The present invention provides a process for in 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 process for in situ synthesis of oligonucleotide sequence fragments on a solid phase microarray platform and subsequent, “on chip” assembly of larger polynucleotides composed of a plurality of smaller oligonucleotide sequence fragments.
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 of the 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 Arrays” and “Gene Chips” but this descriptive name has been attempted to be a trademark) having oligonucleotide content. Most often, 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 claim priority from this priority document, (3) electrochemical in situ synthesis based upon pH based removal of blocking chemical functional groups (see, for example, Montgomery U.S. Pat. No. 6,092,302 the disclosure of which is incorporated by reference herein and Southern U.S. Pat. No. 5,667,667), and (4) electric field attraction/repulsion of fully-formed oligonucleotides (see, for example, Hollis et al., U.S. Pat. No. 5,653,939 and its duplicate Heller U.S. Pat. No. 5,929,208). Only the first three basic techniques can form oligonucleotides in situ e.g., 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 technique loads glass fibers having multiple capillaries drilled through them with different oligonucleotides loaded into each capillary tube. Microarray chips, often simply glass microscope slides, are then stamped out much like a rubber stamp on each sheet of paper of glass slide. It is also possible to use “spotting” techniques to build oligonucleotides in situ. Essentially, this involves “spotting” relevant single nucleotides at the exact location or region on a slide (preferably a glass slide) where a particular sequence of oligonucleotide is to be built. Therefore, irrespective of whether or 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 photomasks to 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 Fodor et al., U.S. Pat. No. 5,445,934 and its various progeny patents. Essentially, this technique provides for “solid-phase chemistry, photolabile protecting groups, and photolithography . . . to achieve light-directed spatially-addressable parallel chemical synthesis.”
The electrochemistry platform (Montgomery U.S. Pat. No. 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) technology to 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 volumes of 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 means for in situ synthesis has to alternate anodes and cathodes in the array in order to generated needed protons (acids) at the anodes so 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 in excess of 1000 bases in length. In vitro 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 polynucleotides of gene length or longer had to turn to nature or gene isolation techniques to obtain polynucleotides of such length. For the purposes of this 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. While it is theoretically possible to synthesize polynucleotides through single base addition, the yield losses make it a practical impossibility beyond 150 bases and 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, which is a copy of an mRNA molecule, can be obtained if the starting material contains the desired mRNA. However, it is not always known if the particular mRNA is present in a sample or the amount of the mRNA might be too low to obtain the corresponding cDNA without significant 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 a polynucleotide of any desired sequence just knowing the primary structure, for a reasonable price, in a short period of time, will significantly move forward several fields of biomedical research and clinical 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 or ligation 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 procedures that are not as cumbersome and labor-intensive as present methods to be able to provide polynucleotides at costs below $1 per base or 1-20 times less than current methods. The present invention was made to address this need.
The present invention provides a process for the assembly of oligonucleotides synthesized on microarrays into a polynucleotide sequence. The desired target polynucleotide sequence is 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 assembles the 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 complementary oligo 1 by extending primer 1, wherein primer 1 is complementary to oligo 1;
(c) disassociating complementary oligo 1 from oligo 1 and annealing complementary oligo 1 to both oligo 1 and to the overlapping region of oligo 2, wherein the annealing of complementary oligo 1 to oligo 2 serves as a primer for extension for forming complementary 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 a sequential 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 bound thereto.
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 sequences are attached to the solid or porous surface, and wherein each oligonucleotide sequence has an overlapping region corresponding to a next oligonucleotide sequence within the sequence and 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 about 50 bases and comprising a primer region and a sequence segment having a restriction enzyme cleavable site;
(b) amplifying each oligonucleotide using the primer regions of the flanking sequence to form double stranded (ds) oligonucleotides;
(c) cleaving the oligonucleotide sequences at the restriction enzyme cleavable site; and
(d) assembling the cleaved oligonucleotide sequences 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 II endonuclease restriction site sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme. Most preferably, the restriction endonuclease class II site corresponds to restriction sites for a restriction endonuclease class II enzyme selected 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 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 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 sequences are attached to the solid or porous surface, and wherein each oligonucleotide sequence has an overlapping region corresponding to a next oligonucleotide sequence within the sequence, and further comprises a sequence segment having a cleavable linker moiety;
(b) cleaving the oligonucleotide sequences at the cleavable linker site to cleave 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.
