PATENT COOPERATION TREATY
`
`PCT
`
`INTERNATIONAL PRELIMINARY REPORT ON PATENTABILITY
`(Chapter I of the Patent Cooperation Treaty)
`
`(PCT Rule 44bis)
`
`Applicant’s or agent’s file reference
`44854-740601
`
`FOR FURTHER ACTION
`
`See item 4 below
`
`International filing date (day/month/year)
`International application No.
`26 October 2018 (26.10.2018)
`PCT/US2018/057857
`International Patent Classification (8th edition unless older edition indicated)
`See relevant information in Form PCT/ISA/237
`
`Priority date (day/month/year)
`27 October 2017 (27.10.2017)
`
`Applicant
`TWIST BIOSCIENCE CORPORATION
`
`This international preliminary report on patentability (Chapter I) is issued by the International Bureau on behalf of the
`International Searching Authority under Rule 44 bis.1(a).
`
`This REPORTconsists of a total of 10 sheets, including this cover sheet.
`
`In the attached sheets, any reference to the written opinion of the International Searching Authority should be read as a
`reterence to the international preliminary report on patentability (Chapter [) instead.
`
`
`
`Box No. III
`
`Xx
`x
`
`LIOIU
`
`
`
`This report contains indications relating to the following items:
`Xx
`[I
`XI
`
`Box No. T
`
`Box No. II
`
`Basis of the report
`
`Priority
`
`Non-establishment of opinion with regard to novelty, inventive step and industrial
`applicability
`
`Lack of unity of invention
`
`Reasoned statement under Article 35(2) with regard to novelty, inventive step or
`industrial applicability; citations and explanations supporting such statement
`
`Certain documents cited
`
`Certain defects in the international application
`
`Certain observations on the international application
`
`The International Bureau will communicate this report to designated Offices in accordance with Rules 44bis.3(c) and 93bis.1
`but not, except where the applicant makes an express request under Article 23(2), before the expiration of 30 months from
`the priority date (Rule 44bis .2).
`
`The International Burcau of WIPO
`34, chemin des Colombettes
`1211 Geneva 20, Switzerland
`Facsimile No. +41 22 338 82 70
`Form PCT/IB/373 (January 2004)
`
`Date of issuance of this report
`28 April 2020 (28.04.2020)
`Authorized officer
`
`Miki Kobayashi
`e-mail: pct.team8@wipo.int
`
`

`

`PCT/US2018/057857 18.03.2019
`
`PATENT COOPERATION TREATY
`
`From the
`INTERNATIONAL SEARCHING AUTHORITY
`To: SEAN A. REED
`WILSON SONSINI GOODRICH & ROSATI
`650 PAGE MILL ROAD
`PALO ALTO, CA 94304-1050
`
`PCT
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`(PCT Rule 43bis.1)
`
`(deyinontniyeor) 18 MAR 2019
`See paragraph 2 below
` Applicant’s or agent’s file reference
`
`
`44854-740601
`
`
`
`International application No.
`Internationalfiling date (day/month/year)
`PCT/US 18/57857
`
`
`26 October 2018 (26.10.2018)
`International Patent Classification (IPC) or both national classification and IPC
`IPC(8)- C12N 15/00, C12N 15/10, C12N 15/09 (2018.01)
`CPC.
