PCT
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`
`
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`(51) International Patent Classification 7 :
`WO 00/53617
`
`(11) International Publication Number:
`
`C07H 21/04, C12P 19/34
`
`(43) International Publication Date:
`
`14 September 2000 (14.09.00)
`
`(21) International Application Number:
`
`PCT/USOO/O6335
`
`(22) International Filing Date:
`
`7 March 2000 (07.03.00)
`
`(30) Priority Data:
`09/264,388
`
`8 March 1999 (08.03.99)
`
`US
`
`(71) Applicant (for all designated States except US): PROTOGENE
`LABORATORIES, INC. [US/US]; R & D Main Building,
`300 Constitution Drive, Menlo Park, CA 94025 (US).
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): BRENNAN, Thomas, M.
`[US/US]; 1998 Broadway #1505, San Francisco, CA 94109
`(US). HEYNEKER, Herbert, L.
`[NL/US]; 2244 Steiner
`Street, San Francisco, CA 94115 (US).
`
`(74) Agent: HALLUIN, Albert, P.; Howrey Simon Arnold &
`White, LLP, Box 34, 1299 Pennsylvania Avenue, N.W.,
`Washington, DC 20004 (US).
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD,
`GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP,
`KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK,
`MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TR, 'IT, UA, UG, US, UZ, VN, YU,
`ZA, ZW, ARIPO patent (GH, GM, KE, LS, MW, SD, SL,
`SZ, TZ, UG, ZW), Eurasian patent (AM, AZ, BY, KG, KZ,
`MD, RU, TJ, TM), European patent (AT, BE, CH, CY, DE,
`DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE),
`OAPI patent (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML,
`MR, NE, SN, TD, TG).
`
`Published
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`or during the assembly into the full—length DNA sequences.
`
`(54) Title: METHODS AND COMPOSITIONS FOR ECONOMICALLY SYNTHESIZING AND ASSEMBLING LONG DNA SE-
`QUENCES
`
`(57) Abstract
`
`the present invention relates to a cost—effective method of assembling long DNA sequences from short synthetic oligonucleotides.
`More specifically, short oligonucleotides are synthesized in situ on a solid support and subsequently cleaved from the solid support prior to
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
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`

`

`W0 00/536l7
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`4
`
`PCT/USOO/06335
`
`METHODS AND COMPOSITIONS FOR ECONOMICALLY
`
`SYNTHESIZING AND ASSEMBLING LONG DNA SEQUENCES
`
`This application is a continuation—in-part application of US. Patent Application
`
`Serial No. 09/264,388, filed on March 8, 1999, which is incorporated herein by reference
`
`in its entirety.
`
`FIELD OF THE INVENTION
`
`The present invention relates to a cost-effective method of assembling long DNA
`
`sequences from short synthetic oligonucleotides. More specifically, short
`
`oligonucleotides are synthesized in situ on a solid support and subsequently cleaved from
`
`the solid support prior to or during the assembly into the full—length sequences.
`
`BACKGROUND OF THE INVENTION
`
`The advent of rapid sequencing technology has created large databases of DNA
`
`sequences containing useful genetic information. The remaining challenges are to find
`
`out what these gene products really do, how they interact to regulate the whole organism,
`
`and ultimately how they may be manipulated to find utility in gene therapy, protein
`
`therapy, and diagnosis. The elucidation of the function of genes requires not only the
`
`knowledge of the wild type sequences, but also the availability of sequences containing
`
`designed variations in order to furthcr the understanding of the roles various genes play
`
`in health and diseases. Mutagenesis is routinely conducted in the laboratory to create
`
`random or directed libraries of interesting sequence variations. However the ability to
`
`manipulate large segments of DNA to perform experiments on the functional effects of
`
`changes in DNA sequences has been limited by the availability of modified enzymes and
`
`their associated costs. For example, the researcher cannot easily control the specific
`
`addition or deletion of certain regions or sequences of DNA via traditional mutagenesis
`
`methods, and must resort to the selection of interesting DNA sequences from libraries
`
`containing genetic variations.
