High-throughput gene synthesis method based on digital micro-fluidic chip

Abstract

The invention provides a high-throughput gene synthesis method based on a digital micro-fluidic chip, and belongs to the technical field of biological chips and gene synthesis. The chip comprises an upper polar plate and a lower polar plate, the surface of the upper polar plate is spin-coated with a hydrophobic layer and is modified with a gene array, the upper polar plate and the lower polar plate form the bipolar plate digital micro-fluidic chip, and liquid drops are controlled to move based on an EWOD technology. Synthesis of high-throughput genes by using the high-throughput gene synthesis chip based on the digital microfluidic system comprises the steps of Oligo pool capturing, cleaning, enzyme digestion reaction, gene synthesis and product amplification. The gene array chip based on the digital micro-fluidic system is designed, the gene array chip is used for improving the flux of gene synthesis, improving the synthesis efficiency and reducing the synthesis cost, non-specific adsorption of enzyme and the surface of the hydrophobic layer is avoided in the synthesis process, and the application range of the digital micro-fluidic chip in the field of gene synthesis is widened.

Description

Technical Field

[0001] The invention belongs to the technical field of biochip and gene synthesis, and in particular relates to a high-throughput gene synthesis method based on a digital microfluidic chip. Background Art

[0002] Currently, the length of long DNA fragments synthesized from scratch is limited, and longer genes or genomes need to be assembled from short oligonucleotide fragments. Commonly used long-fragment DNA assembly methods include ligase chain reaction (LCR) and polymerase cycling assembly (PCA). Regardless of which method is used, traditional synthesis methods require a large amount of reagents and labor costs, which leads to high synthesis costs for long-fragment DNA. Gene chips, also known as DNA arrays or DNA chips, are special glass or silicon-based chips with DNA microarrays. A large number of nucleic acid probes are modified on a tiny area, which can be combined with free DNA, cDNA, RNA, etc., and are used for the capture, detection, and synthesis of target genes. They have the advantages of being fast, accurate, and low-cost.

[0003] Digital microfluidic chips are a new type of micro-droplet manipulation platform, usually developed based on EWOD technology. They consist of two upper and lower plates. By controlling the closure of the electrodes to change the contact angle of the droplets, the micro-droplets can be manipulated to move, split, merge, and other controls. Digital microfluidic chips do not require external pumps or valves, and have the ability to accurately manipulate droplets in the picoliter to microliter range. They are programmable droplet path control and have a high degree of manipulation flexibility. Therefore, digital microfluidic chips are very suitable for micro-droplet manipulation in various biochemical reactions and are widely used in the field of biomedicine.

[0004] The current gene synthesis still has problems such as high synthesis cost, low synthesis flux, long synthesis time, and low degree of automation. Biochips based on digital microfluidics technology can improve synthesis efficiency and reduce synthesis costs, and are an ideal platform for gene synthesis. With the development of micro-nano processing technology and microfluidics technology, the research on digital microfluidic chips has made continuous breakthroughs. However, the current digital microfluidic chips and synthesis methods for applications such as gene synthesis and splicing still have problems such as difficulty in increasing flux and high costs. The main reasons are the high cost of chip consumables and low sample processing volume. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes a high-throughput gene synthesis method based on a digital microfluidic chip.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] One of the technical solutions of the present invention:

[0008] A high-throughput gene synthesis chip based on a digital microfluidic system comprises an upper plate and a lower plate, wherein the upper plate and the lower plate form a bipolar plate digital microfluidic chip, and the movement of droplets is controlled based on the EWOD (electrowetting on dielectric) technology. The surface of the upper plate is spin-coated with a hydrophobic layer and modified with a gene array.

[0009] Furthermore, the lower electrode plate may be a digital microfluidic chip electrode array in any form, which includes a substrate, an electrode layer, a dielectric layer and a hydrophobic layer from bottom to top.

[0010] By switching two adjacent electrodes, the contact angle of the droplet changes under the dielectric wetting effect, and the area above the open electrode becomes hydrophilic, thereby controlling the droplet to move from the hydrophobic area to the hydrophilic area to complete the transfer. The upper electrode provides a closed chamber for the droplet to move, avoid contamination, and enable the droplet to split. At the same time, the upper electrode also serves as a gene chip of the system, with a barcode modified on the surface to capture free oligos.

[0011] Furthermore, the upper plate comprises an upper plate substrate, a hydrophobic layer loaded on the surface of the upper plate substrate, and a gene modification site embedded in the hydrophobic layer, and the preparation method comprises the following steps:

[0012] After cleaning and drying the upper plate substrate, the substrate is fixed on a high-speed spin coater, a hydrophobic material is dripped onto the surface of the substrate, and the hydrophobic material is rapidly thinned and evenly coated on the surface of the substrate by high-speed spin coating. After the spin coating is completed, a hydrophobic layer is prepared, a mask with array micropores is placed above the hydrophobic layer, and plasma treatment is performed to remove the hydrophobic layer at the through holes of the mask to expose the upper plate substrate, thereby obtaining an upper plate substrate with a local hydrophilic area on the surface, modifying chemical groups at the local hydrophilic points of the local hydrophilic areas of the upper plate substrate by covalent connection, obtaining a substrate with a gene array modification point, and incubating and connecting the barcode sequence to the gene array modification point to obtain the upper plate.

[0013] Furthermore, the material of the upper electrode substrate is a smooth and transparent material such as glass, silicon wafer, PMMA (polymethyl methacrylate), PC (polycarbonate), etc., preferably glass.

[0014] Furthermore, the upper plate substrate cleaning step may be acetone-isopropanol-anhydrous ethanol-pure water in sequence. If the upper plate substrate is made of a material that is not resistant to organic solvents, such as PMMA, the acetone cleaning step is removed.

[0015] Furthermore, after the upper plate substrate is cleaned, it is blown dry with nitrogen and baked in an oven or hot plate until the moisture is completely removed. The dried upper plate substrate material is treated with oxygen plasma to further clean stains on the surface of the substrate and activate the functional groups on the surface of the substrate to enhance the cross-linking effect between the hydrophobic layer and the surface of the substrate.

[0016] Preferably, the power of the oxygen plasma treatment is 200W to 300W, and the treatment time is 5 to 10 minutes. Preferably, the power is 300W, and the treatment time is 5 minutes.

[0017] Furthermore, the hydrophobic material is amorphous fluororesin with a concentration of 0.5wt.% to 2wt.%; and / or,

[0018] The spin coating speed is 2000-4000 rpm / min; and / or,

[0019] The thickness of the hydrophobic layer is 30-200 nm, and the thickness of the hydrophobic layer is controlled by controlling the concentration of the hydrophobic material and the rotation speed.

[0020] Preferably, the spin coating speed is 4000 rpm / min.

[0021] Preferably, the hydrophobic material is amorphous fluororesin; and / or,

[0022] The hydrophobic material is amorphous fluororesin such as Teflon AF1600, Teflon AF2400, CYTOP (fluoropolymer), FluoroPelPFC (a fluoropolymer solution produced by Cytonix LLC); and / or,

[0023] The mask is made of non-metallic materials such as glass, silicon wafer, PMMA (polymethyl methacrylate), PEEK (polyetheretherketone), PC (polycarbonate), PET (polyethylene terephthalate), and PI (polyimide).

[0024] Preferably, the hydrophobic material is Teflon AF1600 with a concentration of 2 wt.%.

[0025] Preferably, the mask is made of glass.

[0026] Furthermore, after the spin coating is completed, the substrate is placed on a hot plate at 180° C. and baked for 2 h to completely evaporate the solvent in the hydrophobic material.

[0027] The number of microwell arrays is the system synthesis flux, which can be adjusted according to the size of the digital microfluidic chip to match common well plate consumables. Furthermore, the number of unit arrays of the microwell array on the mask is 96, the unit array is 3×3, the microwell diameter is 0.1-0.2mm, and the microwell spacing is 0.5mm.

[0028] A micropore diameter that is too small will reduce the area of ​​the modification region, resulting in insufficient barcode (nucleotide sequence) modification. A micropore diameter that is too large will make the local hydrophilic area on the hydrophobic layer too large, causing droplet top-up during EWOD pipetting, affecting the pipetting effect.

[0029] Furthermore, the micropore spacing is such that during the plasma treatment, the micropore array on the mask will expose the hydrophobic layer underneath, and the exposed hydrophobic layer will be removed under the action of plasma, thereby transferring the pattern on the mask to the substrate. While etching the hydrophobic layer, the plasma will also activate the substrate surface, thereby introducing functional groups on the surface and turning the substrate surface into a hydrophilic area.

[0030] Furthermore, a mask with array micropores is placed on the hydrophobic layer for plasma treatment, the power and time of which depend on the thickness of the hydrophobic layer. For example, a plasma cleaning machine with a power of 50-300W is required for 1-5 minutes to treat a 1μm thick hydrophobic layer.

