(71) Applicant: COMBIMATRIX CORPORATION—for two-letter codes and other abbreviations, refer to the "Guid-
`
`[US/US]; 6500 Harbour Heights Parkway, Mukilteo, WA=ance Notes on Codes and Abbreviations" appearing at the begin-
`98275 (US).
`ning of each regular issue of the PCT Gazette.
`
`(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`25 March 2004 (25.03.2004)
`
`(43) International Publication Date
`
`(10) International Publication Number
`WO 2004/024886 A2
`
`(51) International Patent Classification’:
`
`C12N
`
`(21) International Application Number:
`PC'L/US2003/028946
`
`(22) International Filing Date:
`12 September 2003 (12.09.2003)
`
`(72) Inventor: OLEINIKOV, Andrew, V.; 16208 32nd Ave.
`SE, Mill Creek, WA 98012 (US).
`
`(74) Agent: OSTER,Jeffrey, B.; Combimatrix Corporation,
`6500 Harbour Heights Parkway, Mukiltco, WA 98275
`(US).
`
`(81) Designated States (national): AU, CA, IL, JP, NO, NZ,
`RU.
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(84) Designated States (regional): European patent (AT, BE,
`BG, CH, CY, CZ, DE, DK, EE, ES, FL FR, GB, GR, HU,
`TE, IT, LU, MC, NL, PT, RO, SE, SI, SK, TR).
`
`(30) Priority Data:
`10/243.367
`,
`
`12 September 2002 (12.09.2002)
`P
`“ —"
`
`Published:
`:
`.
`.
`.
`US — without international search report and to be republished
`upon receipt of that report
`
`(54) Title: MICROARRAY SYNTHESIS AND ASSEMBLY OF GENE-LENGTH POLYNUCLEOTIDES
`
`(57) Abstract: There is disclosed a process for im vitro synthesis and assembly of long, gene-length polynucleotides based upon
`assembly of multiple shorter oligonucleotides synthesized in situ on a microarray platform. Specifically, there is disclosed a process
`for in situ synthesis of oligonucleotide fragments on a solid phase microarray platform and subsequent, "on device" assembly of
`larger polynucleotides composed ofa plurality of shorter oligonucleotide fragments.
`
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`MICROARRAY SYNTHESIS AND ASSEMBLY OF GENE-LENGTH
`
`POLYNUCLEOTIDES
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`Technical Field of the Invention
`The present invention provides a process forin vitro synthesis and assembly of long,
`gene-length polynucleotides based upon assembly of multiple shorter oligonucleotides
`synthesized in situ on a microarray platform. Specifically, the present invention provides a
`processfor in situ synthesis of oligonucleotide sequence fragments on a solid phase microarray
`platform and subsequent, “on chip” assembly of larger polynucleotides composedofaplurality
`10
`of smaller oligonucleotide sequence fragments.
`Background of the Invention
`In the world of microarrays, biological molecules (e.g., oligonucleotides, polypeptides
`and the like) are placed onto surfaces at defined locations for potential binding with target
`samples of nucleotides or receptors. Microarrays are miniaturized arrays of biomolecules
`available or being developed on a variety of platforms. Much ofthe initial focus for these
`microarrays have been in genomics with an emphasis of single nucleotide polymorphisms
`(SNPs) and genomic DNA detection/validation, functional genomics and proteomics
`(Wilgenbus and Lichter, J. Mol. Med. 77:761, 1999; Ashfari et al., Cancer Res. 59:4759, 1999;
`Kurian et al., J. Pathol. 187:267, 1999; Hacia, Nature Genetics 21 suppl.:42, 1999; Hacia et
`al., Mol. Psychiatry 3:483, 1998; and Johnson, Curr. Biol. 26:R171, 1998).