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 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 sequences are attached to the solid or porous surface, and wherein each oligonucleotide sequence has a flanking region at an end attached to 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 sequence on a first oligonucleotide corresponds to a second overlapping sequence of a second oligonucleotide, and wherein the flanking sequence comprises a sequence segment having a restriction endonuclease (RE) recognition sequence capable of being 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; 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 II endonuclease restriction site sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme. Most preferably, the restriction endonuclease class II site corresponds to restriction sites for a restriction endonuclease class II enzyme selected 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 process further comprises a final step of amplifying the polynucleotide sequence using primers located at both ends of the polynucleotide.
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 porous surface, wherein the plurality of oligonucleotide sequences are 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 about 50 bases and comprising a primer region and a sequence segment having a restriction enzyme cleavable site;
(b) amplifying each oligonucleotide using the primer regions of the flanking sequence to form a double stranded (ds) oligonucleotides; and
(c) cleaving the double stranded oligonucleotide sequences at the restriction enzyme cleavable site.
Preferably, the flanking sequence is from about 10 to about 20 bases in length. Preferably, the restriction enzyme cleavable site is a class II endonuclease restriction site sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme. Most preferably, the restriction endonuclease class II site corresponds to restriction sites for a restriction endonuclease class II enzyme selected 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 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 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 porous surface, 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 sequences at 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 combinations thereof.
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 porous surface, wherein the plurality of oligonucleotide sequences are attached to the solid or porous surface, and wherein each oligonucleotide sequence has a flanking region at an end attached to the solid or porous surface, and a specific region, wherein the flanking sequence comprises a sequence segment having a restriction endonuclease (RE) recognition sequence capable of being 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 endonuclease restriction site sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme. Most preferably, the restriction endonuclease class II site corresponds to restriction sites for a restriction endonuclease class II enzyme selected 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.
FIG. 10 shows a sequence of an assembled polynucleotide in Example 4, broken down into its component oligonucleotides (fragments 1-9 (SEQ ID NOs: 14-22) and fragments (1F-29F (SEQ ID NOs: 23-51).
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 sequence of the desired polynucleotide. Overlapping regions refer to sequence regions at 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 sequence or its complementary sequence (second embodiment), and a second oligonucleotide sequence to a 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 (
In the first embodiment the inventive gene/polynucleotide assembly process uses oligonucleotides immobilized on a microarray device. The microarray device itself or a porous reaction layer with immobilized oligonucleotides can be used for the inventive gene/polynucleotide assembly process.
With regard to
In a second embodiment, a plurality of oligonucleotides that together comprise (with overlapping regions) the target polynucleotide sequence are synthesized on a microarray device (or can be synthesized on beads as a solid substrate), wherein each oligonucleotide sequence further comprises flanking short sequence regions, wherein each flanking sequence region comprises one or a plurality of sequence sites for restriction endonuclease, preferably endonuclease class II (ERII) enzymes. Each oligonucleotide is amplified by PCR using appropriate oligonucleotide primers to the flanking sequence regions to form a preparation of a plurality of oligonucleotides. The preparation of oligonucleotides is treated then with appropriate REII enzyme(s) (specific to the restriction sequences in the flanking sequence regions) to produce flanking fragments and overlapping oligonucleotides that, together comprise the desired polynucleotide sequence. Flanking fragments and PCR primers are removed from the mixture, if desired, by different methods based on size or specific labeling of the PCR primers. The oligonucleotides resembling the desired target polynucleotide then assembled into the final target polynucleotide molecule using repetition of the primer extension method and PCR amplification of the final molecule.
