WHATIS CLAIMEDIS:
`
`CLAIMS
`
`1.
`
`A genelibrary, comprising a collection of genes, wherein the collection comprisesat
`
`least 100 different preselected synthetic genes that are each ofat least 0.5 kb length with an error
`
`rate of less than 1 in 3000 bp compared to predetermined sequences comprising the genes.
`
`2.
`
`A genelibrary, comprising a collection of genes, wherein the collection comprisesat
`
`least 100 different preselected synthetic genes that are each ofat least 0.5 kb length, wherein at least
`
`90% of the preselected synthetic genes comprise anerrorrate of less than 1 in 3000 bp comparedto
`
`predetermined sequences comprising the genes.
`
`3.
`
`The genelibrary according to claims 1 or 2, wherein at least 90% ofthe preselected
`
`synthetic genes comprise an errorrate of less than 1 in 5000 bp compared to predetermined
`
`sequences comprising the genes.
`
`4.
`
`The genelibrary according to claims 1 or 2, wherein at least 0.05% of the preselected
`
`synthetic genesareerrorfree.
`
`5.
`
`The genelibrary according to claims | or 2, wherein at least 0.5% of the preselected
`
`synthetic genesareerrorfree.
`
`6.
`
`The genelibrary according to claims 1 or 2, wherein at least 90% ofthe preselected
`
`synthetic genesareerrorfree.
`
`7.
`
`The genelibrary according to claims 1 or 2, wherein the preselected synthetic genes
`
`comprise a deletion rate of less than 1 in 3000 bp comparedto predetermined sequences comprising
`
`the genes.
`
`8.
`
`The genelibrary according to claims 1 or 2, wherein the preselected synthetic genes
`
`comprise an insertion rate of less than 1 in 3000 bp compared to predetermined sequences
`
`comprising the genes.
`
`9.
`
`The genelibrary according to claims 1 or 2, wherein the preselected synthetic genes
`
`comprise a substitution rate of less than 1 in 3000 bp compared to predetermined sequences
`
`comprising the genes.
`
`10.‘
`
`The genelibrary accordingto claims | or 2, further comprising at least 10 copies of
`
`each synthetic gene.
`
`11.
`
`The genelibrary according to claims 1 or 2, further comprising at least 100 copies of
`
`each synthetic gene.
`
`12.
`
`The genelibrary according to claims | or 2, further comprising at least 1000 copies of
`
`each synthetic gene.
`
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`13.
`
`The genelibrary according to claims 1| or 2, further comprising at least 1000000
`
`copies of each synthetic gene.
`
`14.
`
`The genelibrary according to claims 1 or 2, wherein the collection comprisesat least
`
`500 genes.
`
`15.
`
`The genelibrary according to claims 1 or 2, wherein the collection comprisesat least
`
`5000 genes.
`
`16.
`
`The genelibrary according to claims 1 or 2, wherein the collection comprisesat least
`
`10000 genes.
`
`17.
`
`The genelibrary according to claims | or 2, wherein the preselected synthetic genes
`
`are at least 1kb.
`
`18.
`
`The genelibrary according to claims | or 2, wherein the preselected synthetic genes
`
`are at least 2kb.
`
`19.
`
`The genelibrary according to claims | or 2, wherein the preselected synthetic genes
`
`are at least 3kb.
`
`20.
`
`The genelibrary according to claims 1 or 2, wherein the predetermined sequences
`
`comprise less than 20 bp in addition comparedto the preselected synthetic genes.
`
`21.
`
`The genelibrary according to claims | or 2, wherein the predetermined sequences
`
`comprise less than 15 bp in addition comparedto the preselected synthetic genes.
`
`22.
`
`The genelibrary according to claims 1 or 2, wherein at least one of the synthetic
`
`genes differ from any other synthetic gene byat least 0.1%.
`
`23.
`
`The genelibrary according to claims 1 or 2, wherein each of the synthetic genes differ
`
`from any other synthetic gene by at least 0.1%.
`
`24.
`
`The genelibrary according to claims 1 or 2, wherein at least one of the synthetic
`
`genes differ from any other synthetic gene byat least 10%.