`
`It would be most useful if a researcher could systematically synthesize large
`
`regions of DNA to determine the effect of differences in sequences upon the function of
`
`such regions. However, DNA synthesis using traditional methods is impractical because
`
`of the declining overall yield. For example, even with a yield of 99.5% per step in the
`
`phosphoramidite method of DNA synthesis, the total yield of a full length sequence of
`
`l
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`WO 00/53617
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`PCT/USOO/06335
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`500 base pairs long would be less than 1%. Similarly, if one were to synthesize
`
`overlapping strands of, for example, an adenovirus useful as a gene therapy vector, the
`
`50—70 kilobases of synthetic DNA required, even at a recent low price of approximately
`
`$1.00 per base, would cost over $50,000 per full sequence, far too expensive to be
`
`practical.
`
`The recovery of long segments of DNA may be improved when the DNA
`
`chemical synthesis is combined with recombinant DNA technology. Goeddel et al.,
`
`Proc. Natl. Acad. Sci. USA E(1):106-110 (1979); Itakura et al., Science @21056-1063
`
`(1977); and Heyneker et al., Nature @1748—752 (1976). The synthesis ofa long
`
`segment of DNA may begin with the synthesis of several modest—sized DNA fragments
`
`by chemical synthesis and continue with enzymatic ligation of the modest-sized
`
`fragments to produce the desired long segment of DNA. Synthetically made modest—
`
`sized DNA fragments may also be fused to DNA plasmids using restriction enzymes and
`
`ligase to obtain the desired long DNA sequences, which may be transcribed and
`
`translated in a suitable host. Recently, self-priming PCR technology has been used to
`
`assemble large segments of DNA sequences from a pool of overlapping oligonucleotides
`
`by using DNA polymerase without the use of ligase. Dillon et al., Bio Techniques
`
`2(3):298—300 (1990); Prodromou et al., Protein Engineering 5(8):827-829 (1992); Chen
`
`et al., J. Am. Chem. Soc. 116:8799-8800 (1994); and Hayashi et al., BioTechniques
`
`12(2):310-315 (1994). Most recently, DNA shuffling method was introduced to
`
`assemble genes from random fragments generated by paltial DNAaseI digestion or from
`
`a mixture of oligonucleotides. Stemmer et al, Nature 37_0:389-391 (1994); Stemmer et
`
`al, Proc. Natl. Acad. Sci. USA 9_1_:10747—10751 (1994); Stemmer et al, Gene @249—53
`
`(1996); Crameri et al., Nat. Bioteclmol. fiz436—438 (1997); Zhang et al., Proc. Natl.
`
`Acad. Sci. USA fiz4504-4509 (1997); Crameri et al., Nature flz288-291 (1998);
`
`Christians et al., Nat. Biotechnol. fl:259-264 (1999), US. Patent Nos. 5,830,721,
`
`5,811,238, 5,830,721, 5,605,793, 5,834,252, and 5,837,458; and PCT publications WO
`
`98/13487, WO98/27230, WO 98/31837, WO 99/41402, 99/57128, and WO 99/65927.