Specifically, in the second embodiment, the assembly process initially uses oligonucleotides immobilized on a microarray device or beads, via immobilization techniques, such as spotting or ink-jet printing or by direct in situ synthesis of the microarray device using various techniques, such as photolithography or electrochemical synthesis. The overlapping oligonucleotide sequences are designed having an overlapping region and one or two flanking sequence regions comprising a restriction class II recognition site (
The length of flanking sequences is at least the length of REII recognition site. The flanking sequences are designed to have minimal homology to the specific oligonucleotide sequences regions on the microarray device. The flanking sequences can be the same for each oligonucleotide fragment, or be two or more different sequences. For example, a pair of appropriate primers, called Pr1 and Pr2, was designed to amplify each oligonucleotide on a microarray device (
In another embodiment for the assembly step, oligonucleotide sequences that together comprise the target polynucleotide molecule are assembled using a ligase chain reaction as described in Au et al., Biochem. Biophys. Res. Commun. 248:200-3, 1998. Briefly, short oligonucleotides are joined through ligase chain reaction (LCR) in high stringency conditions to make “unit fragments” (Fifty microliters of reaction mixture contained 2.2 mM of each oligo, 8 units Pfu DNA ligase (Stratagene La Jolla, Calif.) and reaction buffer provided with the enzyme. LCR was conducted as follows: 95° C. 1 min; 55° C. 1.5 min, 70° C. 1.5 min, 95° C. 30 sec for 15 cycles; 55° C. 2 min; 70° C. 2 min, which are then fused to form a full-length gene sequence by polymerase chain reaction.
In another embodiment the ds oligonucleotide sequences are assembled after preparation by chain ligation cloning as described in Pachuk et al., Gene 243:19-25, 2000; and U.S. Pat. No. 6,143,527 (the disclosure of which is incorporated by reference herein). Briefly, chain reaction cloning allows ligation of double-stranded DNA molecules by DNA ligases and bridging oligonucleotides. Double-stranded nucleic acid molecules are denatured into single-stranded molecules. The ends of the molecules are brought together by hybridization to a template. The template ensures that the two single-stranded nucleic acid molecules are aligned correctly. DNA ligase joins the two nucleic acid molecules into a single, larger, composite nucleic acid molecule. The nucleic acid molecules are subsequently denatured so that the composite molecule formed by the ligated nucleic acid molecules and the template cease to hybridize to each. Each composite molecule then serves as a template for orienting unligated, single-stranded nucleic acid molecules. After several cycles, composite nucleic acid molecules are generated from smaller nucleic acid molecules. A number of applications are disclosed for chain reaction cloning including site-specific ligation of DNA fragments generated by restriction enzyme digestion, DNAse digestion, chemical cleavage, enzymatic or chemical synthesis, and PCR amplification.
With regard to the second embodiment of the inventive process (illustrated in
In yet another embodiment of the inventive process (illustrated in
In a third embodiment of the inventive process, a plurality of oligonucleotides that can be assembled into a full length polynucleotide are synthesized on a microarray device (or beads having a solid surface) having specific cleavable linker moieties (
Specifically, in the third embodiment and as illustrated in
In alternative B, oligonucleotide sequences are connected to a microarray device through additional flanking sequence regions containing a restriction enzyme (RE) sequence site. A second oligonucleotide fragment, complementary to the flanking sequence, is hybridized to the oligonucleotides on the microarray device. This recreates a ds structure at the flanking sequence region, including the RE recognition site. Digestion of this ds DNA structure with RE enzyme specific to the RE recognition site in the flanking sequence region will release specific oligonucleotides 1 through N into a mixture solution. The oligonucleotides 1 through N are able to assemble into a polynucleotide molecule in solution.
In another example of alternative B, oligonucleotides that together assemble into the polynucleotide are synthesized on a microarray device, each having a flanking sequence on the microarray side. The flanking sequence further comprises a restriction endonuclease (RE) recognition site (see
This example illustrates assembly of 172-mer polynucleotide sequence from non-cleavable oligonucleotide sequences synthesized on a microarray device according to the first embodiment inventive process (
This example illustrates the second embodiment of the inventive process for preparing oligonucleotides for assembly into full-length polynucleotides by PCR and REII (restriction enzyme) digestion. A single oligonucleotide sequence was synthesized on a microarray device according to the procedure in Example 1 (see
This example illustrates the assembly of a 290 bp polynucleotide sequence from 9 oligonucleotide sequences, each having flanking sequences containing a MlyI restriction site. Each of the nine different oligonucleotide sequences was synthesized on a microarray device through an in situ electrochemistry process as described in example 1 herein.