`C©12N 15/1031, C12P 19/34, GOGF 19/22, BO1J 2219/00695, BO1J 2219/00689
`
`Priority date (day/month/year)
`
`27 October 2017 (27.10.2017)
`
`FOR FURTHER ACTION
`
`Applicant TWIST BIOSCIENCE CORPORATION
`
`
`
`1. This opinion contains indicationsrelating to the following items:
`Xx] Box No. I
`Basis of the opinion
`
`[] Box No. II Priority
`
`LIUKk Box No. VII Certain defects in the international application
`
`Box No. HI Non-establishment ofopinion with regard to novelty, inventive step and industrial applicability
`
`Box No. IV Lack ofunity of invention
`
`Box No. V__
`
`Reasoned statement under Rule 43dis.1(a)(i) with regard to novelty, inventive step and industrial applicability;
`citations and explanations supporting such statement
`
`Box No. VI Certain documents cited
`
`[] Box No. VIII Certain observations onthe international application
`
`2. FURTHER ACTION
`
`If a demand for international preliminary examination is made, this opinion will be considered to be a written opinion of the
`International Preliminary Examining Authority (“IPEA”) exceptthat this does not apply where the applicant chooses an Authority
`other than this one to be the IPEA and the chosen IPEA hasnotified the International Bureau under Rule 66.1is(b) that written
`opinionsofthis International Searching Authority will not be so considered.
`If this opinionis, as provided above, considered to be a written opinion ofthe IPEA,the applicantis invited to submit to the IPEA
`a written reply together, where appropriate, with amendments,before the expiration of 3 months from the date of mailing of Form
`PCT/ISA/220 or before the expiration of 22 months from the priority date, whichever expireslater.
`For further options, see Form PCT/ISA/220.
`
`
`
`Nameand mailing address of the ISA/US|Date of completion of this opinion Authorized officer
`
`
`Mail Stop PCT, Attn: ISA/US
`Commissionerfor Patents
`P.O. Box 1450, Alexandria, Virginia 22313-1450
`Facsimile No. 571-273-8300
`
`29 December 2018
`
`Lee W. Young
`PCTHelpdesk: 571-272-4300
`
`PCT OSP:571-272-7774
`
`Form PCT/ISA/237 (cover sheet) (January 2015)
`
`

`

`|. With regard to the language, this opinion has been established on the basis of:
`[Xl]
`the international application in the language in which it wasfiled.
`a translation of the international application into
`furnished for the purposes of international search (Rules 12.3(a) and 23.1(b)).
`
`whichis the languageofa translation
`
`2. [] This opinion has been established taking into accountthe rectification of an obvious mistake authorized byornotified to
`this Authority under Rule 91 (Rule 43 bis. 1(a)).
`
`3. X] With regard to any nucleotide and/or amino acid sequence disclosed in the international application, this opinion has
`been established on the basis of a sequence listing:
`
`a.
`
`4. L_] In addition, in the case that more than one version or copy ofa sequencelisting has been filed or furnished, the required
`statements that the information in the subsequent or additional copiesis identical to that forming part of the application as
`filed or does not go beyond the application asfiled, as appropriate, were furnished.
`
`PCT/US2018/057857 18.03.2019
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/US 18/57857
`
`Box No. I
`
`Basis of this opinion
`
`forming part of the international application as filed:
`NK]
`in the form of an Annex C/ST.25textfile.
`Lj on paperorin the form of an imagefile.
`b. [] furnished together with the international application under PCT Rule 13¢er.1(a) for the purposes ofinternational
`search only in the form of an Annex C/ST.25 text file.
`c. [J furnished subsequentto the international filing date for the purposes ofinternational search only:
`[J in the form of an Annex C/ST.25textfile (Rule 13¢ser. 1(a)).
`[} on paperorin the form of an imagefile (Rule 13¢e7.1(b) and Administrative Instructions, Section 713).
`
`5. Additional comments:
`
`Form PCT/ISA/237 (Box No. 1) (January 2015)
`
`

`

`PCT/US2018/057857 18.03.2019
`
`WRITTENOPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/US 18/57857
`
`Box No. HI
`
`Non-establishmentof opinion with regard to novelty, inventive step and industrial applicability
`
`The questions whetherthe claimed invention appears to be novel, to involve an inventive step (to be non obvious), orto be industrially
`applicable have not been examinedin respectof:
`
`[| the entire international application.
