PCT/USZO18I037161 22.10.2018
`
`PATENT COOPERATION TREATY
`
`PCT
`
`INTERNATIONAL SEARCH REPORT
`
`(PCT Article 18 and Rules 43 and 44)
`
`Applicant’s or agent’s file reference
`44354445501
`
`FOR FURTHER
`ACTION
`
`see Form PCT/IS A020
`as well as, where applicable, item 5 below.
`
`International application No.
`PCT/US18/37161
`
`International filing date (day/monIh/year)
`12 June 2018 (12.06.2018)
`
`(Earliest) Priority Date (day/month/year)
`12 June 2017 (12.06.2017)
`
`Applicant
`TWIST BIOSCIENCE CORPORATION
`
`This international search report has been prepared by this lntemational Searching Authority and is transmitted to the applicant
`according to Article 18. A copy is being transmitted to the lntemational Bureau.
`
`sheets.
`5
`This international search report consists of a total of
`E] It is also accompanied by a copy ot'each prior art document cited in this report.
`
`1. Basis of the report
`a. With regard to the language, the international search was carried out on the basis of:
`E the international application in the language in which it was filed.
`which is the language of
`[3 a translation of the international application into
`a translation furnished for the purposes of international search (Rules 12.3(a) and 23.1(b)).
`
`This international search report has been established taking into account the rectification of an obvious mistake
`authorized by or notified to this Authority under Rule 91 (Rule 43.6bis(a)).
`
`With regard to any nucleotide and/or amino acid sequence disclosed in the international application, see Box No. I.
`
`Certain claims were found unsearchable (see Box No.11).
`
`Unity ofinvention is lacking (see Box No. III).
`
`4. With regard to the title,
`'2 the text is approved as submitted by the applicant.
`[:1 the text has been established by this Authority to read as follows:
`
`b. 1:] none of the figures is to be published with the abstract.
`
`5. With regard to the abstract,
`
`'8 the text is approved as submitted by the applicant.
`[:1 the text has been established, according to Rule 38.2, by this Authority as it appears in Box No. IV. The applicant may,
`within one month from the date of mailing of this international search report, submit comments to this Authority.
`
`6. With regard to the drawings,
`
`a.
`
`the figure of the drawings to be published with the abstract is Figure No. HA)
`[X]
`as suggested by the applicant.
`E] as selected by this Authority, because the applicant failed to suggest a figure.
`[:I as selected by this Authority, because this figure better characterizes the invention.
`
`Form PCT/ISA/210 (first sheet) (January 2015)
`
`

`

`fumished together with the international application under PCT Rule 13ter.1(a) for the purposes of international search
`only in the form ofan Annex C/ST.25 text file.
`
`fumished subsequent to the international filing date for the purposes of international search only:
`
`D in the form ofan Annex C/ST.25 text file (Rule 13ter.1(a)).
`D on paper or in the form ofan image file (Rule 13ter.1(b) and Administrative Instructions, Section 713).
`
`In addition, in the case that more than one version or copy of a sequence listing has been filed or furnished, the required
`statements that the information in the subsequent or additional copies is identical to that forming part of the application as
`filed or does not go beyond the application as filed, as appropriate, were fumished.
`
`PCT/USZO18IO37161 22.10.2018
`
`INTERNATIONAL SEARCH REPORT
`
`lntemational application No.
`PCT/US18/37161
`
`Box No. I
`
`Nucleotide and/or amino acid sequence(s) (Continuation ofitem 1.c ofthe first sheet)
`
`1. With regard to any nucleotide and/or amino acid sequence disclosed in the international application, the intemational search was
`carried out on the basis ofa sequence listing:
`
`forming part of the international application as filed:
`
`E in the form ofan Annex C/ST.25 text file.
`E] on paper or in the form ofan image file.
`
`3. Additional comments:
`
`Form PCT/ISA/ZIO (continuation of first sheet (1)) (January 2015)
`
`