`
`25.
`
`The gene library according to claims 1 or 2, wherein each of the synthetic genes differ
`
`from any other synthetic gene by at least 10%.
`
`26.
`
`The genelibrary according to claims 1 or 2, wherein at least one of the synthetic
`
`genes differ from any other synthetic gene by at least 2 base pairs.
`
`27.
`
`The gene library according to claims 1 or 2, wherein each of the synthetic genes differ
`
`from any other synthetic gene by at least 2 base pairs.
`
`28.
`
`The genelibrary according to claims | or 2, further comprising synthetic genes that
`
`are of less than 2kb with an errorrate of less than 1 in 20000 bp comparedto preselected sequences
`
`of the genes.
`
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`29.
`
`The gene library according to claims 1 or 2, wherein a subsetofthe collection of
`
`genes are covalently linked together.
`
`30.
`
`The gene library according to claims 1 or 2, wherein a first subset of the collection of
`
`genes encode for components ofa first metabolic pathway with one or more metabolic end products.
`
`31.
`
`The genelibrary of claim 30, further comprising selecting of the one or more
`
`metabolic end products, thereby constructing the collection of genes.
`
`32.
`
`The genelibrary of claim 30, wherein the one or more metabolic end products
`
`comprise a biofuel.
`
`33.
`
`The gene library of claim 30, wherein a second subset ofthe collection of genes
`
`encode for components of a second metabolic pathway with one or more metabolic end products.
`
`The genelibrary according to claims 1 or 2, wherein the genelibrary is in a space that
`34.
`is less than 100 m’.
`
`The genelibrary according to claims 1 or 2, wherein the genelibrary is in a space that
`35.
`is less than 1 m’.
`
`The genelibrary according to claims 1 or 2, wherein the genelibrary is in a space that
`36.
`is less than 1 dm’.
`
`37.|A method of constructing a gene library, comprising:
`
`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 500 bp and wherein,
`
`when compiled into a jointlist, the joint list comprises at least 100 genes;
`
`synthesizing more than 90% of the genesin thejoint list before a second timepoint,
`
`thereby constructing a gene library with deliverable genes;
`
`wherein the second timepointis less than a month apart from the first timepoint.
`
`38.
`
`The method ofclaim 37, further comprising delivering at least one gene at a second
`
`timepoint.
`
`39.
`
`The method ofclaim 37, wherein at least one of the genes differ from any other gene
`
`by at least 0.1% in the genelibrary.
`
`40.
`
`The method of claim 37, wherein each of the genes differ from any other gene by at
`
`least 0.1% in the genelibrary.
`
`41.
`
`|The methodof claim 37, wherein at least one of the genes differ from any other gene
`
`by at least 10% in the genelibrary.
`
`42.
`
`The methodof claim 37, wherein each of the genes differ from any other gene by at
`
`least 10% in the genelibrary.
`
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`43.|The method of claim 37, wherein at least one of the genes differ from any other gene
`
`by at least 2 base pairs in the genelibrary.
`
`44.
`
`The method ofclaim 37, wherein each of the genesdiffer from any other gene by at
`
`least 2 base pairs in the genelibrary.
`
`45.
`
`The method of claim 37, wherein at least 90% of the deliverable genesare errorfree.
`
`46.|The method of claim 37, wherein the deliverable genes comprise an error rate of less
`
`than 1/3000 resulting in the generation of a sequence that deviates from the sequenceof a genein the
`
`joint list of genes.
`
`47.
`
`The method of claim 37, wherein at least 90% of the deliverable genes comprise an
`
`error rate of less than 1 in 3000 bp resulting in the generation of a sequence that deviates from the
`
`sequenceof a genein the jointlist of genes.
`
`48.|The method of claim 37, wherein a subsetof the deliverable genes are covalently
`
`linked together.
`
`
`
`49. The methodof claim 37, whereinafirst subset of the jointlist of genes encode for
`
`componentsofa first metabolic pathway with one or more metabolic end products.
`
`50.
`
`The method of claim 49, further comprising selecting the one or more metabolic end
`
`products, thereby constructing thefirst, the second, or the jointlist of genes.