`BC]
`claims Nos. 4-6,8, 10, 12, 14-15, 17, 19, 21, 25, 27, 29, 31, 39-34, 96, 38, 49-45, 47-52, 58, 60, 62-67
`
`because:
`
`the said intemational application, or the said claims Nos.
`subject matter which does not require an international search (specify):
`
`relate to the following
`
`x the description, claims or drawings (indicate particular elements below) or said claims Nos. see below
`are so unclear that no meaningful opinion could be formed (specify):
`Claims 4-6, 8, 10, 12, 14-15, 17, 19, 21, 25, 27, 29, 31, 33-34, 36, 38, 43-45, 47-52, 58, 60, 62-67 are dependent claims and are not
`drafted in accordance with the second andthird sentences of Rule 6.4(a).
`
`[] See Supplemental Box for further details.
`
`[] the claims, or said claims Nos.
`by the description that no meaningful opinion could be formed (specify):
`
`are so inadequately supported
`
`“Claims 4-6, 8, 10, 12, 14-15, 17, 19, 21, 25, 27, 29, 31, 33-34, 36, 38, 43-45, 47-52, 58, 60, 62-67
`
`x|no international search report has been established for said claims Nos. See above*
`
`[] a meaningful opinion could not be formed withoutthe sequencelisting; the applicantdid not, within the prescribed timelimit:
`furnish a sequence listing in the form of an Annex C/ST.25text file, and such listing was not available to the
`International Searching Authority in the form and manner acceptableto it; or the sequencelisting furnished did not
`comply with the standard provided for in Annex C of the Administrative Instructions.
`furnish a sequencelisting on paperor in the form of an image file complying with the standard provided for in Annex
`C of the Administrative Instructions, and suchlisting was not available to the International Searching Authority in the
`form and manneracceptable to it; or the sequence listing furnished did not comply with the standard provided for in
`Annex C of the Administrative Instructions.
`
`pay the required late furnishing fee for the furnishing of a sequence listing in response to an invitation under
`Rule 13yer.1(a) or (b).
`
`Form PCT/ISA/237 (Box No. HI) (January 2015)
`
`

`

`PCT/US2018/057857 18.03.2019
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/US 18/57857
`
`Box No. IV
`
`Lackof unity of invention
`
`1.
`
`In responseto the invitation (Form PCT/ISA/206)to pay additional fees the applicant has, within the applicable timelimit:
`
`paid additional fees.
`
`paid additional fees under protest and, where applicable, the protestfee.
`
`paid additional fees under protest but the applicable protest fee was not paid.
`
`not paid additional fees.
`2. [| This Authority found that the requirement ofunity of invention is not complied with and chose notto invite the applicantto
`pay additional fees.
`
`3. This Authority considers that the requirementof unity of invention in accordance with Rule 13.1, 13.2 and 13.3 is
`L] complied with.
`(X]
`not complied with for the following reasons:
`This application contains the following inventions or groups of inventions which are not so linked as to form a single general inventive
`concept under PCT Rule 13.1. In orderfor all inventions to be searched, the appropriate additional search fees must be paid.
`
`the parts relating to claims Nos. 1-3, 7,9, 11, 13, 16, 18, 20, 22-24, 26, 28, 30, 32, 35 and 37
`
`Group |: Claims 1-3, 7, 9, 11, 13, 16, 18, 20, 22-24, 26, 28, 30, 32, 35 and 37 directed to a system and method for assembling and
`synthesizing a full length polynucleotide by designing overlapping sub-fragments having optimal Tm in overlaps and synthesizing the full-
`length sequence from optimized overlapping polynucleotides.
`
`GroupII: Claims 39-42, 44, 46, 53-57, 59, 61, directed to a system and method for assembling and synthesizingafull-length
`polynucleotide by designing sets of sub-sequences and generating a pass score for each sequence based on GCcontent and presence
`of direct repeats, and synthesizing a set of sequences having a pass score above a threshold value to producethefull-length sequence.
