`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`:
`
`COMMISSIONER FOR PATENTS
`UNITED STATES PATENT AND TRADEMARK OFFICE
`WASHINGTON, D.C. 20231
`.Uspto.
`
`| ATTORNEY DOCKETNO.| mwespiegy
`44854-701.101
`
`
`
`
`| SERIAL NUMBER |REQUEST DATE|
`61/862,445
`8/5/2014
`Title:
`
`FIRST NAMED APPLICANT
`WILLIAM BANYAI
`
`DE NOVO SYNTHESIZED GENELIBRARIES
`
`Correspondence Address:
`
`F. PINAR BAILEY
`WILSON SONSINI GOODRICH & ROSATI
`650 PAGE MILL ROAD
`
`PALO ALTO CA 94304-1050
`
`Ld
`|
`
`
`| Art Unit Paper Number
`
`Licensee under 35 U.S.C. 184 is hereby grantedtofile in any foreign country a patent application and
`any amendmentsthereto corresponding to the subject matter of this U.S. application identified above
`and/or any material accompanying the petition. This license is conditioned upon modification of any
`applicable secrecy order and is subject to revocation without notice.
`
`License Number:
`Grant Date:
`
`554,110.
`06-Aug-14
`
`ApprovedLe)—
`for Commissioner of Patents and Trademarks
`
`This license empowersthefiling, the causation and the authorizationofthe filing of a foreign application
`or applications on the subject matter identified above, subsequent forwarding ofall duplicate and formal
`papers and the prosecution of such aplication or applications.
`
`.
`This license is granted under 37 CFR 5.15(a).
`This license is to be retained by the licensee and may be used at anytime onorafter the date thereof.
`This license is not retroactive unless specifically indicated.
`
`The grantof this license does not in any way lesson the responsibility of the licensee for the security of
`the subject matter as imposed by any Governmentcontractor the provisionsof existing lawsrelating to
`espionage andthe national security or the export of technical data. Licensees should apprise
`themselvesof current regulations, especially with respecct to certain countries, of other agencies,
`particularly the Departmentof the Treasury; Office of Munitions Control, Departmentof State (with
`respect to Arms, Munitions and Implements of War); the Bureau of Trade Regulation, Office of Export
`Adminstration, Department of Commerce; and the Department of Energy.
`
`LICENSE FOR FOREIGN FILING
`[Title 35, United States Code (1952) Sections 184, 185, 186]
`
`

`

`
`
`UNITED STATES PATENT AND TRADEMARK OFFICE
`COMMISSIONER FOR PATENTS
`UNITED STATES PATENT AND TRADEMARK OFFICE
`WASHINGTON, D.C. 20231
`
` SERIAL NUMBER|REQUESTDATE| FIRST NAMED APPLICANT|ATTORNEYDOCKETNO.|www.uspto.gov
`
`61/862,445
`8/5/2014
`WILLIAM BANYAI
`44854-701.101
`
`Title:
`
`DE NOVO SYNTHESIZED GENE LIBRARIES
`
`Correspondence Address:
`F. PINAR BAILEY
`
`WILSON SONSINI GOODRICH & ROSATI
`
`650 PAGE MILL ROAD
`PALO ALTO CA 94304-1050
`
`: |
`
`|
`
`| Paper Number _|
`Art Unit
`
`
`Licensee under 35 U.S.C. 184 is hereby grantedtofile in any foreign country a patent application and
`any amendmentsthereto corresponding to the subject matter of this U.S. application identified above
`and/or any material accompanying the petition. This license is conditioned upon modification of any
`applicable secrecy order and is subject to revocation without notice.
`
`License Number:
`Grant Date:
`
`554, 1 10
`06-Aug-14
`
`rooItem
`
`for Commissioner of Patents and Trademarks
`
`This license empowersthefiling, the causation and the authorization of the filing of a foreign application
`or applications on the subject matteridentified above, subsequentforwardingofall duplicate and formal
`papers and the prosecution of such aplication or applications.
`
`This license is granted under 37 CFR 5.15(a).
`This licenseis to be retained by the licensee and may be usedat anytime on orafter the date thereof.
`This license is not retroactive unless specifically indicated.
`
`The grantofthis license does notin any way lesson the responsibility of the licensee for the security of
`the subject matter as imposed by any Governmentcontract or the provisions of existing laws relating to .
`espionage and the national security or the export of technical data. Licensees should apprise
`themselves of current regulations, especially with respecct to certain countries, of other agencies,
`particularly the Departmentof the Treasury; Office of Munitions Control, Department of State (with
`respect to Arms, Munitions and Implements of War); the Bureau of Trade Regulation, Office of Export
`Adminstration, Department of Commerce; and the Department of Energy.
`
`LICENSE FOR FOREIGN FILING
`[Title 35, United States Code (1952) Sections 184, 185, 186] |
`
`