`

`PCT/USZO18IO37161 22.10.2018
`
`INTERNATIONAL SEARCH REPORT
`
`lntemational application No.
`PCT/US18/37161
`
`
`
`Box No.11
`
`Observations where certain claims were found unsearchable (Continuation ofitem 2 offirst sheet)
`
`This intemational search report has not been established in reSpect of certain claims under Article 17(2)(a) for the following reasons:
`
`1, D ClaimsNos.:
`because they relate to subject matter not required to be searched by this Authority, namely:
`
`
`
`
`
`
`
`
`2. D Claims Nos.:
`
`because they relate to parts of the intemational application that do not comply with the prescribed requirements to such an
`extent that no meaningful international search can be carried out, specifically:
`
`
`3. E] ClaimsNos.:
`
`because they are dependent claims and are not drafled in accordance with the second and third sentences of Rule 6.4(a),
`Box No. III Observations where unity ofinvention is lacking (Continuation ofitem 3 of first sheet)
`
`
`This International Searching Authority found multiple inventions in this international application, as follows:
`-""'-Please See Supplementary Page-“'-
`
`
`As all required additional search fees were timely paid by the applicant, this international search report covers all searchable
`claims.
`
`As all searchable claims could be searched without effort justifying additional fees, this Authority did not invite payment of
`additional fees.
`
`
`
`
`
`As only some of the required additional search fees were timely paid by the applicant, this international search report covers
`only those claims for which fees were paid, specifically claims Nos.:
`
`
`No required additional search fees were timely paid by the applicant, Consequently, this international search report is
`restricted to the invention first mentioned in the claims;
`
`Group I: Claims 1-29, 76, 77
`
`
`it is covered by claims Nos:
`
`
`Remark on Protest
`I: The additional search fees were accompanied by the applicant’s protest and, where applicable, the
`
`payment of a protest fee.
`
`E] The additional search fees were accompanied by the applicant’s protest but the applicable protest
`
`fee was not paid within the time limit specified in the invitation.
`No protest accompanied the payment of additional search fees.
`
`
`
`
`
`Form PCT/ISA/210 (continuation of first sheet (2)) (January 2015)
`
`