[0031] Furthermore, the covalent connection method is to graft 3-aminopropyltriethoxysilane (APTES) to the local hydrophilic points of the local hydrophilic area of ​​the upper plate substrate by covalent connection, and introduce streptavidin as an intermediate carrier to connect the streptavidin to the APTES on the surface of the substrate.

[0032] Furthermore, in order to prevent non-specific adsorption of streptavidin to the surface of the hydrophobic layer, 0.2% (v / v) Tween 20 surfactant was added to the streptavidin solution.

[0033] Furthermore, a filling medium is included between the upper plate and the lower plate. The filling medium between the upper and lower plates will also affect the pipetting effect and nonspecific adsorption. Preferably, the filling medium includes one or more of dimethyl silicone oil, dodecamethylpentasiloxane, n-hexadecane and n-dodecane.

[0034] The second technical solution of the present invention:

[0035] A high-throughput gene synthesis method based on a digital microfluidic chip utilizes the high-throughput gene synthesis chip based on a digital microfluidic system to synthesize high-throughput genes, including the steps of oligo pool (oligonucleotide pool) capture, cleaning, enzyme digestion reaction, gene synthesis and product amplification.

[0036] Furthermore, the method specifically includes the following steps:

[0037] The barcode sequence with biotin (here streptavidin) modification at the end is incubated and connected to the array modification point of the high-throughput gene synthesis chip based on the digital microfluidics system, and the oligo pool droplets are split and moved to the corresponding modified gene site by EWOD. After incubation, the barcode sequence captures the target gene fragment, and the oligo pool droplets after capture are removed, and the cleaning solution is moved to the modification site to clean the residual oligos, and then the cleaning solution is removed and the reaction reagent is moved to the modification gene site, and the oligos are cut off from the upper plate substrate and freed in the reaction reagent by enzyme cleavage reaction, and then gene splicing and error correction are performed in the reaction reagent to obtain the target product, and the splicing of oligos is completed. Part of the product is taken from the reaction reagent droplet containing the target product for PCR amplification to obtain the required DNA long chain.

[0038] Furthermore, the oligo pool used in the present invention is a mixed sample composed of multiple DNA sequences. The process of high-throughput gene synthesis uses multiple oligos to construct a target DNA sequence as a product output. The barcode used is a custom DNA sequence, usually controlled at 5-30bp in length, for the purpose of capturing the target sequence in the free oligo pool. A DNA complementary to the modified barcode will be set on the target sequence, and its sequence and length depend on the target gene sequence, and usually consider factors such as GC ratio, secondary structure, and similar sequence.

[0039] Furthermore, during oligo pool capture, the dielectric wetting force generated by the high-throughput gene synthesis chip based on the digital microfluidics system controls the oligo pool droplets to split and move to the corresponding modified gene sites, so that the droplets completely cover the modified gene sites. The oligo pool is heated to the annealing temperature and incubated for a period of time to allow the barcode array in the microwells to fully capture the target oligos. At this time, the barcode in each unit array captures the oligos that match it.

[0040] Furthermore, the oligos remaining at the reaction sites will seriously affect the specificity of the synthesis. Therefore, the irrelevant oligos adsorbed and remaining at the modification sites must be removed, and the cleaning droplets containing surfactants are moved to each modification site. The oligos remaining on the surface are eluted by repeated movement, and this step is repeated more than three times.

[0041] Furthermore, the cleaning solution comprises NaCl, EDTA, Tris-HCl and Tween 20; and / or,

[0042] The reaction reagents include restriction endonucleases, DNA ligases, dNTPs (deoxynucleotide triphosphates), BSA (bovine serum albumin) and buffers. Furthermore, the reaction reagents may also include error correction enzymes. The amount and type of enzymes are adjusted according to the needs of synthesis, wherein the dNTP concentration is 10 to 50 mM, and the BSA concentration is 0.5 to 1 mg / mL. Preferably, the dNTP concentration is 10 mM, and the BSA concentration is 0.5 mg / mL.

[0043] Furthermore, under the action of temperature, the restriction endonuclease cuts the oligos from the upper plate substrate, making them free in the reaction reagent, and then the reaction reagent is circulated and temperature-controlled, so that the oligos in the reaction reagent are spliced ​​and error-corrected under the action of DNA ligase, and the target product can be obtained after the splicing is completed.

[0044] Furthermore, the cleaning solution contains components with the following concentrations: 2M NaCl, 1mM EDTA, 10mM Tris-HCl and 0.05% (v / v) Tween20.

[0045] The various enzymes and proteins contained in the reaction reagents will non-specifically adsorb to the hydrophobic layer, which is more obvious at high temperatures, causing the surface of the hydrophobic layer to become hydrophilic, making the EWOD pipetting ineffective. In order to solve this problem, the present invention adjusts the buffer composition so that the reaction reagents will not non-specifically adsorb to the hydrophobic layer even at high temperatures. Preferably, the buffer comprises Tris-HCl, MgCl2, DTT (dithiothreitol), PEG8000 (polyethylene glycol, average molecular weight is about 8000) and Tween20; more preferably, the buffer comprises the following components in concentration: 10-250mM Tris-HCl, 50-150mM MgCl2, 50-100mM DTT, 10-100mM PEG8000 and 0.05-0.2% (v / v) Tween20.

[0046] Preferably, the buffer comprises components in the following concentrations: 10 mM Tris-HCl, 50 mM MgCl2, 50 mM DTT, 10 mM PEG8000 and 0.05% (v / v) Tween20.

[0047] Compared with the prior art, the present invention has the following advantages and technical effects:

[0048] The present invention designs a gene array chip based on a digital microfluidic system. Compared with the currently available commercial products, which can only process a gene sample throughput of 8 groups of genes at the same time, the present invention uses the gene array chip to construct no less than 96 groups of gene sequences within 3 hours, and is expected to further increase the throughput by increasing the chip area to improve the synthesis efficiency and reduce the synthesis cost. The synthesis process avoids nonspecific adsorption of enzymes and the hydrophobic layer surface, thereby improving the application scope of digital microfluidic chips in the field of gene synthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The illustrative embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application. In the drawings:

[0050] Figure 1 A schematic diagram of the structure of a high-throughput gene synthesis chip based on a digital microfluidic system prepared in Example 1;

[0051] Figure 2 This is a schematic diagram of the product structure obtained in step (1) during the preparation of a high-throughput gene synthesis chip based on a digital microfluidic system in Example 1;

[0052] Figure 3 Schematic diagram of the process of preparing a high-throughput gene synthesis chip based on a digital microfluidic system in Example 1, step (2);

[0053] Figure 4 This is a schematic diagram of the product structure obtained in step (2) during the preparation of a high-throughput gene synthesis chip based on a digital microfluidic system in Example 1;

[0054] Figure 5 This is a schematic diagram of the product structure obtained in step (4) during the preparation of a high-throughput gene synthesis chip based on a digital microfluidic system in Example 1;

[0055] Figure 6 Schematic diagram of the high-throughput gene synthesis process based on digital microfluidic chip in Application Example 1;

[0056] Figure 7 A top view of the high-throughput gene synthesis chip based on the digital microfluidic system prepared in Example 1;

[0057] Figure 8 A schematic diagram of the process of capturing oligos at local modification points of a high-throughput gene synthesis chip based on a digital microfluidic system prepared in Example 1;

[0058] Fig. 9 This is a schematic diagram of the process of preparing target long-chain DNA using a high-throughput gene synthesis chip based on a digital microfluidics system prepared in Example 1.

[0059] Among them, 1-upper plate substrate; 2-gene modification site; 3-hydrophobic layer; 4-dielectric layer; 5-electrode layer; 6-substrate; 7-droplet; 8-microwell array mask; 9-plasma; 10-barcode; 11-oligos; 12-long-chain DNA; 7-a-oligopool droplet; 7-b-reaction reagent droplet;

[0060] Fig.10 This is the characterization result of the final product of long-chain DNA synthesized by the high-throughput gene synthesis chip based on the digital microfluidic system in Example 1 in Application Example 1. DETAILED DESCRIPTION

[0061] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0062] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0063] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0064] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0065] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0066] The raw materials used in the embodiments of the present invention are all commercially available.

[0067] The technical solution of the present invention is further illustrated by the following embodiments.

[0068] Example 1

[0069] A high-throughput gene synthesis chip based on a digital microfluidic system, the structural diagram is shown in Figure 1 , wherein 1-upper plate substrate; 2-gene modification site; 3-hydrophobic layer; 4-dielectric layer; 5-electrode layer; 6-substrate; 7-droplet; the upper plate includes an upper plate substrate 1, a hydrophobic layer 3 loaded on the surface of the upper plate substrate 1 and a gene modification site 2 embedded in the hydrophobic layer 3, and the lower plate includes a substrate 6, an electrode layer 5, a dielectric layer 4 and a hydrophobic layer 3 from bottom to top. The upper plate and the lower plate form a bipolar plate digital microfluidic chip, which controls the movement of droplets based on EWOD technology. By switching two adjacent electrodes, the contact angle of the droplets changes under the dielectric wetting effect, and the area above the opened electrode becomes hydrophilic, thereby controlling the droplets to move from the hydrophobic area to the hydrophilic area to complete the transfer. The upper plate provides a closed chamber for the movement of droplets to avoid contamination and enable the droplets to split. At the same time, the upper plate also serves as a gene chip of the system, and is modified with a barcode on the surface to capture free oligos.