`

`, 10
`
`Page 3 of 117
`
`A
`
`2014-08-04 16:21:18 PDT
`
`:
`‘A Scope /Jack Keith/SPE 8/5/14
`
`Wilson Sonsini Goodr From: Garcia, Ashley
`
`WeR Wilson Sonsini Goodrich & Rosati
`"
`.
`PROFESSIONAL CORPORATION
`
`12235 Fl CamimoReal, Suite 200
`on
`cae
`San Diego, CA 92130-3002
`rHOnt, 858.350.2300
`vas 858.350.2399
`www.wer.
`
`August4, 2014
`
`iy
`
`Byfacsimile 571-273-0185
`
`Commissioner of Patents
`P.O. Box 1450
`Alexandria, VA. 22313-1450
`
`ATTN:
`
`Licensing and Review
`
`
`
`Re:
`
`PETITION FOR LICENSE FOR FOREIGN FILING
`Under 37 C.F.R. §5.12 and §5.15
`Attorney Docket No. 44854-701.101 and 44854-702.101
`
`Dear Sir or Madam:
`
`Petition is hereby madefor:
`
`x]
`
`A request for a licenseto foreign file a patent application where no corresponding
`' nationalor international application has been filed in the United States is
`requested pursuant to 37 C.F.R. §5.15. A legible copy ofthe material upon which
`a licenseis desired is provided herewith. The applications for which licenses were
`previously obtained or were not required pursuant to 37 C.F.R. §§5.12(a), 5.1 1(c),
`or 5.11(e)(2) are identified as follows:
`
`First named inventor: William Banyai
`
`Application No.:
`
`Filing Date:
`Title:
`
`( 61/862,445 y
`
`5, 2013
`ugust
`DE Novo SYNTHESIZED LIBRARIES
`
`First named inventor: William Banyai
`Application No.:
`61/862,457
`Filing Date:
`August 5, 2013
`Title:
`DEVICES AND METHODSFOR DE NOVO GENE SYNTHESIS
`Please special process the foreign filing license by August 5, 2014.
`
`5442245
`
`PAGE 3/117 * RCVD AT 8/4/2014 7:21:22 PM [Eastern Daylight Time] * SVR:W-PTOFAX-003/3 * DNIS:2709959 * CSID:Wilson Sonsini Goodr * DURATION «mm-ss):95-42
`
`

`

`, To:
`
`Page 4 of 117
`
`2014-08-04 16:21:18 PDT
`
`Wilson Sonsini Goodr From: Garcia, Ashley
`
`Commissionerof Patents
`ATTN: Licensing and Review
`August 4, 2014
`Page 2
`
`Expedited handling ofthis petition for a license is requested and the fee thereforeis to be
`paid as follows:
`
`e Authorization is hereby provided ‘to charge the amount of $200.00 to Deposit
`Account No. 23-2415.
`.
`e Please charge any additional fees required or credit any overpayment to Deposit
`Account No. 23-2415.
`
`Please send a copy ofthe foreignfiling license by facsimile by August 5, 2014 to the
`attention of the undersigned at 650-493-6811.
`
`Respectfully submitted,
`
`/PINAR BAILEY/
`
`F. Pinar Bailey
`Reg. No. 71,605
`
`Wilson Sonsini Goodrich & Rosati
`650 Page Mill Road
`Palo Alto, CA 94304-1050
`Direct Dial
`(650) 565-3650
`Facsimile
`650-493-6811
`
`5442245
`
`PAGE 4/117 * RCVD AT 8/4/2014 7:21:22 PM [Eastern Daylight Time] * SVR:W-PTOFAX-003/3 * DNIS:2709959 * CSID:Wilson Sonsini Goodr * DURATION (mm-ss):95-42
`
`

