the enzyme mixture further comprises endonuclease VIII and exonuclease T.

### Claim 5 (depends on 1)

The method of claim 1 wherein the primer binding sequences are 6-30 nucleotides in length.

### Claim 6 (depends on 5)

The method of claim 5 wherein the 3′ and 5′ primer binding sequences are present on the 3′ and 5′ ends, respectively, of the dsDNA molecule.

### Claim 7 (depends on 1)

The method of claim 1 wherein the dsDNA molecule does not comprise an expressed sequence tag.

### Claim 8 (depends on 1)

The method of claim 1 wherein the non-standard base is selected from the group consisting of: 3-nitropyrrole and 5-nitroindole.

### Claim 9

A composition comprising:
a DNA polymerase, dNTPs, and a plurality of oligonucleotides formed into couplets, wherein each couplet comprises an internal sequence that comprises a portion of the desired nucleic acid sequence;
wherein when the plurality of couplets is arranged in order, adjacent to each other and according to their internal sequences they comprise at least a portion of a desired nucleic acid sequence, and each couplet further comprises a 3′ or a 5′ primer binding sequence, and each couplet contains a sequence that overlaps and is complementary to a portion of a sequence from an adjacent couplet; and
wherein the desired nucleic acid sequence has a 3′ end and a 5′ end, and the couplets that comprise the 3′ and 5′ ends of the desired sequence further comprise a universal 3′ flanking sequence and a universal 5′ flanking sequence, respectively.

### Claim 10 (depends on 9)

The composition of claim 9 comprised in a single container and further comprising an effective amount of a preservative.

### Claim 11 (depends on 10)

The composition of claim 10 wherein the couplets comprise at least 50% of the oligonucleotides in the mixture, and the couplets overlap at least 33% of their sequences.

### Claim 12 (depends on 9)

The composition of claim 9 wherein the universal 3′ and 5′ flanking sequences are comprised to the inside of the 3′ and 5′ primer binding sequences, respectively.

### Claim 13 (depends on 9)

The composition of claim 9 wherein the primer binding sequences comprise at least one non-standard base.

### Claim 14 (depends on 13)

The composition of claim 13 wherein the non-standard base is deoxyuradine.

### Claim 15 (depends on 9)

The composition of claim 9 wherein the 3′ and 5′ primer binding sequences on an oligonucleotide couplet are not complementary to each other.

### Claim 16 (depends on 9)

The composition of claim 9 wherein the oligonucleotides comprise from 60 to 100 nucleotides and the primer binding sequences comprise from 8 to 30 nucleotides.

### Claim 17 (depends on 9)

The composition of claim 9 wherein the 3′ and 5′ primer binding sequences do not comprise a restriction site for a restriction enzyme.

### Claim 18 (depends on 9)

The composition of claim 9 further comprising uracil DNA glycosylase, endonuclease VIII, and exonuclease T.