WSGRDocket No. 44854-701.101
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`U.S. PATENT APPLICATION
`
`DE NOVO SYNTHESIZED GENE LIBRARIES
`
`Inventor(s): William BANYAI,
`Citizen of United States, Residing at
`738 WaylandStreet
`San Francisco, CA 94134
`
`Bill James PECK,
`Citizen of Canada, Residing at
`3086 Carleton Place
`Santa Clara, CA 95051
`
`Assignee:
`
`Twist Bioscience Corporation
`974 RhodeIsland Street
`San Francisco, CA 94107
`
`Entity:
`
`large business concern
`
`WR
`Wilson Sonsini Goodrich & Rosati
`PROPESSTONAL CORPORATION
`
`650 Page Mill Road
`Palo Alto, CA 94304
`(650) 493-9300 (Main)
`(650) 493-6811 (Facsimile)
`
`Filed Electronically on: August 5, 2013
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`WSGRDocket No. 44854-701.101
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`DE NOVO SYNTHESIZED GENE LIBRARIES
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`BACKGROUNDOF THE INVENTION
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`[0001] Highly efficient chemical gene synthesis with high fidelity and low cost has a central role in
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`biotechnology and medicine, and in basic biomedical research.
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`[0002] De novo gene synthesis is a powerful tool for basic biological research and biotechnology
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`applications. While various methods are knownfor the synthesis ofrelatively short fragments in a
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`small scale, these techniques suffer from scalability, automation, speed, accuracy, and cost. There is
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`a need for simple, reproducible, scalable, less error-prone and cost-effective methods that guarantee
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`successful synthesis of desired genes and are amenable to automation.
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`SUMMARYOF THE INVENTION
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`[0003] As noted above,there exists a pressing need for methods, devices and systemsthat can
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`quickly synthesize large gene libraries or relatively longer oligonucleotide fragments efficiently with
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`less error. Similarly, there is also a need for methodsthat can partition and mix liquid reagents in a
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`microfluidic scale for large numbers of individually addressable reactions in parallel. The present
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`invention addresses these needs and provides related advantagesas well.
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`[0004] In one aspect, the present invention provides a genelibrary as described herein. The gene
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`library comprises a collection of genes. In some embodiments, the collection comprisesat least 100
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`different preselected synthetic genes that can be ofat least 0.5 kb length with an errorrate of less
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`than | in 3000 bp compared to predetermined sequences comprising the genes. In another aspect,
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`the present invention also provides a gene library that comprises a collection of genes. The
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`collection may comprise at least 100 different preselected synthetic genes that can be each ofat least
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`0.5 kb length. At least 90% of the preselected synthetic genes may comprise anerrorrate of less than
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`1 in 3000 bp compared to predetermined sequences comprising the genes. Desired predetermined
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`sequences may be supplied by any method,typically by a user, e.g. a user entering data using a
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`computerized system. In various embodiments, synthesized nucleic acids are compared against these
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`predetermined sequences, in some cases by sequencingat least a portion of the synthesized nucleic
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`acids, e.g. using next-generation sequencing methods. In some embodimentsrelated to any of the
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`genelibraries described herein, at least 90% of the preselected synthetic genes comprise an error rate
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`of less than 1 in 5000 bp compared to predetermined sequences comprising the genes. In some
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`embodiments, at least 0.05% of the preselected synthetic genes are error free. In some
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`embodiments,at least 0.5% of the preselected synthetic genesare error free. In some embodiments,
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`at least 90% of the preselected synthetic genes comprise anerrorrate of less than 1 in 3000 bp
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`compared to predetermined sequences comprising the genes. In some embodiments, at least 90% of
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`the preselected synthetic genesare error free or substantially error free. In some embodiments, the
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`preselected synthetic genes comprise a deletion rate of less than 1 in 3000 bp compared to
