WO2020118655A1 - 核酸合成装置和纯化装置、其用途及核酸合成方法和纯化方法 - Google Patents

核酸合成装置和纯化装置、其用途及核酸合成方法和纯化方法 Download PDF

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WO2020118655A1
WO2020118655A1 PCT/CN2018/121092 CN2018121092W WO2020118655A1 WO 2020118655 A1 WO2020118655 A1 WO 2020118655A1 CN 2018121092 W CN2018121092 W CN 2018121092W WO 2020118655 A1 WO2020118655 A1 WO 2020118655A1
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nucleic acid
cpg
acid synthesis
purification device
purified
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French (fr)
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方鑫
黄小罗
沈玥
徐讯
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BGI Shenzhen Co Ltd
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BGI Shenzhen Co Ltd
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Priority to CN201880099717.2A priority Critical patent/CN113348250B/zh
Priority to US17/311,697 priority patent/US12247198B2/en
Priority to PCT/CN2018/121092 priority patent/WO2020118655A1/zh
Publication of WO2020118655A1 publication Critical patent/WO2020118655A1/zh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/40—Apparatus specially designed for the use of free, immobilised, or carrier-bound enzymes, e.g. apparatus containing a fluidised bed of immobilised enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00759—Purification of compounds synthesised

Definitions

  • the invention relates to the technical field of nucleic acid synthesis, in particular to a nucleic acid synthesis device and a purification device, its use, a nucleic acid synthesis method and a purification method.
  • the classic DNA synthesis column contains solid phase carrier, sieve plate and empty column tube.
  • Solid-phase supports usually use Controlled Pore Glass (CPG).
  • CPG Controlled Pore Glass
  • the pore network is huge.
  • the size of the channel is called the pore size.
  • the pore size is stable.
  • the pore size is usually 500 angstroms.
  • the sieve plate is usually sintered with ultra high molecular weight polyethylene (UHMW-PE) or high density polyethylene (HDPE) powder.
  • UHMW-PE ultra high molecular weight polyethylene
  • HDPE high density polyethylene
  • the empty column tube is injection molded from polypropylene.
  • the second-generation DNA general synthesis column is composed of a filter element (Controlled Pore Glass Frits, CPG Frits) containing a controlled pore glass ball and an empty column tube.
  • CPG Frits is sintered from CPG and UHMW-PE or HDPE powder.
  • the PE particles wrap and fix the CPG particles.
  • the bridges between the PE particles form a certain pore size.
  • the CPG particles are evenly embedded in the rigid network structure formed by the PE bridges to form a sieve plate with a determined pore size and porosity.
  • the synthetic reagent flows through the channel formed by the internal channel of the CPG and the PE bridge, so that the reagent channel is long enough to increase the time of the chemical reaction and the chance of the reactant contacting the reaction site on the CPG.
  • the encapsulation effect of the polymer makes the CPG particles fixed and will not rotate due to the flow of the reagent, further prolonging the contact time between the reagent and the reaction site on the CPG, and improving the synthesis efficiency and product purity.
  • the synthesis of primers and other oligonucleotides generally uses the second-generation CPG Frits synthesis column, in which CPG is made of silica and is widely used as a carrier for solid-phase synthesis of oligonucleotides, usually with a universal connection of chemical groups Universal (Linker) modification, the initial step of the synthesis is to remove the DMT protecting group on the connecting arm of CPG with trichloroacetic acid (TCA) to obtain the free 5'-hydroxyl group for the next condensation with the phosphoramidite monomer
  • TCA trichloroacetic acid
  • the oligonucleotide connected to the CPG will be cut off, and it can be used for subsequent experiments after purification, but the effect of fixing the oligonucleotide cannot be achieved.
  • the PE material used has super-hydrophobicity, CPG is wrapped in it, so this synthesis column cannot absorb reaction solutions such as enzyme digestion and PCR.
  • DNA isolation and purification is a key step in molecular biology operations.
  • the isolated DNA can be used for downstream gene cloning and probe preparation experiments.
  • DNA isolation and purification generally includes two types: 1) total DNA extraction. This separation and purification only needs to remove other impurities in the tissue, cells or in vitro DNA synthesis reaction products, and then separate all the DNA genetic material required by physical or chemical methods. 2) Specific DNA extraction. This kind of separation and purification usually requires the synthesis of specific DNA in the amplified product of tissues, cells or in vitro DNA, including DNA of a certain functional type (such as plasmid DNA), DNA of a certain length or a sequence specific according to the purpose of the experiment DNA extraction.
  • DNA of a certain functional type such as plasmid DNA
  • DNA separation and purification methods in the laboratory include silica gel centrifugal adsorption column method and magnetic bead adsorption method.
  • the core principles of the two methods are similar. Both groups are bonded through the surface of the silica gel membrane or magnetic beads. Under certain buffer conditions, they adsorb to the charge on the surface of the DNA, thereby separating and purifying the target DNA. Both of these methods have been developed as mature commercial kits and are commercially available.
  • TIANGEN's universal DNA purification and recovery kit as an example. This kit uses a unique buffer system and a centrifugal adsorption column. It can not only recover DNA fragments from TAE or TBE agarose gel, but also be used to directly purify PCR products.
  • the sol solution contains a pH indicator, and the state of the sol can be judged according to the color.