`There are, in general, three categories of microarrays (also called “biochips” and “DNA
`Axrays” and “Gene Chips”but this descriptive name has been attempted to be a trademark)
`having oligonucleotide content. Mostoften, the oligonucleotide microarrays have a solid
`surface, usually silicon-based and most often a glass microscopic slide. Oligonucleotide
`microarrays are often made by different techniques, including (1) “spotting” by depositing
`single nucleotides for in situ synthesis or completed oligonucleotides by physical means(ink
`jet printing and the like), (2) photolithographic techniques for in situ oligonucleotide synthesis
`(see, for example, Fodor U.S. Patent ‘934 and the additional patents that claimpriority from
`this priority document, (3) electrochemicalin situ synthesis based upon pH based removal of
`blocking chemical functional groups(see, for example, Montgomery U.S. Patent 6,092,302 the
`disclosure of which is incorporated by reference herein and Southern U.S. Patent 5,667,667),
`and (4) electric field attraction/repulsion of fully-formed oligonucleotides (see, for example,
`Hollis et al., U.S. Patent 5,653,939 and its duplicate Heller U.S. Patent 5,929,208). Only the
`first three basic techniques can form oligonucleotides in situ that are, building each
`oligonucleotide, nucleotide-by-nucleotide, on the microarray surface without placing or
`attracting fully formed oligonucleotides.
`With regard to placing fully formed oligonucleotides at specific locations, various
`micro-spotting techniques using computer-controlled plotters or even ink-jet printers have been
`developed to spot oligonucleotides at defined locations. One techniques loadsglass fibers
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`having multiple capillaries drilled through then with different oligonucleotides loaded into
`each capillary tube. Microarray chips, often simply glass microscopeslides, are then stamped
`out muchlike a rubber stamp on cach sheet ofpaperofglass slide. It is also possible to use
`“spotting” techniquesto build oligonucleotidesinsitu. Essentially, this involves “spotting”
`relevant single nucleotides at the exact location or region onaslide (preferably a glass slide)
`where a particular sequence of oligonucleotideis to be built. Therefore, irrespective of
`whetheror not fully formed oligonucleotides or single nucleotides are added for in situ
`synthesis, spotting techniques involve the precise placement of materials at specific sites or
`regions using automated techniques.
`Another technique involves a photolithography process involving photomasksto build
`oligonucleotides in situ, base-by-base, by providing a series of precise photomasks coordinated
`with single nucleotide bases having light-cleavable blocking groups. This technique is
`described in Fodoret al., U.S. Patent 5,445,934 and its various progeny patents. Essentially,
`this technique providesfor “solid-phase chemistry, photolabile protecting groups, and
`photolithography .
`.
`. to achieve light-directed spatially-addressable parallel chemical
`synthesis.”
`The electrochemistry platform (Montgomery U.S. Patent 6,092,302, the disclosure of
`which is incorporated by reference herein) provides a microarray based upon a semiconductor
`chip platform having a plurality of microelectrodes. This chip design uses Complimentary
`Metal Oxide Semiconductor (CMOS)technologyto create high-density arrays of
`microelectrodes with parallel addressing for selecting and controlling individual
`microelectrodes within the array. The electrodes turned on with current flow generate
`electrochemical reagents (particularly acidic protons) to alter the pH in a small “virtual flask”
`region or volume adjacent to the electrode. The microarray is coated with a porous matrix for
`a reaction layer material. Thickness and porosity of the material is carefully controlled and
`biomolecules are synthesized within volumesof the porous matrix whose pH has been altered
`through controlled diffusion of protons generated electrochemically and whose diffusion is
`limited by diffusion coefficients and the buffering capacities of solutions. However, in order to
`function properly, the microarray biochips using electrochemistry meansfor isitu synthesis
`has to alternate anodes and cathodesin the array in order to generated needed protons (acids) at
`the anodesso that the protons and other acidic electrochemically generated acidic reagents will
`cause an acid pH shift and remove a blocking group from a growing oligomer.
`Gene Assembly
`The preparation of arbitrary polynucleotide sequences is useful in a “post-genomic”era
`because it provides any desirable gene oligonucleotide or its fragment, or even whole genome
`material of plasmids, phages and viruses. Such polynucleotides are long, such as excess of
`1000 bases in length. Jvitro synthesis of oligonucleotides (given even the best yield
`conditions of phosphoramidite chemistry) would not be feasible because each base addition
`reaction is less than 100% yield. Therefore, researchers desiring to obtain long
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`polynucleotides of gene length or longer had to turn to nature or gene isolation techniques to
`obtain polynucleotides of such length. For the purposesofthis patent application, the term
`“polynucleotide” shall be used to refer to nucleic acids (either single stranded or double
`stranded) that are sufficiently long so as to be practically not feasible to make in vitro through
`single base addition. In view of the exponential drop-off in yields from nucleic acid synthesis
`chemistries, such as phosphoramidite chemistry, such polynucleotides generally have greater
`than 100 bases and often greater than 200 bases in length. It should be noted that many
`commercially useful gene cDNA’s often have lengths in excess of 1000 bases.