`

`To:
`
`Page 1 of 117
`
`2014-08-04 16:21:18 PDT
`
`Wilson Sonsini Goodr From: Garcia, Ashley
`
`WAGR
`
`Wilson Sonsini Goodrich & Rosati
`PROFESSIONAL CORPORATION
`
`Date: 2014-08-04 16:19:44 PDT
`
`Fax: 15712730185
`
`From: Garcia, Ashley
`
`Subject: Deliver to: Licensing and Review Departmentof U.S. Patent and Trademark Office
`
`
`
`Faxing: Petition for License for Foreign Filing
`Twist Bioscience - 44854-701.101 & 702.101
`
`Best Regards,
`
`Ashley Garcia
`Practice Group Assistant
`WILSON SONSINI GOODRICH & ROSATI a’,
`12235 El Camino Real, Suite 200, San Diego, CA 92130 a’,
`Phone: 858-350-2349 a", Fax: 858-350-2399
`e-mail: asgarcia@wsgr.com<mail to:asgarcia@wsgr.com>
`
`This email and any attachments thereto may contain private, confidential, and privileged material for the sole use of the intended recipient.
`Any review,copying, or distribution of this email (or any attachments thereto) by othersis strictly prohibited.
`If you are not the intended
`recipient, please contact the sender immediately and permanently delete the original and any copies of this email and any attachments
`thereto.
`
`Austin
`900 South Capital of Texas Hwy
`Las CimasIV, Fifth Floor
`Ausiin, TX 78746-5546
`Ph §12.338-5400
`Fx 512-338-5499
`.
`Beijing
`Unit 2901, 29F, Tower C
`Beijing Yintai Centre
`No, 2 Jianguomenwai Avenue
`Chaoyang District, Beijing 100022
`People's Republic of China
`Ph +86 10 65298300
`Fx +86 10 65298399
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`Brussels
`Rue Monloyer 47
`1000 Brussels, Belgium
`Ph +32 (0)}2 274 57 00
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`Ph 302-856-4235
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`Hong Kong
`Unit 1001
`10/F Henley Building
`5 Queen’s Road Central
`Hong Kong
`Ph +852 3972 4988
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`New York
`1301 Avenue of ihe Amencas
`40th Floor
`New York, NY 10019-6022
`Ph 212-999-5600
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`
`Palo Alto
`650 Page Mill Road
`Palo Allo, CA 94304-1050
`Ph 650-493-9300
`Fx 650- 493-6811
`
`San Diego
`12235 El Camino Real
`Suite 200
`San Diego, CA 92130-3002
`Ph 858-350-2300
`Fx 858-350-2399
`
`San Francisco
`One Market Plaza
`Spear Tower, Suite 3300
`San Francisco, CA 94105-1126
`Ph 415-947-2000
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`Seattle
`701 Fiflh Avenue
`Suite 5100
`Seattle, WA 98104-7036
`Ph 206-663-2500
`Fx 206-883-2699
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`Shanghai
`Jin Mao Tower
`SOF, Unit 01
`‘ 88 Century Boulevard
`Pudong, Shanghai 200121
`People's Republic of China
`Ph +86 21 6165 1700
`Fx +86 216165 1799
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`Washington, DC
`1700 K Street NW,Fifth Floor
`Washington, DC 20006-3817
`Ph 202-973-6800
`Fx 202-973-6889
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`This fax may contain confidential and privileged materialfor the sole use of the intended recipient. Any review ordistribution by others is strictly prohibited.
`if you are notthe intended recipient please contact the sender and destroy all copies.
`
`‘Entire Transmission Copyright © 2013 Wilson Sonsini Goodrich & Rosati. All Rights Reserved.”
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`.
`1213434966.4 rtf (785)
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`
`

`

`To:
`
`Page 2of 117
`
`2014-08-04 16:21:18 PDT
`
`Wilson Sonsini Goodr From: Garcia, Ashley
`
`WER.“WilsonSonsiniGoodrich &Rosati
`
`PROFESSIONAL CORPORATION
`
`FACSIMILE TRANSMITTAL SHEET
`
`
`TO:
`
`KROM:
`
`Licensing and Review
`
`.
`COMPANY:
`U.S. Patent and Trademark Office
`FAX NUMBER:
`
`(371) 273-0185
`PITONL NUMBER:
`
`Pinar Bailey
`(650)565-3650
`
`DATF:
`
`8-4-2014
`TOTAL NO. OF PAGHKS INCLUDING COVER:
`116
`SLENDER’S REVERENCE NUMBER:
`
`RE:
`
`YOUR REFERENCE NUMBER:
`
`Twist Bioscieuce —petition for Ifcense
`for Foreign Filing.
`
`44854-701.101 — 61/862,445
`44854-702,101 — 61/862,457
`
`PAGE 2/117 * RCVD AT 8/4/2014 7:21:22 PM [Eastern Daylight Time] * SVR:W-PTOFAX-003/3 * DNIS:2709959 * CSID:Wilson Sonsini Goodr * DURATION (mm-ss):95-42
`
`