`

`PCT/U82018I037161 22.10.2018
`
`INTERNATIONAL SEARCH REPORT
`
`International application No.
`PCT/US1BI37161
`
`A.
`
`CLASSIFICATION OF SUBJECT MATTER
`
`IPC - C12N 15/09, 15/10; BO1J 19/00 (2018.01)
`CPC -
`
`BO1J 19/0046; C12N 15/09, 15/1093
`
`According to International Patent Classification (IPC) or to both national classification and IPC
`B.
`FIELDS SEARCHED
`
`Minimum documentation searched (classification system followed by classification symbols)
`See Search History document
`
`Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched
`See Search History document
`
`Electronic data base consulted during the international search (name of data base and, where practicable, search terms used)
`See Search History document
`
`C. DOCUMENTS CONSIDERED TO BE RELEVANT
`
`Citation of document, with indication, where appropriate, of the relevant passages
`
`Relevant to claim No.
`
`
`
`US 2017/0095785 A1 (TWIST BlOSClENCE CORPORATION) 06 April 2017; Abstract; Figure
`15: Paragraphs [0060], [0080], [0159], [0202], [0367], [0381]-[0383], [0461], [0466], [0474],
`[0486], [0506], [0534]
`
`WU, CH, et al. Sequence-Specific Capture of Protein-DNA Complexes for Mass Spectrometn'c
`Protein Identification. PLoS ONE. 20 October 2011, Vol. 6, No. 10. 626217.
`DOI:10.1371/journal.pone.0026217; Page 3, Left Column, Exonuclease Digestion of DNA
`Protein-DNA Complexes section to Page 3, Right Column, First Paragraph.
`
`ZHOU, D, et al. Establishment and Application of a Loop-Mediated Isothermal Amplification
`(LAMP) System for Detection of cry1 Ac Transgenic Sugarcane. SCIENTIFIC REPORTS. 09
`May 2014. Vol, 4, No. 4912. DOI: 10.1038/srep04912; Abstract; Page 2, Right Column,
`Optimization of the Lamp reaction section, Second Paragraph.
`
`1. 5-7, 13, 17-20, 27-29,
`76, 77
`-_...
`2-4, 8-12, 14-16, 21-26
`
`2. 9, 14, 23, 24
`
`US 9,677,067 82 (TWIST BIOSCIENCE CORPORATION) 13 January 2017; Abstract; Column
`2, Lines 21—51; Column 35, Lines 57-67; Column 36, Table 1; column 44, Tables 2, 4
`
`3, 4, 8, 10, 11, 15, 16, 21,
`22. 25
`
`D Further documents are listed in the continuation of Box C. D See patent family annex.
`Special categories ol‘ cited documents:
`riority
`later document published after the international filing date or
`document defining the general state of the art which is not considered
`date and not in conflict With the apfilication but cited to un erstand
`the principle or theory underlyingt e invention
`to be of particular relevance
`earlier application or patent but published on or afier the international
`document of particular relevance; the claimed'invention cannotbe
`filing date
`conSidered novel or cannot be conSidered to involve an inventive
`step when the document is taken alone
`document which may throw doubts on priority claim(s).or which is
`Cited to establish the publication date of another Citation or other
`document of particular relevance; the claimed invention cannot be
`speCIal reason (as speCified)
`considered to involve an inventive step when the document
`is
`combined with one or more other such documents, such combination
`document referring to an oral disclosure, use, exhibition or other
`means
`being obvious to a person skilled in the art
`document member of the same patent family
`
`document publishedprior to the international filing date but later than
`the priority date claimed
`
`Date of the actual completion of the international search
`
`11 September 2018 (11.09.2018)
`
`Name and mailing address ofthe lSA/
`Mail Stop PCT. Attn: ISA/US. Commissioner for Patents
`PO. Box 1450, Alexandria, Virginia 22313-1450
`Facsimile No. 571-273-3300
`
`Form PCT/lSA/ZIO (second sheet) (January 2015)
`
`Date of mailing of the international search report
`2' 2 OCT 2018
`Authorized officer
`
`Shane Thomas
`
`PCT Helpdesk: 571-272—4300
`PCT OSP: 571-272-7774
`
`

`

`PCT/USZO18I037161 22.10.2018
`
`INTERNATIONAL SEARCH REPORT
`Information on patent family members
`
`International application No.
`PCT/US18/37161
`
`—""-Continued from Box No. III: Observations Where Unity of Invention is Lacking:
`
`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 order for all inventions to be examined, the appropriate additional examination fees must be paid.
`
`Group l: Claims 1-29, 76 and 77 are directed toward methods for nucleic acid assembly. wherein each of a plurality of polynucleotides
`do not comprise a terminal region of sequence homology to another polynucleotide of the plurality of polynucleotides.
`
`Group II: Claims 30-75 are directed toward a method for nucleic acid assembly, comprising two nucleic acids, each comprising a 5'
`flanking adapter sequence, a homology sequence, an insert sequence, and a 3' flanking adapter sequence.
`
`The inventions listed as Groups I and II do not relate to a single general inventive concept under PCT Rule 131 because, under PCT
`Rule 132. they lack the same or corresponding special technical features for the following reasons: the special technical features of
`Group I include wherein the plurality of polynucleotides are annealed in a processive predetermined order. not present in Group II; the
`special technical features of Group II include an insert sequence, not present in Group I.
`
`shared technical features are previously disclosed by the Swift reference, unity of invention is lacking.
`
`Groups I and II share the technical features including: a method for nucleic acid assembly, comprising: (a) providing a first double
`stranded nucleic acid; (b) providing a second double stranded nucleic acid; a 5' flanking adapter sequence; a homology sequence and a
`3' flanking adapter sequence; and mixing the first double stranded nucleic acid, and the second double stranded nucleic acid with a
`reaction mixture comprising an exonuclease, an endonuclease, a polymerase, and a ligase.
`
`However, these shared technical features are previously disclosed by US 2017/0088887 A1 to Swift Biosciences Inc. (hereinafter 'Swift').
`
`Swift discloses a method for nucleic acid assembly (a method for producing a nucleic acid including ligation (a method for nucleic acid
`assembly); paragraph [0040]), comprising: (a) providing a first double stranded nucleic acid (comprising: (a) providing genomic DNA (a
`first double stranded nucleic acid); paragraph [0040]); (b) providing a second double stranded nucleic acid (providing a second double
`stranded nucleic acid; paragraph [0040]): a 5' flanking adapter sequence (a 5' adapter; paragraph [0015]); a homology sequence (a
`terminal adaptor sequence capable of invading the termini (a homology sequence); paragraphs [0115], [01 19]) and a 3' flanking adapter
`sequence (a 3' adapter sequence; paragraph [0014]); and mixing the first double stranded nucleic acid. and the second double stranded
`nucleic acid with a reaction mixture (mixing the first double stranded nucleic acid, and the second double stranded nucleic acid with a
`reaction mixture; paragraphs [0038], [0040], [0264]) comprising an exonuclease, an endonuclease, a polymerase, and a ligase
`(comprising an exonuclease, an endonuclease, a polymerase, and a ligase; paragraphs [0038]; [0040]. [0264], wherein the exonuclease
`is present as an active domain of the polymerase).
`
`Since none of the special technical features of the Groups l+ inventions is found in more than one of the inventions, and since all of the
`
`Form PCT/[SA/ZlO (patent family annex) (January 2015)
`
`

