(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`
`
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`
`
` a
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`(19) World Intellectual Property Organization
`International Bureau
`
`(10) International Publication Number
`(43) International Publication Date
`WO 2011/085075 A2
`14 July 2011 (14.07.2011)
`
`
`(1) International Patent Classification:
`C1I2N 15/10 (2006.01)
`.
`sue
`:
`(21) International Application Numer:rUS>01 1020335
`oe
`(22) International Filing Date:
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`_
`(25) Filing Language:
`(6) Publication Language:
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`6 January 2011 (06.01.2011)
`.
`English
`Fnglish
`
`(81) Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ,
`CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO,
`DZ, EC, EE, EG, ES, FL GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP,
`KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD,
`ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NL
`NO, NZ, OM,PE, PG, PH, PL, PT, RO, RS, RU,SC, SD,
`SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR,
`TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM, ZW.
`.
`en
`(84) Designated States (unless otherwise indicated, for every
`kind ofregional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA,SD, SL, SZ, TZ, UG,
`ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU,TJ,
`TM), European (AL, AT, BE, BG, CH, CY, CZ, DE, DK,
`(71) Applicant (for all designated States except US): GENY,
`EE, ES, FL, FR, GB, GR, HR, HU,IE, IS, IT, LT, LU,
`INC. [US/US]; 500 Technology Square, Suite 130, Cam-
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SL SK,
`bridge, Massachusetts 02139 (US).
`SM, TR), OAPI (BF, BJ, CF, CG, CL CM, GA, GN, GQ,
`Inventors; and
`GW, ML, MR,NE, SN, TD, TG).
`JACOBSON,
`(for US only):
`Inventors/Applicants
`Joseph [US/US]; 223 Grant Avenue, Newton, Mas- Published:
`Sachusetts024°OS).waeaayjivang[ONUSE —_without international search report and to be republished
`02139 (US)
`>
`>
`Se
`-
`upon receipt ofthat report (Rule 48.2(g))
`(74) Agent: | CAMACHO,
`Jennifer A.; Greenberg Traurig — with sequence listingpart ofdescription (Rule 5.2(a))
`*
`nd
`>
`LLP, One International Place, Boston, Massachusetts
`02110(US).
`
`GO) Priority Data:
`61/293.192
`61/310.076
`61/334,416
`
`7 January 2010 (07.01.2010)
`3 March 2010 (03.03.2010)
`13 May 2010 (13.05.2010)
`
`us
`US
`US
`
`(72)
`(75)
`
`’
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`(54) Title: ASSEMBLY OF HIGH FIDELITY POLYNUCLEOTIDES
`
`(57) Abstract: Mcthods and apparatus relate to the synthesis of high fidclity polynucleotides and to the reduction of sequencecr-
`tors generated during synthesis of nucleic acids on a solid support. Specifically, design of support-bound template oligonu-
`cleotides is disclosed. Assembly methods include cycles of annealing, stringent wash and extension of polynucleotides comprising
`a sequence region complementary to immobilized template oligonucleotides. The error free synthetic nucleic acids generated
`therefrom can be used for a variety of applications, including synthesis of biofuels and value-added pharmaceutical products.
`
`
`
`
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`wo2011/085075A2!ANTIAIUMDIMTAIANAAANTAAAT
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`WO 2011/085075
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`PCT/US2011/020335
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`ASSEMBLYOF HIGH FIDELITY POLYNUCLEOTIDES
`
`RELATED APPLICATIONS
`
`[0001] This application claims the benefit of U.S. Provisional Patent Application Ser. No.
`
`61/293,192, filed January 07, 2010; U.S. Provisional Patent Application Ser. No. 61/310,076,
`
`filed March 03, 2010; and U.S. Provisional Patent Application Ser. No. 61/334,416, filed May
`
`13, 2010, cach of which is incorporated herein by referencein its entircty.