  • the principle of the gel recovery kit is: in the gel melt, the gel block is quickly melted and the DNA is released. After adding the high chaotropic sequence solution, the DNA fragments are selectively adsorbed on the silica gel membrane and washed to remove the residue After impurities and high-concentration salt ions on the silica gel membrane, the DNA fragments adsorbed on the silica gel membrane are eluted by elution buffer or water, and can be used for various molecular biology experiments.
  • the magnetic bead adsorption method of nucleic acid extraction is to use nanotechnology to improve the surface of superparamagnetic nanoparticles and modify the surface to prepare superparamagnetic silicon oxide nanoparticles.
  • the magnetic beads can specifically recognize and efficiently bind to nucleic acid molecules on the microscopic interface.
  • DNA and RNA can be extracted from blood, animal tissue, food, pathogenic microorganisms and other samples It can be used in clinical diagnosis, blood transfusion safety, forensic identification, environmental microbiological testing, food safety testing, molecular biology research and other fields.
  • the present invention provides a nucleic acid synthesis device and purification device, its use, and nucleic acid synthesis method and purification method.
  • the nucleic acid synthesis device can be used not only for the synthesis of oligonucleotide sequences, but also for the use of oligonucleotide sequences fixed on CPG Purification of digestion products and PCR products has the advantages of simple structure, small size, light weight, high efficiency, low cost, and diverse functions; the nucleic acid purification device can purify specific nucleic acid sequences, which is efficient, fast, convenient, Safe and non-toxic advantages.
  • an embodiment provides a nucleic acid synthesis device including a solid-phase support, the solid-phase support comprising a controllable pore glass sphere (CPG), CPG is an unmodified bare CPG with hydroxyl groups on its surface, Hydroxyl groups can be linked to phosphoramidite-protected nucleotide monomers or polymers through covalent bonding for nucleic acid synthesis.
  • CPG controllable pore glass sphere
  • CPG Frits synthesis column in which CPG is made of silica, and the surface of the CPG is usually modified with a universal linker (Universal Linker) with chemical groups ,E.g and many more.
  • CPG is an unmodified bare CPG, which means that the surface of the CPG is rich in silicon hydroxyl groups, and the surface of the CPG does not carry any chemical group modifications, such as the modification of the universal link (Universal Linker).
  • the aforementioned nucleic acid is DNA
  • the aforementioned nucleic acid synthesis device is a DNA synthesis device.
  • the pore size of the above CPG is (Angstrom) to
  • the nucleic acid synthesis device further includes a sieve plate, the sieve plate includes an upper sieve plate and a lower sieve plate, and the CPG is disposed between the upper sieve plate and the lower sieve plate.
  • the above upper sieve plate is subjected to hydrophilic treatment or selected from hydrophilic materials, and the above lower sieve plate is selected from hydrophobic materials.
  • the solid phase carrier further includes a thermoplastic polymer resin, and the thermoplastic polymer resin is sintered with CPG to form a filter element (CPG Frits) containing glass balls with controllable pore diameters.
  • CPG Frits filter element
  • thermoplastic polymer resin is selected from polyethylene, preferably ultra high molecular weight polyethylene (UHMW-PE) or high density polyethylene (HDPE).
  • UHMW-PE ultra high molecular weight polyethylene
  • HDPE high density polyethylene
  • the above sintering temperature is 160°C to 250°C, preferably 180°C.
  • the diameter of the above CPG Frits is ⁇ 3.5 mm, and the thickness is ⁇ 1.5 mm.
  • the diameter of the CPG Frits is 3 mm, and the thickness is 4 mm.
  • each CPG Frits contains 0.1-10 mg CPG, preferably 4 mg CPG.
  • the above CPG Frits are hydrophilic CPG Frits.
  • hydrophilic CPG Frits are CPG Frits obtained by hydrophilic treatment.
  • the above-mentioned hydrophilic treatment is first wetted with an alcohol solvent, and then bonded with a surfactant.
  • the alcoholic solvent is a C1-10 linear or branched alcoholic solvent, preferably methanol, ethanol, propanol, n-butanol, ethylene glycol, glycerin, and their aqueous solvents or Their combination is more preferably 95% (v/v) ethanol;
  • surfactants are anionic surfactants, cationic surfactants, zwitterionic surfactants or nonionic surfactants, preferably sodium dodecyl sulfate, benzalkonium bromide, polysorbate, fatty acid sorbitan, quaternary Ammonium compounds, lecithin, amino acids, or a combination thereof.
  • the nucleic acid synthesis device further includes a storage device, and the solid phase carrier is placed in the storage device.
  • the above-mentioned receiving device is an empty column tube.
  • At least one assembly position is reserved in the empty column tube, the assembly position is a cylinder, and the solid phase carrier is cylindrical, so that the solid phase carrier completely fits the assembly position, and the height of the assembly position is greater than The thickness of the solid support.
  • the length of the empty column tube is 70 mm
  • the upper inner diameter of the empty column tube is 4.52 mm
  • the upper part of the assembly position gradually closes to 3 mm
  • the inner diameter of the assembly position is 2.95 mm
  • the height of the assembly position is 5mm.
  • an embodiment provides a nucleic acid synthesis apparatus including the nucleic acid synthesis device as in the first aspect.
  • an embodiment provides a use of the nucleic acid synthesis device of the first aspect in nucleic acid synthesis.
  • the above-mentioned nucleic acid synthesis is performed using the solid-phase phosphoramidite triester method.