`

`PCT/USZO18I037161 22.10.2018
`
`From the
`INTERNATIONAL SEARCHING AUTHORITY
`
`PATENT COOPERATION TREATY
`
`T0: David Harburger
`Wilson Sonsini Goodrich & Rosati
`650 Page Mill Road
`Palo Alto, California 94304
`United States of America
`
`PCT
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`Applicant’s or agent’s file reference
`44854-746601
`
`(day/month/year) 2 2 O C T 2018
`FOR FURTHER ACTION
`See paragraph '2 below
`
`lntemational application No.
`
`International filing date (day/monrh/year)
`
`Priority date (day/monIh/year)
`
`PCT/US18/37161
`
`12 June 2018 (12.06.2018)
`
`12 June 2017 (12.06.2017)
`
`lntemational Patent Classification (IPC) or both national classification and [PC
`IPC - C12N 15/09, 15/10; BO1J 19/00 (2018.01)
`CPC -
`
`BO1J 19/0046; C12N 15/09, 15/1093
`
`Applicant TWIST BIOSCIENCE CORPORATION
`
`1. This opinion contains indications relating to the following items:
`
`Box No. I
`
`Basis of the opinion
`
`Box No. II
`
`Priority
`
`(PCT Rule 43bis. l) Date of mailing
`
`
`DUE]EEDDK Box No. VIII Certain observations on the international application
`
`Box No. “I Non-establishment ofopinion with regard to novelty, inventive step and industrial applicability
`
`Box No. IV Lack of unity of invention
`
`Box No. V
`
`Box No. VI
`
`Reasoned statement under Rule 43bis.1(a)(i) with regard to novelty, inventive step and industrial applicability;
`citations and explanations supporting such statement
`Certain documents cited
`
`Box No. Vll Certain defects in the 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
`lntemational Preliminary Examining Authority (“IPEA”) except that this does not apply where the applicant chooses an Authority
`other than this one to be the IPEA and the chosen IPEA has notified the Intemational Bureau under Rule 66.1bis(b) that written
`opinions of this lntemational Searching Authority will not be so considered.
`If this opinion is, as provided above, considered to be a written opinion of the IPEA, the applicant is invited to submit to the lPEA
`a written reply together, where appropriate, with amendments, before the expiration of 3 months fl'om the date of mailing of Form
`PCT/ISA/220 or before the expiration of 22 months from the priority date, whichever expires later.
`For firrther options, see Form PCT/lSA/220.
`
`Name and mailing address of the ISA/US Date ofcompletion ofthis opinion
`Mail Sto PCT, Attn: ISA/US
`p
`Commissime' f°r ”items
`PO. Box 1450. Alexandria, Virginia 22313—1450
`Facsimile No. 571-273-8300
`
`11 September 2018 (11.09.2018)
`
`PCT 05?: 571-272-7774
`
`Authorized officer
`Shane Thomas
`pc-r Helmgsk: 5714724300
`
`Form PCT/ISA/237 (cover sheet) (January 2015)
`
`