`

`To:
`
`Page3 of 17
`
`i
`wt
`‘A’ Scope /Jack Keith/SPE 8/5/14
`
`2014-08-04 16:21:18 PDT
`
`Wilson Sonsini Goodr From: Garcia, Ashley
`
`WeR Wilson Sonsini Goodrich & Rosati
`
`PROFESSIONAL CORPORATION
`
`12235 F) Cunino Real, Suite 200
`San Phego, CA 92130-3002
`pron, 858.350.2300
`ax 858.350.2399
`www.wsgr.com
`
`ean
`
`August 4, 2014
`
`Byfacsimile 571-273-0185
`
`Commissioner of Patents
`;
`P.O. Box 1450
`Alexandria, VA 22313-1450
`
`ATIN:
`
`Licensing and Review
`
`Re:
`
`PETITION FOR LICENSE FOR FOREIGN FILING
`Under37 C.F.R. §5.12 and §5.15
`Attorney Docket No, 44854-701.101 and 44854-702.101
`
`Dear Sir or Madam: —
`
`Petition is hereby madefor:
`
`~
`
`A requestfor a license to foreign file a patent application where no corresponding
`' nationalor international application has been filed in the United Statesis
`requested pursuant to 37 C.F.R. §5.15. A legible copy of the material upon which
`a license is desired is provided herewith. The applications for which licenses were
`previously obtained or were not required pursuant to 37 C.F.R. §§5.12(a), 5.11(c),
`or 5.11¢(e)(2) are identified as follows:
`
`
`
`Application No.: CoussoaasJFiling Date:
`
`First named inventor: William
`
`Banyaj
`
`Title:
`
`9, 2013
`ugust
`DE Novo SYNTHESIZED LIBRARIES
`
`
`
`First named inventor;
`Application No.:
`
`August 5, 2013
`Filing Date:
`DEVICES AND METHODS FOR DE NOVO GENE SYNTHESIS
`.
`Title:
`Please special process the foreign filing license by August 5, 2014.
`
`5442245
`
`PAGE 3/117 * RCVD AT8/4/2014 7:21:22 PM [Eastern Daylight Time] * SVR:W-PTOFAX-003/3 * DNIS:2709959 * CSID:Wilson Sonsini Goodr * DURATION (mm-ss):95-42
`
`

`

`To:
`
`Page 4 of 117
`
`2014-08-04 16:21:18 PDT
`
`Wilson Sonsini Goodr From: Garcia, Ashley
`
`Commissioner of Patents
`ATTN: Licensing and Review
`August 4, 2014
`Page 2
`
`Expedited handling ofthis petition for a license is requested and the fee thereforeis to be
`paid as follows:
`
`¢ Authorization is hereby provided to charge the amount of $200.00 to Deposit
`Account No, 23-2415,
`® Please charge any additional fees required or credit any overpayment to Deposit
`Account No. 23-2415.
`
`|
`
`Please send a copy ofthe foreign filing license by facsimile by August 5, 2014 to the
`attention of the undersigned at 650-493-6811.
`
`Respectfully submitted,
`
`_ (PINAR BAILEY/
`
`F. Pinar Bailey
`Reg. No. 71,605
`
`Wilson Sonsini Goodrich & Rosati
`650 Page Mill Road
`Palo Alto, CA 94304-1050
`Direct Dial
`(650) 565-3650
`Facsimile
`650-493-6811
`
`5442745
`
`PAGE 4/117 * RCVD AT 8/4/2014 7:21:22 PM [Eastern Daytight Time] * SVR:W-PTOFAX-003/3 * DNIS:2709959 * CSID:Wilson Sonsini Goodr * DURATION (mm-ss):95-42
`
`