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`predetermined sequences comprising the genes. In some embodiments,the preselected synthetic
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`genes comprise an insertion rate of less than 1 in 3000 bp compared to predetermined sequences
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`comprising the genes. In some embodiments, the preselected synthetic genes comprise a substitution
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`rate of less than 1 in 3000 bp compared to predetermined sequences comprising the genes. In some
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`embodiments, the gene library as described herein further comprisesat least 10 copies of each
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`synthetic gene. In some embodiments, the genelibrary as described herein further comprises at least
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`100 copies of each synthetic gene. In some embodiments, the gene library as described herein further
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`comprises at least 1000 copies of each synthetic gene. In some embodiments, the gene library as
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`described herein further comprises at least 1000000 copies of each synthetic gene. In some
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`embodiments, the collection of genes as described herein comprisesat least 500 genes. In some
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`embodiments, the collection comprises at least 5000 genes. In some embodiments,the collection
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`comprises at least 10000 genes. In some embodiments, the preselected synthetic genesare at least
`
`1kb. In some embodiments, the preselected synthetic genesare at least 2kb. In some embodiments,
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`the preselected synthetic genesare at least 3kb. In some embodiments, the predetermined sequences
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`comprise less than 20 bp in addition comparedto the preselected synthetic genes. In some
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`embodiments, the predetermined sequences comprise less than 15 bp in addition compared to the
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`preselected synthetic genes. In some embodiments, at least one of the synthetic genes differs from
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`any other synthetic gene by at least 0.1%. In some embodiments, each of the synthetic genes differs
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`from any other synthetic gene byat least 0.1%. In some embodiments, at least one of the synthetic
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`genesdiffers from any other synthetic gene by at least 10%. In some embodiments, each of the
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`synthetic genes differs from any other synthetic gene byat least 10%. In some embodiments,at least
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`one of the synthetic genes differs from any other synthetic gene byat least 2 base pairs. In some
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`embodiments, each of the synthetic genes differs from any other synthetic gene by at least 2 base
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`pairs. In some embodiments, the gene library as described herein further comprises synthetic genes
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`that are of less than 2kb with an errorrate of less than 1 in 20000 bp comparedto preselected
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`sequencesofthe genes. In some embodiments, a subset of the deliverable genes is covalently linked
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`together. In some embodiments,afirst subset of the collection of genes encodes for components of a
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`first metabolic pathway with one or more metabolic end products. In some embodiments, the gene
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`library as described herein further comprises selecting of the one or more metabolic end products,
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`thereby constructing the collection of genes. In some embodiments, the one or more metabolic end
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`products comprise a biofuel. In some embodiments, a second subset of the collection of genes
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`encodes for components of a second metabolic pathway with one or more metabolic end products. In
`some embodiments, the genelibrary is in a spacethat is less than 100 m-*. In some embodiments, the
`genelibrary is in a spacethat is less than 1 m°. In some embodiments, the genelibrary is in a space
`that is less than 1 m’.
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`[0005] In another aspect, the present invention also provides a methodof constructing a genelibrary.
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`The method comprisesthe steps of: entering before a first timepoint, in a computer readable non-
`
`transient medium atleast a first list of genes and a secondlist of genes, wherein the genesare at least
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`500 bp and when compiled intoajointlist, the joint list comprises at least 100 genes; synthesizing
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`more than 90% of the genesin the joint list before a second timepoint, thereby constructing a gene
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`library with deliverable genes. In some embodiments, the second timepoint is less than a month
`
`apart from the first timepoint.