`

`1, With regard to the language, this opinion has been established on the basis of:
`
`E] the international application in the language in which it was filed.
`D a translation of the international application into
`fumished for the purposes ofintemational search (Rules 12.3(a) and 23.1(b)).
`
`which is the language of a translation
`
`This opinion has been established taking into account the rectification ofan obvious mistake authorized by or notified to
`this Authority under Rule 9] (Rule 43bis. 1(a)).
`
`With regard to any nucleotide and/or amino acid sequence disclosed in the international application, this opinion has
`been established on the basis ofa sequence listing:
`
`a.
`
`forming part ofthe international application as filed:
`
`
`
`E in the form ofan Annex C/ST.25 text file.
`E] on paper or in the form ofan image file.
`b. El furnished together with the international application under PCT Rule l3teri1(a) for the purposes ofintemational
`search only in the form of an Annex C/ST.25 text file.
`
`
`
`PCT/USZO18IO37161 22.10.2018
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`
`
`International application No.
`PCT/US18/37161
`
`Box No. I
`
`Basis of this opinion
`
`
`
`
`
`
`
`
`
`
`
`c. C] fumished subsequent to the international filing date for the purposes of international search only:
`
`
`C] in the form ofan Annex C/ST.25 text file (Rule l3ler.l(a)).
`D on paper or in the form ofan image file (Rule l3ter. 1(b) and Administrative Instructions, Section 713)
`
` 4. [:1 In addition, in the case that more than one version or copy ofa sequence listing has been filed or fumished, the required
`
`
`statements that the information in the subsequent or additional copies is identical to that forming part of the application as
`filed or does not go beyond the application as filed, as appropriate, were fiimished.
`
`5. Additional comments:
`
`
`
`Form PCT/ISA/237 (Box No. I) (January 2015)
`
`