  • an embodiment provides a nucleic acid synthesis method including nucleic acid synthesis using the nucleic acid synthesis apparatus of the first aspect or the nucleic acid synthesis apparatus of the second aspect, wherein the hydroxyl group on the surface of the CPG in the nucleic acid synthesis apparatus is used as a The starting point for the synthesis of synthetic nucleic acids is linked to phosphoramidite-protected nucleotide monomers or polymers by covalent bonding.
  • the above method is a solid phase phosphoramidite triester method.
  • an embodiment provides a nucleic acid purification device including a solid-phase carrier, the solid-phase carrier includes a controllable pore glass sphere (CPG), CPG is an unmodified bare CPG, and its surface has hydroxyl groups, hydroxyl groups An oligonucleotide sequence is connected by covalent bonding, and the oligonucleotide sequence and the nucleic acid to be purified are all or partially specifically complementary or randomly matched.
  • CPG controllable pore glass sphere
  • all or part of the oligonucleotide sequence and the region at the 3'end and/or 5'end of the nucleic acid to be purified are specifically complementary paired or randomly paired.
  • the length of the aforementioned oligonucleotide sequence is 6 to 12 bases in length.
  • the aforementioned nucleic acid to be purified is DNA, and accordingly, the aforementioned nucleic acid purification device is a DNA purification device.
  • the nucleic acid to be purified is a biological reaction product, preferably an enzyme digestion product or a PCR product.
  • the pore size of the above CPG is (Angstrom) to
  • the nucleic acid purification device further includes a sieve plate, the sieve plate includes an upper sieve plate and a lower sieve plate, and the CPG is placed between the upper sieve plate and the lower sieve plate.
  • the above upper sieve plate is subjected to hydrophilic treatment or selected from hydrophilic materials, and the above lower sieve plate is selected from hydrophobic materials.
  • the solid phase carrier further includes a thermoplastic polymer resin, and the thermoplastic polymer resin is sintered with CPG to form a filter element (CPG Frits) containing glass balls with controllable pore diameters.
  • CPG Frits filter element
  • thermoplastic polymer resin is selected from polyethylene, preferably ultra high molecular weight polyethylene (UHMW-PE) or high density polyethylene (HDPE).
  • UHMW-PE ultra high molecular weight polyethylene
  • HDPE high density polyethylene
  • the diameter of the above CPG Frits is ⁇ 3.5 mm, and the thickness is ⁇ 1.5 mm.
  • the nucleic acid purification device further includes a containing device, and the solid phase carrier is placed in the containing device.
  • the above-mentioned receiving device is an empty column tube.
  • At least one assembly position is reserved in the empty column tube, the assembly position is a cylinder, and the solid phase carrier is cylindrical, so that the solid phase carrier completely fits the assembly position, and the height of the assembly position is greater than The thickness of the solid support.
  • an embodiment provides a nucleic acid purification device, including the nucleic acid purification device as in the fifth aspect.
  • an embodiment provides a nucleic acid purification method, including adding a solution containing the nucleic acid to be purified to the nucleic acid purification device of the fifth aspect to incubate, so that the nucleic acid to be purified and the nucleic acid purification device described above All or part of the oligonucleotide sequences are specifically complementary paired or randomly paired; then the nucleic acid to be purified is eluted from the nucleic acid purification device.
  • the nucleic acid to be purified is a single-stranded nucleic acid.
  • the nucleic acid purification device performs ammonia deprotection treatment before incubating with the solution.
  • the ammonia deprotection treatment refers to the removal of the protecting group on the heterocyclic base amino group of the monomer or polymer used in the synthesis of the oligonucleotide and the cyanoethyl group on the phosphoester hydroxyl group under the aminolysis conditions Base protecting group to restore the structure of the oligonucleotide itself.
  • the above ammonia deprotection treatment is carried out at 90°C for 2 hours; then, using acetonitrile/aqueous solution and ultrapure water as the mobile phase for washing and centrifugation to obtain a nucleic acid purification device connected with the above oligonucleotide sequence Then incubate with the above solution.
  • washing is carried out with ethanol/aqueous solution as a mobile phase to remove unbound nucleic acids and/or enzymes and/or buffers, and then the nucleic acid to be purified is eluted from the nucleic acid purification device Down.
  • the above-mentioned elution of the nucleic acid to be purified from the nucleic acid purification device specifically includes: denaturing and melting the paired combined nucleic acid to be purified and the oligonucleotide sequence under heating conditions, and heating to Pure water elutes the unwound single-stranded nucleic acid to be purified.
  • the nucleic acid to be purified is eluted from the nucleic acid purification device and then annealed to restore the complementary single strand to the double helix structure.
  • an embodiment provides a use of the nucleic acid purification device of the fifth aspect in nucleic acid purification.
  • the CPG is an unmodified bare CPG, without a universal linker (Universal Linker), the surface is exposed to a certain number of hydroxyl groups, and the oligonucleotide primers and other nucleic acids are synthesized and then processed after ammonia hydrolysis. Nucleic acids such as oligonucleotide primers attached to the CPG will not be cut off, but will still be attached to the solid support.
  • Universal Linker Universal Linker
  • the nucleic acid synthesis device of the present invention can achieve the purpose of synthesizing nucleic acids and immobilizing nucleic acids at the same time, and can be used not only for the synthesis of nucleic acids such as oligonucleotide primers, but also for enzymes using nucleic acids such as oligonucleotide primers fixed on CPG Cut products, purification of PCR products, etc. have the advantages of simple structure, small size, light weight, high efficiency, low cost, and diverse functions.