`Moreover, the term “oligonucleotides” or shorter term “oligos” shall be used to refer to
`shorter length single stranded or double stranded nucleic acids capable of in vitro synthesis and
`generally shorter than 150 bases in length. Whileit is theoretically possible to synthesize
`polynucleotides through single base addition, the yield losses madeit a practical impossibility
`beyond 150 basesand certainly longer than 250 bases.
`However, knowledge of the precise structure of the genetic material is often not
`sufficient to obtain this material from natural sources. Mature cDNA, whichis a copy of an
`mRNA molecule, can be obtained if starting material contains this desired mRNA. However,
`it is not always knownifthis particular mRNAis present in a sample or the amountofthis
`mRNAmight be too low to obtain the corresponding cDNA withoutsignificant difficulties.
`Also, different levels of homology or splice variants may interfere with obtaining one
`particular species of mRNA. On the other hand many genomic materials might be not
`appropriate to prepare mature gene (CDNA)due to exon-intron structure of genes in many.
`different genomes.
`In addition, there is a need in the art for polynucleotides not existing in nature to
`improve genomic research performance. In general, the ability to obtain polynucleotide of any
`desired sequence just knowing the primary structure for the reasonable price in short period of
`time will significantly move forward several fields of biomedical research andclinical practice.
`Assembly of long arbitrary polynucleotides from oligonucleotides synthesized by
`organic synthesis and individually purified has other problems. The assembly can be
`performed using PCR orligation methods. The synthesis and purification of many different
`oligonucleotides by conventional methods (even using multi-channel synthesizers) are
`laborious and expensive procedures. The current price of assembled polynucleotide on the
`market is about $12-25 per base pair, which can be considerable for assembling larger
`polynucleotides. Very often the amount of conventionally synthesized oligonucleotides would
`be excessive. This also contributes to the cost of the final product.
`Therefore, there is a need in the art to provide cost-effective polynucleotides by
`proceduresthat are not as cumbersomeandlabor-intensive as present methodsto be able to
`provide polynucleotides at costs below $1 per base or 1-20 times less than current methods.
`The present invention was madeto address this need.
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`Summary of the Invention
`The present invention provides a process for the assembly of oligonucleotides
`synthesized on microarrays into a polynucleotide sequence. The desired target polynucleotide
`sequenceis dissected into pieces of overlapping oligonucleotides. In the first embodiment
`these oligonucleotides are synthesized in situ, in parallel on a microarray chip in a non-
`cleavable form. A primer extension process assemblesthe target polynucleotides. The primer
`extension process uses starting primers that are specific for the appropriate sequences. The last
`step is PCR amplification of the final polynucleotide product. Preferably, the polynucleotide
`product is a cDNA suitable for transcription purposes and further comprising a promoter
`sequence for transcription.
`The present invention provides a process for assembling a polynucleotide from a
`plurality of oligonucleotides comprising:
`(a)
`synthesizing or spotting a plurality of oligonucleotide sequences on a
`microarray device or bead device having a solid or porous surface, wherein a first
`oligonucleotide is oligo 1 and a second oligonucleotide is oligo 2 and so on, wherein the
`plurality of oligonucleotide sequences are attached to the solid or porous surface, and wherein
`the first oligonucleotide sequence has an overlapping sequence region of from about 10 to
`about 50 bases that is the same or substantially the same as a region of a second
`oligonucleotide sequence, and wherein the second oligonucleotide sequence has an
`overlapping region with a third oligonucleotide sequence and so on;
`(b)
`forming complimentary oligo 1 by extending primer 1, wherein primer 1 is
`complimentary to oligo 1;
`(c)
`disassociating complimentary oligo 1 from oligo 1 and annealing
`complimentary oligo 1 to both oligo 1 and to the overlapping region of oligo 2, wherein the
`annealing of complimentary oligo 1 to oligo 2 serves as a primer for extension for forming
`complimentary oligo 1+-2;
`(d)
`repeating the primer extension cycles of step (c) until a full-length
`polynucleotide is produced; and
`(e)
`amplifying the assembled complementary full length polynucleotide to produce
`a full length polynucleotide in desired quantities.