The microarray device containing the nine specific oligonucleotide sequences (with flanking sequences as shown in
This example illustrates the creation of a cDNA polynucleotide sequence capable of coding on expression for fusion protein MIP-GFP-FLAG (Macrophage Inflammation Protein—Green Fluorescence Protein—FLAG peptide) using thirty-eight overlapping oligonucleotide sequences (
Number | Name | Date | Kind |
---|---|---|---|
4683195 | Mullis et al. | Jul 1987 | A |
4683202 | Mullis | Jul 1987 | A |
4800159 | Mullis et al. | Jan 1989 | A |
4965188 | Mullis et al. | Oct 1990 | A |
4999294 | Looney et al. | Mar 1991 | A |
5082767 | Hatfield et al. | Jan 1992 | A |
5093251 | Richards et al. | Mar 1992 | A |
5096825 | Barr et al. | Mar 1992 | A |
5104789 | Permar et al. | Apr 1992 | A |
5132215 | Jayarama et al. | Jul 1992 | A |
5143854 | Pirrung et al. | Sep 1992 | A |
5356802 | Chandrasegaran | Oct 1994 | A |
5395750 | Dillon et al. | Mar 1995 | A |
5405783 | Pirrung et al. | Apr 1995 | A |
5424186 | Fodor et al. | Jun 1995 | A |
5436150 | Chandrasegaran | Jul 1995 | A |
5436327 | Southern et al. | Jul 1995 | A |
5459039 | Modrich et al. | Oct 1995 | A |
5498531 | Jarrell | Mar 1996 | A |
5556750 | Modrich et al. | Sep 1996 | A |
5605793 | Stemmer | Feb 1997 | A |
5641658 | Adams et al. | Jun 1997 | A |
5661028 | Foote | Aug 1997 | A |
5667667 | Southern | Sep 1997 | A |
5679522 | Modrich et al. | Oct 1997 | A |
5695940 | Drmanac et al. | Dec 1997 | A |
5700637 | Southern | Dec 1997 | A |
5702894 | Modrich et al. | Dec 1997 | A |
5750335 | Gifford | May 1998 | A |
5766550 | Kaplan et al. | Jun 1998 | A |
5780272 | Jarrell | Jul 1998 | A |
5834233 | Molin et al. | Nov 1998 | A |
5834252 | Stemmer et al. | Nov 1998 | A |
5858754 | Modrich et al. | Jan 1999 | A |
5861482 | Modrich et al. | Jan 1999 | A |
5912129 | Vinayagamoorthy et al. | Jun 1999 | A |
5916794 | Chandrasegaran | Jun 1999 | A |
5922539 | Modrich et al. | Jul 1999 | A |
5928905 | Stemmer et al. | Jul 1999 | A |
5942609 | Hunkapiller et al. | Aug 1999 | A |
5953469 | Zhou | Sep 1999 | A |
6008031 | Modrich et al. | Dec 1999 | A |
6013440 | Lipshutz et al. | Jan 2000 | A |
6017696 | Heller | Jan 2000 | A |
6027877 | Wagner, Jr. | Feb 2000 | A |
6027910 | Klis et al. | Feb 2000 | A |
6093302 | Montgomery | Jul 2000 | A |
6110668 | Strivhov et al. | Aug 2000 | A |
6114147 | Frenken et al. | Sep 2000 | A |
6136568 | Hiatt et al. | Oct 2000 | A |
6143527 | Pachuk et al. | Nov 2000 | A |
6150102 | Mills, Jr. et al. | Nov 2000 | A |
6150141 | Jarrell | Nov 2000 | A |
6165793 | Stemmer et al. | Dec 2000 | A |
6261797 | Sorge et al. | Jul 2001 | B1 |
6271957 | Quate et al. | Aug 2001 | B1 |
6277632 | Harney | Aug 2001 | B1 |
6280595 | Montgomery | Aug 2001 | B1 |