`
`Methods for synthesizing a large variety of short or modest—sized
`
`oligonucleotides have been extensively described. One of the methods is to use
`
`microarray technology, where a large number of oligonucleotides are synthesized
`
`simultaneously on the surface of a solid support. The microarray technology has been
`
`described in Green et al., Curr. Opin. in Chem. Biol. 2:404—410 (1998), Gerhold et al.,
`
`TIBS, 24:168—173 (1999), US. Patent Nos. 5,510,270, 5,412,087, 5,445,934, 5,474,796,
`2
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`WO 00/53617
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`PCT/US00/06335
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`5,744,305, 5,807,522, 5,843,655, 5,985,551, and 5,927,547. One method for
`
`synthesizing high density arrays of DNA fragments on glass substrates uses light—
`
`directed combinatorial synthesis. However, the photolithographic synthesis method
`
`provides oligonucleotides which are neither pure enough for later enzymatic assembly
`
`nor a method which is flexible and cost effective. For example, due to the low chemical
`
`coupling yield of in situ synthesis using photolithography, each oligonucleotide may
`
`contain a substantial number of truncated products in addition to the desired length
`
`oligonucleotides. For example, in lO-mers and 20-mers, only about 40% and 15% ofthc
`
`oligonucleotides are ofthe full length respectively. Forman, I, er (1]., Molecular
`
`Modeling of Nucleic Acids, Chapter 13, pp 206—228, American Chemical Society
`
`(1998)) and McGall et (11., J. Am. Chem. Soc, 119:5081-5090 (1997). In addition,
`
`several thousands of dollars of masks specific to any given series of sequences are
`
`required for practical assembly.
`
`Existing methods for the synthesis of long DNA sequences also have many
`
`drawbacks, for example, the length limitations of conventional solid phase DNA
`
`synthesis, the requirement of synthesizing both strands of DNA, and the complexity of
`
`multiple enzymatic reactions for stepwise assembly. These drawbacks inevitably add to
`
`the cost of obtaining long DNA sequences. There is a need in the art to economically
`
`synthesize multiple oligonucleotides and subsequently assemble them into long DNA
`
`sequences. Such an inexpensive and custom synthesis and assembly process has many
`
`uses. Gene sequences of interest can be assembled and tested for a variety of
`
`functionalities, for example, the function of relative position of promoter to gene coding
`
`sequence, the role of introns versus exons, the minimization of gene sequence necessary
`
`for function, the role of polymorphisms and mutations, the effectiveness of sequence
`
`changes to gene therapy vectors, the optimization of the gene coding for a protein for a
`
`specific experiment or industrial application, among others. These functional analysis
`
`may be explored with the DNA designs truly under the control of the researcher.
`
`In
`
`other cases, specific variations in assembled sequence can be used to create structured
`
`libraries containing many possible genetic variations for testing of the function or the
`
`inhibition of the function. Eventually entire genomes could be easily synthesized,
`
`assembled, and functionally tested in this manner. In short, any experiment in which a
`
`model system of synthetic genes or genomes could be changed in a specific way under
`
`the control of a researcher, could be performed easily and less expensively.
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`WO 00/5361‘7
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`SUMMARY OF THE INVENTION
`
`The present method for synthesizing and assembling long DNA sequences from
`
`short oligonucleotides comprises the steps of: (a) synthesizing on a solid support an array
`
`of oligonucleotide sequences wherein the oligonucleotides collectively encode both
`
`strands of the target DNA and are covalently attached to the solid support using a
`
`cleavable moiety; (b) cleaving the oligonucleotides from the solid support; and (c)
`
`assembling the oligonucleotides into the target full—length sequence. The target long
`
`DNA sequences contemplated in the present invention may be a regulatory sequence, a
`
`gene or a fragment thereof, a vector, a plasmid, a virus, a full genome of an organism, or
`
`any other biologically functional DNA sequences which may be assembled from
`
`overlapping oligonucleotides, either directly or indirectly by enzymatic ligation, by using
`
`a DNA polymerase, by using a restriction enzyme, or by other suitable assembly
`methods known in the art.
`
`In preferred embodiments, oligonucleotides may be prepared by in siru synthesis
`
`on a solid support.
`
`In particular, the in Situ synthesis of oligonucleotides may employ the
`
`“drop-on—demand” method, which uses technology analogous to that employed in ink-jet
`
`printers. In addition, hydrophilic/hydrophobic arrays or surface tension arrays, which
`
`consist of pattemed regions of hydrophilic and hydrophobic surfaces, may be employed.