`
`FIELD OF THE INVENTION
`
`[0002] Methods and apparatuses provided herein relate to the synthesis and assembly of high
`
`fidelity nucleic acids and nucleic acid libraries having a predefined sequence. Moreparticularly,
`
`methods and apparatuses are provided for polynucleotide synthesis, error reduction, and/or
`
`sequence verification on a solid support.
`
`In some embodiments, picoliter and sub-picoliter
`
`dispensing and droplet moving technologies are applied to access and manipulate the
`
`oligonucleotides on DNA microarrays.
`
`BACKGROUND
`
`[0003] Using the techniques of recombinant DNA chemistry,
`
`it is now common for DNA
`
`sequences to be replicated and amplified from nature and then disassembled into component
`
`parts. As component parts, the sequences are then recombined or reassembled into new DNA
`
`sequences.
`
`However,
`
`reliance on naturally available sequences significantly limits the
`
`possibilities that may be explored by researchers. While it is now possible for short DNA
`
`sequences to be directly synthesized from individual nucleosides,
`
`it has been generally
`
`impractical
`
`to directly construct
`
`large segments or assemblies of polynucleotides, 1.c.,
`
`polynucleotide sequences longer than about 400 basepairs.
`
`[0004] Oligonucleotide synthesis can be performed through massively parallel custom syntheses
`
`on microchips (Zhou et al. (2004) Nucleic Acids Res. 32:5409; Fodor et al. (1991) Science
`
`251:767). However, current microchips have very low surface areas and hence only small
`
`amounts of oligonucleotides can be produced. When released into solution, the oligonucleotides
`
`are present at picomolar or
`
`lower concentrations per sequence, concentrations that are
`
`insufficiently high to drive bimolecular priming reactions efficiently. Current methods for
`
`assembling small numbers of variant nucleic acids cannot be scaled up in a cost-effective manner
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`to generate large numbers of specified variants. As such, a need remains for improved methods
`
`and devices for high-fidelity gene assembly andthe like.
`
`[0005] Furthermore, oligonucleotides on microchips are generally synthesized via chemical
`
`reactions. Spurious chemical reactions cause random baseerrors in oligonucleotides. One of the
`
`critical
`
`limitations in chemical nucleic acid synthesis is the error-rate. The error rate of
`
`chemically-synthesized oligonucleotides (e.g. deletions at a rate of 1
`
`in 100 bases and
`
`mismatches and inscrtions at about |
`
`in 400 bascs) cxeeeds the crror rate obtainable through
`
`enzymatic means of replicating an existing nucleic acid (e.g., PCR). Therefore, there is an
`
`urgent need for new technology to produce high-fidelity polynucleotides.
`
`SUMMARY
`
`[0006] Aspects of the invention relate to methods and apparatuses for preparing and/or
`
`assembling high fidelity polymers. Also provided herein are devices and methods for processing
`
`nucleic acid assembly reactions and assembling nucleic acids.
`
`It is an object of this invention to
`
`provide practical, economical methods of synthesizing custom polynucleotides.
`
`It is a further
`
`object to provide methods of producing synthetic polynucleotides that have lowererror rates than
`
`synthetic polynucleotides made by methods knowninthe art.
`
`[0007] According to one embodiment,
`
`the invention provides a method for producing a
`
`polynucleotide having a predetermined sequence on a solid support.
`
`In some embodiments,
`
`pluralities of support-boundsingle-stranded oligonucleotides are provided at different features of
`
`a solid support, each plurality of oligonucleotides having a predefined sequence and each
`
`plurality being bound to a different discrete feature of the support.
`
`In some embodiments, each
`
`plurality of oligonucleotides comprises a sequence region at its 5’ end that is the same as a
`
`sequence region of a 3’ end of another oligonucleotide and a sequence region at its 3’ end that is
`
`the same as a sequence region at a 5’ end of a different oligonucleotide and wherein the first
`
`plurality of oligonucleotides has a 3’ end that is complementary to a 3’ end ofa first input single-
`
`stranded oligonucleotide.