6284463 | Hasebe et al. | Sep 2001 | B1 |
6287825 | Weissman et al. | Sep 2001 | B1 |
6287861 | Stemmer et al. | Sep 2001 | B1 |
6291242 | Stemmer et al. | Sep 2001 | B1 |
6315958 | Singh-Gasson et al. | Nov 2001 | B1 |
6322971 | Chetverin et al. | Nov 2001 | B1 |
6326489 | Church et al. | Dec 2001 | B1 |
6333153 | Fishel et al. | Dec 2001 | B1 |
6346399 | Weissman et al. | Feb 2002 | B1 |
6355412 | Stewart et al. | Mar 2002 | B1 |
6358712 | Jarrell et al. | Mar 2002 | B1 |
6365355 | McCutchen-Maloney | Apr 2002 | B1 |
6372429 | Sharon | Apr 2002 | B1 |
6372434 | Weissman et al. | Apr 2002 | B1 |
6375903 | Cerrina et al. | Apr 2002 | B1 |
6376246 | Crameri et al. | Apr 2002 | B1 |
6406847 | Cox et al. | Jun 2002 | B1 |
6410220 | Hodgson et al. | Jun 2002 | B1 |
6426184 | Gao et al. | Jul 2002 | B1 |
6444111 | Montgomery | Sep 2002 | B1 |
6444175 | Singh-Gasson et al. | Sep 2002 | B1 |
6472184 | Hegemann | Oct 2002 | B1 |
6480324 | Quate et al. | Nov 2002 | B2 |
6489146 | Stemmer et al. | Dec 2002 | B2 |
6495318 | Harney | Dec 2002 | B2 |
6509156 | Stewart et al. | Jan 2003 | B1 |
6521427 | Evans | Feb 2003 | B1 |
6586211 | Stahler et al. | Jul 2003 | B1 |
6593111 | Baric et al. | Jul 2003 | B2 |
6650822 | Zhou | Nov 2003 | B1 |
6664112 | Mulligan et al. | Dec 2003 | B2 |
6670127 | Evans | Dec 2003 | B2 |
6897025 | Cox et al. | May 2005 | B2 |
6921636 | Brennan | Jul 2005 | B1 |
6921818 | Sproat | Jul 2005 | B2 |
6936470 | Liang et al. | Aug 2005 | B2 |
6946296 | Patten et al. | Sep 2005 | B2 |
6955883 | Margus et al. | Oct 2005 | B2 |
6969587 | Taylor | Nov 2005 | B2 |
6969847 | Davis et al. | Nov 2005 | B2 |
20010031483 | Sorge et al. | Oct 2001 | A1 |
20010049125 | Stemmer et al. | Dec 2001 | A1 |
20020012616 | Zhou et al. | Jan 2002 | A1 |
20020058275 | Fishel et al. | May 2002 | A1 |
20020081582 | Gao et al. | Jun 2002 | A1 |
20020132259 | Wagner, Jr. et al. | Sep 2002 | A1 |
20030017552 | Jarrell et al. | Jan 2003 | A1 |
20030044980 | Mancebo et al. | Mar 2003 | A1 |
20030050437 | Montgomery | Mar 2003 | A1 |
20030050438 | Montgomery | Mar 2003 | A1 |
20030068633 | Belshaw et al. | Apr 2003 | A1 |
20030068643 | Brennan et al. | Apr 2003 | A1 |
20030087298 | Green et al. | May 2003 | A1 |
20030091476 | Zhou et al. | May 2003 | A1 |
20030099952 | Green et al. | May 2003 | A1 |
20030118485 | Singh-Gasson et al. | Jun 2003 | A1 |
20030118486 | Zhou et al. | Jun 2003 | A1 |
20030120035 | Gao et al. | Jun 2003 | A1 |
20030134807 | Hardin et al. | Jul 2003 | A1 |
20030140255 | Ricchetti et al. | Jul 2003 | A1 |
20030143550 | Green et al. | Jul 2003 | A1 |
20030143724 | Cerrina et al. | Jul 2003 | A1 |
20030171325 | Gascoyne et al. | Sep 2003 | A1 |
20030186226 | Brennan et al. | Oct 2003 | A1 |