`

`To:
`
`Page 5 of 117
`
`2014-08-04 16:21:18 PDT
`
`Wilson Sonsini Goodr From: Garcia, Ashley
`
`WSGRDocket No. 44854-701.711
`
`PEOPLE'S REPUBLIC OF CHINA UTILITY MODEL PATENT APPLICATION
`
`THREE DIMENSIONAL MICROFLUIDIC DEVICE
`
`Inventor(s):
`
`William BANYAIT,
`Citizen of United States, Residing at
`738 Wayland Street
`San Francisco, CA 94134
`
`_ Bill James PECK,
`Citizen of Canada, Residingat
`3086 Carleton Place
`Santa Clara, CA 95051
`
`Andres FERNANDEZ,
`Citizen of United States Residing at
`400 Beale Street, 1406 San Francisco 94105
`
`Siyuan CHEN,
`Citizen of Peoples Republic of China, Residing at
`1021 Coriander Walkway, San Jose, CA 95133
`
`Pierre INDERMUHLE,
`Citizen of United States, Residing at
`1817 A Oregon Street
`Berkeley, CA, 94703
`
`Assignee:
`
`Twist Bioscience Corporation
`974 RhodeIsland Street
`San Francisco, CA 94107
`
`Entity:
`
`large business concern
`
`Wilson Sonsini Goodrich & Rosati
`PROFESSIONAL CORPORATION
`
`650 Page Mill Road
`Palo Alto, CA 94304
`
`6530586.DOC
`
`WSGR Docket No. 44854-701.711
`
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`
`

`

`To:
`
`Page 6 of 117
`
`2014-08-04 16:21:18 PDT
`
`Witson Sonsini Goodr From: Garcia, Ashley
`
`(650) 493-9300 (Main)
`(650) 493-6811 (Facsimile)
`
`Filed Electronically on: XXX, 2014
`
`6530586.DOC
`
`WSGR Docket No. 44854-701.711
`
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`
`

`

`To:
`
`Page 7 of 117
`
`2014-08-04 16:21:18 PDT
`
`Wilson Sonsini Goodr From: Garcia, Ashley
`
`THREE DIMENSIONAL MICROFLUIDIC DEVICE
`
`BACKGROUND OF THE INVENTION
`
`[0001] Highly efficient chemical gene synthesis with high fidelity and low cost has a centralrole in
`biotechnology and medicine, and in basic biomedicalresearch.
`[0002] De novo gene synthesis is a powerfultool for basic biological research and biotechnology
`applications. While various methods are knownfor the synthesis of relatively short fragments in a small
`scale, these techniques suffer from scalability, automation, speed, accuracy, and cost. There is a need for
`devices for simple, reproducible, scalable, less error-prone and cost-effective methods that guarantee
`successful synthesis of desired genes and are amenable to automation.
`
`SUMMARYOF THE INVENTION
`
`[0003] As noted above, there exists a pressing need for methods, devices and systemsthat can quickly
`synthesize large genelibraries or relatively longer oligonucleotide fragments efficiently with less error.
`Similarly, there is also a need for methods thatcan partition and mix liquids reagents in a microfluidic scale
`for large numbers of individually addressable reactions in parallel. The present invention addresses these
`needs and provides related advantages as well.
`.
`[0004]
`In some embodiments related to the array of enclosures as describedherein, the resolved loci reside
`on microstructures fabricated into a support surface. In some embodiments, the microstructures comprise at
`
`least two channels in fluidic communication with each other. In some embodiments, the at least two channels
`
`comprise two channels with different width. In some embodiments, the at least two channels comprise two
`
`channels with different length. In some embodiments,at least one of the channels is longer than 100 pm, In
`
`some embodiments, at least one of the channelsis shorter than 1000 pm. In some embodiments,at least one
`of the channels is wider than 50 um in diameter. In some embodiments,at least one of the channelsis
`narrower than 100 pm in diameter. In some embodiments, the microstructures comprise a nominal arclength
`
`ofthe perimeter of the at least two channels that has a density of at least 0.01 pm // square pm. In some
`embodiments, the microstructures comprise a nominal arclength of the perimeter of the at least two channels
`that has a density of at least 0.001 pm / square um. In some embodiments, the resolved reactors are separated
`
`with a releasable seal. In some embodiments, the seal comprises a capillary burst valve.
`[0005]
`In some embodimentsrelated to the array of enclosures as described herein, the plurality of resolved
`loci of the first substrate comprise a coating of reagents. In some embodiments, the plurality of resolved loci
`
`of the second substrate comprises a coating of reagents. In some embodiments, the coating of reagents is
`
`covalently linked to the first or second surface. In some embodiments, the coating of reagents comprises
`oligonucleotides. In some embodiments, the coating of reagents has a surface areaofat least 1 um’per 1.0
`um” ofplanar surface area. In some embodiments,the coating of reagents has a surface area of at least 1.25
`ym” per 1.0 um?of planar surface area. In some embodiments, the coating of reagents has a surface area of at
`
`6530586.DOC .
`
`-3-
`
`WSGRDocket No. 44854-701.711
`
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`
`