`
`Preferably, the size of the long DNA sequence ranges from about 200 to 10,000 bases,
`
`more preferably, from about 400 to 5,000 bases. Preferably, the length of each
`
`oligonucleotide may be in the range of about 10 to 200 bases long, more preferably, in
`
`the range of about 20 to 100 bases long. Preferably, the number of oligonucleotides
`
`synthesized on the solid support is from about 10 to 2000, more preferably, from about
`10 to 500.
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`Figure 1 shows hydroxyl-group bearing non—cleavable linkers used for
`
`hybridization directly on the glass chip.
`
`Figure 2 shows the coupling of a chemical phosphorylation agent as the special
`
`amidite to allow cleavage of the oligonucleotide after synthesis.
`
`Figure 3 shows the amidite (TOPS) used to prepare universal CPG-support to
`
`allow cleavage of the oligonucleotide after synthesis.
`
`Figure 4A illustrates the formation of an array surface that is ready for solid
`
`phase synthesis.
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`Figure 4B illustrates O—Nitrocarbamate array making chemistry.
`
`Figure 5 illustrates surface tension wall effect at the dot—interstice interface. The
`
`droplet containing solid phase synthesis reagents does not spread beyond the perimeter
`of the dot due to the surface tension wall.
`
`Figure 6 illustrates hydrogen—phosphonate solid phase oligonucleotide synthesis
`
`on an array surface.
`
`Figure 7A illustrates the top view ofa piezoelectric impulsejet ofthe type used
`
`to deliver solid phase synthesis reagents to individual dots in the array plate synthesis
`methods.
`
`Figure 7B illustrates the side view of a piezoelectric impulse jet of the type used
`
`to deliver solid phase synthesis reagents to individual dots in the array plate synthesis
`methods.
`
`Figure 8 illustrates use of a piezoelectric impulse jet head to deliver blocked
`
`nucleotides and activating agents to individual dots on an array plate. The configuration
`
`shown has a stationary head/moving plate assembly.
`
`Figure 9 illustrates an enclosure for array reactions showing array plate, sliding
`
`cover and manifolds for reagent inlet and outlet.
`
`Figure 10 illustrates the gene assembly process from short synthetic
`
`oligonucleotides.
`
`DETAILED DESCRIPTION OF THE INVENTION
`
`The present invention relates to a cost—effective method for assembling long
`
`DNA sequences from short synthetic oligonucleotides. In general, the present method
`
`for synthesizing and assembling long DNA sequences from synthetic oligonucleotides
`
`comprises the steps of: (a) synthesizing on a solid support an array of oligonucleotide
`
`sequences wherein the oligonucleotides collectively encode both strands of the target
`
`DNA and are covalently attached to the solid support using a cleavable moiety; (b)
`
`cleaving the oligonucleotides from the solid support; and (c) assembling the
`
`oligonucleotides into the target full—length sequence. The target long DNA sequences
`
`contemplated in the present invention may be a regulatory sequence, a gene or a
`
`fragment thereof, a vector, a plasmid, a virus, a full genome of an organism, or any other
`
`DNA sequences which may be assembled from overlapping oligonucleotides, either
`
`directly or indirectly by enzymatic ligation, by using a DNA polymerase, by using a
`
`restriction enzyme, or by other suitable assembly methods known in the art.