`
`In some embodiments, the first plurality of oligonucleotide comprises
`
`at its 5’ cnd a scquence region that is the same as a scquence region at the 3’ end of a second
`
`oligonucleotide and the Nth plurality of oligonucleotide comprisesat its 3’ end a sequence region
`
`that is the same as a sequence region of the (N-1) oligonucleotide.
`
`In some embodiments, a first
`
`input oligonucleotide is provided in solution at
`
`the feature where the first plurality of
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`oligonucleotides is immobilized. A first plurality of complementary oligonucleotides is
`
`synthesized by template-dependent synthesis in which each ofthe first plurality of support-bound
`
`oligonucleotides is hybridized with the first input oligonucleotide thereby forming an extension
`
`product duplex. The extension product duplex is dissociated to release the first plurality of
`
`complementary oligonucleotides. The first plurality of complementary oligonucleotides (e.g.,
`
`second input oligonucleotide) may then anneal to a second plurality of support-bound single
`
`stranded oligonucleotides wherein the anncaling of the first plurality of complementary
`
`oligonucleotides to the second plurality of support-bound oligonucleotides serves as a primer for
`
`extension of the first plurality of complementary oligonucleotides. The cycles of primer
`
`extension, dissociation and annealing can be repeated until
`
`the target polynucleotide is
`
`synthesized. The target polynucleotide can be amplified.
`
`In some embodiments,the first input
`
`oligonucleotide is a primer, for example a universal primer or a unique primer.
`
`In other
`
`embodiments, the first input oligonucleotide is a synthetic oligonucleotide or a single stranded
`
`nucleic acid fragment. The plurality of support bound oligonucleotides may be synthesized on
`
`the solid support or synthetic oligonucleotides can be spotted on the solid support.
`
`In some
`
`embodiments, the solid support is a microarray device.
`
`[0008] Some aspects of the invention relates to a method for producing at
`
`least one
`
`polynucleotide having a predefined sequence, the method comprising providing at least a first
`
`and a second plurality of support-bound single-stranded oligonucleotides, wherein each first and
`
`secondplurality of oligonucleotides has a predefined sequence and is boundto a discrete feature
`
`of the support, each first plurality of oligonucleotides comprising a sequence region at its 5’ end
`
`that is the same as a sequence region of a 3’ end of the second plurality of oligonucleotides. A
`
`plurality of first input single-stranded oligonucleotides is provided wherein the 3’ end of the
`
`plurality of the first input oligonucleotide is complementary to the 3’ end ofthe first plurality of
`
`oligonucleotides. The plurality of first input oligonucleotides is hybridized to the first plurality
`
`of support-bound oligonucleotides at a first feature and a first plurality of complementary
`
`oligonucleotides is generated in a chain extension reaction, thereby forming an extension product
`
`duplex. The extension product duplex is dissociated, thereby producing a first plurality of
`
`complementary oligonucleotides. The first plurality of complementary oligonucleotides is
`
`transferred from the first feature to a second feature, thereby bringing into contact the first
`
`plurality of complementary oligonucleotides
`
`to the second plurality of support-bound
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`oligonucleotides. The first plurality of complementary oligonucleotides is then annealed to the
`
`second plurality of support-bound single stranded oligonucleotides at the second feature, wherein
`
`the annealing of the first plurality of complementary oligonucleotides to the second plurality of
`
`support-bound oligonucleotides serves as a primer for extension of the first plurality of
`
`complementary oligonucleotides, thereby producing the polynucleotide.
`
`[0009] In some embodiments, a third plurality of support-bound single-stranded oligonucleotides
`
`is provided wherein cach third plurality of oligonuclcotidcs has a predefined scquence and is
`
`bound to a third discrete feature of the support, each third plurality of oligonucleotides
`
`comprising a sequence region at its 3’ end that is the same as a sequence region of a 5’ end of the
`
`second plurality of oligonucleotides, and repeating annealing, chain extension, denaturation and
`
`transferring steps to produce a longer polynucleotide.