[0070] A method for preparing a high-throughput gene synthesis chip based on a digital microfluidic system comprises the following steps:

[0071] (1) The upper plate substrate (glass) was cleaned with acetone, isopropanol, anhydrous ethanol and pure water in sequence. After cleaning, it was blown dry with nitrogen and baked in an oven until the moisture was completely removed. The dried upper plate substrate material was treated with oxygen plasma (power of 300 W, treatment time of 5 min) to further clean the stains on the substrate surface. Then, the substrate was fixed on a high-speed spin coater, and a hydrophobic material (Teflon AF1600, concentration of 2 wt.%) was added dropwise on the substrate surface. At a rotation speed of 4000 rpm / min, the hydrophobic material was quickly thinned and evenly coated on the substrate surface by high-speed spin coating. After spin coating, it was placed on a hot plate at 180°C and baked for 2 h to prepare a hydrophobic layer with a thickness of 200 nm. The schematic diagram of the prepared product structure is shown in FIG. Figure 2 , wherein 1-upper plate substrate; 3-hydrophobic layer.

[0072] (2) Place a mask with array micropores (96 cells in the cell array, 3×3 cells, a micropore diameter of 0.1 mm, and a micropore spacing of 0.5 mm) on top of the hydrophobic layer and perform plasma treatment (power of 300 W, treatment time of 5 min). See the flow diagram of this step. Figure 3 , wherein 1-upper plate substrate; 3-hydrophobic layer; 8-mask with array micropores; 9-plasma, the schematic diagram of the prepared substrate-hydrophobic layer structure is shown in Figure 4 , wherein 1-upper plate substrate; 3-hydrophobic layer.

[0073] (3) The hydrophobic layer at the through hole of the mask was removed to expose the upper electrode substrate, and the upper electrode substrate with a local hydrophilic area on the surface was obtained. APTES, isopropanol and pure water were prepared into a solution at a volume ratio of 1:39:10, and the solution was dripped onto the local hydrophilic point of the local hydrophilic area of ​​the upper electrode substrate. After standing at room temperature for 15 minutes, the solution was washed with pure water. At this time, the APTES grafting of the modified point substrate was completed. Streptavidin (SA) was dissolved in PBS solution (pH = 7) at a concentration of 1 mg / mL, and 0.1 % (v / v) Tween20 to prevent nonspecific adsorption with the hydrophobic layer, prepare SA-PBS solution, add the SA-PBS solution dropwise to the modified area, let it stand at 4°C for 2 hours to allow streptavidin and APTES to connect. After the modification is completed, the substrate surface is washed three times with PBS (pH=7) solution containing 0.1% (v / v) Tween20 to completely remove the residual streptavidin. At this time, the chemical modification of the substrate surface is completed, and the barcode gene can be modified.

[0074] (4) 1 pmol / ul of barcode reagent was dripped onto the modified area to completely cover the modified area. Different barcode solutions were prevented from cross contamination. After standing at 4°C for 1 hour, the substrate surface was cleaned three times with a cleaning solution (2M NaCl, 1mM EDTA, 10mM Tris-HCl) containing 0.2% (v / v) Tween20, and then cleaned with pure water and dried to obtain a high-throughput gene synthesis chip based on a digital microfluidic system. The schematic diagram of the structure is shown in Figure 5 , wherein 1-upper plate substrate; 3-hydrophobic layer;

[0075] On the APTES substrate surface, a substrate with gene array modification points is obtained. The schematic diagram of the structure is shown in Figure 5 , wherein 1-upper plate substrate; 2-gene modification site; 3-hydrophobic layer; 10-barcode.

[0076] Application Example 1

[0077] A high-throughput gene synthesis method based on a digital microfluidic chip, using the high-throughput gene synthesis chip based on a digital microfluidic system to synthesize high-throughput genes, including the steps of oligo pool capture, cleaning, enzyme digestion reaction, gene synthesis and product amplification, specifically including the following steps (see the flowchart for a schematic diagram) Figure 6 ):

[0078] The barcode sequence modified with biotin (here streptavidin) at the end is incubated and connected to the array modification point of the high-throughput gene synthesis chip based on the digital microfluidic system prepared in Example 1, and the oligo pool droplet is split and moved to the corresponding modified gene site by EWOD. After incubation, the barcode sequence captures the target gene fragment, and the oligo pool droplet after capture is removed, and the cleaning solution is moved to the modification site to clean the residual oligos. This step is repeated more than three times, and then the cleaning solution is removed and the reaction reagent is moved to the modified gene site. The restriction endonuclease cuts the oligos from the upper plate substrate to make it free in the reaction reagent, and then the reaction reagent is heated or cooled by a metal semiconductor for cyclic temperature control, so that the oligos in the reaction reagent are spliced ​​and error-corrected under the action of DNA ligase. After the splicing is completed, the target product can be obtained, and the splicing of the oligos is completed. Part of the product is taken from the reaction reagent droplet containing the target product for PCR amplification to obtain the desired DNA long chain;

[0079] The cleaning solution used contained the following components: 2M NaCl, 1mM EDTA, 10mM Tris-HCl and 0.05% (v / v) Tween20;

[0080] The reaction reagents used included 10 U restriction endonuclease, 2 U DNA ligase, 10 mM dNTP, 0.5 mg / mL BSA and buffer (10 mM Tris-HCl, 50 mM MgCl2, 50 mM DTT, 10 mM PEG8000 and 0.05% (v / v) Tween20).

[0081] The long-chain DNA sequences prepared according to Application Example 1 are shown in SEQ ID NO: 1 to SEQ ID NO: 150.

[0082] AAGACCCCACACCATTGAGCAGTGATCGCTTCCATGACGCAGAAGTTAACACTTTC GGATATTTCTGATGAGTCGAAAAATTATCTTGATAAAGCAGGAATTACTACTGCTTGTTTA CGAA(SEQ ID NO:1)

[0083] TTCGTAAACAAGCAGTAGTAATTCCTGCTTTATCAAGATAATTTTTCGACTCATCAGA AATATCCGAAAGTGTTAACTTCTGCGTCATGGAAGCGATCACTGC(SEQ ID NO:2)

[0084] AAGACCCCACACCATTGAGCAGTGAAGAGCTTCTCGAGCTGCGCAAGGATAGGTC GAATTTTCTCATTTTCCGCCAGCAGTCCACTTCGATTTAATTCGTAAACAAGCAGTAGT (SEQ ID NO: 3)

[0085] ACTACTGCTTGTTTACGAATTAAATCGAAGTGGACTGCTGGCGGAAAATGAGAAAA TTCGACCTATCCTTGCGCAGCTCGAGAAGCTCTTCACTGC(SEQ ID NO:4)

[0086] AAGACCCCACACCATTGAGCAGTGCGCAGCTCGAGAAGCTCTTACTTTGCGACCTT TCGCCATCAACTAACGATTCTGTCAAAAACTGACGCGTTGGATGAGGAGAAGTGGCTT (SEQ ID NO: 5)

[0087] AAGCCACTTCTCCTCATCCAACGCGTCAGTTTTTGACAGAATCGTTAGTTGATGGCG AAAGGTCGCAAAGTAAGAGCTTCTCGAGCTGCGCACTGC(SEQ ID NO:6)

[0088] AAGACCCCACACCATTGAGCAGTGAGAGAATCTCTACCATGAACAAAATGTGACTC ATATCTAAACCAGTCCTTGACGAACGTGCCAAGCATATTAAGCCACTTCTCCTCATCC(SEQ ID NO:7)

[0089] GGATGAGGAGAAGTGGCTTAATATGCTTGGCACGTTCGTCAAGGACTGGTTTAGATATGAGTCACATTTTGTTCATGGTAGAGATTCTCTCACTGC(SEQ ID NO:8)

[0090] AAGACCCCACACCATTGAGCAGTGTTCATGGTAGAGATTCTCTTGTTGACATTTTAAAAGAGCGTGGATTACTATCTGAGTCCGATGCTGTTCAACCACTAATAGGTAAGAAAT(SEQ ID NO:9)

[0091] ATTTCTTACCTATTAGTGGTTGAACAGCATCGGACTCAGATAGTAATCCACGCTCTTTTAAAATGTCAACAAGAGAATCTCTACCATGAACACTGC(SEQ ID NO:10)

[0092] AAGACCCCACACCATTGAGCAGTGACGGCAGAAGCCTGAATGAGCTTAATAGAGGCCAAAGCGGTCTGGAAACGTACGGATTGTTCAGTAACTTGACTCATGATTTCTTACCTATTAGTGG(SEQ ID NO:11)