`

`PCT/USZO18IO37161 22.10.2018
`
`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
`
`
`
`lntemational application No.
`PCT/US18/37161
`
`Box No. IV
`
`Lack of unity ofinvention
`
`
`
`l. E In response to the invitation (Form PCT/lSA/206) to pay additional fees the applicant has, within the applicable time limit:
`
`paid additional fees.
`
`paid additional fees under protest and, where applicable, the protest fee.
`
`paid additional fees under protest but the applicable protest fee was not paid.
`
`
`
`
`
`not paid additional fees.
`
`2. E] This Authority found that the requirement of unity of invention is not complied with and chose not to invite the applicant to
`pay additional fees.
`
`3. This Authority considers that the requirement of unity of invention in accordance with Rule 13.1, 13.2 and 13.3 is
`
`D complied with.
`'2'
`not complied with for the following reasons:
`
`
`
` Group II: Claims 30—75 are directed toward a method for nucleic acid assembly. comprising two nucleic acids. each comprising a 5'
`
`flanking adapter sequence. a homology sequence. an insert sequence, and a 3' flanking adapter sequence.
`The inventions listed as Groups I 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 same or corresponding special technical features for the following reasons: the special technical features of
`Group I include wherein the plurality of polynucleotides are annealed in a processive predetermined order. not present in Group II; the
`special technical features of Group II include an insert sequence, not present in Group I.
`
`
`Groups I and II share the technical features including: a method for nucleic acid assembly. comprising: (a) providing a first double
`
`
`stranded nucleic acid: (b) providing a second double stranded nucleic acid; a 5' flanking adapter sequence; a homology sequence and a
`
`
`3' flanking adapter sequence; and mixing the first double stranded nucleic acid. and the second double stranded nucleic acid with a
`reaction mixture comprising an exonuclease, an endonuclease. a polymerase. and a ligase.
`
`However, these shared technical features are previously disclosed by US 2017/0088887 A1 to Swift Biosciences Inc. (hereinafter 'Swifl').
`
`
`Swift discloses a method for nucleic acid assembly (a method for producing a nucleic acid including ligation (a method for nucleic acid
`
`
`assembly); paragraph [0040]). comprising: (a) providing a first double stranded nucleic acid (comprising: (a) providing genomic DNA (a
`first double stranded nucleic acid); paragraph [0040]); (b) providing a second double stranded nucleic acid (providing a second double
`
`
`stranded nucleic acid; paragraph [0040]); a 5' flanking adapter sequence (a 5' adapter; paragraph [0015]); a homology sequence (a
`terminal adaptor sequence capable of invading the termini (a homology sequence): paragraphs [0115], [0119]) and a 3' flanking adapter
`
`
`sequence (a 3' adapter sequence; paragraph [0014]): and mixing the first double stranded nucleic acid. and the second double stranded
`nucleic acid with a reaction mixture (mixing the first double stranded nucleic acid. and the second double stranded nucleic acid with a
`
`
`reaction mixture; paragraphs [0038], [0040], [0264]) comprising an exonuclease. an endonuclease. a polymerase, and a ligase
`(comprising an exonuclease. an endonuclease. a polymerase, and a ligase; paragraphs [0038]; [0040]. [0264]. wherein the exonuclease
`
`is present as an active domain of the polymerase).
` Since none of the special technical features of the Groups |+ inventions is found in more than one of the inventions, and since all of the
`shared technical features are previously disclosed by the Swift reference. unity of invention is lacking.
`
`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 order for all inventions to be examined. the appropriate additional examination fees must be paid.
`
`Group I: Claims 1-29. 76 and 77 are directed toward methods for nucleic acid assembly. wherein each of a plurality of polynucleotides do
`not comprise a terminal region of sequence homology to another polynucleotide of the plurality of polynucleotides.
`
`
`
`4.
`
`
`
`Consequently, this opinion has been established in respect of the following parts of the international application:
`
`
`E] all parts.
`the parts relating to claims Nos. GrOUp I: Claims 1'29 76' 77
`
`
`Form PCT/ISA/237 (Box No, 1V) (January 2015)
`
`