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`Attachment ofa cleavable moiety to the oligonucleotides and the solid support
`
`In solid phase or microarray oligonucleotide synthesis designed for diagnostic
`
`and other hybridization—based analysis, the final oligonucleotide products remain
`
`attached to the solid support such as controlled—pore glass (CPG) or chips. A non-
`
`cleavable linker such as the hydroxyl linker I or II in Figure l is typically used. These
`
`hydroxyl linkers remain intact during the deprotection and purification processes and
`
`during the hybridization analysis. Synthesis of a large number of overlapping
`
`oligonucleotide for the eventual assembly into a longer DNA segment, however, is
`
`performed on a linker which allows the cleavage of the synthesized oligonucleotide. The
`
`cleavable moiety is removed under conditions which do not degrade the
`
`oligonucleotides. Preferably the linker may be cleaved using two approaches, either (a)
`
`simultaneously under the same conditions as the deprotection step or (b) subsequently
`
`utilizing a different condition or reagent for linker cleavage after the completion of the
`
`deprotection step. The former approach may be advantageous, as the cleavage of the
`
`linker is performed at the same time as the deprotection of the nucleoside bases. Time
`
`and effort are saved to avoid additional post-synthesis chemistry. The cost is lowered by
`
`using the same reagents for deprotection in the linker cleavage. The second approach
`
`may be desirable, as the subsequent linker cleavage may serve as a pre-purification step,
`
`eliminating all protecting groups from the solution prior to assembly.
`
`Any suitable solid supports may be used in the present invention. These
`
`materials include glass, silicon, wafer, polystyrene, polyethylene, polypropylene,
`
`polytetrafluorethylene, among others. Typically, the solid supports are functionalized to
`
`provide cleavable linkers for covalent attachment to the oligonucleotides. The linker
`
`moiety may be of six or more atoms in length. Alternatively, the cleavable moiety may
`
`be Within an oligonucleotide and may be introduced during in situ synthesis. A broad
`
`variety of cleavable moieties are available in the art of solid phase and microarray
`
`oligonucleotide synthesis. Pon, R., “Solid—Phase Supports for Oligonucleotide
`
`Synthesis” in “Protocols for oligonucleotides and analogs; synthesis and properties,”
`
`Methods Mol. Biol. 2_O:465-496 (1993); Verma el‘ al., Annu. Rev. Biochem. 6199—134
`
`(1998); and US. Patent Nos. 5,739,386, 5,700,642 and 5,830,655. A suitable cleavable
`
`moiety may be selected to be compatible with the nature of the protecting group of the
`
`nucleoside bases, the choice of solid support, the mode of reagent delivery, among
`
`others. The cleavage methods may include a variety of enzymatic, or non—enzymatic
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`means, such as chemical, thermal, or photolytic cleavage. Preferably, the
`
`oligonucleotides cleaved from the solid support contain a free 3’—OH end. The free 3’—
`
`OH end may also be obtained by chemical or enzymatic treatment, following the
`
`cleavage of oligonucleotides.
`
`The covalent immobilization site may either be at the 5’ end of the
`
`oligonucleotide or at the 3’end of the oligonucleotide.
`
`In some instances, the
`
`immobilization site may be within the oligonucleotide (i. e. at a site other than the 5’ or 3’
`
`end of the oligonucleotide). The cleavable site may be located along the oligonucleotide
`
`backbone, for example, a modified 3’—5’ internucleotide linkage in place of one of the
`
`phosphodiester groups, such as ribose, dialkoxysilane, phosphorothioate, and
`
`phosphoramidate internucleotide linkage. The cleavable oligonucleotide analogs may
`
`also include a substituent on or replacement of one of the bases or sugars, such as 7—
`
`deazaguanosine, 5-methylcytosine, inosine, uridine, and the like.
`
`In one embodiment, cleavable sites contained within the modified
`
`oligonucleotide may include chemically cleavable groups, such as dialkoxysilane, 3'—(S)—
`
`phosphorothioate, 5'-(S)-phosphorothioate, 3'-(N)—phosphoramidate, 5'—(N)-
`
`phosphoramidate, and ribose. Synthesis and cleavage conditions of chemically cleavable
`
`oligonucleotides are described in US. Patent Nos. 5,700,642 and 5,830,655. For
`
`example, depending upon the choice of cleavable site to be introduced, either a
`
`functionalized nucleoside or a modified nucleoside dimer may be first prepared, and then
`
`selectively introduced into a growing oligonucleotide fragment during the course of
`
`oligonucleotide synthesis. Selective cleavage of the dialkoxysilane may be effected by
`
`treatment with fluoride ion. Phosphorothioate internucleotide linkage may be selectively
`
`cleaved under mild oxidative conditions. Selective cleavage of the phosphoramidate
`
`bond may be carried out under mild acid conditions, such as 80% acetic acid. Selective
`
`cleavage of ribose may be carried out by treatment with dilute ammonium hydroxide.