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`[0006] In practicing any of the methodsof constructing a gene library as provided herein, the
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`method as described herein further comprises delivering at least one gene at a second timepoint. In
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`some embodiments,at least one of the genes differs from any other gene by at least 0.1% in the gene
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`library. In some embodiments, each of the genes differs from any other gene byat least 0.1% in the
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`genelibrary. In some embodiments, at least one of the genes differs from any other gene byat least
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`10% in the genelibrary. In some embodiments, each of the genes differs from any other gene byat
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`least 10% in the gene library. In some embodiments, at least one of the genes differs from any other
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`gene byat least 2 base pairs in the gene library. In some embodiments, each of the genesdiffers
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`from any other gene byat least 2 base pairs in the gene library. In some embodiments, at least 90%
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`of the deliverable genes are error free. In some embodiments,the deliverable genes comprises an
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`error rate of less than 1/3000 resulting in the generation of a sequencethat deviates from the
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`sequenceof a genein the jointlist of genes. In some embodiments,at least 90% of the deliverable
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`genes comprise an errorrate of less than 1 in 3000 bp resulting in the generation of a sequencethat
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`deviates from the sequence of a genein the jointlist of genes. In some embodiments, genes in a
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`subset of the deliverable genes are covalently linked together. In some embodiments, a first subset
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`of the joint list of genes encode for componentsofa first metabolic pathway with one or more
`
`metabolic end products. In some embodiments, any of the methods of constructing a genelibrary as
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`described herein further comprises selecting of the one or more metabolic end products, thereby
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`constructing thefirst, the secondor the jointlist of genes. In some embodiments, the one or more
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`metabolic end products comprise a biofuel. In some embodiments, a second subsetof the jointlist of
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`genes encode for components of a second metabolic pathway with one or more metabolic end
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`products. In some embodiments, the joint list of genes comprisesat least 500 genes. In some
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`embodiments, the joint list of genes comprises at least 5000 genes. In some embodiments,the joint
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`list of genes comprises at least 10000 genes. In some embodiments, the genes can beat least Ikb. In
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`some embodiments, the genesare at least 2kb. In some embodiments, the genesare at least 3kb. In
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`some embodiments, the second timepoint is less than 25 days apart from the first timepoint. In some
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`embodiments, the second timepointis less than 5 days apart from thefirst timepoint. In some
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`embodiments, the second timepointis less than 2 days apart from thefirst timepoint.It is noted that
`
`any of the embodiments described herein can be combined with any of the methods, devices or
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`systems provided in the current invention.
`
`[0007] In another aspect, a method of constructing a genelibrary is provided herein. The method
`
`comprises the steps of: enteringat a first timepoint, in a computer readable non-transient medium a
`
`list of genes; synthesizing more than 90% ofthe list of genes, thereby constructing a gene library
`
`with deliverable genes; and delivering the deliverable genesat a second timepoint. In some
`
`embodiments, the list comprises at least 100 genes and the genes can beat least 500 bp.
`
`Instill yet
`
`some embodiments, the second timepoint is less than a month apart from thefirst timepoint.
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`[0008] In practicing any of the methods of constructing a gene library as provided herein, in some
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`embodiments, the method as described herein further comprises delivering at least one gene at a
`
`second timepoint. In some embodiments, at least one of the genes differs from any other gene by at
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`least 0.1% in the gene library. In some embodiments, each of the genes differs from any other gene
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`by at least 0.1% in the genelibrary. In some embodiments, at least one of the genes differs from any
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`other gene byat least 10% in the gene library. In some embodiments, each of the genes differs from
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`any other gene byat least 10% in the genelibrary. In some embodiments, at least one of the genes
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`differs from any other geneby at least 2 base pairs in the gene library. In some embodiments, each
`
`of the genes differs from any other gene byat least 2 base pairs in the genelibrary. In some
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`embodiments, at least 90% of the deliverable genesare error free. In some embodiments, the
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`deliverable genes comprises an error rate of less than 1/3000 resulting in the generation of a
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`sequencethat deviates from the sequence ofa genein thelist of genes. In some embodiments,at
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`least 90% of the deliverable genes comprise an error rate of less than 1 in 3000 bp resulting in the
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`generation of a sequence that deviates from the sequence ofa genein the list of genes. In some
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`embodiments, genes in a subsetof the deliverable genes are covalently linked together. In some
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`embodiments,a first subset of the list of genes encode for componentsofa first metabolic pathway
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`with one or more metabolic end products. In some embodiments, the method of constructing a gene
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`library further comprises selecting of the one or more metabolic end products, thereby constructing
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`the list of genes. In some embodiments, the one or more metabolic end products comprise a biofuel.
`
`In some embodiments, a second subset of the list of genes encode for components of a second
`
`metabolic pathway with one or more metabolic end products. It is noted that any of the embodiments
`
`described herein can be combined with any of the methods, devices or systems provided in the
`
`current invention.