  • the nucleic acid purification device of the present invention can purify specific nucleic acid sequences, has the advantages of high efficiency, fast, convenient, safe and non-toxic, and overcomes the existing nucleic acid separation and purification methods that cannot purify specific DNA sequences, the operation is complicated, the efficiency is low, and the separation The speed is low, the economic cost is high, and the need to use toxic chemical reagents.
  • FIG. 1 is a schematic diagram of the design of a nucleic acid synthesis column or a nucleic acid purification column in an embodiment of the present invention, in which 1- empty column tube; 2- CPG Frits assembled in the empty column tube;
  • FIG. 2 is a schematic cross-sectional view of a CPG Frits cross section of a nucleic acid synthesis column or a nucleic acid purification column in an embodiment of the present invention, in which a rigid network structure formed by a bridge between 3-PE particles; 4- without a universal linker (Universal Linker), Unmodified bare CPG particles;
  • FIG 3 is a schematic diagram of a CPG structure design principle of a nucleic acid synthesis column in an embodiment of the present invention, in which the surface of unmodified bare CPG particles has hydroxyl groups (-OH);
  • FIG. 4 is a schematic diagram of a CPG structure design principle of a nucleic acid purification column in an embodiment of the present invention, in which an unmodified naked CPG particle surface is connected with an oligonucleotide sequence;
  • FIG. 5 is a schematic flowchart of a nucleic acid purification method in an embodiment of the present invention.
  • FIG. 6 is a diagram of electrophoresis results of DNA fragments obtained by purification in an example of the present invention on a 1.2% agarose gel, in which 1- using a DNA purification column connected with a 6nt degenerate oligonucleotide sequence to recover PCR products; 2 -Use a DNA purification column connected with an 8nt degenerate oligonucleotide sequence to recover PCR products; 3- Use a DNA purification column connected with a 10nt degenerate oligonucleotide sequence to recover PCR products; 4- Use a 12nt degenerate oligo oligonucleotide DNA purification column of nucleotide sequence recovers PCR products; M-DL2000 DNA Marker.
  • An embodiment of the present invention provides a nucleic acid synthesis device.
  • the device includes a solid-phase carrier.
  • the solid-phase carrier includes a controllable pore glass sphere (CPG).
  • the CPG is an unmodified bare CPG whose surface does not carry chemicals.
  • the universal linker of the group has a hydroxyl group on its surface. The hydroxyl group is used as the starting point for nucleic acid synthesis and is used to connect the phosphoramidite-protected nucleotide monomer or polymer for nucleic acid through covalent bonding. synthesis.
  • the covalent bonding connection between the hydroxyl group and the nucleotide on the surface of the CPG can be achieved by the classic solid-phase phosphoramidite triester method.
  • the CPG used in the embodiment of the present invention It is an unmodified bare CPG, and the hydroxyl group on its surface acts as a 5'-hydroxyl group in the solid-phase phosphoramidite triester reaction. It bonds with the added phosphoramidite-protected nucleotide monomer or polymer. .
  • the nucleic acid synthesis device of the present invention is suitable for synthesis of any suitable nucleic acid, including but not limited to DNA and RNA, etc., preferably DNA synthesis. Therefore, in a preferred embodiment, the nucleic acid synthesis device of the present invention is a DNA synthesis device.
  • the controllable pore glass sphere (CPG) sphere has many irregular pores inside, the pore network is huge, the size of the pores is called the pore diameter, and the pore diameter is stable, usually choose the pore diameter (Angstrom) to (Preferably ) Is used for the preparation of the nucleic acid synthesis device of the embodiment of the present invention, and other pore sizes can also be selected.
  • the aperture of the controllable aperture glass ball (CPG) can be determined according to the specific situation.
  • the nucleic acid synthesis device may be designed in various suitable forms.
  • the present invention provides two exemplary forms.
  • the nucleic acid synthesis device includes a controlled pore glass ball (CPG) and a sieve plate
  • the sieve plate includes an upper sieve plate and a lower sieve plate
  • the controlled pore glass sphere (CPG) is placed on the upper sieve plate and the lower sieve
  • the sieve plate can be made by sintering thermoplastic polymer resin powder such as ultra high molecular weight polyethylene (UHMW-PE) or high density polyethylene (HDPE).
  • UHMW-PE ultra high molecular weight polyethylene
  • HDPE high density polyethylene
  • the role of the sieve plate is to prevent the CPG from leaking and the synthetic reagent can pass through.
  • the upper screen plate can be treated with hydrophilic or hydrophilic material, and the lower screen plate can use hydrophobic material.
  • the nucleic acid synthesis device further includes a containing device, a solid-phase carrier such as a controlled pore glass ball (CPG) and an optional sieve plate are placed in the containing device, the containing device may be an empty column tube, and the empty column tube may be used Polypropylene is injection molded.
  • a solid-phase carrier such as a controlled pore glass ball (CPG) and an optional sieve plate
  • the nucleic acid synthesis device includes a controlled pore glass ball (CPG) and a filter core (CPG Frits) formed by sintering a thermoplastic polymer resin as a solid phase carrier.
  • the sintering temperature may be 160°C to 250°C, preferably 180°C.
  • the thermoplastic polymer resin may be selected from polyethylene or polypropylene, etc., preferably ultra high molecular weight polyethylene (UHMW-PE) or high density polyethylene (HDPE).
  • Polyethylene (PE) particles wrap and fix the CPG particles.
  • the bridges between the PE particles form a certain pore size.
  • the CPG particles are evenly embedded in the rigid network structure formed by the PE bridges to form CPG Frits with a defined pore size and porosity.