`Preferably, the solid or porous surface is in the form of a microarray device. Most
`preferably, the microarray device is a semiconductor device having a plurality of electrodes for
`synthesizing oligonucleotides in situ using electrochemical means to couple and decouple
`nucleotide bases. Preferably, the primer extension reaction is conducted through asequential
`process of melting, annealing and then extension. Most preferably, the primer extension
`reaction is conducted in a PCR amplification device using the microarray having the plurality
`of oligonucleotides boundthereto.
`The present invention further provides a process for assembling a polynucleotide from
`a plurality of oligonucleotides comprising:
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`synthesizing in situ or spotting a plurality of oligonucleotide sequences on a
`(a)
`microarray device or bead device each having a solid or porous surface, wherein the plurality
`of oligonucleotide sequencesare attached to the solid or porous surface, and wherein each
`oligonucleotide sequence has an overlapping region correspondingto a next oligonucleotide
`sequence within the sequence and further comprises two flanking sequences, oneat the 3’ end
`and the otherat the 5’ end of each oligonucleotide, wherein each flanking sequence is from
`about 7 to about 50 bases and comprising a primer region and a sequence segment having a
`testriction enzyme cleavable site;
`(b)
`amplifying cach oligonucleotide using the primer regionsofthe flanking
`sequence to form double stranded (ds) oligonucleotides;
`(c)
`cleaving the oligonucleotide sequencesat the restriction enzymecleavablesite;
`
`and
`
`assembling the cleaved oligonucleotide sequences through the overlapping
`(d)
`regions to form a full length polynucleotide.
`Preferably, the flanking sequence is from about 10 to about 20 basesin length.
`Preferably, the restriction enzyme cleavable site is a class II endonucleaserestriction site
`sequence capable of being cleaved by its corresponding class II restriction endonuclease
`enzyme. Most preferably, the restriction endonuclease class IT site corresponds to restriction
`sites for a restriction endonuclease class II enzymeselected from the group consisting of Mly I,
`BspM I, Bae I, BsaX I, Bsr I, Bmr I, Btr I, Bts I, Fok I, and combinations thereof. Preferably,
`the flanking sequence further comprises a binding moiety usedto purify cleaved
`oligonucleotides from flanking sequences. Preferably, the process further comprises the step
`of labeling the flanking sequence during the amplification step (b) using primer sequences
`labeled with binding moieties. Most preferably, a binding moiety is a small molecule able to
`be captured, such as biotin captured by avidin or streptavidin, or fluorescéin able to be
`captured by an anti-fluorescein antibody.
`The present invention further provides a process for assembling a polynucleotide from
`a plurality of oligonucleotides comprising:
`(a)
`synthesizing in situ or spotting a plurality of oligonucleotide sequences on a
`microarray device or bead device each havinga solid or porous surface, wherein the plurality
`of oligonucleotide sequencesare attached to the solid or porous surface, and wherein each
`oligonucleotide sequence has an overlapping region correspondingto a next oligonucleotide
`sequence within the sequence, and further comprises a sequence segment having a cleavable
`linker moiety;
`cleaving the oligonucleotide sequencesat the cleavable linkersite to cleave
`(b)
`each oligonucleotide complex from the microarray or bead solid surface to form a soluble
`mixture of oligonucleotides, each having an overlapping sequence; and
`(c)
`assembling the oligonucleotide sequences through the overlapping regions to
`form a full length polynucleotide.
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`Preferably, the cleavable linker is a chemical composition having a succinate moiety
`bound to a nucleotide moiety such that cleavage produces a 3”*hydroxy nucleotide. Most
`preferably, the cleavable linker is selected from the group consisting of 5’-dimethoxyitrityl-
`thymidine-3 succinate, 4-N-benzoyl-5’-dimethoxytrityl-deoxycytidine-3’~succinate, 1-N-
`benzoyl-5’-dimethoxytrityl-deoxyadenosine-3’-succinate, 2-N-isobutyryl-5’-dimethoxytrityl-
`deoxyguanosone-3’-succinate, and combinations thereof.