`
`[0010] In some aspects of the invention, the reaction steps are performed within discrete droplet
`
`volumes (nanoliter, picoliter or subpicoliter droplets volumes).
`
`In some embodiments, the
`
`annealing and extension steps are performed within a first droplet volume at a first feature and
`
`the first plurality of complementary oligonucleotides is released within the first droplet volume.
`
`The first droplet volume may be moved to a second feature comprising a second plurality of
`
`support-bound oligonucleotides. Droplets volumes may be moved at specific locations of the
`
`solid support by different
`
`techniques such as electrowetting or following a hydrophilicity
`
`gradient.
`
`In some embodiments,
`
`the whole support
`
`is subjected to conditions promoting
`
`annealing or primer extension or denaturing. In some embodiments,
`
`the whole support or
`
`selected features are subjected to thermocycling conditions. In other embodiments, selected
`
`features are subjected to conditions promoting annealing or primer extension or denaturing.
`
`[0011] Aspects of the invention relate to the synthesis of at
`
`least one high fidelity target
`
`polynucleotide having a predetermined sequence.
`
`In some embodiments, the method comprises
`
`the steps of providing pluralities of different support-bound single-stranded oligonucleotides at
`
`different features of a solid support, wherein each plurality of support-bound oligonucleotides
`
`has at least two sequence regions, a first sequence region at its 5’ end that is the same as a
`
`sequence region of the 3’ end of another oligonucleotide and a second sequence regionatits 3’
`
`end that is the same as a sequenceregion at a 5’ end of a different oligonucleotide and wherein
`
`each plurality of oligonucleotides has a 3° end that is complementary to a 3’ end of a different
`
`input single-stranded polynucleotide. A first input polynucleotide is provided in solution at the
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`feature of a first plurality of support-bound oligonucleotides wherein the input polynucleotide 1s
`
`generated from a previous extension step. Thefirst input polynucleotide is hybridized to the first
`
`plurality of support-bound oligonucleotides under hybridizing conditions thereby forming
`
`duplexes.
`
`In some embodiments, the duplexes may comprise duplexes having at least one
`
`mismatch in a complementary region and/or duplexes that do not comprise a mismatch in the
`
`complementary region. The duplexes having at least one mismatch in the complementary region
`
`are unstable duplexes that can be denatured understringent melt conditions. The stringent melt
`
`conditions (e.g., stringent melt temperature) do not denature the duplexes that do not comprise a
`
`mismatch
`
`in
`
`the
`
`complementary region
`
`(stable
`
`duplexes).
`
`Error-containing input
`
`polynucleotides are then released in solution and removed. The remaining stable duplexes can
`
`then be subjected to primer extension conditions, generating a first plurality of complementary
`
`oligonucleotides by template-dependent synthesis, thereby forming an extension product duplex.
`
`The extension product duplex is dissociated to release a second input polynucleotide (or
`
`complementary polynucleotide). The second input polynucleotide can be allowed to anneal to a
`
`second plurality of support-bound single stranded oligonucleotides. Cycles of stringent melt,
`
`extension, dissociation and annealing are repeated until the target polynucleotide is synthesized.
`
`[0012] In some embodiments, the annealing and stringent melt steps can be performed within a
`
`first droplet volumeat a first feature thereby releasing the error-containing polynucleotides in the
`
`first droplet volume. The first droplet volume can be discarded and a second droplet volume
`
`comprising reagent for primer extension can be added to the first feature under condition
`
`promoting primer extension. Complementary strands are released into the second droplet volume
`
`and the second droplet volume may be movedto a second feature comprising a secondplurality
`
`of support bound oligonucleotides.