  • CPG Frits is cylindrical, and its diameter can be any suitable diameter, such as diameter ⁇ 3.5mm; the thickness can be any suitable thickness, such as thickness ⁇ 1.5mm. Preferably, the diameter of CPG Frits is 3 mm and the thickness is 4 mm.
  • Each CPG Frits contains 0.1-10 mg CPG, preferably 4 mg CPG.
  • hydrophilic treatment can achieve The absorption of aqueous solution by this material will expand its wide application in synthetic biology. Therefore, in order to improve the hydrophilicity of CPG Frits, CPG Frits can adopt hydrophilic CPG Frits. Hydrophilic treatment may be wetted with alcohol first, and then bonded with a surfactant.
  • the alcohol solvent may be a C1-10 linear or branched chain alcohol solvent, preferably methanol, ethanol, propanol, n-butanol, ethylene glycol, glycerin, and their aqueous solvents or combinations thereof, more It is preferably 95% (v/v) ethanol.
  • the surfactant may be an anionic surfactant, a cationic surfactant, a zwitterionic surfactant or a nonionic surfactant, preferably sodium dodecyl sulfate, benzalkonium bromide, polysorbate, fatty acid sorbitan, quaternary ammonium Compounds, lecithin, amino acids, or combinations thereof.
  • the nucleic acid synthesis device of the present invention further includes a receiving device, and solid phase carriers such as CPG Frits are placed in the receiving device.
  • the receiving device may be an empty column tube, which may be injection molded from polypropylene .
  • the accommodating device is an empty column tube
  • the solid phase carrier CPG Frits is made into a cylindrical shape and placed in the empty column tube
  • at least one assembly position is reserved in the empty column tube
  • the assembly position is a cylinder
  • the solid-phase carrier is cylindrical and completely fits the empty column tube
  • the height of the assembly position is greater than the thickness of the solid-phase carrier.
  • the empty column tube is injection molded from polypropylene, with a gradient straight port design, and the reserved CPG Frits assembly position is a cylinder with no slope.
  • the length of the empty column tube is 70mm, the inner diameter of the upper part of the empty column tube is 4.52mm, and the upper part of the empty position is gradually closed to 3mm, the inner diameter of the assembly position is 2.95mm, and the height of the assembly position is 5mm.
  • the receiving device in addition to the use of empty column tubes, can also use other suitable forms, such as multi-well plates, such as 24-well plates, 96-well plates, 384-well plates, etc., each CPG Frits, or The CPG and the sieve plate are placed in each well of the multiwell plate, respectively, to form a nucleic acid synthesis device in the form of a multiwell plate, which can perform nucleic acid synthesis in batches.
  • multi-well plates such as 24-well plates, 96-well plates, 384-well plates, etc.
  • each CPG Frits or The CPG and the sieve plate are placed in each well of the multiwell plate, respectively, to form a nucleic acid synthesis device in the form of a multiwell plate, which can perform nucleic acid synthesis in batches.
  • the nucleic acid synthesis device is a nucleic acid synthesis column
  • the nucleic acid synthesis column includes: an empty column tube 1 and a CPG assembled in the empty column tube Frits 2.
  • CPG is an unmodified bare CPG carrier without Universal Linker, with a certain number of hydroxyl groups exposed on the surface, and the pore size of CPG is CPG Frits 2 is formed by sintering unmodified bare CPG particles 4 and a thermoplastic polymer resin (for example, PE), and the bridges between the PE particles form a rigid network structure 3.
  • the sintering temperature may be 160°C to 250°C, preferably 180°C.
  • the sintering time may be from 5 minutes to 10 hours, etc., and it can be determined according to specific needs.
  • Figure 3 shows that the surface of the bare CPG carrier is exposed to a certain number of hydroxyl groups (-OH), through which hydroxyl groups can form a covalent bond with the phosphoramidite-protected nucleotide monomer or polymer to be added For nucleic acid synthesis.
  • An embodiment of the present invention provides a nucleic acid synthesis device, which includes the nucleic acid synthesis device of the embodiment of the present invention.
  • the nucleic acid synthesis device of the present invention other parts such as a liquid circuit system, a gas circuit system, a control system, a transmission system, etc., can be implemented according to the prior art.
  • the nucleic acid synthesis device used in the Dr. Oligo 192 nucleic acid synthesizer can be replaced with the nucleic acid synthesis device of the present invention as an example of the nucleic acid synthesis device of the present invention.
  • the use of the nucleic acid synthesis device of the present invention in a method for synthesizing nucleic acids is provided.
  • the synthetic nucleic acid may be any suitable nucleic acid, including but not limited to DNA and RNA, etc., preferably DNA.
  • the solid phase phosphoramidite triester method is used for nucleic acid synthesis.
  • the nucleic acid synthesis device of the present invention as a synthesis column in the Dr. Oligo 192 nucleic acid synthesizer, and using the classical solid-phase phosphoramidite triester method to synthesize an oligonucleotide primer sequence on the synthesizer, The purpose of the present invention is achieved.
  • An embodiment of the present invention provides a nucleic acid synthesis method which uses the nucleic acid synthesis device or nucleic acid synthesis device of the present invention to perform nucleic acid synthesis, and the hydroxyl group on the surface of CPG in the nucleic acid synthesis device is used as the synthesis starting point of the nucleic acid to be synthesized,
  • the phosphoramidite-protected nucleotide monomer or polymer is connected by covalent bonding.
  • the nucleic acid synthesis method is a solid phase phosphoramidite triester method.