`
`51.|The method of claim 49, wherein the one or more metabolic end products comprise a
`
`biofuel.
`
`52.|The method of claim 37, wherein a second subsetofthe joint list of genes encode for
`
`components of a second metabolic pathway with one or more metabolic end products.
`
`53.|The method of claim 37, wherein the joint list comprises at least 500 genes.
`
`54.|The method of claim 37, wherein the joint list comprises at least 5000 genes.
`
`55.|The method of claim 37, wherein the joint list comprises at least 10000 genes.
`
`56.|The method ofclaim 37, wherein the genesareat least 1kb.
`
`57.|The method ofclaim 37, wherein the genesareat least 2kb.
`
`58.|The method ofclaim 37, wherein the genesareat least 3kb.
`
`59.|The method ofclaim 37, wherein the second timepoint is less than 25 days apart from
`
`the first timepoint.
`
`60.|The method ofclaim 37, wherein the second timepointis less than 5 days apart from
`
`the first timepoint.
`
`61.|The methodof claim 37, wherein the second timepointis less than 2 days apart from
`
`the first timepoint.
`
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`62.
`
`A methodof constructing a gene library, comprising:
`
`entering at a first timepoint, in a computer readable non-transient mediumalist of
`
`genes, wherein the list comprises at least 100 genes and wherein the genesare at least 500 bp;
`
`synthesizing more than 90% ofthe list of genes, thereby constructing a gene library
`
`with deliverable genes;
`
`delivering the deliverable genes at a second timepoint, wherein the second timepoint
`
`is less than a month apart from thefirst timepoint.
`
`63.|The method ofclaim 62, further comprising delivering at least one gene at a second
`
`timepoint.
`
`64.|The method ofclaim 62, wherein at least one of the genes differ from any other gene
`
`by at least 0.1% in the genelibrary.
`
`65.|The methodof claim 62, wherein each ofthe genes differ from any other gene byat
`
`least 0.1% in the genelibrary.
`
`66.|The method ofclaim 62, wherein at least one of the genes differ from any other gene
`
`by at least 10% in the genelibrary.
`
`67.
`
`|The methodof claim 62, wherein each of the genes differ from any other gene byat
`
`least 10% in the genelibrary.
`
`68.|The methodof claim 62, wherein at least one of the genes differ from any other gene
`
`by at least 2 base pairs in the genelibrary.
`
`69.
`
`The method ofclaim 62, wherein each ofthe genes differ from any other gene byat
`
`least 2 base pairs in the genelibrary.
`
`70.
`
`71.
`
`The method ofclaim 62, wherein at least 90% ofthe deliverable genesare errorfree.
`
`The methodof claim 62, wherein the deliverable genes comprise an errorrate ofless
`
`than 1/3000 resulting in the generation of a sequence that deviates from the sequenceof a genein the
`
`list of genes.
`
`72.
`
`The method of claim 62, wherein at least 90% of the deliverable genes comprise an
`
`error rate of less than 1 in 3000 bp resulting in the generation of a sequence that deviates from the
`
`sequenceof a genein the list of genes.
`
`73.
`
`The method ofclaim 62, wherein a subset of the deliverable genes are covalently
`
`linked together.
`
`
`
`74. |The method ofclaim 62, whereinafirst subset of the list of genes encode for
`
`componentsofa first metabolic pathway with one or more metabolic end products.
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`75.
`
`The method ofclaim 62, further comprising selecting of the one or more metabolic
`
`end products, thereby constructing thelist of genes.
`
`76.
`
`The method ofclaim 62, wherein the one or more metabolic end products comprise a
`
`biofuel.
`
`77.
`
`The method ofclaim 62, wherein a secondsubsetofthelist of genes encode for
`
`components of a second metabolic pathway with one or more metabolic end products.
`
`78.
`
`79.
`
`The method ofclaim 62, wherein the list comprises at least 500, genes.
`
`The method ofclaim 62, wherein the list comprises at least 5000 genes.
`
`80.|The method of claim 62, wherein the list comprisesat least 10000 genes.
`
`81.|The method of claim 62, wherein the genesare at least 1kb.
