# US 2016/0250613 A1 · Methods and Devices for Nucleic Acid Synthesis

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- Number: US 2016/0250613 A1
- Document ID: US20160250613A1
- Status: Published application
- Kind: A1
- Application number: 15/054,038
- Filed: 2016-02-25
- Published: 2016-09-01
- Earliest claimed: 2010-11-12
- Assignees: Gen9, Inc.
- Applicants: Gen9, Inc.
- Inventors: Joseph Jacobson; Li-yun A. Kung; Andrew Kirk Wilson; Senthil Ramu; Daniel Schindler; Michael Hudson
- Web page: https://patentgrep.com/patent/US20160250613A1

## CPC

- B01J 19/0046: {Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays (synthesis methods per se C40B50/00)}
- 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}
- 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
- C12Q 1/6834: Enzymatic or biochemical coupling of nucleic acids to a solid phase
- B01J 2219/00585: Parallel processes
- B01J 2219/00596: Solid-phase processes
- B01J 2219/00722: Nucleotides
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- 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
- 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)}

## Abstract

Methods and apparatus relate to the synthesis of polynucleotides having a predefined sequence on a support. Assembly methods include primer extension to generate overlapping construction oligonucleotides and assembly of the polynucleotides of interest onto an anchor support-bound oligonucleotides. Methods and apparatus for selection of polynucleotides having the predefined sequence and/or length are disclosed.

## Claims

### Claim at position 1

1-41. (canceled)

### Claim 42

A nucleic acid array system comprising:
a. one or more solid support;
b. a plurality of discrete features associated with the one or more solid support wherein each feature comprises a plurality of support-bound oligonucleotides having a predefined sequence wherein a first support-bound oligonucleotide comprises a terminal region that is complementary to a terminal region of a second support-bound oligonucleotide, wherein a Nth support-bound oligonucleotide comprises a terminal sequence complementary to a terminal sequence region of a (N-1)th support-bound oligonucleotide, wherein N is no less than 3; and
c. at least a first plurality of anchor oligonucleotides comprising a terminal sequence that is identical to a sequence region of the first support-bound oligonucleotide.

### Claim 43 (depends on 42)

The nucleic acid array system of claim 42 further comprising a second plurality of support-bound anchor oligonucleotide having a sequence region that is identical to the Nth support-bound oligonucleotide.

### Claim at position 4

44-65. (canceled)

### Claim 66 (depends on 42)

The nucleic acid array system of claim 42 wherein the first to the Nth support-bound oligonucleotides together comprise a target nucleic acid having a predetermined sequence.

### Claim 67 (depends on 66)

The nucleic acid array system of claim 66 wherein each solid support comprises a plurality of features for assembling two or more target nucleic acids in parallel.

### Claim 68 (depends on 42)

The nucleic acid array system of claim 42 further comprising an anchor support wherein the anchor oligonucleotides are immobilized on the anchor support.

### Claim 69 (depends on 68)

The nucleic acid array system of claim 68 further comprising a microvolume of reagents localized on each feature for synthesizing construction oligonucleotides in a chain extension reaction using the support-bound oligonucleotides as template.

### Claim 70 (depends on 69)

The nucleic acid array system of claim 69 further comprising a fluid medium for transferring the construction oligonucleotides to the anchor support.

### Claim 71

A system for producing at least one target nucleic acid, comprising:
a. one or more solid supports having at least a first and a second features thereon, the first feature having a first plurality of support-bound oligonucleotides, the second feature having a second plurality of support-bound oligonucleotides, wherein each of the first plurality of support-bound oligonucleotides comprises a terminal sequence region complementary to that of the second plurality of support-bound oligonucleotides;
b. an anchor support comprising a plurality of anchor oligonucleotides, each having a terminal region identical to that of the first plurality of support-bound oligonucleotides; and
c. a solution of reagents for synthesizing construction oligonucleotides in a chain extension reaction using the support-bound oligonucleotides as template.

### Claim 72 (depends on 71)

The system of claim 71 further comprising a Nth plurality of support-bound oligonucleotides comprising a terminal sequence complementary to that of a (N-1)th plurality of support-bound oligonucleotides, wherein N>=3.

### Claim 73 (depends on 72)

The system of claim 72 wherein the first to the Nth pluralities of support-bound oligonucleotides together comprise a target nucleic acid having a predetermined sequence.

### Claim 74 (depends on 73)

The system of claim 73 wherein each solid support comprises a plurality of features for assembling two or more target nucleic acids in parallel.

### Claim 75 (depends on 71)

The system of claim 71 wherein the solution comprises a plurality of microvolumes each localized on a feature.

### Claim 76 (depends on 71)

The system of claim 71 further comprising a fluid medium for transferring the construction oligonucleotides to the anchor support.

### Claim 77 (depends on 71)

The system of claim 71 wherein the support-bound oligonucleotides are synthesized or spotted on the one or more solid supports.

### Claim 78 (depends on 42)

A method for producing at least one target nucleic acid, comprising:
a. providing the nucleic acid array system of claim 42;
b. synthesizing a first to Nth construction oligonucleotides in a chain extension reaction using the support-bound oligonucleotides as template;
c. transferring the first to Nth construction oligonucleotides to the first plurality of anchor oligonucleotides; and
d. hybridizing the first to Nth construction oligonucleotides with the first plurality of anchor oligonucleotides, thereby producing at least one target nucleic acid.

### Claim 79 (depends on 71)

A method for producing at least one target nucleic acid, comprising:
a. providing the nucleic acid array system of claim 71;
b. synthesizing a first and second construction oligonucleotides using the support-bound oligonucleotides as template;
c. transferring the first and second construction oligonucleotides to the anchor support; and
d. hybridizing the first and second construction oligonucleotides with the anchor oligonucleotides, thereby producing at least one target nucleic acid.

### Claim 80 (depends on 73)

A method for producing at least one target nucleic acid, comprising:
a. providing the nucleic acid array system of claim 73;
b. synthesizing a first to a Nth construction oligonucleotides using the support-bound oligonucleotides as template;
c. transferring the first to the Nth construction oligonucleotides to the anchor support; and
d. hybridizing the first to the Nth construction oligonucleotides with the anchor oligonucleotides, thereby producing at least one target nucleic acid.