`
`The inventions listed as Groups | and II do not relate to a single general inventive concept under PCT Rule 13.1 because, under PCT
`Rule 13.2, they lack the sameor corresponding special technical features for the following reasons:
`
`Special Technical Features
`
`Groups|| does not require designing overlapping fragmentsofa full-length sequence with optimal TMsin overlapping regions, as
`required by groupI.
`
`Group | does not require generating sets of sub-sequences, assigning a pass score to each sub-sequence, and generating a score for
`eachset of sub-sequence scores to determine sets that meet a threshold score, as required by group Il.
`
`----Please see continuation in first supplemental box----------------
`
`Consequently, this opinion has been established in respect of the following parts of the international application:
`[| all parts,
`Xx]
`
`Form PCT/ISA/237 (Box No. IV) (January 2015)
`
`

`

`PCT/US2018/057857 18.03.2019
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCTIUS 18/57857
`
`Box No. V
`
`Reasoned statement under Rule 434is.1(a)(i) with regard to novelty, inventive step or industrial applicability;
`citations and explanations supporting such statement
`
`Statement
`
`Novelty (N)
`
`Claims
`Claims
`
`1-3, 7,9, 11, 13, 16, 18, 20, 22-24, 26, 28, 30, 32, 35, 37
`None
`
`Inventive step (IS)
`
`Claims
`Claims
`
`None
`1-3, 7, 9, 11, 13, 16, 18, 20, 22-24, 26, 28, 30, 32, 35, 37
`
`Industrial applicability (IA)
`
`Claims
`Claims
`
`1-3, 7, 9, 11, 13, 16, 18, 20, 22-24, 26, 28, 30, 32, 35, 37
`None
`
`----Please see continuation in second supplemental box----------------
`
`Regarding claim 1, Twist teaches a computerized system for polynucleotide assembly (Abstract - "Methods and compositions are provided
`for assembly of large nucleic acids"; para [0132] - "The methods and systems described herein may comprise and/or are performed using
`a software program on a computer system.") comprising:
`a general purpose computer(para [0015] - "FiG. 7 illustrates an example of a computer system."); and a computer readable medium
`comprising functional modutes including instructions for the general purpose computer(para [0137] - "The computer system 700illustrated
`in FIG. 7 depicts a logical apparatus that readsinstructions from media 711 and/or a network port 705, which is optionally be connected to
`server 709 havingfixed media 712. In some cases, a computer system, such as shownin FIG. 7, includes a CPU 701, disk drive 703,
`optional input devices such as keyboard 715 and/or mouse 716 and optional monitor 707."), wherein said computerized system is
`configured for operating in a methodof:
`i} receiving operating instructions, wherein the operating instructions comprisea full length polynucleotide sequence; ii) automatically
`generatinga plurality of designs each comprising a plurality of polynucleotide sequences (para [0132] - "Accordingly, computerized control
`for the optimization of design algorithms described herein and the synthesis and assemblyof nucleic acids are within the boundsofthis
`disctosure.... In some instances, a computer system described herein accepts as an input one or more orders for one or more nucleic
`acids of predetermined sequence,devises an algorithm(s) for the synthesis and/or assembly of the one or more nucleic acid fragments,
`provides an outputin the form of instructions to a peripheral device(s) for the synthesis and/or assemblyof the one or more nucleic acid
`fragments, and/orinstructs for the production of the one or more nucleic acid fragments by the peripheral devices to form the desired
`nucleic acid of predetermined sequence."; para [0138] - "The example computer system of FIG. 8 includes a processor 802 for processing
`instructions."); wherein the plurality of polynucleotide sequences each comprisesat least one overlap region of 1 to 10 or more basesin
`length (para (0061] - "An overhang is capable of annealing to a complementary overhang undersuitable reaction conditions. In some
`cases, "sticky end" and "overhang'are usedinterchangeably. Non-limiting examples of overhang lengthsinclude 1, 2, 3, 4, 5, 6, 7, 8, 9,
`10 or more bases."),
`wherein each overlap region is complementary to another overlap region (para [0132] - "In some instances, a computer system is
`programmedto searchfor sticky end motifs in a user specified predetermined nucleic acid sequence, interface these motifs withalist of
`suitable nicking enzymes, and/or determine one or more assembly algorithms to assemble fragments defined by the sticky end motifs”),
`and wherein each of the at least one overlap regions does not comprise a homopolymeric sequence (para [0135]- “Site candidates are
`evaluated so as to reduce the presence ofat least one of palindromic sequences, homopolymers").