`
`82.|The method of claim 62, wherein the genesare at least 2kb.
`
`83.|The method of claim 62, wherein the genesare at least 3kb.
`
`84.|The method of claim 62, wherein the second timepointis less than 25 days apart from
`
`the first timepoint.
`
`85.|The method of claim 62, wherein the second timepointis less than 5 days apart from
`
`the first timepoint.
`
`86.|The method of claim 62, wherein the second timepointis less than 2 days apart from
`
`the first timepoint.
`
`87.|A method of synthesizing n-meroligonucleotides on a substrate, comprising:
`
`providing a substrate with resolved loci that are functionalized with a chemical
`
`moiety suitable for nucleotide coupling; and
`
`coupling at least two building blocksto a plurality of growing oligonucleotide chains
`
`each residing on oneofthe resolved loci at a rate of at least 12 nucleotides per hour according to a
`
`locus specific predetermined sequence, thereby synthesizing a plurality of oligonucleotides that are n
`
`basepairs long.
`
`88.|The method of claim 87, further comprising coupling at least two building blocks to a
`
`plurality of growing oligonucleotide chains each residing on one ofthe resolved loci at a rate ofat
`
`least 15 nucleotides per hour
`
`89.|The method of claim 87, further comprising coupling at least two building blocks to a
`
`plurality of growing oligonucleotide chains each residing on one ofthe resolved loci at a rate of at
`
`least 20 nucleotides per hour.
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`90.|The method ofclaim 87, further comprising coupling at least two building blocksto a
`
`plurality of growing oligonucleotide chains each residing on one of the resolved loci at a rate of at
`
`least 25 nucleotides per hour.
`
`91.
`
`|The method of claim 87, wherein at least one resolved locus comprises n-mer
`
`oligonucleotides deviating from the locus specific predetermined sequence with anerror rate of less
`
`than 1/500 bp.
`
`92.|The method of claim 87, wherein at least one resolved locus comprises n-mer
`
`oligonucleotides deviating from the locus specific predetermined sequence with anerror rate of less
`
`than 1/1000 bp.
`
`93.|The method ofclaim 87, wherein at least one resolved locus comprises n-mer
`
`oligonucleotides deviating from the locus specific predetermined sequence with anerror rate of less
`
`than 1/2000 bp.
`
`94.
`
`The methodofclaim 87, wherein the plurality of oligonucleotides on the substrate
`
`deviate from respective locus specific predetermined sequencesat an error rate of less than 1/500 bp.
`
`95.|The method ofclaim 87, wherein the plurality of oligonucleotides on the substrate
`
`deviate from respective locus specific predetermined sequencesat an error rate of less than 1/1000
`
`bp.
`
`96.|The method ofclaim 87, wherein the plurality of oligonucleotides on the substrate
`
`deviate from respective locus specific predetermined sequencesat an error rate of less than 1/2000
`
`bp.
`
`97.|The method ofclaim 87, wherein the building blocks comprise adenine, guanine,
`
`thymine, cytosine, or uridine.
`
`98.
`
`The method ofclaim 87, wherein the building blocks comprise a modified nucleotide.
`
`99.|The methodofclaim 87, wherein the building blocks comprise dinucleotides or
`
`trinucleotides.
`
`100.
`
`101.
`
`102.
`
`103.
`
`104.
`
`105.
`
`The methodof claim 87, wherein the building blocks comprise phosphoramidite.
`
`The methodof claim 87, wherein n is at least 100.
`
`The methodof claim 87, wherein n is at least 200.
`
`The method of claim 87, wherein n is at least 300.
`
`The methodof claim 87, wherein n is at least 400.
`
`The method of claim 87, wherein the substrate comprises at least 100,000 resolved
`
`loci and wherein at least two ofthe plurality of growing oligonucleotides are different from each
`
`other.
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`106.
`
`The method ofclaim 87, further comprising vacuum drying the substrate before
`
`coupling.
`
`107.
`
`108.
`
`109.
`
`The method of claim 87, wherein the building blocks comprise a blocking group.
`
`The method of claim107, wherein the blocking group comprises an acid-labile DMT.