`

`To:
`
`Page 8 of 117
`
`2014-08-04 16:21:18 PDT
`
`Wilson Sonsini Goodr From: Garcia, Ashley
`
`least 1.45 pm? per 1.0 um? ofplanar surface area. In some embodiments,the plurality of resolved loci of the
`first substrate comprises a high energy surface. In some embodiments,the first and second substrates
`comprise a different surface tension with a given liquid. In some embodiments, the surface energy
`correspondsto a water contactangle of less than 20 degree. In some embodiments, the plurality of resolved
`loci or the reactor capsare located on a solid substrate comprising a material selected from the group
`consisting ofsilicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides, PDMS,and glass.
`Itis noted that any of the embodiments described herein can be combined with any of the methods, devices,
`arrays or systems provided in the current invention.
`[0006] Provided herein, in some embodiments,is a microfluidic device for nucleic acid synthesis,
`- comprising a substantially planar substrate portion comprising n groupings of mmicrofluidic connections
`between opposite surfaces, wherein each one of the n*mmicrofluidic connections comprises a first channel
`_ and a second channel, and wherein the first channel! within each of the n groupings is commonto all m
`microfluidic connections, wherein the plurality ofmicrofluidic connections span the substantially planar
`substrate portion along the smallest dimensionof the substrate, and wherein n and m are atleast 2. In some
`embodiments, the second channelis functionalized with a coating thatis capable of facilitating the attachment
`of an oligonucleotideto the device. In some embodiments, the device further comprisesa first oligonucleotide
`that is attached to the second channels in k of the n groupings. In some embodiments, k is 1. In some
`embodiments, the device further comprises a second oligonucleotidethat is attached to ] of the n groupings. In
`some embodiments,| is 1. In some embodiments, the none of the groupingsin the | groupingsare in the k
`groupings.
`— .
`[0007]
`In some embodiments,the oligonucleotideis at least 10 nucleotides, 25 nucleotides, 50 nucleotides,
`75 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, or 200 nucleotides long.
`[0008]
`In some embodiments,
`the first and the second oligonucleotides differ by at least 2 nucleotides, 3
`nucleotides, 4 nucleotides, 5 nucleotides, or 10 nucleotides.
`[0009]
`In some embodiments, the n*m microfluidic connections are at most 5 mim, 1,5 mm, 1.0 mm,or 0.5
`mm long. In some embodiments, the first channel within each ofthe n groupingsis at most 5 mm, 1.5 mm,
`1.0 mm, or 0.5 mm long. In some embodiments,the first channel within eachof the n groupingsis at least
`0.05 mm, 0.75 mm, 0.1 mm, 0.2 mm, 0.3 mm, or 0.4 mm long. In some embodiments, the second channel in
`each of the n*m microfluidic connections is at most 0.2 mm, 0.1 mm, 0.05 mm, 0.04 mm, or 0.03 mm long.
`In some embodiments, the second channelin each of the n*m microfluidic connectionsis at least 0.001 oun,
`0.005 mum, 0.01 mm, 0.02 mm,or 0.03 mm long. In some embodiments, the cross section of the first channel
`within each of the n groupingsis at least 0.01 mm, 0.025 mm, 0.05 mm, or 0.075 mm. In some embodiments,
`the cross section ofthe first channel within each of the n groupingsis at most | mm, 0.5 mm, 0.25 mm, 0.1
`mim, or 0.075 mm. In some embodiments, the cross section of the second channel in each of the n*¥m
`microfluidic connectionsis at least 0.001 mm, 0.05 mm, 0.01 mm, 0.015 mm,or 0.02 mm. In some
`embodiments,the cross section of the second channelin each of the n*m microfluidic connectionsis at most
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`0.25 mm, 0.125 mm,0.050 mm, 0.025 mm, 0.02 mm. In some embodiments,the standard deviation in the
`cross section of the second channels in each of the n*m microfluidic connectionsis less than 25%, 20%, 15%,
`
`10%, 5%, or 1% of the mean of the cross section. In some embodiments, the variation in the cross section
`within at least 90% of the second channels of the n*m microfluidic connections is at most 25%, 20%, 15%,
`
`10%, 5%, or 1%.
`[0010]
`In some embodiments,n is at least 10, 25, 50, 100, 1000, or 10000. In some embodiments,m is at
`
`least 3, 4, or 5.
`[0011]
`In some embodiments, the substrate comprises at least 5 %, 10%, 25%, 50%, 80%, 90%, 95%, or
`99% silicon.
`
`In some embodiments, at least 90% of the second channels of the n*m microfluidic connections is
`[0012]