  • An embodiment of the present invention provides a nucleic acid purification device.
  • the device includes a solid-phase carrier.
  • the solid-phase carrier includes a controlled pore glass ball (CPG).
  • the CPG is an unmodified bare CPG without a universal connecting arm. (Universal Linker), the surface of which has hydroxyl groups, and these hydroxyl groups are connected by a covalent bond to an oligonucleotide sequence that is specifically complementary or randomly matched with all or part of the nucleic acid to be purified.
  • CPG controlled pore glass ball
  • the covalent bonding of the hydroxyl group on the surface of the CPG and the oligonucleotide sequence can be achieved by the classic solid-phase phosphoramidite triester method.
  • the embodiment of the present invention uses The CPG is an unmodified naked CPG, and the hydroxyl group on the surface acts as a 5'-hydroxyl group in the solid-phase phosphoramidite triester reaction. It bonds with the added phosphoramidite-protected nucleotide monomer or polymer. ⁇ He reaction.
  • the oligonucleotide sequence and the nucleic acid to be purified are all or partially specifically complementary paired or randomly paired. Therefore, the specific nucleic acid sequence can be purposefully purified, when the sequence of the nucleic acid to be purified is known It is easy to design the above oligonucleotide sequence according to the principle of base complementary pairing.
  • the pairing position of the oligonucleotide sequence on the nucleic acid to be purified is selected at the 3′ end or/and 5′ end region of the nucleic acid to be purified, specifically, it may be a region at the 3′ end of the nucleic acid to be purified It can also be a section of the 5'end of the nucleic acid to be purified, or a section of each of the 3'end and 5'end of the nucleic acid to be purified.
  • the length of the oligonucleotide sequence is not particularly limited, and any length can be designed according to specific needs, but according to the test effect, it is determined that the length of the oligonucleotide sequence can achieve excellent results within the range of 6 to 12 bases. For example, 6 bases, 8 bases, 10 bases, or 12 bases.
  • nucleic acid purification device means that the device of the present invention can purify any suitable nucleic acid, including but not limited to DNA and RNA.
  • the nucleic acid to be purified is DNA. Therefore, the nucleic acid purification device of the present invention is a DNA purification device.
  • the nucleic acid to be purified is a biological reaction product, which can be the product of any biological, biochemical, etc. reaction, and the product requires specific purification of specific fragments.
  • biological reaction products are typical but not limited Examples of sex are digested products or PCR products.
  • the CPG sphere used has many irregular pores, the pore network is huge, the size of the pores is called the pore diameter, and the pore diameter is stable.
  • the pore diameter is selected (Angstrom) to (Preferably ) Is used for the preparation of the nucleic acid purification device of the embodiment of the present invention, and other pore sizes can also be selected.
  • the pore size of the CPG used can be determined according to the specific conditions of the nucleic acid to be purified.
  • the nucleic acid purification device may be designed in various suitable forms.
  • the present invention provides two exemplary forms.
  • the nucleic acid purification device includes a CPG and a sieve plate.
  • the sieve plate includes an upper sieve plate and a lower sieve plate.
  • the CPG is placed between the upper sieve plate and the lower sieve plate.
  • the sieve plate may use ultra-high molecular weight polyethylene ( UHMW-PE) or high density polyethylene (HDPE) and other thermoplastic polymer resin powder sintered.
  • UHMW-PE ultra-high molecular weight polyethylene
  • HDPE high density polyethylene
  • the role of the sieve plate is to prevent CPG from leaking and various reagents can pass through.
  • the upper screen plate can be treated with hydrophilic or hydrophilic material, and the lower screen plate can use hydrophobic material.
  • the nucleic acid purification device further includes a containing device, a solid phase carrier such as CPG and an optional sieve plate are placed in the containing device, the containing device may be an empty column tube, and the empty column tube may be injection molded from polypropylene.
  • the nucleic acid purification device includes a CPG and a filter element (CPG Frits) formed by sintering a thermoplastic polymer resin as a solid phase carrier.
  • the thermoplastic polymer resin may be selected from polyethylene or polypropylene, etc., preferably ultra high molecular weight polyethylene (UHMW-PE) or high density polyethylene (HDPE).
  • UHMW-PE ultra high molecular weight polyethylene
  • HDPE high density polyethylene
  • Polyethylene (PE) particles wrap and fix the CPG particles.
  • the bridges between the PE particles form a certain pore size.
  • the CPG particles are evenly embedded in the rigid network structure formed by the PE bridges to form CPG Frits with a defined pore size and porosity.
  • CPG Frits can be cylindrical, and its diameter can be any suitable diameter, such as diameter ⁇ 3.5mm; thickness can be any suitable thickness, such as thickness ⁇ 1.5mm.
  • the diameter of CPG Frits is 3 mm and the thickness is 4 mm.
  • the nucleic acid purification device may further include a containing device, and solid phase carriers such as CPG Frits are placed in the containing device.
  • the containing device may be an empty column tube, and the empty column tube may be injection molded from polypropylene.
  • the accommodating device is an empty column tube
  • the solid phase carrier CPG Frits is made into a cylindrical shape and placed in the empty column tube
  • at least one assembly position is reserved in the empty column tube
  • the assembly position is a cylinder
  • the solid-phase carrier is cylindrical and completely fits the empty column tube
  • the height of the assembly position is greater than the thickness of the solid-phase carrier.