`The present invention further provides a process for assembling a polynucleotide from
`a plurality of oligonucleotides comprising:
`(a)
`synthesizing in situ or spotting a plurality of oligonucleotide sequences on a
`microarray device or bead device each having a solid or porous surface, wherein the plurality
`of oligonucleotide sequencesare attached to the solid or porous surface, and wherein each
`oligonucleotide sequence has a flanking region at an end attachedto the solid or porous
`surface, and a specific region designed by dissecting the polynucleotide sequence into a
`plurality of overlapping oligonucleotides, wherein a first overlapping sequenceona first
`oligonucleotide corresponds to a second overlapping sequence of a second oligonucleotide, and
`wherein the flanking sequence comprises a sequence segment havinga restriction
`endonuclease (RE) recognition sequence capable of being cleaved by a corresponding RE
`
`enzyme;
`hybridizing an oligonucleotide sequence complementary to the flanking region
`(b)
`to form a double stranded sequence capable of interacting with the corresponding RE enzyme;
`(c)
`digesting the plurality of oligonucleotides to cleave them from the microarray
`device or beads into a solution; and
`(d)
`assembling the oligonucleotide mixture through the overlapping regions to form
`a full length polynucleotide.
`Preferably, the flanking sequence is from about 10 to about 20 bases in length.
`Preferably, the restriction enzyme cleavable site is a class If endonucleaserestriction site
`sequence capable of being cleaved by its corresponding class II restriction endonuclease
`enzyme. Most preferably, the restriction endonucleoseclass II site correspondsto restriction
`sites for a restriction endonuclease class II enzyme selected from the group consisting of MlyI,
`BspM I, Bae I, BsaX I, Bsr I, Bmr I, BtrI, Bts I, Fok I, and combinations thereof. Preferably,
`the process further comprisesa final step of amplifying the polynucleotide sequence using
`primers located at both ends ofthe polynucleotide.
`.
`The present invention further provides a process for creating a mixture of
`oligonucleotide sequencesin solution comprising:
`(a)
`synthesizing in situ or spotting a plurality of oligonucleotide sequences on a
`microarray device or bead device each having a solid or porous surface, wherein the plurality
`of oligonucleotide sequencesare attached to the solid or porous surface, and wherein each
`oligonucleotide sequence further comprises two flanking sequences, one at the 3’ end and the
`other at the 5’ end of each oligonucleotide, wherein each flanking sequence is from about 7 to
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`about 50 bases and comprising a primer region and_a sequence segment havinga restriction
`enzyme cleavablesite;
`(b)
`amplifying each oligonucleotide using the primerregions of the flanking
`sequence to form a doublestranded (ds) oligonucleotides, and
`(c)
`cleaving the doublestranded oligonucleotide sequencesat the restriction
`enzyme cleavablesite.
`Preferably, the flanking sequenceis from about 10 to about 20 bases in length.
`Preferably, the restriction enzyme cleavablesite is a class IT endonucleaserestriction site
`sequence capable ofbeing cleaved by its corresponding class II restriction endonuclease
`enzyme. Mostpreferably, the restriction endonucleoseclass IT site correspondsto restriction
`sites for a restriction endonucleaseclass II enzymeselected from the group consisting of Mly I,
`BspM I, BaeI, BsaX I, Bsr I, BmrI, Bir I, Bis I, Fok I, and combinationsthereof. Preferably,
`the flanking sequence further comprises a binding moiety used to purify cleaved
`oligonucleotides from flanking sequences. Preferably, the process further comprises the step
`of labeling the flanking sequence during the amplification step (b) using primer sequences
`labeled with binding moieties. Most preferably, a binding moiety is a small molecule able to
`be captured, such as biotin captured by avidin or streptavidin, or fluorescein able to be
`captured by an anti-fluorescein antibody.
`The present invention further provides a processfor creating a mixture of
`oligonucleotide sequencesin solution comprising:
`(a)
`synthesizing in situ or spotting a plurality of oligonucleotide sequences on a
`microarray device or bead device each having a solid or poroussurface, wherein the plurality
`of oligonucleotide sequences are attached to the solid or porous surface, and wherein each
`oligonucleotide sequence has a sequence segment having a cleavable linker moiety;
`(b)
`cleaving the oligonucleotide sequencesat the cleavable linker site to cleave
`each oligonucleotide sequence from the microarray or bead solid surface to form a soluble
`mixture of oligonucleotides.
`Preferably, the cleavable linker is a chemical composition having a succinate moiety
`bound to a nucleotide moiety such that cleavage produces a 3’”hydroxy nucleotide. Most
`preferably, the cleavable linker is selected from the group consisting of 5’-dimethoxytrityl-
`thymidine-3’succinate, 4-N-benzoyl-5 *_dimethoxytrityl-deoxycytidine-3’-succinate, 1-N-
`benzoyl-5’-dimethoxytrityl-deoxyadenosine-3’-succinate, 2-N-isobutyryl-5’-dimethoxytrityl-
`deoxyguanosone-3’-succinate, and combinationsthereof.