20030186233 | Chesnut et al. | Oct 2003 | A1 |
20030198948 | Stahler et al. | Oct 2003 | A1 |
20030215856 | Church et al. | Nov 2003 | A1 |
20030224521 | Court et al. | Dec 2003 | A1 |
20040005673 | Jarrell et al. | Jan 2004 | A1 |
20040009520 | Albert et al. | Jan 2004 | A1 |
20040014083 | Yuan et al. | Jan 2004 | A1 |
20040092016 | Court et al. | May 2004 | A1 |
20040096891 | Bennett | May 2004 | A1 |
20040101444 | Sommers et al. | May 2004 | A1 |
20040101894 | Albert et al. | May 2004 | A1 |
20040101949 | Green et al. | May 2004 | A1 |
20040110211 | McCormick et al. | Jun 2004 | A1 |
20040110212 | McCormick et al. | Jun 2004 | A1 |
20040126757 | Cerrina | Jul 2004 | A1 |
20040132029 | Sussman et al. | Jul 2004 | A1 |
20040166567 | Santi et al. | Aug 2004 | A1 |
20040224336 | Wagner | Nov 2004 | A1 |
20040259146 | Friend et al. | Dec 2004 | A1 |
20040259256 | Monahan et al. | Dec 2004 | A1 |
20050053979 | Livak et al. | Mar 2005 | A1 |
20050053997 | Evans | Mar 2005 | A1 |
20050079618 | Court et al. | Apr 2005 | A1 |
20050106606 | Parker et al. | May 2005 | A1 |
20050112631 | Piepenburg et al. | May 2005 | A1 |
20050118628 | Evans | Jun 2005 | A1 |
20050196760 | Pemov et al. | Sep 2005 | A1 |
20050196865 | Frazer | Sep 2005 | A1 |
20050208503 | Yowanto et al. | Sep 2005 | A1 |
20050208536 | Schultz et al. | Sep 2005 | A1 |
20050221340 | Evans | Oct 2005 | A1 |
20050227235 | Carr et al. | Oct 2005 | A1 |
20050240352 | Liang | Oct 2005 | A1 |
20050255477 | Carr et al. | Nov 2005 | A1 |
20060008833 | Jacobson | Jan 2006 | A1 |
20060024733 | Wong et al. | Feb 2006 | A1 |
20070269870 | Church et al. | Nov 2007 | A1 |
Number | Date | Country |
---|---|---|
199 07 080.6 | Feb 1999 | DE |
0259160 | Mar 1988 | EP |
0385410 | Sep 1990 | EP |
1 180 548 | Feb 2002 | EP |
WO-9000626 | Jan 1990 | WO |
WO-9627025 | Sep 1996 | WO |
WO-9633207 | Oct 1996 | WO |
WO-9941007 | Aug 1999 | WO |
WO-0134847 | May 2001 | WO |
WO-0202227 | Jan 2002 | WO |
WO-0204597 | Jan 2002 | WO |
WO-0204680 | Jan 2002 | WO |
WO-0244425 | Jun 2002 | WO |
WO-02072791 | Sep 2002 | WO |
WO-02081490 | Oct 2002 | WO |
WO-02095073 | Nov 2002 | WO |
WO-02103446 | Dec 2002 | WO |
WO-03040410 | May 2003 | WO |
WO-03046223 | Jun 2003 | WO |
WO-0354232 | Jul 2003 | WO |
WO-03064026 | Aug 2003 | WO |
WO-03064027 | Aug 2003 | WO |
WO-03064699 | Aug 2003 | WO |
WO-03065038 | Aug 2003 | WO |
WO-03066212 | Aug 2003 | WO |
WO-03072832 | Sep 2003 | WO |
WO-03085094 | Oct 2003 | WO |
WO-03100012 | Dec 2003 | WO |
WO-2004024886 | Mar 2004 | WO |
WO-2004029586 | Apr 2004 | WO |
WO-2004031351 | Apr 2004 | WO |
WO-2004031399 | Apr 2004 | WO |
WO-2004039953 | May 2004 | WO |
WO-2005089110 | Sep 2005 | WO |
Number | Date | Country | |
---|---|---|---|
20060035218 A1 | Feb 2006 | US |