`Twist does not expressly teach overlap region of 30 to 50 basesin length, oriii) automatically selecting a design from theplurality of
`designs that comprises polynucleotide sequences having the lowest variance in Tm betweenthe at least one overlap regions. However,
`since Twist teaches sticky end overhangs comprising overlap regions can beof variable tength, from 1-10 or more bases (para [0061] -
`“Non-limiting examples of overhang lengthsinclude 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bases."), it would have been obviousto one of
`ordinary skill in the art that the overlap region can be of variable length, and could be easily optimized by routine experimentation. Further,
`since Twist teaches consideration of melting temperature for annealing of complementary sticky ends understringent hybridization
`conditions (para [0031] - "The annealing andligation reactions 150 can include rounds of annealing, ligating and melting under conditions
`suchthatonly desired sticky ends 140 a-140 d are able to anneal andligate, while cleaved end fragments remain unligated."; para [0047]-
`"Sticky ends of cleaved precursor nucleic acid fragments are allowed to anneal to one another under conditions promoting stringent
`hybridization, such that in some cases, only perfectly reverse complementary sticky ends anneal."; para [0127] - "In some cases, the
`mixture is temperature cycled to allow for the removal of cleaved sticky ended distal fragments from precursor fragments at elevated
`temperatures and to allow for the annealing of the fragments with complementary sticky ends at a lower temperature."), it would have
`been obvious to one of ordinary skill in the art that since Tm of a sequence could be determined based on the sequence composition
`using art known methods, the computersoftware algorithm could automatically select a design from the plurality of designs that comprises
`polynucleotide sequences having the lowest variance in Tm betweenthe atleast one overlap regions because such sequences would
`hybridize correctly at or near their Tm forming a stable double stranded region under stringent hybridization conditions according to Twist,
`comparedto other polynucleotides with higher variance in Tm that would not hybridize correctly at the same Tm, or would form incomplete
`or unstable hybrids.
`
`Citations and explanations:
`2.
`Claims 1-3, 7, 9, 11, 13, 16, 18, 20, 22-24, 26, 28, 30, 32, 35 and 37lack an inventive step under PCT Article 33(3) as being obvious over
`US 2016/0264958 A1 to Twist Bioscience Corporation (hereinafter ‘Twist').
`
`Form PCT/ISA/237 (Box No. V) (January 2015)
`
`

`

`
`
`
`
`
`In case the spacein any of the preceding boxesis not sufficient.