`
`The method of claim 108, wherein the acid-labile DMT comprises4,4'-
`
`dimethoxytrityl.
`
`110.
`
`111.
`
`The method ofclaim 87, further comprising oxidation or sulfurization.
`
`The method of claim 87, further comprising chemically capping uncoupled
`
`oligonucleotide chains.
`
`112.
`
`The method of claim 107, further comprising removing the blocking group, thereby
`
`deblocking the growing oligonucleotide chain.
`
`113.
`
`The method of claim 106, wherein the position of the substrate during the coupling
`
`step is within 10 cm ofthe position of the substrate during the vacuum dryingstep.
`
`114.
`
`The method of claim 110, wherein the position of the substrate during the coupling
`
`step is within 10 cm ofthe position of the substrate during the oxidation step.
`
`115.
`
`The method of claim 111, wherein the position of the substrate during the coupling
`
`step is within 10 cm ofthe position of the substrate during the cappingstep.
`
`116.
`
`The method of claim 112, wherein the position of the substrate during the coupling
`
`step is within 10 cm ofthe position of the substrate during the deblockingstep.
`
`117.
`
`The method of claim 105, wherein the substrate comprisesat least 10,000 vias
`
`providing fluid communication betweena first surface of the substrate and a second surface of the
`
`substrate.
`
`118.
`
`The method of claim 105, wherein the substrate comprisesat least 100,000 vias
`
`providing fluid communication betweena first surface of the substrate and a second surface of the
`
`substrate.
`
`119.
`
`The method of claim 105, wherein the substrate comprisesat least 1,000,000 vias
`
`providing fluid communication betweena first surface of the substrate and a second surface of the
`
`substrate.
`
`120. A method for synthesizing oligonucleotides on a substrate having a functionalized
`
`surface, comprising:
`
`a) applying throughat least one inkjet pumpat least one dropofa first reagent to
`
`a first locus ofa plurality ofloci;
`
`b) applying negative pressure to the substrate; and
`
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`c) applying throughat least one inkjet pump at least one drop of a second reagent
`
`to the first locus.
`
`121.
`
`122.
`
`The methodof claim 120, wherein the first and second reagentsare different.
`
`The method of claim 120, wherein the first locus is functionalized with a moiety that
`
`increases their surface energy.
`
`123.
`
`124.
`
`The method of claim 122, wherein the moiety is a chemically inert moiety.
`
`The methodof claim 120, wherein the plurality of loci reside on microstructures
`
`fabricated into the substrate surface.
`
`125.
`
`The methodof claim 124, wherein the microstructures comprise at least two channels
`
`in fluidic communication with each other.
`
`126.
`
`The method of claim 125, wherein the at least two channels comprise two channels
`
`with different width.
`
`127.
`
`The method of claim 125, wherein the at least two channels comprise two channels
`
`with different length.
`
`128.
`
`129.
`
`um.
`
`The method of claim 125, wherein at least one of the channels is longer than 100 um.
`
`The method of claim 125, wherein at least one of the channels is shorter than 1000
`
`130.
`
`The method of claim 125, wherein at least one of the channels is wider than 50 wm in
`
`diameter.
`
`131.
`
`The method of claim 125, wherein at least one of the channels is narrower than 100
`
`um in diameter.
`
`132.
`
`The method of claim 124, wherein the substrate surface comprises a material selected
`
`from the group consisting of silicon, polystyrene, agarose, dextran, cellulosic polymers,
`
`polyacrylamides, PDMS, andglass.
`
`133.
`
`134.
`
`135.
`
`The method of claim 120, wherein the volumeofthe dropis at least 2 pl.
`
`The method of claim 120, wherein the volumeofthe drop is about 40pl.
`
`The method of claim 120, wherein the volumeofthe drop is at most 100 pl.
`
`The methodof claim 120, wherein the plurality of loci comprise a density of the
`136.
`nominal arclength ofthe perimeterofat least 0.01 um/um’.
`
`The methodof claim 120, wherein the plurality of loci comprise a density of the
`137.
`nominal arclength ofthe perimeter ofat least 0.001 um/um?.