`
`[0009] In practicing any of the methods of constructing a gene library as provided herein, in some
`
`embodiments, the list of genes comprises at least 500 genes. In some embodiments,the list
`
`comprises at least 5000 genes. In some embodiments, the list comprises at least 10000 genes. In
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`some embodiments, the genesare at least 1kb. In some embodiments, the genesare at least 2kb. In
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`some embodiments, the genesare at least 3kb. In some embodiments, the second timepoint as
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`described in the methods of constructing a genelibrary is less than 25 days apart from the first
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`timepoint. In some embodiments, the second timepointis less than 5 days apart from the first
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`timepoint. In some embodiments, the second timepointis less than 2 days apart from the first
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`timepoint. It is noted that any of the embodiments described herein can be combined with any ofthe
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`methods, devices or systems provided in the current invention.
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`[0010] In another aspect, the present invention also provides a method of synthesizing n-mer
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`oligonucleotides on a substrate. The method comprises a) providing a substrate with resolved loci
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`that are functionalized with a chemical moiety suitable for nucleotide coupling; and b) coupling at
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`least two building blocksto a plurality of growing oligonucleotide chains each residing on one ofthe
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`resolved loci at a rate of at least 12 nucleotides per hour according to a locus specific predetermined
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`sequence, thereby synthesizing a plurality of oligonucleotides that are n basepairs long. Various
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`embodiments related to the method of synthesizing n-mer oligonucleotides on a substrate are
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`described herein.
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`[0011] In any of the methods of synthesizing n-meroligonucleotides on a substrate as provided
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`herein, in some embodiments, the methods further comprise coupling at least two building blocks to
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`a plurality of growing oligonucleotide chains each residing on one of the resolvedloci at a rate ofat
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`least 15 nucleotides per hour. In some embodiments, the method further comprises coupling at least
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`two building blocksto a plurality of growing oligonucleotide chains each residing on one of the
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`resolved loci at a rate of at least 20 nucleotides per hour. In some embodiments, the method further
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`comprises coupling at least two building blocksto a plurality of growing oligonucleotide chains each
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`residing on one of the resolved loci at a rate of at least 25 nucleotides per hour. In some
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`embodiments, at least one resolved locus comprises n-meroligonucleotides deviating from the locus
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`specific predetermined sequencewith an error rate of less than 1/500 bp. In some embodiments,at
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`least one resolved locus comprises n-meroligonucleotides deviating from the locus specific
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`predetermined sequence with an error rate of less than 1/1000 bp. In some embodiments, at least one
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`resolved locus comprises n-meroligonucleotides deviating from the locus specific predetermined
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`sequence with an error rate of less than 1/2000 bp. In some embodiments,the plurality of
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`oligonucleotides on the substrate deviate from respective locus specific predetermined sequencesat
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`an error rate of less than 1/500 bp. In some embodiments, the plurality of oligonucleotides on the
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`substrate deviate from respective locus specific predetermined sequencesat an error rate of less than
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`1/1000 bp. In some embodiments, the plurality of oligonucleotides on the substrate deviate from
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`respective locus specific predetermined sequencesat an error rate of less than 1/2000 bp.
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`[0012] In practicing any of the methods of synthesizing n-mer oligonucleotides on a substrate as
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`provided herein, in some embodiments, the building blocks comprise adenine, guanine, thymine,
`
`cytosine, or uridine. In some embodiments, the building blocks comprise a modified nucleotide. In
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`some embodiments, the building blocks comprise dinucleotides or trinucleotides. In some
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`embodiments, the building blocks comprise phosphoramidite. In some embodiments, n of the n-mer
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`oligonucleotidesis at least 100. In some embodiments, n is at least 200. In some embodiments, n is
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`at least 300. In some embodiments,n is at least 400. In some embodiments, the surface comprises at
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`least 100,000 resolved loci and at least two ofthe plurality of growing oligonucleotides can be
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`different from each other.