`functionalized with a moiety that increases surface energy. In some embodiments, the surface energy is
`increased to a level corresponding to a water contactangleofless than 75, 50, 30, or 20 degrees.
`[0013]
`In some embodiments, the aspectratio for at least 90% of the second channels of the n*m
`microfluidic connectionsis less than |, 0.5, or 0.3. In some embodiments, the aspectratio for at least 90% of
`
`:
`the first channels in the n groupingsis less than 0.5, 0.3, or 0.2.
`[0014]
`In some embodiments, the total length ofat least 10%, 25%, 50%, 75%, 90%, or 95% of the n*¥m
`fluidic connections are within 10%, 20%, 30%, 40%, 50%, 100%, 200%, 500%, or 1000% of the smallest
`
`dimension of the substantially planar substrate.
`[0015]
`In some embodiments,the substantially planar portion of the device is fabricated from a SOI wafer.
`
`INCORPORATION BY REFERENCE
`
`[0016] All publications, patents, and patent applications mentioned inthis specification are herein
`incorporated by reference to the same extentas if each individual publication, patent, or patent application
`was specifically and individually indicated to be incorporated by reference.
`
`BRIEF DESCRIPTION OF THE DRAWINGS
`[0017] The novelfeatures of the invention are set forth with particularity in the appended claims. A better
`understanding of the features and advantages of the presentinvention will be obtained by reference to the
`following detailed description thatsets forth illustrative embodiments, in which the principles of the invention
`are utilized, and the accompanying drawings of which:
`[0018] Figure 1 demonstrates an example process outlining the gene synthesis and nanoreactor technologies.
`Figure 1A illustrates an example process for oligonucleotide synthesis on a substrate using an inkjet printer,
`Figure 1B illustrates an example process for gene amplification in a resolved enclosure, or a nanoreactor.
`Figure 1C illustrates an exampleofthe use of a plurality of wafers linking microfluidic reactions for
`oligonucleotide synthesis and gene assembly in parallel. The gene printing and NanoReactor technology
`shown in Figure 1C may be advantageous in that, the throughput of inkjet DNA printing may be high and the
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`error rate may below,andthe silicon nanoreactor gene assembly may achievea precise control and a low
`
`reagentuse.
`[0019] Figures 2 A-C are block diagrams demonstrating exemplary business process flows. Cloningof the
`
`synthesized genes may be skipped (Figure 2B). Figure 2A and Figure 2B illustrates an exemplary process
`
`“flow chart-all correct” with an error less than 1 in 10kb. In Figure 2C, synthesized genes ure cloned prior to
`
`shipment (Figure 2C).
`
`[0020] Figure 3 demonstrates an exemplary outline of a system for oligonucleotide synthesis, including a
`
`printer, e.g. inkjet printer, for reagent deposition, a substrate (wafer), schematicsoutlining the alignment of
`the system elements in multiple directions, and exemplary setups for reagent flow.
`
`[0021] Figure 4 illustrates an example of the-design microstructures built into a substrate (oligonucleotide
`
`wafer reactor).
`
`[0022] Figure 5 is a diagram demonstrating an exemplary process for reagent deposition into the
`microstructuresillustrated in Figure 4. The selected area for surface functionalization allows reagent
`spreading into the smaller functionalized wells under wetting conditions.
`[0023] Figure 6A areillustrations further exemplifying the microstructures illustrated in Figure 4. Figure 6B
`
`are illustrations of various alternative designs for the microstructures. Figure 6C illustrates a layout design for
`
`the microstructures on the substrate (wafer).
`
`[0024] Figure 7 illustrates an exemplary layout of reactor caps on a capping element..
`[0025] Figure 8 is a diagram demonstrating an exemplary process workflow for gene synthesis to shipment.
`
`[0026] Figure 9A showillustrations of an exemplary flowcell with lid opened or closed. Figure 9B illustrates
`a cross-sectional view of an exemplary flowcell and waste collector assembly. Figure 9C illustrates a
`magnified cross-sectional view of an exemplary flowcell and waste collector assembly.
`
`[0027] Figure 10A illustrates an example of a single groove vacuum chuck with a single 1-5mm groove,
`
`198mm diameter. Figure 10B illustrates a sintered metal insert in between a substrate (wafer) and the vacuum
`chuck and an optional thermal control element incorporated into the receiving element. Figure 10C illustrates