[0093] CCACTAATAGGTAAGAAATCATGAGTCAAGTTACTGAACAATCCGTACGTTTCCAGACCGCTTTGGCCTCTATTAAGCTCATTCAGGCTTCTGCCGTCACTGC(SEQ ID NO:12)

[0094] AAGACCCCACACCATTGAGCAGTGTCATTCAGGCTTCTGCCGTTTTGGATTTAACCGAAGATGATTTCGATTTTCTGACGAGTAACAAAGTTTGGATTGCTACTGACCGCTCTCGTGCTCG(SEQ ID NO:13)

[0095] CGAGCACGAGAGCGGTCAGTAGCAATCCAAACTTTGTTACTCGTCAGAAAATCGAAATCATCTTCGGTTAAATCCAAAACGGCAGAAGCCTGAATGACACTGC(SEQ ID NO:14)

[0096] AAGACCCCACACCATTGAGCAGTGTAAACTCAACAGGAGCAGGAAAGCGAGGGTATCCCACAAAGTCCAGCGTACCATAAACGCAAGCCTCAACGCAGCGACGAGCACGAGAGCGGTCAG(SEQ ID NO:15)

[0097] CTGACCGCTCTCGTGCTCGTCGCTGCGTTGAGGCTTGCGTTTATGGTACGCTGGACTTTGTGGGATACCCTCGCTTTCCTGCTCCTGTTGAGTTTACACTGC(SEQ ID NO:16)

[0098] GCTGTGGGGAACTTGACAGCAGTGCCTGCTCCTGTTGAGTTTATTGCTGCCGTCATTGCTTATTATGTTCATCCCGTCAACATTCAAACGGCCTGTCTCATCATGGAAGGCGCTG(SEQ ID NO:17)

[0099] CAGCGCCTTCCATGATGAGACAGGCCGTTTGAATGTTGACGGGATGAACATAATAAGCAATGACGGCAGCAATAAACTCAACAGGAGCAGGCACTGC(SEQ ID NO:18)

[0100] GCTGTGGGGAACTTGACAGCAGTGTACACGCAAGGTAAACGCGAACAATTCAGCGGCTTTAACCGGACGCTCGACGCCATTAATAATGTTTTCCGTAAATTCAGCGCCTTCCATGATGAG(SEQ ID NO:19)

[0101] CTCATCATGGAAGGCGCTGAATTTACGGAAAACATTATTAATGGCGTCGAGCGTCCGGTTAAAGCCGCTGAATTGTTCGCGTTTACCTTGCGTGTACACTGC(SEQ ID NO:20)

[0102] GCTGTGGGGAACTTGACAGCAGTGCGCGTTTACCTTGCGTGTACGCGCAGGAAACACTGACGTTCTTACTGACGCAGAAGAAAACGTGCGTCAAAAATTACGTGCGGAAGGAGTGATGTA(SEQ ID NO:21)

[0103] TACATCACTCCTTCCGCACGTAATTTTTGACGCACGTTTTCTTCTGCGTCAGTAAGAACGTCAGTGTTTCCTGCGCGTACACGCAAGGTAAACGCGCACTGC(SEQ ID NO:22)

[0104] GCTGTGGGGAACTTGACAGCAGTGTTACGCTTGCCTTTAGTACCTCGCAACGGCTGCGGACGACCAGGGCGAGCGCCAGAACGTTTTTTACCTTTAGACATTACATCACTCCTTCCGCAC(SEQ ID NO:23)

[0105] GTGCGGAAGGAGTGATGTAATGTCTAAAGGTAAAAAACGTTCTGGCGCTCGCCCTGGTCGTCCGCAGCCGTTGCGAGGTACTAAAGGCAAGCGTAACACTGC(SEQ ID NO:24)

[0106] GCTGTGGGGAACTTGACAGCAGTGGTACTAAAGGCAAGCGTAAAGGCGCTCGTCTTTGGTATGTAGGTGGTCAACAATTTTAATTGCAGGGGCTTCGGCCCCTTACTTGAGGATAAATT(SEQ ID NO:25)

[0107] AATTTATCCTCAAGTAAGGGGCCGAAGCCCCTGCAATTAAAATTGTTGACCACCTACATACCAAAGACGAGCGCCTTTACGCTTGCCTTTAGTACCACTGC(SEQ ID NO:26)

[0108] GCTGTGGGGAACTTGACAGCAGTGCCAATCTGACCAGCAAGGAAGCCAAGATGGGAAAGGTCATGCGGCATACGCTCGGCGCCAGTTTGAATATTAGACATAATTTATCCTCAAGTAAGG(SEQ ID NO:27)

[0109] CCTTACTTGAGGATAAATTATGTCTAATATTCAAACTGGCGCCGAGCGTATGCCGCATGACCTTTCCCATCTTGGCTTCCTTGCTGGTCAGATTGGCACTGC(SEQ ID NO:28)

[0110] GCTGTGGGGAACTTGACAGCAGTGTCCTTGCTGGTCAGATTGGTCGTCTTATTACCATTTCAACTACTCCGGTTATCGCTGGCGACTCCTTCGAGATGGACGCCGTTGGCGCTCT(SEQ ID NO:29)

[0111] AGAGCGCCAACGGCGTCCATCTCGAAGGAGTCGCCAGCGATAACCGGAGTAGTTGAAATGGTAATAAGACGACCAATCTGACCAGCAAGGACACTGC(SEQ ID NO:30)

[0112] GCTGTGGGGAACTTGACAGCAGTGGACGATGAGGGACATAAAAGTAAAATGTCTACAGTAGAGTCAATAGCAAGGCCACGACGCAATGGAGAAAGACGGAGAGCCAACGGCGTCCA(SEQ ID NO:31)

[0113] TGGACGCCGTTGGCGCTCTCCGTCTTTCTCCATTGCGTCGTGGCCTTGCTATTGACTCTACTGTAGACATTTTTACTTTTTGTCCCTCATCGTCCACTGC(SEQ ID NO:32)

[0114] AGAACCGGGTGCTGTAGTGCAGTGTTTTATGTCCCTCATCGTCACGTTTATGGTGAACAGTGGATTAAGTTCATGAAGGATGGTGTTAATGCCACTCCTCTCCCGACTGTTAACACTACT(SEQ ID NO:33)

[0115] AGTAGTGTTAACAGTCGGGAGAGGAGTGGCATTAACACCATCCTTCATGAACTTAATCCACTGTTCACCATAAACGTGACGATGAGGGACATAAAACACTGC(SEQ ID NO:34)

[0116] AGAACCGGGTGCTGTAGTGCAGTGCTGAAACAAATGCTTAGGGATTTTATTGGTATCAGGGTTAATCGTGCCAAGAAAAGCGGCATGGTCAATAATAACCAGTAGTGTTAACAGTCGGG(SEQ ID NO:35)

[0117] CCCGACTGTTAACACTACTGGTTATATTGACCATGCCGCTTTTCTTGGCACGATTAACCCTGATACCAATAAAATCCCTAAGCATTTGTTTCAGCACTGC(SEQ ID NO:36)

[0118] AGAACCGGGTGCTGTAGTGCAGTGCCCTAAGCATTTGTTTCAGGGTTATTTGAATATCTATAACAACTATTTTAAAGCGCCGTGGATGCCTGACCGTACCGAGGCTAACCCTAAT(SEQ ID NO:37)

[0119] ATTAGGGTTAGCCTCGGTACGGTCAGGCATCCACGGCGCTTTAAAATAGTTGTTATAGATATTCAAATAACCCTGAAACAAATGCTTAGGGCACTGC(SEQ ID NO:38)

[0120] AGAACCGGGTGCTGTAGTGCAGTGAAGCGGAGCAGTCCAAATGTTTTTGAGATGGCAGCAACGGAAACCATAACGAGCATCATCTTGATTAAGCTCATTAGGGTTAGCCTCGGTA(SEQ ID NO:39)

[0121] TACCGAGGCTAACCCTAATGAGCTTAATCAAGATGATGCTCGTTATGGTTTCCGTTGCTGCCATCTCAAAAACATTTGGACTGCTCCGCTTCACTGC(SEQ ID NO:40)

[0122] AGAACCGGGTGCTGTAGTGCAGTGAACATTTGGACTGCTCCGCTTCCTCCTGAGACTGAGCTTTCTCGCCAAATGACGACTTCTACCACATCTATTGACATTATGGGTCTGCAAG(SEQ ID NO:41)

[0123] CTTGCAGACCCATAATGTCAATAGATGTGGTAGAAGTCGTCATTTGGCGAGAAAGCTCAGTCTCAGGAGGAAGCGGAGCAGTCCAAATGTTCACTGC(SEQ ID NO:42)

[0124] AGAACCGGGTGCTGTAGTGCAGTGATAACATCATGGTAACGCTGCATGAAGTAATCACGTTCTTGGTCAGTATGCAAATTAGCATAAGCAGCTTGCAGACCCATAATGTCAATAG(SEQ ID NO:43)