  • the receiving device in addition to the use of empty column tubes, can also use other suitable forms, such as multi-well plates, such as 24-well plates, 96-well plates, 384-well plates, etc., each CPG Frits, or The CPG and the sieve plate are placed in each well of the multi-well plate respectively to form a nucleic acid purification device in the form of a multi-well plate, which can perform nucleic acid purification in batches.
  • multi-well plates such as 24-well plates, 96-well plates, 384-well plates, etc.
  • each CPG Frits or The CPG and the sieve plate are placed in each well of the multi-well plate respectively to form a nucleic acid purification device in the form of a multi-well plate, which can perform nucleic acid purification in batches.
  • the nucleic acid purification device is a nucleic acid purification column
  • the nucleic acid purification column includes: an empty column tube 1 and a CPG assembled in the empty column tube Frits 2.
  • CPG is an unmodified bare CPG carrier without Universal Linker, with a certain number of hydroxyl groups exposed on the surface, and the pore size of CPG is CPG Frits 2 is formed by sintering unmodified bare CPG particles 4 and a thermoplastic polymer resin (for example, PE), and the bridges between the PE particles form a rigid network structure 3.
  • the sintering temperature may be 160°C to 250°C, preferably 180°C.
  • the sintering time may be from 5 minutes to 10 hours, etc., and it can be determined according to specific needs.
  • Fig. 4 shows that the surface of the naked CPG carrier is exposed with a certain number of hydroxyl groups (-OH), which are linked by a covalent bond to an oligonucleotide sequence, which is specific to all or part of the nucleic acid to be purified Sexual complementary pairing or random pairing.
  • -OH hydroxyl groups
  • CPG Frits is formed by mixing CPG and UHMW-PE powder uniformly and sintering at 180°C.
  • the diameter of CPG Frits is 3 mm, and the thickness of CPG Frits is 4 mm.
  • CPG Frits are hydrophilically treated, and the content of CPG in each CPG Frits is 4mg. Among them, the hydrophilic treatment process of CPG Frits can be wetted with alcohol first, and then bonded with a surfactant.
  • the alcohol solvent may be a C1-10 linear or branched chain alcohol solvent, preferably methanol, ethanol, propanol, n-butanol, ethylene glycol, glycerin, and their aqueous solvents or combinations thereof, more It is preferably 95% (v/v) ethanol.
  • the surfactant may be an anionic surfactant, a cationic surfactant, a zwitterionic surfactant or a nonionic surfactant, preferably sodium dodecyl sulfate, benzalkonium bromide, polysorbate, fatty acid sorbitan, quaternary ammonium Compounds, lecithin, amino acids, or combinations thereof.
  • the empty column tube can be made of polypropylene injection molding, using a gradient straight mouth design, the reserved CPG Frits assembly position is a cylinder, no slope.
  • the length of the empty column tube is 70mm, the inner diameter of the upper part of the empty column tube is 4.52mm, and the upper part of the empty position is gradually closed to 3mm, the inner diameter of the assembly position is 2.95mm, and the height of the assembly position is 5mm.
  • An embodiment of the present invention provides a nucleic acid purification device, including the nucleic acid purification device as the embodiment of the present invention.
  • An embodiment of the present invention provides a nucleic acid purification method.
  • the method includes incubating a solution containing the nucleic acid to be purified into the nucleic acid purification device of the present invention to make the nucleic acid sequence to be purified and the oligonucleotide sequence in the nucleic acid purification device specific sexual complementary pairing or random pairing; then elute the nucleic acid to be purified from the nucleic acid purification device.
  • the nucleic acid to be purified may be any suitable nucleic acid, including but not limited to DNA and RNA.
  • the nucleic acid to be purified is DNA.
  • the nucleic acid to be purified is a biological reaction product, which can be the product of any biological, biochemical, etc. reaction, and the product requires specific purification of specific fragments.
  • biological reaction products are typical but not limited Examples of sex are digested products or PCR products.
  • the nucleic acid to be purified may be a nucleic acid containing at least a part of single strands, including but not limited to a nucleic acid to be purified with a protruding single strand and a double strand in the middle, or a nucleic acid to be purified with a double strand and a single strand in the middle
  • the strand, or the entire strand of nucleic acid to be purified is single stranded.
  • the nucleic acid to be purified is a single-stranded nucleic acid.
  • the nucleic acid purification device performs ammonia deprotection treatment before incubating with the solution to be purified.
  • the ammonia deprotection treatment for example, can be carried out at 90°C for 2 hours; then, using acetonitrile/aqueous solution and ultrapure water as the mobile phase for washing and centrifugation to obtain a pure nucleic acid purification device connected with the oligonucleotide sequence, and then Incubate with the solution to be purified.
  • the nucleic acid to be purified is a PCR product, which is denatured and untwisted at high temperature (for example, 95°C) to form a single strand, with an oligonucleotide sequence connected to the surface of CPG in the nucleic acid purification device at both ends.
  • Complementary sequence load the denatured and unwound PCR product into the nucleic acid purification device and incubate.
  • the oligonucleotide sequence linked to the CPG surface is complementary to the sequence segment at both ends of the single strand; wash (for example, ethanol/water solution as mobile phase Washing) to remove unbound DNA and enzymes, buffers and other impurities in the reaction system; then elute the nucleic acid to be purified from the nucleic acid purification device, for example, under heating, the paired bound nucleic acid to be purified and The oligonucleotide sequence is denatured and melted, and the unstranded nucleic acid to be purified is eluted by heating ultrapure water; finally, the eluted nucleic acid is denatured and annealed again to restore the complementary single strand to the double helix structure and be purified DNA product.