`Continuation of:
`
`Box No.IV. 3. Lack of unity of invention
`Common Technical Features
`
`
`
`
`The commontechnical feature shared by Groups| andII, is a computerized system for polynucleotide assembly, comprising a general
`purpose computer; and a computer readable medium comprising functional modules including instructions for the general purpose
`computer, wherein said computerized system is configured for operating in a method ofreceiving a full length polynucleotide sequence,
`
`
`automatically generating a plurality of designs each comprising a plurality of polynucleotide sequences, and automatically selecting a
`design from the plurality of designs. However, this shared technical feature does not represent a contribution over prior art, because the
`shared technical feature is anticipated by US 2016/0264958 A1 to Twist Bioscience Corporation (hereinafter 'Twist'). Twist teaches a
`computerized system for polynucleotide assembly (Abstract- ‘Methods and compositions are provided for assembly oflarge nucleic acids’;
`para [0132] - 'The methods and systems described herein may comprise and/or are performed using a software program on a computer
`system.') comprising: a general purpose computer (para [0015] - 'FIG. 7 illustrates an example of a computer system.'); and a computer
`readable medium comprising functional modules including instructions for the general purpose computer (para [0137] - ‘The computer
`system 700illustrated in FIG. 7 depicts a togical apparatus that reads instructions from media 711 and/or a network port 705, whichis
`optionally be connected to server 709 having fixed media 712. In some cases, a computer system, such as shownin FIG. 7, includes a
`CPU 701, disk drive 703, aptional input devices such as keyboard 715 and/or mouse 716 and optianal monitor 707.'), wherein said
`computerized system is configured for operating in a methodof: receiving operating instructions, wherein the operating instructions
`comprise a full length polynucleotide sequence and automatically generatesa plurality of designs each comprising a plurality of
`polynucleotide sequences[fragments], (para [0132] - ‘Accordingly, computerized control for the optimization of design algorithms
`described herein and the synthesis and assembly of nucleic acids are within the boundsofthis disclosure. ... a computer system described
`herein accepts as an input one or more orders for one or more nucleic acids of predetermined sequence,devises an algorithm(s) for the
`synthesis and/or assembly of the one or more nucleic acid fragments, provides an outputin the form ofinstructions to a peripheral
`device(s) for the synthesis and/or assembly of the one or more nucleic acid fragments, and/orinstructs for the production of the one or
`more nucleic acid fragments by the peripheral devices to form the desired nucleic acid of predetermined sequence. ... a computer system
`operates without humanintervention during one or more of steps for the production of a target nucleic acid of predetermined sequence or
`nucleic acid fragment thereof.', para [0135] - ‘breakpoint selection [ends of fragments] is continued for sites up to ... including each
`breakpoint or near each breakpoint. Site candidates are evaluated so as to reduce the presenceofat least one of palindromic sequences,
`homopolymers, extreme GC content, and extreme AT content.'). Twist does not expressly recite automatically selecting a design from the
`plurality of designs, however, it would have been obvious to one of ordinary skill in the art that Twists computer algorithms operates
`without human intervention, and would by necessity generate multiple designs for a set of fragments by reviewing breakpointsite
`candidates, thus, a computerized system of Twist would include selecting a design from the plurality of designs.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
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`
`
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`
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`
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`
`
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`
`
`
`
`PCT/US2018/057857 18.03.2019
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/US 18/57857
`
`Supplemental Box
`
`
`
`Another commontechnical feature shared by Groups | andII, is a method for polynucleotide synthesis ofthe full-length sequence inputted
`by the computer system discussed above, wherein the selected design(s) generated by the computerized system is synthesized. However,
`
`this shared technical feature does not rapresent a contribution overprior art, because the shared technical feature is anticipated by Twist
`
`(para [0132] - ‘Accordingly, computerized control for the optimization of design algorithms described herein and tha synthesis and
`
`assembly of nucleic acids are within the boundsofthis disclosure. ... a computer system described herein accepts as an input one or more
`orders for one or more nucleic acids of predetermined sequence, devises an algorithm(s)for the synthesis and/or assembly of the one or
`
`more nucleic acid fragments, provides an outputin the form ofinstructions to a peripheral device(s) for the synthesis and/or assembly of
`
`the one or more nucleic acid fragments, and/or instructs for the production of the one or more nucleic acid fragments by the peripheral
`
`devices to form the desired nucleic acid of predetermined sequence. ... a computer system operates without human intervention during
`
`one or more of steps for the production of a target nucleic acid of predetermined sequence or nucleic acid fragmentthereof.').
`
`
`
`
`
`
`
`
`
`
`Asthe technical feature was knownin the art at the timeof the invention, this cannot be considered a special technical feature that would
`otherwise unify the groups.