`
`In preferred embodiments, in order to convert the non-cleavable hydroxyl linker
`
`(Figure 1) into a cleavable linker, a special phosphoramidite may be coupled to the
`
`hydroxyl group prior to the phophoramidite or H—phosphonate oligonucleotide synthesis.
`
`One preferred embodiment of such special phophoramidite, a chemical phosphorylation
`
`agent, is shown in Figure 2. The reaction conditions for coupling the hydroxyl group
`
`with the chemical phosphorylation agent are known to those skilled in the art. The
`
`cleavage of the chemical phosphorylation agent at the completion of the oligonucleotide
`
`synthesis yields an oligonucleotide bearing a phosphate group at the 3' end. The 3'—
`
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`phosphate end may be converted to a 3' hydroxyl end by a treatment with a chemical or
`
`an enzyme, such as alkaline phosphatase, which is routinely carried out by those skilled
`in the art.
`
`Another class of cleavable linkers is described by McLean, et a]. in PCT
`
`publication WO 93/20092. This class ofcleavable linker, also known as TOPS for two
`
`oligonucleotides per synthesis, was designed for generating two oligonucleotides per
`
`synthesis by first synthesizing an oligonucleotide on a solid support, attaching the
`
`cleavable TOPS linker to the first oligonucleotide, synthesizing a second oligonucleotide
`
`on the TOPS linker, and finally cleaving the linker from both the first and second
`
`oligonucleotides.
`
`In the present invention however, the TOPS phosphoramidite may be
`
`used to convert a non-cleavable hydroxyl group on the solid support to a cleavable
`
`linker, suitable for synthesizing a large number of overlapping oligonucleotides. A
`
`preferred embodiment of TOPS reagents is the Universal TOPSTM phosphoramidite,
`
`which is shown in Figure 3. The conditions for Universal TOPSTM phosphoramidite
`
`preparation, coupling and cleavage are detailed in Hardy et (1]., Nucleic Acids Research
`
`203229988 004 (1994), which is incorporated herein by reference. The Universal
`
`TOPSTM phosphoramidite yields a cyclic 3' phosphate that may be removed under basic
`
`conditions, such as the extended amonia and/or ammonia/methylamine treatment,
`
`resulting in the natural 3' hydroxy oligonucleotide.
`
`A cleavable amino linker may also be employed in the synthesis of overlapping
`
`oligonucleotides. The resulting oligonucleotides bound to the linker via a
`
`phosphoramidite linkage may be cleaved with 80% acetic acid yielding a 3'—
`
`phosphorylated oligonucleotide.
`
`In another embodiment, cleavable sites contained Within the modified
`
`oligonucleotide may include nucleotides cleavable by an enzyme such as nucleases,
`
`glycosylases, among others. A wide range of oligonucleotide bases, e.g. uracil, may be
`
`removed by DNA glycosylases, which cleaves the N—glycosylic bond between the base
`
`and deoxyribose, thus leaving an abasic site. Krokan er. al., Biochem. J. $21—16
`
`(1997). The abasic site in an oligonucleotide may then be cleaved by Endonuclease IV,
`
`leaving a free 3’—OH end. In another embodiment, the cleavable site may be a restriction
`
`endonuclease cleavable site, such as class Ils restriction enzymes. For example, Bpml,
`
`Bsgl, BseRI, BsmFI, and Fokl recognition sequence may be incorporated in the
`
`immobilized oligonucleotides and subsequently cleaved to release oligonucleotides.