`a cross-sectional view of the single groove vacuum chuck(sintered metal chuck) exemplified in Figure 10A.
`[0028] Figure 11 illustrates exemplary application standard phosphoramidite chemistry for oligonucleotide
`synthesis.
`
`[0029] Figure 12 illustrates an example for an inkjet assembly, with 10 inkjet heads that havesilicon orifice
`
`plates with 256 nozzles on 254 pm centers (each head configured to have access to each wellthatit traverses),
`and 100 pmfly height.
`,
`[0030] Figures 13 A-C depict different views of a cluster comprising a high density of groupings. Figures 13
`
`D-E depict different views of a diagram of a microfluidic device comprising a substantially planar substrate
`
`portion. Figure 13F depicts the device view of a diagram of a microfluidic device comprising a substrantially
`
`planar substrate portion having 108 clusters and a designated area for a label. Figure 13G is a device view
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`showing well locations wthin individual cluster, and depicts the device view of a cluster comprising 109
`groupings.
`[0031] Figure 14A depicts a cross-section view of a diagram of a nanoreactor, where the view shows a row
`of the nanoreactor comprising 11 wells, along A-A of Figure 14B. Figure 14B depicts a device view of a
`diagram of a nanoreactor comprising 108 raised wells. The detail F depicts a detailed view of one well of the
`' nanoreactor. Figure 14C depicts an angled device view of the nanoreactor diagram shownin Figure 14 B.
`Figure 14D depicts a handle view of a diagram of a nanoreactor. The detail H depicts a detailed view of a
`fiducial marking on the handle side of the nanoreactor. Figure 14E depicts a device view of a diagram of
`nanoreactor comprising 108 wells and a label, showing the reaction well locations.
`[0032] Figure 15 illustrates in detail the design features of an exemplary oligonucleotide synthesis device
`that is differentially functionalized. Figure 15abis a partial enlarged view of Figure 15a. In Figure 15, the
`device is of 30 pm thick with 20 um holes, the oxideis of 1 pm, and the handleis of 400 pm thick with 75
`pm holes.
`
`[0033] Figure 16 illustrates a workflowfor the front-end manufacturing process for the exemplary device in
`Figure 15. Figure 16aillustrates a process of oxidation of a SOI substrate. Figure 16billustrates a process of
`device-side photolithography. Figure 16c illustrates a process of device-side deep RIE. Figure 16d illustrates a
`processofphotoresist strip. Figure 16e illustrates a process of handle-side photolithography. Figure 16f
`illustrates a process of handle-side deep RIE. Figure 16gillustrates a process of photoresist strip and BOX
`. etch. Figure 16h illustrates aprocess of oxidation/strip/oxidation.
`[0034] Figure 17 illustrates an exemplary baseline processflow for the back-end manufacturing of the
`exemplary oligonucleotide synthesis device of Figure 15 for differential functionalization. Figure 17a
`illustrates a process of cleaning a chip (piranha+O, plasma). Figure 17b illustrates a process of coating the
`chip with photoresist. Figure 17c illustrates a process of photolithography and descum. Figure 17d illustrates
`a process of functionalization (e.g., with fluorosilane CVD). Figure 17e illustrates a process ofresist
`stripping. Figure 17f illustrates a process of active functionalization.
`[0035] Figure 18 illustrates a functionalized surface with a controlled density of active groupsfor nucleic
`acid synthesis. In the active functionalization of Figure 18, the concentration of hydroxy] groupsin a sea of
`passive groups is diluted by applying mixed silanes.
`[0036] Figure 19 shows an image of a device manufactured according to the methods described herein.
`[0037] Figure 20 illustrates the design details (nanowell chip structure) of an exemplary nanoreactor device.
`[0038] Figure 21 illustrates an exemplary baseline process flow for the front-end manufacturing of the
`exemplary device described in Figure 20. Figure 21aillustrates a process of oxidation of substrate. Figure 21b
`illustrates a process of back-side photolithography. Figure 21c illustrates a process of back-side etch. Figure
`21d illustrates a processof photoresist strip. Figure 21e illustrates a process offront-side photolithography.
`Figure 21fillustrates a process of DRIE wells. Figure 21g illustrates a process of photoresist strip. Figure 21h
`illustrates a process of oxide growth/strip/oxidation.
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`[0039] Figure 22 illustrates an exemplary baseline proc