`
` Groups| andIt therefore lack unity under PCT Rule 13 because they do not share a same or corresponding special technical feature.
`
`
`Item 4, continued: claims 4-6, 8, 10, 12, 14-15, 17, 19, 21, 25, 27, 29, 31, 33-34, 36, 38, 43, 45, 47-52, 58, 60 and 62-67 are held
`unsearchable becausethey are not drafted in accordance with the second and third sentences of Rule 6.4(a) regarding multiply dependent
`
`
`claims.
`
`Form PCT/ISA/237 (Supplemental Box) (January 2015)
`
`

`

`PCT/US2018/057857 18.03.2019
`
`WRITTENOPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`International application No.
`PCT/US 18/57857
`
`Supplemental Box
`
`In case the space in any of the preceding boxesis notsufficient.
`Continuationof.
`Box No. V2. Citations and Explanations
`
`Regarding claim 2, Twist teaches the computerized system of claim 1, yet does not expressly teach wherein assembly of the
`polynucleotide sequences having the lowest variance in Tm betweentheatleast one overlap regions results in the full length
`polynucleotide sequence.
`Since Twist teaches consideration of melting temperature for annealing of complementary sticky ends understringent hybridization
`conditions (para [0031] - "The annealing andligation reactions 150 can include rounds of annealing, ligating and melting underconditions
`such that only desired sticky ends 140 a-140 d are able to anneal andligate, while cleaved end fragments remain unligated.”; para [0127] -
`“In some cases, the mixture is temperature cycled to allow for the removalof cleaved sticky endeddistal fragments from precursor
`fragments at elevated temperatures and to allow for the annealing of the fragments with complementary sticky ends at a lower
`tamperature."), and further teaches annealed sticky ends areligated to form a complete target nucleic acid molecule (para [0047]- "Sticky
`ends of cleaved precursor nucleic acid fragments are allowed to anneal to one another under conditions promoting stringent hybridization,
`suchthat in some cases, only perfectly reverse complementary sticky ends anneal. In somecases,less stringent annealing is permitted.
`Annealedsticky endsareligated to form either complete target nucleic acid molecules,or larger fragmenttarget nucleic acid molecules."),
`it would have been obviousto oneofordinary skill in the art that assembly of the polynucleotide sequences having the lowestvariancein
`Tm betweentheat least one overlap regions would occur because such sequences would hybridize correctly at or near their Tm forming a
`stable double stranded region understringent hybridization conditions according to Twist resulting in the creation offull length
`polynucleotide sequence, in contrast to other polynucleotides with higher variance in Tm that would not hybridize correctly at the same Tm,
`or would form incomplete or unstable hybrids, and thus would notlead to the creation of fult length polynucleotide sequence.
`
`----Please see continuation in next supplemental box----------------
`
`Regarding claim 3, Twist teaches the computerized system of claim 1 or 2, wherein the full length polynucleotide sequenceis at least 500
`basesin length (para [0048] "Methods and compositions described herein allow assemblyof large nucleic acid target molecules with a
`high degree of confidence as to sequenceintegrity. The target molecules are assembled from precursornucleic acid fragments that are in
`manycases synthesized to a length ...ln some cases,this length is 100, 150, 200, 250, 300, 350, 400, 450, or 500 nucleic acid bases.").
`
`Regardingclaim 7, Twist teaches the computerized system of claim 1, wherein thefull length polynucleotide sequenceis at least 1,000
`basesin length (Claim 36 - "The method ofclaim 25, wherein the predetermined nucleic acid sequenceis 1 kb to 100 kbin length.").