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`[0013] In some embodiments, the method of synthesizing n-mer oligonucleotides on a substrate as
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`described herein further comprises vacuum drying the substrate before coupling. In some
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`embodiments, the building blocks comprise a blocking group. In some embodiments, the blocking
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`group comprises an acid-labile DMT. In some embodiments, the acid-labile DMT comprises 4,4'-
`
`dimethoxytrityl. In some embodiments, the method of synthesizing n-mer oligonucleotides on a
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`substrate as described herein further comprises oxidation or sulfurization. In some embodiments, the
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`methodof synthesizing n-mer oligonucleotides on a substrate as described herein further comprises
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`chemically capping uncoupled oligonucleotide chains. In some embodiments, the method of
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`synthesizing n-meroligonucleotides on a substrate as described herein further comprises removing
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`the blocking group, thereby deblocking the growing oligonucleotide chain. In some embodiments,
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`the position of the substrate during the coupling step is within 10 cm ofthe position of the substrate
`
`during the vacuum drying step. In some embodiments, the position of the substrate during the
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`coupling step is within 10 cm ofthe position of the substrate during the oxidation step. In some
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`embodiments, the position of the substrate during the coupling step is within 10 cm ofthe position of
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`the substrate during the capping step. In some embodiments, the position of the substrate during the
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`coupling step is within 10 cm ofthe position of the substrate during the deblocking step. In some
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`embodiments, the substrate comprises at least 10,000 vias providing fluid communication between a
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`first surface of the substrate and a second surface of the substrate. In some embodiments, the
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`substrate comprisesat least 100,000 vias providing fluid communication betweenafirst surface of
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`the substrate and a second surface of the substrate. In some embodiments, the substrate comprisesat
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`least 1,000,000 vias providing fluid communication betweena first surface of the substrate and a
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`second surface of the substrate. It is noted that any of the embodiments described herein can be
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`combined with any of the methods, devices or systems provided in the current invention.
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`[0014] In another aspect of the present invention, a system for conducting a set of parallel reactions
`
`is provided herein. The system comprises: a first surface with a plurality of resolved loci; a capping
`
`elementwith a plurality of resolved reactor caps. In some embodiments, the system aligns the
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`plurality of resolved reactor caps with the plurality of resolved loci on the first surface forming a
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`temporary seal betweenthefirst surface and the capping element, thereby physically dividing the
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`loci on the first surface into groupsofat least two loci into a reactor associated with each reactor
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`cap. In some embodiments, each reactor holdsa first set of reagents.
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`[0015] In some embodiments related to any of the systems for conductingaset of parallel reactions
`
`as described herein, upon release from the first surface, the reactor caps retain at least a portion of
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`the first set of reagents. In some embodiments, the portion is about 30%. In some embodiments, the
`
`portion is about 90%. In some embodiments, the plurality of resolved loci resides on microstructures
`
`fabricated into a support surface. In some embodiments,the plurality of resolved loci is at a density
`of at least 1 per mm?. In some embodiments,the plurality of resolved lociis at a density ofat least
`10 per mm?. In some embodiments,the plurality of resolved loci are at a density ofat least 100 per
`mm?. In some embodiments, the microstructures comprise at least two channelsin fluidic
`
`communication with each other. In some embodiments, the at least two channels comprise two
`
`channels with different width. In some embodiments, at least two channels comprise two channels
`
`with different length. In some embodiments, at least one of the channels is longer than 100 um. In
`
`some embodiments, at least one of the channels is shorter than 1000 um. In some embodiments,at
`
`least one of the channels is wider than 50 um in diameter. In some embodiments, at least one ofthe
`
`channels is narrower than 100 um in diameter. In some embodiments, the system further comprises a
`second surface with a plurality of resolvedloci at a density ofat least 0.1 per mm’. In some
`
`embodiments, the system further comprises a second surface with a plurality of resolved loci at a
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`density ofat least 1 per mm’. In some embodiments, the system further comprises a second surface
`with a plurality of resolved loci at a density of at least 10 per mm’.