`
`In some embodiments, the support-bound oligonucleotides
`
`comprise a third sequence region at the 3’ end of the oligonucleotide. In some embodiments, the
`
`plurality of different support-bound single-stranded oligonucleotides at different features of a
`
`solid support comprises at least three sequence regions: a 5° end sequence region N,at least two
`
`sequence regions (N-1) and (N-2)
`
`that are complementary to the 3’ end of an input
`
`polynucleotide, and a 3’ end sequence region. The (N-1) sequence region is adjacent to the 5’
`
`end sequence region and the (N-2) sequenceregion is adjacent to the (N-1) sequence region.
`
`In
`
`some embodiments, a first input polynucleotide is provided in solution at the feature of a first
`
`plurality of support-bound oligonucleotides wherein the first input polynucleotide comprises
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`sequences regions complementary to the at least two sequences regions (N-1) and (N-2). The
`
`first input polynucleotide is hybridized with the first plurality of support-bound oligonucleotides
`
`under hybridizing conditions wherein the 3’ end of the first input polynucleotide hybridizes, at
`
`least in part, to the at least two sequence regions (N-1) and (N-2) of the oligonucleotides thereby
`
`forming duplexes,
`
`the duplexes comprising a first duplex having at least one mismatch in a
`
`complementary region and a second duplex that does not comprise a mismatch in the
`
`complementary region. The first duplex may be denatured under stringent melt conditions
`
`without denaturing the second duplex.
`
`In this fashion, error-contaming input polynucleotides are
`
`released in solution and may be removed.
`
`In subsequentstep, a first plurality of complementary
`
`oligonucleotides is generated by template-dependent synthesis under condition promoting
`
`extension of the input polynucleotides thereby forming an extension product duplex. The
`
`extension product is dissociated, releasing a second input polynucleotide. The second input
`
`
`
`polynucleotide may anneal support-bound—single-strandedto a second plurality of
`
`
`
`
`
`
`
`oligonucleotides and by repeating the cycles of stringent melt, extension, dissociation and
`
`annealing, the target polynucleotide is synthesized.
`
`[0013] Aspects of the invention relate to a method of removing error-containing polynucleotides
`
`synthesized on a solid support, the method comprising the following steps. A plurality of
`
`support-bound single
`
`stranded oligonucleotides
`
`is provided on a
`
`solid support;
`
`the
`
`oligonucleotides comprising a 5° end sequence region, a 3° end sequence region and at least two
`
`different sequences regions (N-1) and (N-2) between the 5’ end and the 3’ end sequence regions.
`
`An input polynucleotide, the input polynucleotide being a product of at least two cycles (N-2)
`
`and (N-1) of chain extension reaction is provided. The input polynucleotide is hybridized to the
`
`plurality of support-boundoligonucleotides, thereby forming duplexes in which the 3’ end of the
`
`input polynucleotide hybridizes to the (N-1) and (N-2) sequences regions of the support-bound
`
`oligonucleotide.
`
`In some embodiments, the duplexes comprise duplexes having at least one
`
`mismatch in a complementary region and duplexes that do not comprise a mismatch in the
`
`complementary region. The duplexes having at least one mismatch in the complementary region
`
`are denatured. under stringent melt conditions releasing error-containing input polynucleotides.
`
`In some embodiment,
`
`the support-bound oligonucleotides comprise at
`
`least three different
`
`sequences regions (N-1), (N-2) and (N-3) between the 5’ end and the 3’ end sequenceregions,
`
`and the input polynucleotide hybridizes to the (N-1), (N-2) and (N-3) sequences regions of the
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`support-bound oligonucleotides.
`
`In some embodiments,
`
`the 3’ end sequence is a spacer
`
`sequence and may comprise a primerbindingsite.
`
`[0014] In some embodiments, the (N-1) sequence is adjacent to the 5’ end sequence region and
`
`the (N-2) sequence region is adjacent to the (N-1) sequence region and so on.