`
`Regarding claim 9, Twist teaches the computerized system of claim 1, yet does not expressly teach wherein the at least one overlap
`regions comprises an average of 40 percent to 60 percent GC content. Since Twist teaches selecting against extreme GC or extreme AT
`content regions (para [0135] - "In some cases, breakpoint selection is continued for sites up to and In some cases, including each
`breakpoint or near each breakpoint. Site candidates are evaluated so as to reduce the presenceofat teast one of palindromic sequences,
`homopolymers, extreme GC content, and extreme AT content."), it would have been obviousto one of ordinary skill in the art that the at
`least one overlap region comprises a moderate GC content such as around 40-60% GC content, becauseit was well knownin the art that
`extreme GC or extreme AT content would result in a correspondingly extreme melting temperatures (too low ortoo high) that would have a
`negative impact on the assembly reaction.
`
`Regarding claim 11, Twist teaches the computerized system of claim 1, yet does not expressly teach wherein each ofthe at least one
`overlap regions comprises 40 percent to 70 percent GC content. Since Twist teaches selecting against extreme GC or extreme AT content
`regions (para [0135] - "In some cases, breakpointselection is continued for sites up to and In some cases, including each breakpoint or
`near each breakpoint. Site candidates are evaluated so as to reduce the presenceofat least one ofpalindromic sequences,
`homopolymers, extreme GC content, and extreme AT content.”), it would have been obvious to one of ordinary skill in the art that each of
`the at least one overlap region comprises a GC content such as around 40-70% GC content, becauseit was well knownin the art that
`extreme GC or extreme AT content would result in a correspondingly extreme melting temperatures (too low ortoo high) that would have a
`negative impact on the assembly reaction.
`
`Regarding claim 13, Twist teaches the computerized system of claim 1, yet does not expressly teach wherein eachofthe at least one
`overlap regionsis 25 to 40 basesin length. However, since Twist teaches sticky end overhangs comprising overlap regions can be of
`variable tength, from 1-10 or more bases (para [0061] - “An overhangis capable of annealing to a complementary overhang undersuitable
`reaction conditions. In some cases,"sticky end" and “overhang” are used interchangeably. Non-limiting examples of overhang lengths
`include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more bases."),it would have been obviousto one of ordinary skill in the art that the overlap region can
`be of variable length, and could be easily optimized by routine experimentation.
`
`Regarding claims 16 and 18, Twist teaches the computerized system of claim 1, wherein the plurality of polynucleotide sequences
`comprisesat least 10 polynucleotide sequencesor 10 to 30 polynucleotide sequences {para [0089] - “In various embodiments, the
`fragmentlibrary comprises about or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 ... or more nucleic acid
`fragments. In some instances,the fragmentlibrary comprises 2-75 fragments").
`
`Regarding claim 20, Twist teaches the computerized system of claim 1, wherein each polynucleotide sequence is 50 to 150 basesin
`length (para [0133] - "Givena final target sequenceoflength I, a desired target fragment of J, and a desired sticky end overhang iength of
`K (for 5.. ANNNNT 3..(SEQ ID NO.: 2), K=6) and a maximum desired similarity between sites of L, assembly parameters are in some
`instances calculated asfollows. In some cases, J is about 200. In some cases,J is about 1000. In some cases,J is a number selected
`from about 50, 100, 150").
`
`Form PCT/ISA/237 (Supplemental Box) (January 2015)
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`PCT/US2018/057857 18.03.2019
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`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
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`Supplemental Box
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`In case the spacein any of the preceding boxesis notsufficient.
`Continuationof.
`;
`Box No. V2. Citations and Explanations
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` International application No.
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`PCT/US 18/57857
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`Regarding claim 22, Twist teaches a method for polynucleotide synthesis (Abstract - "Methods and compositions are provided for
`assembly of large nucleic acids"; para [0132] - "The methods and systems described herein may comprise and/or are performed using a
`
`software program on a computer system."), comprising:
`
`
`a) receiving operating instructions, wherein the operating instructions comprisea full length polynucleotide sequence;
`
`
`b) automatically generating a plurality of designs each comprising a plurality of polynucleotide sequences (para [0132] - “Accordingly,
`computerized control for the optimization of design algorithms described herein and the synthesis and assembly of nucleic acids a