`The present invention further provides a process for creating a mixture of
`oligonucleotide sequences in solution comprising:
`(a)
`synthesizing in situ or spotting a plurality of oligonucleotide sequences on a
`microarray device or bead device each having a solid or poroussurface, wherein the plurality
`of oligonucleotide sequences are attached to the solid or porous surface, and wherein each
`oligonucleotide sequence hasa flanking region at an end attached to the solid or porous
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`surface, and a specific region, wherein the flanking sequence comprises a sequence segment
`having a restriction endonuclease (RE) recognition sequence capable ofbeing cleaved by a
`corresponding RE enzyme;
`(b)
`hybridizing an oligonucleotide sequence complementary to the flanking region
`to form a double stranded sequence capable of interacting with the corresponding RE enzyme;
`(c)
`digesting the plurality of oligonucleotides to cleave them from the microarray
`device or beads into a solution.
`.
`Preferably, the flanking sequence is from about 10 to about 20 bases in length.
`Preferably, the restriction enzyme cleavable site is a class II endonucleaserestriction site
`sequence capable ofbeing cleaved by its correspondingclass II restriction endonuclease
`enzyme. Mostpreferably, the restriction endonucleoseclass II site correspondsto restriction
`sites for a restriction endonuclease class II enzymeselected from the group consisting ofMly 1,
`BspMI, BaeI, BsaX I, Bsr I, BurIJ, Btr IJ, Bis I, Fok I, and combinations thereof.
`Brief Description of the Drawings
`Figure 1 shows a schematic of gene assembly on a microarray device surface or porous
`matrix. In Figure 1A, the target gene sequence is dissected into number of overlapping
`oligonucleotides. The 3’ and 5’ are the ends ofthe shown strand. Figure 1A also shows,
`relative to the target sequence, primer Pri; extension product ofprimer Pr1, whichis
`complementary oligonucleotide 1; and extension product of complimentary oligonucleotide 1,
`which is complementary oligonucleotides 1+2. Figure 1B illustrates one embodiment ofthe
`initial steps of an assembly process. In step 1 of assembly, Primer Pr1 is annealed to
`oligonucleotide 1 and extended by appropriate polymerase enzymeinto product
`complementary to oligonucleotide 1. The second step is melting, re-annealing and extension
`(i.e., amplification) to lead to productionoflarger amountofPrl extension product
`(complementary oligonucleotide 1), re-association ofthe complementary oligonucleotide 1
`with oligonucleotide 1, and to annealing ofthe complementary oligonucleotide 1 with
`oligonucleotide 2 followedby its extension into product complementary to oligonucleotides
`1+2. Figure 1C shows a continuation ofthe assembly process from Figure 1B. Specifically,
`step 3 ofthe process(i.e., melting, re-annealing and extension) leads to the same products as
`step 2 plus a product complementary to oligonucleotides 1+2+3. Cycles (steps) are repeated
`until a full-length complementary polynucleotide is formed. Thefinal step is preparation of
`the final target polynucleotide molecule in desirable amounts by amplification (i.e., PCR)
`using two primers complementary to the ends of this molecule (PrX and PrY).
`Figure 2 shows a second embodiment ofthe inventive gene assembly process using
`oligonucleotides synthesized in situ onto a microarray device, each having a flanking sequence
`region containing a restriction enzyme cleavage site, followed by a PCR amplification step and
`followed by a REIIrestriction enzyme cleavagestep.
`Figure 3 shows a schematic for gene assembly using oligos synthesized and then
`cleaved from a microarray device. Specifically, in the upper panel marked “A”,
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`oligonucleotide sequences are connected to the microarray device through a cleavable linker
`(CL) moiety. An example of a cleavable linker moiety is provided in Figure 3A. The
`cleavable linkers are molecules that can withstand the oligonucleotide synthesis process(i.2.,
`phosphoramidite chemistry) and then can be cleaved to release oligonucleotide fragments.
`Chemical cleavage at cleavable linker CL recreates usual 3’ end of specific oligos | through N.