[0125] CTATTGACATTATGGGTCTGCAAGCTGCTTATGCTAATTTGCATACTGACCAAGAACGTGATTACTTCATGCAGCGTTACCATGATGTTATCACTGC(SEQ ID NO:44)

[0126] AGAACCGGGTGCTGTAGTGCAGTGCAGCGTTACCATGATGTTATTTCTTCATTTGGAGGTAAAACCTCTTATGACGCTGACAACCGTCCTTTACTTGTCATGCGCTCTAATCTCT(SEQ ID NO:45)

[0127] AGAGATTAGAGCGCATGACAAGTAAAGGACGGTTGTCAGCGTCATAAGAGGTTTTACCTCCAAATGAAGAAATAACATCATGGTAACGCTGCACTGC(SEQ ID NO:46)

[0128] AGAACCGGGTGCTGTAGTGCAGTGTCTGTTGAACACGACCAGAAAACTGGCCTAACGACGTTTGGTCAGTTCCATCAACATCATAGCCAGATGCCCAGAGATTAGAGCGCATGAC(SEQ ID NO:47)

[0129] GTCATGCGCTCTAATCTCTGGGCATCTGGCTATGATGTTGATGGAACTGACCAAACGTCGTTAGGCCAGTTTTCTGGTCGTGTTCAACAGACACTGC(SEQ ID NO:48)

[0130] AGAACCGGGTGCTGTAGTGCAGTGTTTCTGGTCGTGTTCAACAGACCTATAAACATTCTGTGCCGCGTTTCTTTGTTCCTGAGCATGGCACTATGTTTACTCTTGCGCTTGTTCG(SEQ ID NO:49)

[0131] CGAACAAGCGCAAGAGTAAACATAGTGCCATGCTCAGGAACAAAGAAACGCGGCACAGAATGTTTATAGGTCTGTTGAACACGACCAGAAACACTGC(SEQ ID NO:50)

[0132] AATCGGCTGTCCTAGAGGGCAGTGTTACTCTTGCGCTTGTTCGTTTTCCGCCTACTGCGACTAAAGAGATTCAGTACCTTAACGCTAAAGGTGCTTTGACTTATACCGATATTGC(SEQ ID NO:51)

[0133] GCAATATCGGTATAAGTCAAAGCACCTTTAGCGTTAAGGTACTGAATCTCTTTAGTCGCAGTAGGCGGAAAACGAACAAGCGCAAGAGTAACACTGC(SEQ ID NO:52)

[0134] AATCGGCTGTCCTAGAGGGCAGTGCCAGAACGGAAAACATCCTTCATAGAAATTTCACGCGGCGGCAAGTTGCCATACAAAACAGGGTCGCCAGCAATATCGGTATAAGTCAAAG(SEQ ID NO:53)

[0135] CTTTGACTTATACCGATATTGCTGGCGACCCTGTTTTGTATGGCAACTTGCCGCCGCGTGAAATTTCTATGAAGGATGTTTTCCGTTCTGGCACTGC(SEQ ID NO:54)

[0136] AATCGGCTGTCCTAGAGGGCAGTGAAGGATGTTTTCCGTTCTGGTGATTCGTCTAAGAAGTTTAAGATTGCTGAGGGTCAGTGGTATCGTTATGCGCCTTCGTATGTTTCTCCTG(SEQ ID NO:55)

[0137] CAGGAGAAACATACGAAGGCGCATAACGATACCACTGACCCTCAGCAATCTTAAACTTCTTAGACGAATCACCAGAACGGAAAACATCCTTCACTGC(SEQ ID NO:56)

[0138] AATCGGCTGTCCTAGAGGGCAGTGTAAGTACGCGTTCTTGCAAATCACCAGAAGGCGGTTCCTGAATGAATGGGAAGCCTTCAAGAAGGTGATAAGCAGGAGAAACATACGAAGG(SEQ ID NO:57)

[0139] CCTTCGTATGTTTCTCCTGCTTATCACCTTCTTGAAGGCTTCCCATTCATTCAGGAACCGCCTTCTGGTGATTTGCAAGAACGCGTACTTACACTGC(SEQ ID NO:58)

[0140] AATCGGCTGTCCTAGAGGGCAGTGTGATTTGCAAGAACGCGTACTTATTCGCCACCATGATTATGACCAGTGTTTCCAGTCCGTTCAGTTGTTGCAGTGGAATAGTCAGGTTAAA(SEQ ID NO:59)

[0141] TTTAACCTGACTATTCCACTGCAACAACTGAACGGACTGGAAACACTGGTCATAATCATGGTGGCGAATAAGTACGCGTTCTTGCAAATCACACTGC(SEQ ID NO:60)

[0142] AATCGGCTGTCCTAGAGGGCAGTGTCTTTTATCACGAAGTCATGATTGAATCGCGAGTGGTCGGCAGATTGCGATAAACGGTCACATTAAATTTAACCTGACTATTCCACTGCAA(SEQ ID NO:61)

[0143] TTGCAGTGGAATAGTCAGGTTAAATTTAATGTGACCGTTTATCGCAATCTGCCGACCACTCGCGATTCAATCATGACTTCGTGATAAAAGACACTGC(SEQ ID NO:62)

[0144] AATCGGCTGTCCTAGAGGGCAGTGCAATCATGACTTCGTGATAAAAGATTGAGTGTGAGGTTATAACGCCGAAGCGGTAAAAATTTTAATTTTTGCCGCTGAGGGGTTGACCAAG(SEQ ID NO:63)

[0145] CTTGGTCAACCCCTCAGCGGCAAAAATTAAAATTTTTACCGCTTCGGCGTTATAACCTCACACTCAATCTTTTATCACGAAGTCATGATTGCACTGC(SEQ ID NO:64)

[0146] AATCGGCTGTCCTAGAGGGCAGTGGAATTATGGCGAGAAATAAAAGTCTGAAACATGATTAAACTCCTAAGCAGAAAACCTACCGCGCTTCGCTTGGTCAACCCCTCAGCGGCAA(SEQ ID NO:65)

[0147] TTGCCGCTGAGGGGTTGACCAAGCGAAGCGCGGTAGGTTTTCTGCTTAGGAGTTTAATCATGTTTCAGACTTTTATTTCTCGCCATAATTCCACTGC(SEQ ID NO:66)

[0148] AATCGGCTGTCCTAGAGGGCAGTGGACTTTTATTTCTCGCCATAATTCAAACTTTTTTTCTGATAAGCTGGTTCTCACTTCTGTTACTCCAGCTTCTTCGGCACCTGTTTTACAGACACC(SEQ ID NO:67)

[0149] GGTGTCTGTAAAACAGGTGCCGAAGAAGCTGGAGTAACAGAAGTGAGAACCAGCTTATCAGAAAAAAAGTTTGAATTATGGCGAGAAATAAAAGTCCACTGC(SEQ ID NO:68)

[0150] ACCTACTAGGCCCGTCTAGCAGTGCACCTGTTTTACAGACACCTAAAGCTACATCGTCAACGTTATATTTTGATAGTTTGACGGTTAATGCTGGTAATGGTGGTTTTCTTCATTGCATTC(SEQ ID NO:69)

[0151] GAATGCAATGAAGAAAACCACCATTACCAGCATTAACCGTCAAACTATCAAAATATAACGTTGACGATGTAGCTTTAGGTGTCTGTAAAACAGGTGCACTGC(SEQ ID NO:70)

[0152] ACCTACTAGGCCCGTCTAGCAGTGTAGGGTCGGCATCAAAAGCAATATCAGCACCAACAGAAACAACCTGATTAGCGGCGTTGACAGATGTATCCATCTGAATGCAATGAAGAAAACCAC(SEQ ID NO:71)

[0153] GTGGTTTTCTTCATTGCATTCAGATGGATACATCTGTCAACGCCGCTAATCAGGTTGTTTCTGTTGGTGCTGATATTGCTTTTGATGCCGACCCTACACTGC(SEQ ID NO:72)

[0154] ACCTACTAGGCCCGTCTAGCAGTGGCTTTTGATGCCGACCCTAAATTTTTTGCCTGTTTGGTTCGCTTTGAGTCTTCTTCGGTTCCGACTACCCTCCCGACTGCCTATGATGTTT(SEQ ID NO:73)

[0155] AAACATCATAGGCAGTCGGGAGGGTAGTCGGAACCGAAGAAGACTCAAAGCGAACCAAACAGGCAAAAAATTTAGGGTCGGCATCAAAAGCCACTGC(SEQ ID NO:74)

[0156] ACCTACTAGGCCCGTCTAGCAGTGGGGGAAGGACGTCAATAGTCACACAGTCCTTGACGGTATAATAACCACCATCATGGCGACCATTCAAAGGATAAACATCATAGGCAGTCGG(SEQ ID NO:75)

[0157] CCGACTGCCTATGATGTTTATCCTTTGAATGGTCGCCATGATGGTGGTTATTATACCGTCAAGGACTGTGTGACTATTGACGTCCTTCCCCCACTGC(SEQ ID NO:76)