`
`In some
`
`embodiments, each input polynucleotide is the product of a chain extension reaction. For
`
`example, the input polynucleotide may be the productof at least one, at least two, at least three,
`
`ctc.. extension chain reactions, cach extension chain reaction using a different plurality of
`
`support-boundoligonucleotides as a template. In some embodiments, the input polynucleotideis
`
`the product of at least two extension chain reactions, each extension chain reaction adding a
`
`sequenceat the 3’ end of the input polynucleotide. For example, a first extension chain reaction
`
`results in the addition of a first sequence complementary to sequence 1, the (n-2) extension
`
`reaction results in the addition of a sequence complementary to sequence (N-2),
`
`the (n-1)
`
`extension reaction results in the addition of a sequence complementary to sequence (N-1) and so
`
`on.
`
`[0015] In some embodiments, the extension duplexes are subjected to shuffling process before
`
`undergoing a next cycle of extension. The shuffling process comprises the steps of denaturing
`
`extension duplexes such as single-stranded extension products are released into solution; re-
`
`annealing single-stranded extension products to the support-bound oligonucleotides thereby
`
`producing re-annealed duplexes; subjecting the re-annealed duplexes to stringent melt conditions
`
`to dissociate error-containing duplexes; removing error-containing single-stranded extension
`
`products; and dissociating the error-free duplexes thereby releasing error-free extension products
`
`in solution.
`
`[0016] In some embodiments, each plurality of oligonucleotides is designed to serve as a
`
`template to a different polymerase extension reaction, thereby forming pluralities of extension
`
`duplexes, wherein each plurality of extension duplexes has a substantially identical melting
`
`temperature. In some embodiments, the difference of melting temperature between the plurality
`
`of duplexes is less than 10°C, less than 5°C, less than 1°C.
`
`[0017] In some aspects, methods for producing at least one double-stranded polynucleotide
`
`having a predefined sequence are provided.
`
`In some embodiments,
`
`in a first step, a
`
`polynucleotide is synthesized on a discrete feature of a support. The polynucleotide comprises a
`
`3’ terminal sequence region complementary to a 5’ region of an oligonucleotide at a discrete
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`feature and a 5’ terminal region that is not complementaryto the oligonucleotide. Atleast a first
`
`plurality of support-bound oligonucleotides is provided, wherein the at least first plurality of
`
`oligonucleotides has a predefined sequence and is boundtoafirst discrete feature of the support,
`
`each first plurality of oligonucleotides comprising a primer binding sequenceat its 3’ end and a
`
`sequence region at its 5’ end substantially identical to a 5’ end of the polynucleotide.
`
`In a
`
`subsequent step, the primer is annealed to the first plurality of oligonucleotides at the first
`
`discrete feature, wherein the anncaling of the primer to the first plurality of support-bound
`
`oligonucleotides serves as a primer for extension of the first plurality of complementary
`
`oligonucleotides,
`
`thereby generating a first extension product duplex. The primer is then
`
`removed from the extension duplex. Preferably, the primer sequence comprises at least one
`
`Uracil and the primer is removed using a mixture of Uracil DNA glycosylase (UDG) and the
`
`DNA glycosylase-lyase Endonuclease VIII. The first extension product duplex is dissociated
`
`thereby producinga first plurality of complementary oligonucleotides which are then transferred
`
`to a the discrete feature comprising the polynucleotide thereby bringing into contact the first
`
`plurality of oligonucleotides with the polynucleotide, wherein the
`
`first plurality of
`
`oligonucleotides is complementary to the 5’ end of the polynucleotide. The first plurality of
`
`complementary oligonucleotides is then annealed to the polynucleotide, wherein the annealing of
`
`the oligonucleotides serves as a primer for extension of the polynucleotide, thereby producing a
`
`double stranded polynucleotide.