`
`[0016] In some embodiments related to any of the systems for conductingaset of parallel reactions
`
`as described herein, the resolved loci of the first surface comprise a coating of reagents. In some
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`embodiments, the resolved loci of the second surface comprise a coating of reagents. In some
`
`embodiments, the coating of reagents is covalently linked to the first or second surface. In some
`
`embodiments, the coating of reagents comprises oligonucleotides. In some embodiments, the coating
`of reagents has a surface area ofat least 1.45 um’ per 1.0 um?ofplanar surface area. In some
`embodiments, the coating of reagents has a surface area ofat least 1.25 um” per 1.0 wm’ ofplanar
`surface area. In some embodiments, the coating of reagents has a surface area ofat least 1 um? per
`1.0 um’ ofplanar surface area. In some embodiments,the resolvedloci in the plurality of resolved
`loci comprise a nominal arclength of the perimeter at a density ofat least 0.001 um/ um’. In some
`
`embodiments, the resolved loci in the plurality of resolved loci comprise a nominal arclength ofthe
`perimeter at a density ofat least 0.01 um/um”. In some embodiments,the resolved loci in the
`
`plurality of resolved loci of the first surface comprise a high energy surface. In some embodiments,
`
`the first and second surfaces comprise a different surface tension with a given liquid. In some
`
`embodiments, the high surface energy correspondsto a water contact angle of less than 20 degree. In
`
`some embodiments, the plurality of resolved loci are located on a solid substrate comprising a
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`material selected from the group consisting ofsilicon, polystyrene, agarose, dextran, cellulosic
`
`polymers, polyacrylamides, PDMS, and glass. In some embodiments, the capping elements comprise
`
`a material selected from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic
`
`polymers, polyacrylamides, PDMS, andglass. It is noted that any of the embodiments described
`
`herein can be combined with any of the methods, devices or systems provided in the current
`
`invention.
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`[0017] In yet another aspect, the present invention also provides an array of enclosures. The array of
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`enclosures comprise: a plurality of resolved reactors comprisinga first substrate and a second
`
`substrate comprising reactor caps; at least 2 resolved loci in each reactor. In somecases, the
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`resolved reactors are separated with a releasable seal. In some cases, the reactor capsretain at least a
`
`part of the contents of the reactors upon release of the second substrate from the first substrate. In
`some embodiments, the reactor caps on the second substrate have a density ofat least 0.1 per mm’.
`In some embodiments, reactor caps on the second substrate have a density ofat least 1 per mm”.In
`some embodiments, reactor caps on the second substrate have a density ofat least 10 per mm’.
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`[0018] In some embodimentsrelated to the array of enclosures as provided herein, the reactor caps
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`retain at least 30% of the contents of the reactors. In some embodiments, the reactor caps retain at
`
`least 90% of the contents of the reactors. In some embodiments, the resolved loci are at a density of
`at least 2/mm?. In some embodiments,the resolvedlociare at a density of at least 100/mm7”. In some
`
`embodiments, the array of enclosures further comprises at least 5 resolved loci in each reactor. In
`
`some embodiments, the array of enclosures as described herein further comprisesat least 20 resolved
`
`loci in each reactor. In some embodiments, the array of enclosures as described herein further
`
`comprises at least 50 resolved loci in each reactor. In some embodiments, the array of enclosures as
`
`described herein further comprises at least 100 resolved loci in each reactor.
`
`[0019] In some embodimentsrelated to the array of enclosures as described herein, the resolved loci
`
`reside on microstructures fabricated into a support surface. In some embodiments, the
`
`microstructures comprise at least two channels in fluidic communication with each other. In some
`
`embodiments, the at least two channels comprise two channels with different width. In some
`
`embodiments, the at least two channels comprise two channels with different length. In some
`
`embodiments,at least one of the channels is longer than 100 um. In some embodiments,at least one
`
`of the channels is shorter than 1000 um. In some embodiments, at least one of the channels is wider
`
`than 50 um in diameter. In some embodiments,at least one of the channels is narrower than 100 um
`
`in diameter. In some embodiments, the microstructures comprise a nominal arclength of the
`
`perimeter of the at least two channels that has a density of at least 0.01 um / square um. In some
`
`embodiments, the microstructures comprise a nominalarclength of the perimeter of the at least two
`
`channels that has a density of at least 0.001 um / square um. In some embodiments, the resolved
`
`reactors are separated with a releasable seal. In some embodiments, the seal comprises a capillary
`
`burst valve.