`

`Statement
`
`Novelty (N)
`
`inventive step (IS)
`
`Claims
`Claims
`
`Claims
`Claims
`
`2—4, 8-12, 14-16. 21-26, 77
`1, 5-7, 13, 17-20, 27-29. 76
`
`NONE
`1-29. 76. 77
`
`Industrial applicability (IA)
`
`Claims
`Claims
`
`1-29. 76. 77
`NONE
`
`Citations and explanations:
`
`Claims 1, 5-7, 13, 17-20, 27-29. and 76 lack novelty under PCT Article 33(2) as being anticipated by US 2017/0095785 A1 (TWIST
`BIOSCIENCE CORPORATION) (hereinafter 'TWIST Bioscience'785)
`
`As per claim 1, TWIST Bioscience'785 discloses a method for nucleic acid assembly (longer nucleic acids can be synthesized in parallel
`using microfluidic assemblies; Abstract), comprising: (a) providing a plurality of polynucleotides (the term “oligomef' is meant to
`encompass any polynucleotide or polypeptide or other chemical compound with repeating moieties such as nucleotides, amino acids,
`carbohydrates and the like; devices for the manufacturing of large libraries of long and high—quality nucleic acids; Abstract; Paragraph
`[0367]), wherein each of the polynucleotides do not comprise a terminal region of sequence homology to another polynucleotide of the
`plurality of polynucleotides (homologous repeats or high GC regions in a sequence to be assembled may introduce errors associated with
`the correct order and hybridization of smaller oligonucleotides; longer oligonucleotides can circumvent these problems by reducing the
`number of oligonucleotides to be ordered and aligned. by avoiding problematic sequences. such as homology repeats; clue to its reliance
`on a homologous template, this method is suitable to the synthesis of only a limited number of sequences with similarity to an existing
`polynucleotide molecule; Paragraphs [0474]. [0486]); and (b) mixing the plurality of polynucleotides with an exonuclease (the cleavage
`products resulting from the treatment of heteroduplexes may be subjected to PCA after the error at the cleavage site is chewed out. e.g..
`by an exonuclease. to generate error depleted products; Figure 15; Paragraph [0506]), an endonuclease (the sequence element
`comprises a recognition site for a restriction endonuclease: Paragraph [0060]). a polymerase (implementation of polymerase chain
`assembly (PCA); oligonucleotide synthesis typically in situ on a DNA synthesis wafer, may be followed by a gene assembly reaction.
`such as polymerase cycling assembly (PCA); Figure 12; Paragraphs [0080], [0202]), and a ligase (ligation comprises the use of a ligase;
`Paragraph [0060]), wherein the plurality of polynucleotides are annealed in a processive predetermined order based on a complementary
`sequence between adjacent polynucleotides (one or more auxiliary oligonucleotides may be provided to anneal to the single stranded
`amplicon nucleic acids; the auxiliary oligonucleotides may be partially or completely complementary to the adaptor copies; homologous
`repeats or high GC regions in a sequence to be assembled may introduce errors associated with the correct order and hybridization of
`smaller oligonucleotides; longer oligonucleotides can circumvent these problems by reducing the number of oligonucleotides to be
`ordered and aligned, by avoiding problematic sequences. such as homology repeats; Paragraphs [0377]. [0474]. [0486]).
`
`As per claim 5, TWIST Bioscience'785 discloses the method of claim 1, and TWIST Bioscience’785 further discloses wherein the
`polymerase comprises 5' to 3' polymerase activity (the amplification product may be cleaved by non-Type IIS endonucleases which
`cleave at the 5' end of the recognition site; Paragraphs [0381]-[0383]).
`
`As per claim 6, TWIST Bioscience'785 discloses the method of claim 1, and TWIST Bioscience'785 further disclose wherein the
`polymerase is a DNA polymerase (implementation of polymerase chain assembly (PCA); oligonucleotide synthesis typically in situ on a
`DNA synthesis wafer, may be followed by a gene assembly reaction, such as polymerase cycling assembly (PCA); Figure 12;
`Paragraphs [0080]. [0202]).
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`As per claim 7, TWIST Bioscience'785 discloses the method of claim 1, and TWIST Bioscience'785 further discloses wherein the ligase
`catalyzes joining of at least two nucleic acids (ligation comprises the use of a ligase for circularizing nucleic acids; Paragraph [0060]).
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`PCT/USZO18I037161 22.10.2018
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`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
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`Intematlonal application No
`PCT/US18/37161
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`Box No. V
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`Reasoned statement under Rule 43bis.l(a)(i) with regard to novelty, inventive step and industrial applicability;
`citations and explanations supporting such statement
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`-""—Continued Within the Next Supplemental Box-"'-
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`Form PCT/ISA/237 (Box No. V) (January 2015)
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`PCT/USZO18IO37161 22.10.2018
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`WRITTEN OPINION OF THE
`INTERNATIONAL SEARCHING AUTHORITY
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`lntemational application No‘