`
`The method of claim 120, wherein the functionalized surface comprises a nominal
`138.
`surface area ofat least 1 um? per 1.0 um’ ofplanar surface area ofthe substrate.
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`The array of claim 120, wherein the functionalized surface comprises a nominal
`139.
`surface area ofat least 1.25 um”per 1.0 wm’ ofplanar surface area of the substrate.
`
`The method of claim 120, wherein the functionalized surface comprises a nominal
`140.
`surface area ofat least 1.45 um”per 1.0 wm’ ofplanar surface area of the substrate.
`
`141.
`
`The methodof claim 120, wherein the pressure surrounding the substrate can be
`
`reduced to less than 1 mTorr..
`
`142.
`
`The methodof claim 120, further comprising couplingat least a first building block
`
`originating from the first drop to a growing oligonucleotide chain onthefirst locus.
`
`143.
`
`144.
`
`145.
`
`The method of claim 120, wherein the building blocks comprise a blocking group.
`
`The methodof claim 143, wherein the blocking group comprises an acid-labile DMT.
`
`The method of claim 144, wherein the acid-labile DMT comprises 4,4'-
`
`dimethoxytrityl.
`
`146.
`
`147.
`
`The methodof claim 120, further comprising oxidation or sulfurization.
`
`The method of claim 120, further comprising chemically capping uncoupled
`
`oligonucleotide chains.
`
`148.
`
`The method of claim 143, further comprising removing the blocking group, thereby
`
`deblocking the growing oligonucleotide chain.
`
`149.
`
`The method of claim 142, wherein the position of the substrate during the negative
`
`pressure application is within 10 cm ofthe position of the substrate during the coupling step.
`
`150.
`
`The method of claim 146, wherein the position of the substrate during the negative
`
`pressure application is within 10 cm ofthe position of the substrate during the oxidation step.
`
`151.
`
`The method of claim 147, wherein the position of the substrate during the negative
`
`pressure application is within 10 cm ofthe position of the substrate during the cappingstep.
`
`152.
`
`The method of claim 148, wherein the position of the substrate during the negative
`
`pressure application is within 10 cm ofthe position of the substrate during the deblockingstep.
`
`153.
`
`The method of claim 142, wherein the first locus resides on a microstructure
`
`fabricated into the substrate surface.
`
`154.
`
`The methodof claim 146, wherein at least one reagent for the oxidation step is
`
`provided by flooding the microstructure with a solution comprising the at least one reagent.
`
`155.
`
`The method of claim 147, wherein at least one reagent for the capping step is
`
`provided by flooding the microstructure with a solution comprising the at least one reagent.
`
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`156.
`
`The method of claim 148, wherein the first locus resides on a microstructure
`
`fabricated into the substrate surface and wherein at least one reagent for the deblockingstep is
`
`provided by flooding the microstructure with a solution comprising the at least one reagent.
`
`157.
`
`The methodof claim 120, further comprising enclosing the substrate within a sealed
`
`chamber.
`
`158.
`
`The method of claim 157, wherein the sealed chamberallows for purging of liquids
`
`from thefirst locus.
`
`159.
`
`The methodof claim 120, further comprising draining a liquid througha drain thatis
`
`operably linkedto the first locus.
`
`160.
`
`The methodof claim 120, wherein after step b) the moisture content on the substrate
`
`is less than 1 ppm.
`
`161.
`
`The method of claim 122, wherein the surface energy is increased corresponding to a
`
`water contact angle of less than 20 degree.
`
`162. A methodof synthesizing n-mer oligonucleotides on a substrate, comprising:
`
`providing a substrate with resolved loci that are functionalized with a chemical
`
`moiety suitable for nucleotide coupling; and
`
`coupling at least two building blocksto a plurality of growing oligonucleotide chains
`
`each residing on oneofthe resolved loci according to a locus specific predetermined sequence
`
`without transporting the substrate between the couplingsofthe at least two building blocks, thereby
`
`synthesizing a plurality of oligonucleotides that are n basepairs long.
`
`163.
`
`The methodof claim 162, further comprising coupling at least two building blocksto
`
`a plurality of growing oligonucleotide chains each residing on one ofthe resolvedloci at a rate ofat
`
`least 12 nucleotides per hour.