`
`[0020] In some embodiments related to the array of enclosures as described herein, the plurality of
`
`resolved loci ofthe first substrate comprise a coating of reagents. In some embodiments, the
`
`plurality of resolved loci of the second substrate comprises a coating of reagents. In some
`
`embodiments, the coating of reagents is covalently linked to the first or second surface. In some
`
`embodiments, the coating of reagents comprises oligonucleotides. In some embodiments, the coating
`of reagents has a surface area ofat least 1 um” per 1.0 wm’ ofplanar surface area. In some
`embodiments, the coating of reagents has a surface area ofat least 1.25 um” per 1.0 wm’ ofplanar
`surface area. In some embodiments, the coating of reagents has a surface areaofat least 1.45 um?
`per 1.0 um? of planar surface area. In some embodiments, the plurality of resolved loci of the first
`
`substrate comprises a high energy surface. In some embodiments,the first and second substrates
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`comprise a different surface tension with a given liquid. In some embodiments, the surface energy
`
`corresponds to a water contact angle of less than 20 degree. In some embodiments,the plurality of
`
`resolved loci or the reactor caps are located on a solid substrate comprising a material selected from
`
`the group consisting ofsilicon, polystyrene, agarose, dextran, cellulosic polymers, polyacrylamides,
`
`PDMS, andglass. It is noted that any of the embodiments described herein can be combined with
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`any of the methods, devices, arrays or systems providedin the current invention.
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`[0021] In still yet another aspect, the present invention also provides a method of conducting a set of
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`parallel reactions. The method comprises: (a) providing a first surface with a plurality of resolved
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`loci; (b) providing a capping element with a plurality of resolved reactor caps; (c) aligning the
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`plurality of resolved reactor caps with the plurality of resolved loci on the first surface and forming a
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`temporary seal betweenthefirst surface and the capping element, thereby physically dividing the
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`loci on the first surface into groupsofat least two loci; (d) performinga first reaction, thereby
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`forminga first set of reagents; and (e) releasing the capping element from the first surface, wherein
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`each reactor cap retainsat least a portion ofthe first set of reagents in a first reaction volume. In
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`some embodiments, the portion is about 30%. In some embodiments, the portion is about 90%.
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`[0022] In some embodiments, the method of conductinga set of parallel reactions as described
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`herein further comprisesthe steps of: (f) providing a second surface with a plurality of resolved loci;
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`(g) aligning the plurality of resolved reactor caps with the plurality of resolved loci on the second
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`surface and forming a temporary seal between the second surface and the capping element, thereby
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`physically dividing the loci on the second surface; (h) performing a second reaction using the portion
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`of the first set of reagents, thereby forming a secondset of reagents; and(i) releasing the capping
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`element from the second surface, wherein each reactor cap can retain at least a portion of the second
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`set of reagents in a second reaction volume. In some embodiments, the portion is about 30%. In
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`some embodiments, the portion is about 90%.
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`[0023] In practicing any of the methods of conducting a set of parallel reactions as described herein,
`the plurality of resolved loci can have a density ofat least 1 per mm” onthefirst surface. In some
`embodiments,the plurality of resolved loci have a density ofat least 10 per mm’onthefirst surface.
`In some embodiments, the plurality of resolved loci have a density ofat least 100 per mm”on the
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`first surface. In some embodiments, the plurality of resolved reactor caps have a density ofat least
`0.1 per mm? on the capping element. In some embodiments, the plurality of resolved reactor caps
`have a density ofat least 1 per mm’ on the capping element. In some embodiments,the plurality of
`resolved reactor caps have a density ofat least 10 per mm’ on the capping element. In some
`embodiments,the plurality of resolved loci have a density of more than 0.1 per mm’ on the second
`5570740.DOC
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`WSGRDocket No. 44854-701.101
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`surface. In some embodiments,the plurality of resolved loci have a density of more than 1 per mm?
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`on the second surface. In some embodiments,the plurality of resolved loci have a density of more
`than 10 per mm’onthe secondsurface.
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`[0024] In practicing any of the methods of conducting a set of parallel reactions as described herein,
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`the releasing of the capping elements from the surface steps such as the releasing steps in (e) and (i)
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`as described herein can be performedat a different velocity. In some embodiments, the resolved loci
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`of the first surface comprise a coating of reagents for thefirst reaction. In some embodiments, the
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`resolved loci of the second surface comprise a coating of reag