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`In another embodiment, the cleavable site within an immobilized oligonucleotide
`
`may include a photocleavable linker, such as ortho—nitrobenzyl class of photocleavable
`
`linkers. Synthesis and cleavage conditions of photolabile oligonucleotides on solid
`
`support are described in Venkatesan er a]. J. of Org. Chem. (31:525-529 (1996), Kahl et
`
`al., J. ofOrg. Chem. fiz507—510 (1999), Kahl et al., J. ofOrg. Chem. @24870—4871
`
`(1998), Greenberg et al., J. ofOrg. Chem. 2:746—753 (1994), Holmes er al., J. ofOrg.
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`Chem. Q:2370—2380 (1997), and US. Patent No. 5,739,386. Ortho—nitobenzyl—based
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`linkers, such as hydroxymethyl, hydroxyethyl, and Fmoc—aminoethyl carboxylic acid
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`linkers, may also be obtained commercially.
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`Determination of overlapping oligonucleotides encoding the long DNA sequence of
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`inter—est
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`The present invention represents a general method for synthesizing and
`
`assembling any long DNA sequence from an array of overlapping oligonucleotides.
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`Preferably, the size of the long DNA region ranges from about 200 to 10,000 bases.
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`More preferably, the size of the long DNA region ranges from about 400 to 5,000 bases.
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`The long DNA sequence of interest may be split into a series of overlapping
`
`oligonucleotides. With the enzymatic assembly of the long DNA sequence, it is not
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`necessary that every base, both the sense and antisense strand, of the long DNA sequence
`
`of interest be synthesized. The overlapping oligonucleotides are typically required to
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`collectively encode both strands of the DNA region of interest. The length of each
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`overlapping oligonucleotide and the extent of the overlap may vary depending on the
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`methods and conditions of oligonucleotide synthesis and assembly. Several general
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`factors may be considered, for example, the costs and errors associated with synthesizing
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`modest size oligonucleotides, the annealing temperature and ionic strength of the
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`overlapping oligonucleotides, the formation of unique overlaps and the minimization of
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`non—specific binding and intramolecular base pairing, among others. Although, in
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`principle, there is no inherent limitation to the number of overlapping oligonucleotides
`
`that may be employed to assemble them in to the target sequence, the number of
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`overlapping oligonucleotides is preferably from about 10 to 2000, and more preferably,
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`from about 10 to 500.
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`In particular, for the assembly method using a DNA polymerase, 21 unique
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`overlap is preferred in order to produce the correct size of long DNA sequence after
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`assembly. Unique overlaps may be achieved by increasing the degree of overlap.
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`However, increasing the degree of overlap adds the number of bases required, which
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`naturally incurs additional cost in oligonucleotide synthesis. Those skilled in the art Will
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`select the optimal length ofthe overlapping oligonucleotides and the optimal length of
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`the overlap suitable for oligonucleotide synthesis and assembly methods.
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`In particular, a
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`computer search of both strands of the target sequence with the sequences of each of the
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`overlap regions may be used to show unique design of oligonucleotides with the least
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`likelihood of give nonspecific binding. Preferably, the length of each oligonucleotide
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`may be in the range of about 10 to 200 bases long. More preferably, the length of each
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`oligonucleotide is in the range of about 20 to 100 bases long. Preferably,
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`oligonucleotides overlap their complements by about 10 to 100 bases. The lowest end of
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`the range, at least a 10—base overlap, is necessary to create stable priming of the
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`polymerase extension of each strand. At the upper end, maximally overlapped
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`oligonucleotides of 200 bases long would contain 100 bases of complementary overlap.
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`Most preferably, the overlapping regions, in the range of about 15—20 base pairs in length
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`may be designed to give a desired melting temperature, 6. g. , in the range of 52-56 0C, to
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`ensure oligonucleotide specificity. It may also be preferred that all overlapping
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`oligonucleotides have a similar extent of overlap and thus a similar annealing
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`temperature, which will normalize the annealing conditions during PCR cycles.