`
`164.
`
`The methodof claim 162, further comprising coupling at least two building blocksto
`
`a plurality of growing oligonucleotide chains each residing on oneofthe resolvedlociat a rate ofat
`
`least 15 nucleotides per hour.
`
`165.
`
`The methodof claim 162, further comprising coupling at least two building blocksto
`
`a plurality of growing oligonucleotide chains each residing on one ofthe resolvedloci at a rate ofat
`
`least 20 nucleotides per hour.
`
`166.
`
`The methodof claim 162, further comprising coupling at least two building blocksto
`
`a plurality of growing oligonucleotide chains each residing on one ofthe resolvedloci at a rate ofat
`
`least 25 nucleotides per hour.
`
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`167.
`
`The method of claim 162, wherein at least one resolved locus comprises n-mer
`
`oligonucleotides deviating from the locus specific predetermined sequence with anerror rate of less
`
`than 1/500 bp.
`
`168.
`
`The method of claim 162, wherein at least one resolved locus comprises n-mer
`
`oligonucleotides deviating from the locus specific predetermined sequence with anerror rate of less
`
`than 1/1000 bp.
`
`169.
`
`The method of claim 162, wherein at least one resolved locus comprises n-mer
`
`oligonucleotides deviating from the locus specific predetermined sequence with anerror rate of less
`
`than 1/2000 bp.
`
`170.
`
`The methodof claim 162, wherein the plurality of oligonucleotides on the substrate
`
`deviate from respective locus specific predetermined sequencesat an error rate of less than 1/500 bp.
`
`171.
`
`The methodof claim 162, wherein the plurality of oligonucleotides on the substrate
`
`deviate from respective locus specific predetermined sequencesat an errorrate of less than 1/1000
`
`bp.
`
`172.
`
`The method of claim 162, wherein the plurality of oligonucleotides on the substrate
`
`deviate from respective locus specific predetermined sequencesat an error rate of less than 1/2000
`
`bp.
`
`173.
`
`The methodof claim 162, wherein the building blocks comprise adenine, guanine,
`
`thymine, cytosine, or uridine.
`
`174.
`
`The method of claim 162, wherein the building blocks comprise a modified
`
`nucleotide.
`
`175.
`
`176.
`
`177.
`
`178.
`
`179.
`
`180.
`
`181.
`
`The method of claim 162, wherein the building blocks comprise dinucleotides.
`
`The methodof claim 162, wherein the building blocks comprise phosphoramidite.
`
`The methodof claim 162, wherein n is at least 100.
`
`The method of claim 162, wherein n is at least 200.
`
`The method of claim 162, wherein n is at least 300.
`
`The method of claim 162, wherein n is at least 400.
`
`The method of claim 162, wherein the substrate comprisesat least 100,000 resolved
`
`loci and wherein at least two ofthe plurality of growing oligonucleotides are different from each
`
`other.
`
`182.
`
`The method of claim 162, further comprising vacuum drying the substrate before
`
`coupling.
`
`183.
`
`The method of claim 162, wherein the building blocks comprise a blocking group.
`
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`184.
`
`185.
`
`The methodof claim183, wherein the blocking group comprises an acid-labile DMT.
`
`|The method of claim 184, wherein the acid-labile DMT comprises4,4'-
`
`dimethoxytrityl.
`
`186.
`
`187.
`
`The methodof claim 162, further comprising oxidation or sulfurization.
`
`The method of claim 162, further comprising chemically capping uncoupled
`
`oligonucleotide chains.
`
`188.
`
`The method of claim 183, further comprising removing the blocking group, thereby
`
`deblocking the growing oligonucleotide chain.
`
`189.
`
`The method of claim 181, wherein the substrate comprisesat least 10,000 vias
`
`providing fluid communication betweena first surface of the substrate and a second surface of the
`
`substrate.
`
`190.
`
`The method of claim 181, wherein the substrate comprisesat least 100,000 vias
`
`providing fluid communication betweena first surface of the substrate and a second surface of the
`
`substrate.
`
`191.