`These oligonucleotides are released into a mixture. The mixture of oligonucleotides is
`subsequently assembledinto full-length polynucleotide molecules. In the lower panel marked
`“B” of Figure 3, oligonucleotide sequences are connectedto the microarray device through
`additional flanking sequence containinga restriction enzyme (RE) sequencesite. Another
`oligonucleotide sequence, complementary to the flanking sequence region, is hybridized to the
`oligonucleotides on the microarray device. This recreates a “ds” or double-stranded
`oligonucleotide structure, each having a RE sequence recognition region in the flanking
`sequence region. Digestion of this ds oligonucleotides with the corresponding RE enzymesat
`the RE recognition sites in the flanking sequenceregions releases the specific oligonucleotides
`1 through N. When assembled,oligonucleotide sequences 1 through N form a full-length
`polynucleotide molecule.
`Figure 4 shows the assembly of a polynucleotide from three oligonucleotide fragments
`wherein each oligonucleotide fragment was synthesized in situ on a microarray device. The
`fully assembled polynucleotide was 172 mers in length, a length not practically achievable by
`in situ synthesis. The first embodiment inventive process was used in this example.
`Figure 5 showsthe oligonucleotide sequences used to assemble the 1’72-mer
`polynucleotide of Figure 4. The sequencesof primers X and Z are underlined. The Hpa II
`restriction site is indicated by italic underlinedletters.
`Figure 6 shows a schemefor preparing the sequences of flanking regions and primers
`used for preparation of specific oligonucleotide for assembly using the REI enzyme Mbt.
`Primer 1 is complementary to the oligonucleotide strand on a microarray device and contains a
`Biotin-TEG(triethylene glycol) moiety. Primer 2 is the same strand as the oligonucleotide
`strand on microarray device and contains Biotin-TEG moiety. Any sequence between the
`primers can be usedandis just designated by a string ofN’s.
`Figure 7 showsthe results of PCR and MiyI digestion of an oligonucleotide sequence as
`described in Figure 6. The clean bands showtheability to obtain pure oligonucleotides using
`the second embodimentofthe inventive process to cleave off oligonucleotide sequences using
`appropriate restriction enzymes.
`Figure 8 shows the sequences from nine oligonucleotides fragments (consecutively
`numbered 1-9) used to assemble a 290 bp polynucleotide. The flanking regions are shown in
`bold and underlined. The process used for polynucleotide assembly was the second
`embodiment. The overlapping regionsfurther contained a cleavable site as the Miyl
`recognition site for the M/yI classII restriction endonuclease.
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`WO 2004/024886
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`PCT/US2003/028946
`
`Figure 9 shows a schematic in the top panel for assembling a polynucleotide from nine
`oligonucleotides. Nine oligonucleotide sequences, shown in Figure 8, were amplified by PCR
`using primers 1 and 2 (as described in Figure 6) into ds DNA fragments containing the same
`flanking regions and specific overlapping sequences, digested with A¢/)I enzyme to remove
`flanking sequences, and used for assembly of 290 bp DNA fragment. The columnsin the gel
`shown are M — markers, 1 — negative control, assembly without primers FP1 and FP2, 2 —
`negative control, assembly without specific oligos, 3 —- assembly of 290 bp fragment from
`specific oligos plus amplification with FP1 and FP2 primers. The band in column 3 shows a
`high efficiency of the inventive polynucleotide assembly process.
`Figure 10 shows a sequence of an assembled polynucleotide in Example 4, broken
`down into its component oligonucleotides.
`Detailed Description of the Invention
`The present invention describes the preparation of a polynucleotide sequence (also
`called “gene”) using assembly of overlapping shorter oligonucleotides synthesized or spotted
`on microarray devices or on solid surface bead devices. The shorter oligonucleotides include
`sequence regions having overlapping regions to assist in assembly into the sequenceofthe
`desired polynucleotide. By overlapping regions,it is referred to sequence regionsat either a 3’
`end or a 5’ end of a first oligonucleotide sequence that is the same as part of the second
`oligonucleotide and has the same direction (relative to 3’ to 5° or 5’ to 3’ direction), and will
`hybridize to the 5’ end or 3’ end of a second oligonucleotide sequenceor its complimentary
`sequence (second embodiment), and s second oligonucleotide sequenceto third
`oligonucleotide sequence, and so on. In order to design or develop a microarray device or bead
`device to be used for polynucleotide assembly, the polynucleotide sequence is divided (or
`dissected) into a number of overlapping oligonucleotides segments, each with lengths
`preferably from 20 to 1000 bases, and most preferably from 20 to 200 bases (Figure 1A). The
`overlap between oligonucleotid