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`Oligonucleotide synthesis
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`Synthesis of oligonucleotides may be best accomplished using a variety of chip
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`or microarray based oligonucleotide synthesis methods. Traditional solid phase
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`oligonucleotide synthesis on controlled—pore glass may be employed, in particular when
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`the number of oligonucleotides required to assemble the desired DNA sequence is small.
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`Oligonucleotides may be synthesized on an automated DNA synthesizer, for example, on
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`an Applied Biosystems 380A synthesizer using 5—dimethoxytritylnucleoside B-
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`cyanoethyl phosphoramidites. Synthesis may be carried out on a 0.2 uM scale CPG
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`solid support with an average pore size of 1000 A. Oligonucleotides may be purified by
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`gel electrophoresis, HPLC, or other suitable methods known in the art.
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`In preferred embodiments, oligonucleotides may be prepared by in situ synthesis
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`on a solid support in a step—wise fashion. With each round of synthesis, nucleotide
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`building blocks may be added to growing chains until the desired sequence and length
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`are achieved in each spot. In particular, the in situ synthesis of oligonucleotides may
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`WO 00/53617
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`employ the “drop—on-demand” method, which uses technology analogous to that
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`employed in ink-jet printers. US. Patent Nos. 5,474,796, 5,985,551, 5,927,547,
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`Blanchard er a[., Biosensors and Bioelectronics 11:687-690 (1996), and Schena el al.,
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`TIBTECH162301—306 (1998). This approach typically utilizes piezoelectric or other
`
`forms of propulsion to transfer reagents from miniature nozzles to solid surfaces. For
`
`example, the printer head travels across the array, and at each spot, electric field
`
`contracts, forcing a microdroplet of reagents onto the array surface. Following washing
`and deproteetion, the next cycle of oligonucleotide synthesis is carried out. The step
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`yields in piezoelectric printing method typically equal to, and even exceed, traditional
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`CPG oligonucleotide synthesis. The drop—on—demand technology allows high—density
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`gridding of virtually any reagents of interest. It is also easier using this method to take
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`advantage of the extensive chemistries already developed for oligonucleotide synthesis,
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`for example, flexibility in sequence designs, synthesis of oligonucleotide analogs,
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`synthesis in the 5’—3’ direction, among others. Because ink jet technology does not
`
`require direct surface contact, piezoelectric delivery is amendable to very high
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`throughput.
`
`In preferred embodiments, a piezoelectric pump may be used to add reagents to
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`the in situ synthesis of oligonucleotides. Microdroplets of 50 picoliters to 2 microliters
`
`of reagents may be delivered to the array surface. The design, construction, and
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`mechanism of a piezoelectric pump are described in US. Patent Nos. 4,747,796 and
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`5,985,551. The piezoelectric pump may deliver minute droplets of liquid to a surface in
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`a very precise manner. For example, a picopump is capable of producing picoliters of
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`reagents at up to 10,000 Hz and accurately hits a 250 micron target at a distance of 2 cm.
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`In preferred embodiments of the instant invention, hydrophilic/hydrophobic
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`arrays or surface tension arrays, which consist of patterned regions of hydrophilic and
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`hydrophobic surfaces, may be employed. US. Patent Nos. 4,747,796 and 5,985,551. A
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`hydrophilic/hydrophobic array may contain large numbers of hydrophilic regions against
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`a hydrophobic background. Each hydrophilic region is spatially segregated from
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`neighboring hydrophilic region because of the hydrophobic matrix between hydrophilic
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`spots. Surface tension arrays described in may be employed in the present invention.
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`Typically the support surface has about 10—50 x 10'15 moles of functional binding sites
`
`per mm2 and each functionalized binding site is about 50—2000 microns in diameter.
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`There are significant advantages to making arrays by surface tension localization
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`and reagent microdelivery. The lithography and chemistry used to pattern the substrate
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