`
`The method of claim 181, wherein the substrate comprisesat least 1,000,000 vias
`
`providing fluid communication betweena first surface of the substrate and a second surface of the
`
`substrate.
`
`192. A method of constructing a gene library, comprising:
`
`entering at a first timepoint, in a computer readable non-transient mediumalist of
`
`genes, wherein the list comprises at least 100 genes and wherein the genesare at least 500 bp;
`
`synthesizing more than 90% ofthelist of genes, thereby constructing a genelibrary
`
`with deliverable genes;
`
`preparing a sequencing library that represents the genelibrary;
`
`obtaining sequence information;
`
`selecting at least a subset of the deliverable genes based on the sequence information;
`
`and
`
`delivering the the selected deliverable genes at a second timepoint, wherein the
`
`second timepoint is less than a month apart from thefirst timepoint.
`
`193.
`
`The method of claim 192, wherein the sequence information is obtained bia next-
`
`generation sequencing.
`
`194.
`
`The methodof claim 192, wherein the sequence information is obtained by Sanger
`
`sequencing.
`
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`195.
`
`The methodof claim 192, further comprising delivering at least one gene at a second
`
`timepoint.
`
`196.
`
`The methodof claim 192, wherein at least one of the genes differ from any other gene
`
`by at least 0.1% in the genelibrary.
`
`197.
`
`The methodof claim 192, wherein each of the genes differ from any other gene by at
`
`least 0.1% in the genelibrary.
`
`198.
`
`The methodof claim 192, wherein at least one of the genes differ from any other gene
`
`by at least 10% in the genelibrary.
`
`199.
`
`The methodof claim 192, wherein each of the genes differ from any other gene by at
`
`least 10% in the genelibrary.
`
`200.
`
`The method of claim 192, wherein at least one of the genes differ from any other gene
`
`by at least 2 base pairs in the genelibrary.
`
`201.
`
`The method of claim 192, wherein each of the genes differ from any other gene by at
`
`least 2 base pairs in the genelibrary.
`
`202.
`
`203.
`
`The method of claim 192, wherein at least 90% of the deliverable genesare errorfree.
`
`The method of claim 192, wherein the deliverable genes comprise anerror rate of less
`
`than 1/3000 resulting in the generation of a sequence that deviates from the sequence of a genein the
`
`list of genes.
`
`204.
`
`The method of claim 192, wherein at least 90% of the deliverable genes comprise an
`
`error rate of less than 1 in 3000 bp resulting in the generation of a sequence that deviates from the
`
`sequenceof a genein the list of genes.
`
`205.
`
`The method of claim 192, wherein a subset of the deliverable genes are covalently
`
`linked together.
`
`
`
`206. The method of claim 192, whereinafirst subset of the list of genes encode for
`
`componentsofa first metabolic pathway with one or more metabolic end products.
`
`207.
`
`The methodof claim 192, further comprising selecting of the one or more metabolic
`
`end products, thereby constructing thelist of genes.
`
`208.
`
`The method of claim 192, wherein the one or more metabolic end products comprise
`
`a biofuel.
`
`209.
`
`The method of claim 192, wherein a second subsetofthe list of genes encode for
`
`components of a second metabolic pathway with one or more metabolic end products.
`
`210.
`
`211.
`
`5570740.DOC
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`The method of claim 192, wherein the list comprises at least 500, genes.
`
`The method of claim 192, wherein the list comprises at least 5000 genes.
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`212.
`
`213.
`
`214.
`
`215.
`
`216.
`
`The method of claim 192, wherein the list comprises at least 10000 genes.
`
`The method of claim 192, wherein the genesare at least 1kb.
`
`The method of claim 192, wherein the genesare at least 2kb.
`
`The method of claim 192, wherein the genesare at least 3kb.
`
`The method of claim 192, wherein the second timepointis less than 25 days apart
`
`from thefirst timepoint.
`
`217.
`
`The method of claim 192, wherein the second timepointis less than 5 days apart from
`
`the first timepoint.
`
`218.
`
`The method of claim 192, wherein the second timepointis less than 2 days apart from
`
`the first timepoint.
`
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`