[0158] ACCTACTAGGCCCGTCTAGCAGTGACTATTGACGTCCTTCCCCGTACGCCGGGCAATAACGTTTATGTTGGTTTCATGGTTTGGTCTAACTTTACCGCTACTAAATGCCGCGGAT(SEQ ID NO:77)

[0159] ATCCGCGGCATTTAGTAGCGGTAAAGTTAGACCAAACCATGAAACCAACATAAACGTTATTGCCCGGCGTACGGGGAAGGACGTCAATAGTCACTGC(SEQ ID NO:78)

[0160] ACCTACTAGGCCCGTCTAGCAGTGCCAAACATAAATCACCTCACTTAAGTGGCTGGAGACAAATAATCTCTTTAATAACCTGATTCAGCGAAACCAATCCGCGGCATTTAGTAGC(SEQ ID NO:79)

[0161] GCTACTAAATGCCGCGGATTGGTTTCGCTGAATCAGGTTATTAAAGAGATTATTTGTCTCCAGCCACTTAAGTGAGGTGATTTATGTTTGGCACTGC(SEQ ID NO:80)

[0162] ACCTACTAGGCCCGTCTAGCAGTGAGTGAGGTGATTTATGTTTGGTGCTATTGCTGGCGGTATTGCTTCTGCTCTTGCTGGTGGCGCCATGTCTAAATTGTTTGGAGGCGGTCAA(SEQ ID NO:81)

[0163] TTGACCGCCTCCAAACAATTTAGACATGGCGCCACCAGCAAGAGCAGAAGCAATACCGCCAGCAATAGCACCAAACATAAATCACCTCACTCACTGC(SEQ ID NO:82)

[0164] ACCTACTAGGCCCGTCTAGCAGTGCACCCATGCCTACAGTATTGTTATCGGTAGCAAGCACATCACCTTGAATGCCACCGGAGGCGGCTTTTTGACCGCCTCCAAACAATTTAGA(SEQ ID NO:83)

[0165] TCTAAATTGTTTGGAGGCGGTCAAAAAGCCGCCTCCGGTGGCATTCAAGGTGATGTGCTTGCTACCGATAACAATACTGTAGGCATGGGTGCACTGC(SEQ ID NO:84)

[0166] CCTCGTGTCGAACGATGTGCAGTGAATACTGTAGGCATGGGTGATGCTGGTATTAAATCTGCCATTCAAGGCTCTAATGTTCCTAACCCTGATGAGGCCGCCCCTAGTTTTGTTT(SEQ ID NO:85)

[0167] AAACAAAACTAGGGGCGGCCTCATCAGGGTTAGGAACATTAGAGCCTTGAATGGCAGATTTAATACCAGCATCACCCATGCCTACAGTATTCACTGC(SEQ ID NO:86)

[0168] CCTCGTGTCGAACGATGTGCAGTGAAACGGCAGAAGTGCCAGCCTGCAACGTACCTTCAAGAAGTCCTTTACCAGCTTTAGCCATAGCACCAGAAACAAAACTAGGGGCGGCCTC(SEQ ID NO:87)

[0169] GAGGCCGCCCCTAGTTTTGTTTCTGGTGCTATGGCTAAAGCTGGTAAAGGACTTCTTGAAGGTACGTTGCAGGCTGGCACTTCTGCCGTTTCACTGC(SEQ ID NO:88)

[0170] CCTCGTGTCGAACGATGTGCAGTGCAGGCTGGCACTTCTGCCGTTTCTGATAAGTTGCTTGATTTGGTTGGACTTGGTGGCAAGTCTGCCGCTGATAAAGGAAAGGATACTCGTG(SEQ ID NO:89)

[0171] CACGAGTATCCTTTCCTTTATCAGCGGCAGACTTGCCACCAAGTCCAACCAAATCAAGCAACTTATCAGAAACGGCAGAAGTGCCAGCCTGCACTGC(SEQ ID NO:90)

[0172] CCTCGTGTCGAACGATGTGCAGTGCAGCAGAGGAAGCATCAGCACCAGCACGCTCCCAAGCATTAAGCTCAGGAAATGCAGCAGCAAGATAATCACGAGTATCCTTTCCTTTATC(SEQ ID NO:91)

[0173] GATAAAGGAAAGGATACTCGTGATTATCTTGCTGCTGCATTTCCTGAGCTTAATGCTTGGGAGCGTGCTGGTGCTGATGCTTCCTCTGCTGCACTGC(SEQ ID NO:92)

[0174] CCTCGTGTCGAACGATGTGCAGTGTGGTGCTGATGCTTCCTCTGCTGGTATGGTTGACGCCGGATTTGAGAATCAAAAAGAGCTTACTAAAATGCAACTGGACAATCAGAAAGAG(SEQ ID NO:93)

[0175] CTCTTTCTGATTGTCCAGTTGCATTTTAGTAAGCTCTTTTTGATTCTCAAATCCGGCGTCAACCATACCAGCAGAGGAAGCATCAGCACCACACTGC(SEQ ID NO:94)

[0176] CCTCGTGTCGAACGATGTGCAGTGCTTTCGTATTCTGGCGTGAAGTCGCCGACTGAATGCCAGCAATCTCTTTTTGAGTCTCATTTTGCATCTCGGCAATCTCTTTCTGATTGTCCAGTT(SEQ ID NO:95)

[0177] AACTGGACAATCAGAAAGAGATTGCCGAGATGCAAAATGAGACTCAAAAAGAGATTGCTGGCATTCAGTCGGCGACTTCACGCCAGAATACGAAAGCACTGC(SEQ ID NO:96)

[0178] CCTCGTGTCGAACGATGTGCAGTGTCACGCCAGAATACGAAAGACCAGGTATATGCACAAAATGAGATGCTTGCTTATCAACAGAAGGAGTCTACTGCTCGCGTTGCGTCTATTATGGAA(SEQ ID NO:97)

[0179] TTCCATAATAGACGCAACGCGAGCAGTAGACTCCTTCTGTTGATAAGCAAGCATCTCATTTTGTGCATATACCTGGTCTTTCGTATTCTGGCGTGACACTGC(SEQ ID NO:98)

[0180] CCTCGTGTCGAACGATGTGCAGTGTGACCAGCCGTTTGAGCTTGAGTAAGCATTTGGCGCATAATCTCGGAAACCTGCTGTTGCTTGGAAAGATTGGTGTTTTCCATAATAGACGCAACG(SEQ ID NO:99)

[0181] CGTTGCGTCTATTATGGAAAACACCAATCTTTCCAAGCAACAGCAGGTTTCCGAGATTATGCGCCAAATGCTTACTCAAGCTCAAACGGCTGGTCACACTGC(SEQ ID NO:100)

[0182] CCTCGTGTCGAACGATGTGCAGTGAAGCTCAAACGGCTGGTCAGTATTTTACCAATGACCAAATCAAAGAAATGACTCGCAAGGTTAGTGCTGAGGTTGACTTAGTTCATCAGCAAACG(SEQ ID NO:101)

[0183] CGTTTGCTGATGAACTAAGTCAACCTCAGCACTAACCTTGCGAGTCATTTCTTTGATTTGGTCATTGGTAAAATACTGACCAGCCGTTTGAGCTTCACTGC(SEQ ID NO:102)

[0184] CACGACGCATTGCTCGTAGCAGTGCTTAGTTCATCAGCAAACGCAGAATCAGCGGTATGGCTCTTCTCATATTGGCGCTACTGCAAAGGATATTTCTAATGTCGTCACTGATGCT(SEQ ID NO:103)

[0185] AGCATCAGTGACGACATTAGAAATATCCTTTGCAGTAGCGCCAATATGAGAAGAGCCATACCGCTGATTCTGCGTTTGCTGATGAACTAAGCACTGC(SEQ ID NO:104)

[0186] CACGACGCATTGCTCGTAGCAGTGCCAGAAATTGTTCCAAGTATCGGCAACAGCTTTATCAATACCATGAAAAATATCAACCACACCAGAAGCAGCATCAGTGACGACATTAGAA(SEQ ID NO:105)

[0187] TTCTAATGTCGTCACTGATGCTGCTTCTGGTGTGGTTGATATTTTTCATGGTATTGATAAAGCTGTTGCCGATACTTGGAACAATTTCTGGCACTGC(SEQ ID NO:106)

[0188] CACGACGCATTGCTCGTAGCAGTGCGATACTTGGAACAATTTCTGGAAAGACGGTAAAGCTGATGGTATTGGCTCTAATTTGTCTAGGAAATAACCGTCAGGATTGACACCCTCC(SEQ ID NO:107)

[0189] GGAGGGTGTCAATCCTGACGGTTATTTCCTAGACAAATTAGAGCCAATACCATCAGCTTTACCGTCTTTCCAGAAATTGTTCCAAGTATCGCACTGC(SEQ ID NO:108)