`
`[0018] In some embodiments,
`
`the method for producing at
`
`least one double-stranded
`
`polynucleotide having a predefined sequence comprises the following steps: a) providingat least
`
`a first, a second and a third plurality of support-bound single-stranded oligonucleotides, each
`
`first, second and third plurality of oligonucleotides having a predefined sequence and being
`
`bound to a discrete feature of the support. Each first and second plurality of oligonucleotides
`
`comprise a primer bindingsite at its 3’ end that is complementary to a primer sequence and the
`
`first plurality of oligonucleotide has a sequence 5’ sequence region that is complementary to the
`
`5’ sequence region of the secondplurality of oligonucleotides and a sequence region between the
`
`primer binding site and the 5’ sequence region that is identical to a 5’ end of the third plurality of
`
`oligonucleotides, and the secondplurality of oligonucleotides comprises a primer bindingsite at
`
`its 3° end; b) annealing the primers to the primer binding sites of the first and the second
`
`plurality of oligonucleotides, wherein the annealing of the primerto the first and second plurality
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`of support-bound oligonucleotides serves as a primer for extension of the first and second
`
`plurality of complementary oligonucleotides, thereby producing a first and second plurality of
`
`extension product duplexes; c) removing the primer sequences from the extension product
`
`duplexes; d) dissociating the extension product duplexes, thereby producinga first and second
`
`plurality of complementary oligonucleotides; e) hybridizing the first plurality of complementary
`
`oligonucleotides to the third plurality of oligonucleotides; and f) hybridizing the second plurality
`
`of complementary oligonucleotides to the first plurality of oligonuclcotides, thereby producing
`
`the polynucleotide.
`
`In some embodiments, the method further provides a fourth plurality of
`
`support-boundsingle-stranded oligonucleotides wherein each fourth plurality of oligonucleotides
`
`has a predefined sequence and is bound to a fourth discrete feature of the support, each fourth
`
`plurality of oligonucleotides comprising a primer binding site at its 3’ end that is complementary
`
`to a primer sequence and a sequence region that
`
`is complementary to a 5° end of the
`
`polynucleotide, and repeating steps b) through f) thereby producing a longer polynucleotide.
`
`In
`
`some embodiments, the primers hybridizing to the first and second plurality of oligonucleotides
`
`are the same. The primers may comprise at least one Uracil and the primer is removed using a
`
`mixture of Uracil DNA glycosylase (UDG) and the DNA glycosylase-lyase Endonuclease VIII.
`
`[0019] In some embodiments,
`
`the method for producing at
`
`least one double-stranded
`
`polynucleotide having a predefined sequence comprises the following steps: a) providingat least
`
`a first and a second plurality of support-bound single-stranded oligonucleotides, each first and
`
`secondplurality of oligonucleotides having a predefined sequence and being boundtoafirst and
`
`second discrete feature of the support, each first plurality of oligonucleotides comprising a
`
`primer binding site at its 3’ end which is complementary to a primer sequence, a first sequence
`
`region at the 5’ end of the primer bindingsite and a second 3’ end sequence region and wherein
`
`the second plurality of oligonucleotides comprises a sequence region at its 5’ end thatis identical
`
`to the first sequence region of the first plurality of oligonucleotides; b) annealing the primer to
`
`the primer binding sites of the first plurality of oligonucleotides at the first feature, wherein the
`
`annealing of the primer to the first plurality of support-bound oligonucleotides serves as a primer
`
`for extension of the first plurality of complementary oligonucleotides, thereby producing a first
`
`plurality of extension product duplexes; c) removing the primer sequences from the extension
`
`
`
`product duplexes; d) dissociating the extension product duplexes, thereby producingafirst
`
`plurality of complementary oligonucleotides; e) hybridizing the first plurality of complementary
`
`-9.