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`PCT/US18/37161
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`Supplemental Box
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`In case the space in any of the preceding boxes is not sufficient.
`Continuation of:
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`-"“-Continued from Box V: Citations and Explanations-""-
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`As per claim 13, TW|ST Bioscience’785 discloses a method for nucleic acid assembly (longer nucleic acids can be synthesized in
`parallel using microfluidic assemblies; Abstract), comprising: (a) providing a first double stranded nucleic acid (the double stranded
`nucleic acids comprise the first adapter and the first auxiliary oligonucleotide: Paragraph [0060]); (b) providing a second double stranded
`nucleic acid (the double stranded nucleic acids comprise the first adapter and the first auxiliary oligonucleotide; Paragraph [0060]); (c)
`providing a third double stranded nucleic acid (Paragraph [0060]) comprising in 5' to 3' order: a 5' flanking adapter sequence (the
`different target sequences in the n linear single stranded nucleic acids are flanked by a first and a second adaptor hybridization
`sequence; Paragraph [0060]), a first homology sequence to the first double stranded nucleic acid (a first adaptor sequence is
`homologous with the first auxiliary oligonucleotide; Paragraph [0060]), a second homology sequence to the second double stranded
`nucleic acid (a second adaptor sequence which is homologous to a different target sequence; Paragraph [0060]), and a 3' flanking
`adapter sequence (the different target sequences in the n linear single stranded nucleic acids are flanked by a first and a second adaptor
`hybridization sequence; Paragraph [0060]), wherein the first double stranded nucleic acid (the double stranded nucleic acids comprise
`the first adapter and the first auxiliary oligonucleotide; Paragraph [0060]), the second double stranded nucleic acid (the double stranded
`nucleic acids comprise the first adapter and the first auxiliary oligonucleotide; Paragraph [0060]), and the third double stranded nucleic
`acid (Paragraph [0060]) comprise non-homologous sequences at terminal regions (homologous repeats or high GC regions in a
`sequence to be assembled may introduce errors associated with the correct order and hybridization of smaller oligonucleotides; longer
`oligonucleotides can circumvent these problems by reducing the number of oligonucleotides to be ordered and aligned, by avoiding
`problematic sequences, such as homology repeats; due to its reliance on a homologous template, this method is suitable to the
`synthesis of only a limited number of sequences with similarity to an existing polynucleotide molecule; Paragraphs [0474], [0486]): and
`(d) mixing the first double stranded nucleic acid (the double stranded nucleic acids comprise the first adapter and the first auxiliary
`oligonucleotide; Paragraph [0060]), the second double stranded nucleic acid (the double stranded nucleic acids comprise the first
`adapter and the first auxiliary oligonucleotide; Paragraph [0060]), and the third double stranded nucleic acid (Paragraph [0060]) with a
`reaction mixture comprising an exonuclease (the cleavage products resulting from the treatment of heteroduplexes may be subjected to
`PCA after the error at the cleavage site is chewed out, e.g., by an exonuclease, to generate error depleted products; Figure 15;
`Paragraph [0506]). an endonuclease (the sequence element comprises a recognition site for a restriction endonuclease; Paragraph
`[0060]). a polymerase (implementation of polymerase chain assembly (PCA); oligonucleotide synthesis typically in situ on a DNA
`synthesis wafer, may be followed by a gene assembly reaction. such as polymerase cycling assembly (PCA): Figure 12; Paragraphs
`[0080], [0202]). and a ligase (ligation comprises the use of a ligase for circularizing nucleic acids; Paragraph [0060]).
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`As per claim 17, TW|ST Bioscience'785 discloses the method of claim 13, and 1W|ST Bioscience'785 further discloses wherein the
`polymerase comprises 5' to 3' polymerase activity (the amplification product may be cleaved by non-Type IIS endonucleases which
`cleave at the 5' end of the recognition site; Paragraphs [0381]-[0383]).
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`As per claim 18, TW|ST Bioscience'785 discloses the method of claim 13, and TWIST Bioscience'785 further disclose wherein the
`polymerase is a DNA polymerase (implementation of polymerase chain assembly (PCA); oligonucleotide synthesis typically in situ on a
`DNA synthesis wafer, may be followed by a gene assembly reaction, such as polymerase cycling assembly (PCA); Figure 12;
`Paragraphs [0080], [0202]),
`
`As per claim 19, TW|ST Bioscience'785 discloses the method of claim 13, and TW|ST Bioscience’785 further discloses wherein the
`ligase catalyzes joining of at least two nucleic acids (ligation comprises the use of a ligase for circularizing nucleic acids; Paragraph
`[0060]).
`
`As per claim 20, TW|ST Bioscience'785 discloses the method of claim 13, and TW|ST Bioscience'785 further discloses wherein the
`homology sequence of the first double stranded nucleic acid (the double stranded nucleic acids comprise the first adapter and the first
`auxiliary