[0190] CACGACGCATTGCTCGTAGCAGTGTCAGAAGGGTAATAAGAACGAACCATAAAAAAGCCTCCAAGATTTGGAGGCATGAAAACATACAATTGGGAGGGTGTCAATCCTGACGGTT(SEQ ID NO:109)

[0191] AACCGTCAGGATTGACACCCTCCCAATTGTATGTTTTCATGCCTCCAAATCTTGGAGGCTTTTTTATGGTTCGTTCTTATTACCCTTCTGACACTGC(SEQ ID NO:110)

[0192] CACGACGCATTGCTCGTAGCAGTGCGTTCTTATTACCCTTCTGAATGTCACGCTGATTATTTTGACTTTGAGCGTATCGAGGCTCTTAAACCTGCTATTGAGGCTTGTGGCATTT(SEQ ID NO:111)

[0193] AAATGCCACAAGCCTCAATAGCAGGTTTAAGAGCCTCGATACGCTCAAAGTCAAAATAATCAGCGTGACATTCAGAAGGGTAATAAGAACGCACTGC(SEQ ID NO:112)

[0194] CACGACGCATTGCTCGTAGCAGTGTCTCTTCCAAGAGCTTGATGCGGTTATCCATCTGCTTATGGAAGCCAAGCATTGGGGATTGAGAAAGAGTAGAAATGCCACAAGCCTCAAT(SEQ ID NO:113)

[0195] ATTGAGGCTTGTGGCATTTCTACTCTTTCTCAATCCCCAATGCTTGGCTTCCATAAGCAGATGGATAACCGCATCAAGCTCTTGGAAGAGACACTGC(SEQ ID NO:114)

[0196] CACGACGCATTGCTCGTAGCAGTGACCGCATCAAGCTCTTGGAAGAGATTCTGTCTTTTCGTATGCAGGGCGTTGAGTTCGATAATGGTGATATGTATGTTGACGGCCATAAGGC(SEQ ID NO:115)

[0197] GCCTTATGGCCGTCAACATACATATCACCATTATCGAACTCAACGCCCTGCATACGAAAAGACAGAATCTCTTCCAAGAGCTTGATGCGGTCACTGC(SEQ ID NO:116)

[0198] CACGACGCATTGCTCGTAGCAGTGGCATTGTGCCAATTCATCCATTAACTTCTCAGTAACAGATACAAACTCATCACGAACGTCAGAAGCAGCCTTATGGCCGTCAACATACATA(SEQ ID NO:117)

[0199] TATGTATGTTGACGGCCATAAGGCTGCTTCTGACGTTCGTGATGAGTTTGTATCTGTTACTGAGAAGTTAATGGATGAATTGGCACAATGCCACTGC(SEQ ID NO:118)

[0200] TTCACCTGTTCCCGGATAGCAGTGGGATGAATTGGCACAATGCTACAATGTGCTCCCCCAACTTGATATTAATAACACTATAGACCACCGCCCCGAAGGGGACGAAAAATGGTTT(SEQ ID NO:119)

[0201] AAACCATTTTTCGTCCCCTTCGGGGCGGTGGTCTATAGTGTTATTAATATCAAGTTGGGGGAGCACATTGTAGCATTGTGCCAATTCATCCCACTGC(SEQ ID NO:120)

[0202] TTCACCTGTTCCCGGATAGCAGTGTATCCTTAAGAGGGCGTTCAGCAGCCAGCTTGCGGCAAAACTGCGTAACCGTCTTCTCGTTCTCTAAAAACCATTTTTCGTCCCCTTCGGG(SEQ ID NO:121)

[0203] CCCGAAGGGGACGAAAAATGGTTTTTAGAGAACGAGAAGACGGTTACGCAGTTTTGCCGCAAGCTGGCTGCTGAACGCCCTCTTAAGGATACACTGC(SEQ ID NO:122)

[0204] TTCACCTGTTCCCGGATAGCAGTGCTGCTGAACGCCCTCTTAAGGATATTCGCGATGAGTATAATTACCCCAAAAAGAAAGGTATTAAGGATGAGTGTTCAAGATTGCTGGAGGC(SEQ ID NO:123)

[0205] GCCTCCAGCAATCTTGAACACTCATCCTTAATACCTTTCTTTTTGGGGTAATTATACTCATCGCGAATATCCTTAAGAGGGCGTTCAGCAGCACTGC(SEQ ID NO:124)

[0206] TTCACCTGTTCCCGGATAGCAGTGATGAGCCTGTCGCATTGCATTCATCAAACGCTGAATAGCAAAGCCTCTACGCGATTTCATAGTGGAGGCCTCCAGCAATCTTGAACACTCA(SEQ ID NO:125)

[0207] TGAGTGTTCAAGATTGCTGGAGGCCTCCACTATGAAATCGCGTAGAGGCTTTGCTATTCAGCGTTTGATGAATGCAATGCGACAGGCTCATCACTGC(SEQ ID NO:126)

[0208] TTCACCTGTTCCCGGATAGCAGTGATGAATGCAATGCGACAGGCTCATGCTGATGGTTGGTTTATCGTTTTTGACACTCTCACGTTGGCTGACGACCGATTAGAGGCGTTTTATG(SEQ ID NO:127)

[0209] CATAAAACGCCTCTAATCGGTCGTCAGCCAACGTGAGAGTGTCAAAAACGATAAACCAACCATCAGCATGAGCCTGTCGCATTGCATTCATCACTGC(SEQ ID NO:128)

[0210] TTCACCTGTTCCCGGATAGCAGTGCGACCCTCGGCAGCAAGAACCATACGACCAATATCACGAAAATAGTCACGCAAAGCATTGGGATTATCATAAAACGCCTCTAATCGGTCGT(SEQ ID NO:129)

[0211] ACGACCGATTAGAGGCGTTTTATGATAATCCCAATGCTTTGCGTGACTATTTTCGTGATATTGGTCGTATGGTTCTTGCTGCCGAGGGTCGCACTGC(SEQ ID NO:130)

[0212] TTCACCTGTTCCCGGATAGCAGTGTATGGTTCTTGCTGCCGAGGGTCGCAAGGCTAATGATTCACACGCCGACTGCTATCAGTATTTTTGTGTGCCTGAGTATGGTACAGCTAAT(SEQ ID NO:131)

[0213] ATTAGCTGTACCATACTCAGGCACACAAAAATACTGATAGCAGTCGGCGTGTGAATCATTAGCCTTGCGACCCTCGGCAGCAAGAACCATACACTGC(SEQ ID NO:132)

[0214] TTCACCTGTTCCCGGATAGCAGTGAATTAGGGTCAACGCTACCTGTAGGAAGTGTCCGCATAAAGTGCACCGCATGGAAATGAAGACGGCCATTAGCTGTACCATACTCAGGCAC(SEQ ID NO:133)

[0215] GTGCCTGAGTATGGTACAGCTAATGGCCGTCTTCATTTCCATGCGGTGCACTTTATGCGGACACTTCCTACAGGTAGCGTTGACCCTAATTCACTGC(SEQ ID NO:134)

[0216] CCGCCCTTGTAATGCACAGCAGTGGGTAGCGTTGACCCTAATTTTGGTCGTCGGGTACGCAATCGCCGCCAGTTAAATAGCTTGCAAAATACGTGGCCTTATGGTTACAGTATGC(SEQ ID NO:135)

[0217] GCATACTGTAACCATAAGGCCACGTATTTTGCAAGCTATTTAACTGGCGGCGATTGCGTACCCGACGACCAAAATTAGGGTCAACGCTACCCACTGC(SEQ ID NO:136)

[0218] CCGCCCTTGTAATGCACAGCAGTGTTAGCATCAACAGGCCACAACCAACCAGAACGTGAAAAAGCGTCCTGCGTGTAGCGAACTGCGATGGGCATACTGTAACCATAAGGCCACG(SEQ ID NO:137)

[0219] CGTGGCCTTATGGTTACAGTATGCCCATCGCAGTTCGCTACACGCAGGACGCTTTTTCACGTTCTGGTTGGTTGTGGCCTGTTGATGCTAACACTGC(SEQ ID NO:138)

[0220] CCGCCCTTGTAATGCACAGCAGTGGTTGTGGCCTGTTGATGCTAAAGGTGAGCCGCTTAAAGCTACCAGTTATATGGCTGTTGGTTTCTATGTGGCTAAATACGTTAACAAAAAG(SEQ ID NO:139)

[0221] CTTTTTGTTAACGTATTTAGCCACATAGAAACCAACAGCCATATAACTGGTAGCTTTAAGCGGCTCACCTTTAGCATCAACAGGCCACAACCACTGC(SEQ ID NO:140)

[0222] CCGCCCTTGTAATGCACAGCAGTGGCTTGGTTTTTAGTGAGTTGTTCCATTCTTTAG CTCCTAGACCTTTAGCAGCAAGGTCCATATCTGACTTTTTGTTAACGTATTTAGCCAC(SEQ ID NO:141)