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`WO 2011/085075
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`PCT/US2011/020335
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`oligonucleotides to the second plurality of oligonucleotides at the second feature;
`
`f) providing a
`
`stem-loop oligonucleotide, wherein the 3’ end of the stem structure is complementary to the 3’
`
`end of the extension product; g) hybridizing the stem-loop oligonucleotide to the first plurality
`
`of oligonucleotides at the second feature; and h) ligating the stem-loop oligonucleotide to the
`
`first extension product, thereby generating the double-stranded stem and loop polynucleotide. In
`
`some embodiments, the method further comprises a) providing at least a third and a fourth
`
`plurality of support-bound single-stranded oligonucleotides, cach third and fourth plurality of
`
`oligonucleotides having a predefined sequence and being boundto a third and fourth discrete
`
`feature of the support, each third plurality of oligonucleotides comprising a primer bindingsite at
`
`its 3’ end which is complementary to a primer sequence,a first sequence region at the 5’ end of
`
`the primer binding site, the first region sequence being substantially identical to the S’ end of the
`
`double-stranded stem-loop polynucleotide and a second 3° end sequence region, wherein the
`
`fourth plurality of oligonucleotides comprises a sequence region at
`
`its 5’ end which is
`
`substantially identical to the first sequence region ofthe third plurality of oligonucleotides; b)
`
`annealing the primer to the primer binding sites of the third plurality of oligonucleotides at the
`
`third feature, wherein the annealing of the primer to the third plurality of support-bound
`
`oligonucleotides serves as a primer for extension of the third plurality of complementary
`
`oligonucleotides, thereby producing a third plurality of extension product duplexes; c) removing
`
`the primer sequences from the extension product duplexes; d) dissociating the extension product
`
`duplexes, thereby producinga third plurality of complementary oligonucleotides; e) hybridizing
`
`the third plurality of complementary oligonucleotides to the fourth plurality of oligonucleotides
`
`at the fourth feature; f) dissociating the double-stranded stem-loop polynucleotide from the
`
`second feature; g) transferring the stem-loop polynucleotide to the fourth feature; h) hybridizing
`
`the stem-loop polynucleotide to the fourth plurality of oligonucleotides at the fourth feature,
`
`thereby extending the stem-loop polynucleotide; and h) ligating the 3’ end of the stem-loop
`
`polynucleotide with the 5’ end of the third plurality of oligonucleotides, thereby forming a longer
`
`double-stranded polynucleotide.
`
`In some embodiments, steps a) through h) may berepeated to
`
`produce a longer polynucleotide.
`
`In some embodiments, the primers hybridizingto the first and
`
`second plurality of oligonucleotides are the same. The primers may comprise at least one Uracil
`
`and the primer is removed using a mixture of Uracil DNA glycosylase (UDG) and the DNA
`
`glycosylase-lyase Endonuclease VIII.
`
`-10-
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`WO 2011/085075
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`PCT/US2011/020335
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`[0020]
`
`In some embodiments,
`
`the method for producing at
`
`least one double-stranded
`
`polynucleotide having a predefined sequence comprises a) synthesizing a polynucleotide at a
`
`first discrete feature; b) synthesizing a complementary oligonucleotide at a second discrete
`
`feature, wherein the 3’ terminal region of the complementary oligonucleotide is complementary
`
`to the
`
`5’
`
`terminal
`
`region of the polynucleotide;
`
`cc)
`
`transferring the complementary
`
`oligonucleotide to the first feature; and d) hybridizing the complementary oligonucleotide to the
`
`polynucleotide.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`[0021] Fig. 1 illustrates an exemplary method of for the elongation of polynucleotides on a solid
`
`support using repeated polymerase extension reactions.
`
`[0022] Fig. 2 illustrates a non-limiting example screening of extension junctions formed during
`
`the (n-1) and (n-2) extensionssteps.
`
`[0023] Tig. 3 illustrates a non-limiting example of different design strategies of screening of
`
`error-containing polynucleotides.
`
`[0024] Fig. 4 illustrates a non-limiting exemplary method for polynucleotide extension and
`
`screening of error-containing polynucleotides.
`
`[0025] Fig. 5 illustrates a non-limiting ex