[0223] GTGGCTAAATACGTTAACAAAAAGTCAGATATGGACCTTGCTGCTAAAGGTCTAGG AGCTAAAGAATGGAACAACTCACTAAAAACCAAGCCACTGC(SEQ ID NO:142)

[0224] CCGCCCTTGTAATGCACAGCAGTGAACTCACTAAAAACCAAGCTGTCGCTACTTCC CAAGAAGCTGTTCAGAATCAGAATGAGCCGCAACTTCGGGATGAAAATGCTCACAATG A(SEQ ID NO:143)

[0225] TCATTGTGAGCATTTTCATCCCGAAGTTGCGGCTCATTCTGATTCTGAACAGCTTCTT GGGAAGTAGCGACAGCTTGGTTTTTAGTGAGTTCACTGC(SEQ ID NO:144)

[0226] CCGCCCTTGTAATGCACAGCAGTGTATCTGGTTGAACGGCGTCGCGTCGTAACCCA GCTTGGTAAGTTGGATTAAGCACTCCGTGGACAGATTTGTCATTGTGAGCATTTTCATC (SEQ ID NO: 145)

[0227] GATGAAAATGCTCACAATGACAAATCTGTCCACGGAGTGCTTAATCCAACTTACCA AGCTGGGTTACGACGCGACGCCGTTCAACCAGATACACTGC(SEQ ID NO:146)

[0228] CCGCCCTTGTAATGCACAGCAGTGGACGCCGTTCAACCAGATATTGAAGCAGAACG CAAAAAGAGAGATGAGATTGAGGCTGGGAAAAGTTACTGTAGCCGACGTTTTGGCGGC G (SEQ ID NO: 147)

[0229] CGCCGCCAAAACGTCGGCTACAGTAACTTTTCCCAGCCTCAATCTCATCTCTCTTTT TGCGTTCTGCTTCAATATCTGGTTGAACGGCGTCCACTGC(SEQ ID NO:148)

[0230] CCGCCCTTGTAATGCACAGCAGTGTGCAGGTTGGATACGCCAATCATTTTTATCGAA GCGCGCATAAATTTGAGCAGATTTGTCGTCACAGGTTGCGCCGCCAAAACGTCGGCTA (SEQ ID NO: 149)

[0231] TAGCCGACGTTTTGGCGGCGCAACCTGGTGACGACAAATCTGCTCAAATTTATGCGCGCTTCGATAAAAATGATTGGCGTATCCAACCTGCACACTGC(SEQ ID NO:150)

[0232] The top view of the high-throughput gene synthesis chip based on the digital microfluidic system prepared in Example 1 is shown in Figure 7, where 2-gene modification site; 5-electrode layer; 7-droplet; the grid is a schematic diagram of the electrodes on the digital microfluidic chip, each cell represents a pixel electrode, and the droplet can move between pixel electrodes. The black dots in the figure represent the barcode modification points on the upper electrode plate;

[0233] Schematic diagram of local modification point capture oligos is shown in Figure 8 , where 7-a is an oligo pool droplet; 11 is oligos; the oligo pool solution is moved to the local modification point by EWOD, and under the influence of the incubation temperature, the free target oligos in the oligo pool will be captured by the barcode and fixed at the local modification point;

[0234] The reaction reagent is moved to the reaction site. Under the action of the endonuclease and annealing temperature, the oligos connected to the barcode are cut off and released in the reaction reagent. After the enzyme cutting is completed, the reagent enters the thermal cycle and reacts. The free oligos are connected end to end under the action of the ligase and gradually spliced ​​into the target long-chain DNA. The schematic diagram of this step is shown in Fig. 9 , where 7-b is a reaction reagent droplet; 12 is a long-chain DNA.

[0235] The characterization results of the final product of synthesizing long-chain DNA using the high-throughput gene synthesis chip based on the digital microfluidic system in Example 1 are shown in Fig.10 It can be seen that the technical solution of the present invention realizes high-throughput DNA splicing preparation.

[0236] As can be seen from Application Example 1, compared with the existing 96-well plate-based splicing solution, it requires at least 25uL reaction system; the technical solution of the present invention controls the solution volume to <0.5μL, and reduces the cost of enzyme reagents and oligo by at least 2 orders of magnitude. In addition, the present invention can construct no less than 96 sets of gene sequences using the gene array chip, and is expected to further increase the flux by increasing the chip area to improve the synthesis efficiency and reduce the synthesis cost. The synthesis process avoids nonspecific adsorption of enzymes and the hydrophobic layer surface, and improves the application scope of digital microfluidic chips in the field of gene synthesis.

[0237] The above are only preferred specific implementations of the present application, but the protection scope of the present application is not limited thereto. Any changes or substitutions that can be easily thought of by a person skilled in the art within the technical scope disclosed in the present application should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A high-throughput gene synthesis chip based on a digital microfluidic system, comprising an upper plate and a lower plate, characterized in that: The surface of the upper electrode plate is spin-coated with a hydrophobic layer and modified with a gene array. The upper electrode plate and the lower electrode plate form a bipolar plate digital microfluidic chip, which controls the movement of droplets based on the dielectric electrowetting technology.

2. The high-throughput gene synthesis chip based on the digital microfluidic system according to claim 1, characterized in that: The upper plate comprises an upper plate substrate, a hydrophobic layer loaded on the surface of the upper plate substrate, and a gene modification site embedded in the hydrophobic layer. The preparation method comprises the following steps: After cleaning and drying the upper electrode plate substrate, a hydrophobic material is spin-coated on the surface to prepare a hydrophobic layer, a mask with array micropores is placed on the hydrophobic layer, and plasma treatment is performed to remove the hydrophobic layer at the through holes of the mask plate to expose the upper electrode plate substrate to obtain an upper electrode plate substrate with a local hydrophilic area on the surface, and chemical groups are modified at the local hydrophilic points of the local hydrophilic areas of the upper electrode plate substrate by covalent connection to obtain a substrate with gene array modification points, and a barcode sequence is incubated and connected to the gene array modification points to obtain the upper electrode plate.

3. The high-throughput gene synthesis chip based on the digital microfluidic system according to claim 2, characterized in that: The hydrophobic material is amorphous fluororesin with a concentration of 0.5wt.% to 2wt.%; and / or, The spin coating speed is 2000-4000 rpm; and / or, The thickness of the hydrophobic layer is 30-200 nm.

4. The high-throughput gene synthesis chip based on a digital microfluidic system according to claim 2, characterized in that: The number of unit arrays of the microhole array on the mask is 96, the unit array is 3×3, the diameter of the microhole is 0.1-0.2 mm, and the microhole spacing is 0.5 mm.

5. The high-throughput gene synthesis chip based on the digital microfluidic system according to claim 2, characterized in that: The covalent connection method is to graft 3-aminopropyltriethoxysilane to the local hydrophilic points of the local hydrophilic area of ​​the upper plate substrate by covalent connection, and then connect streptavidin to the 3-aminopropyltriethoxysilane on the surface of the substrate.

6. The high-throughput gene synthesis chip based on a digital microfluidic system according to claim 1, characterized in that: A filling medium is contained between the upper electrode plate and the lower electrode plate, and the filling medium includes one or more of dimethyl silicone oil, dodecamethylpentasiloxane, n-hexadecane and n-dodecane.

7. A high-throughput gene synthesis method based on a digital microfluidic chip, characterized in that: High-throughput gene synthesis using the high-throughput gene synthesis chip based on the digital microfluidic system according to any one of claims 1 to 6 comprises the steps of oligo pool capture, cleaning, enzyme digestion reaction, gene synthesis and product amplification.

8. The high-throughput gene synthesis method based on a digital microfluidic chip according to claim 7, characterized in that: The specific steps include: The array modification points of a high-throughput gene synthesis chip based on a digital microfluidics system with oligo pool droplets on the surface are connected to the barcode sequence to capture the target gene fragment, and the oligo pool droplets after capture are removed, and the cleaning solution is moved to the modification site to clean the residual oligo pool, and then the cleaning solution is removed and the reaction reagent is moved to the modification gene site, and the oligos are cut off from the upper plate substrate and freed in the reaction reagent by enzyme cleavage reaction, and then gene splicing and error correction are performed in the reaction reagent to obtain the target product, and the splicing of oligos is completed. Part of the product is taken from the reaction reagent droplet containing the target product for PCR amplification to obtain the required DNA long chain.

9. The high-throughput gene synthesis method based on a digital microfluidic chip according to claim 8, characterized in that: The cleaning solution comprises NaCl, EDTA, Tris-HCl and Tween20; and / or, The reaction reagents include restriction endonuclease, DNA ligase, dNTP, BSA and buffer.

10. The high-throughput gene synthesis method based on a digital microfluidic chip according to claim 9, characterized in that: The buffer comprises Tris-HCl, MgCl2, DTT, PEG8000 and Tween20; and / or, The dNTP concentration in the reaction reagent is 10-50 mM, and the BSA concentration is 0.5-1 mg / mL.

Images