  • wash for example, ethanol/water solution as mobile phase Washing
  • the use of the nucleic acid purification device of the present invention in nucleic acid purification is provided, in which an oligonucleotide sequence is connected to the hydroxyl group on the CPG surface of the nucleic acid purification device, and the oligonucleotide sequence is to be purified All or part of the nucleic acids are specifically complementary or randomly matched.
  • step (3) (4) Quickly load the denatured DNA aqueous solution obtained in step (3) into the DNA purification column, be absorbed by CPG Frits, and incubate at 25°C for 5 minutes to bind with the oligonucleotide on the column;
  • a NanoDrop UV spectrophotometer was used to detect the purified PCR product, and the ratio of OD 260 /OD 280 was 1.8-2.0.
  • Table 1 shows the comparison results of the purification results of the PCR products purified by DNA purification columns connected with oligonucleotides of different lengths (both random sequences of degenerate bases N) under the same conditions.
  • FIG. 6 is a diagram of the electrophoresis results of the DNA fragments purified in the above examples on a 1.2% agarose gel, where lane 1 represents the use of a DNA purification column connected to a 6nt degenerate oligonucleotide sequence to recover PCR products; lane 2 represents the use of a DNA purification column connected with an 8nt degenerate oligonucleotide sequence to recover the PCR product; lane 3 represents the use of a DNA purification column connected with a 10nt degenerate oligonucleotide sequence to recover the PCR product; lane 4 represents the use of a 12nt connected The DNA purification column of the degenerate oligonucleotide sequence recovers the PCR product; M stands for DL2000 DNA Marker.

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Abstract

一种核酸合成装置和纯化装置、其用途及核酸合成方法和纯化方法，该核酸合成装置包括固相载体，该固相载体包括可控孔径玻璃球(CPG)，该CPG是未被修饰的裸CPG，其表面具有羟基，羟基用作核酸合成的起始点，用于通过共价键合连接亚磷酰胺保护的核苷酸单体或多聚体以进行核酸合成。本发明的核酸合成装置，不仅可以用于寡核苷酸引物的合成，还可以利用固定在CPG上的寡核苷酸引物进行酶切、PCR产物的纯化等，具有结构简单、体积小、重量轻、效率高、成本低、功能多样的优点。

Description

核酸合成装置和纯化装置、其用途及核酸合成方法和纯化方法 技术领域
本发明涉及核酸合成技术领域，具体涉及一种核酸合成装置和纯化装置、其用途及核酸合成方法和纯化方法。
背景技术
经典的DNA合成柱，包含固相载体、筛板和空柱管。固相载体通常用可控孔径玻璃球(Controlled Pore Glass，CPG)，CPG球体内部有很多不规则的孔道，孔隙网络庞大，孔道的大小称为孔径，孔径稳定，通常选择孔径500埃
Figure PCTCN2018121092-appb-000001
和
Figure PCTCN2018121092-appb-000002
的CPG用于DNA合成，也可以选择其它孔径。筛板通常用超高分子量聚乙烯(UHMW-PE)或高密度聚乙烯(HDPE)粉末烧结而成。在DNA合成柱中，筛板的作用是阻挡CPG不漏下来而合成试剂能够通过。空柱管为聚丙烯注塑而成。
第二代DNA通用合成柱由含可控孔径玻璃球的滤芯(Controlled Pore Glass Frits,CPG Frits)和空柱管构成。CPG Frits是CPG和UHMW-PE或HDPE粉末烧结而成。PE颗粒包裹并固定CPG颗粒，PE颗粒之间搭桥形成一定的孔径，CPG颗粒均匀地嵌入PE搭桥而成的刚性网状结构中，形成具有确定孔径和孔隙率的筛板。引物合成过程中，合成试剂流经CPG内部孔道与PE搭桥形成的孔道，使试剂流道足够长，从而增加化学反应的时间以及反应物与CPG上的反应位点接触的机会。聚合物的包裹作用使CPG颗粒固定，不会因为试剂的流动而发生转动，进一步延长了试剂和CPG上的反应位点的接触时间，提高了合成效率和产物纯度。
目前引物等寡核苷酸的合成普遍采用第二代CPG Frits合成柱，其中CPG为二氧化硅材质，被广泛用作固相合成寡核苷酸的载体，通常带有化学基团的通用连接臂(Universal Linker)修饰，合成起始步骤为用三氯乙酸(TCA)去除CPG所连的连接臂上的DMT保护基，获得游离的5’-羟基供下一步与亚磷酰胺单体发生缩合反应，合成完毕经过氨解后处理，连接在CPG上的寡核苷酸会被切下来，经过纯化可用于后续实验，而达不到固定寡核苷酸的效果。由于所使用的PE材料具有超强的疏水性，CPG被包裹其中，因此这种合成柱也不能吸收酶切、PCR等反应溶液。
DNA的分离纯化是分子生物学操作中的关键步骤，分离的DNA可以用于下游基因克隆、 探针制备等实验。DNA的分离纯化通常包括两种类型：1)总DNA提取。这种分离纯化，只需要将组织、细胞或体外DNA合成反应产物中的其它杂质去除，然后通过物理或者化学的方法分离出所需要的所有DNA遗传物质即可。2)特异DNA提取。这种分离纯化通常需要根据实验目的，对组织、细胞或者体外DNA合成扩增产物中的特异DNA，包括某一功能类型的DNA(比如质粒DNA)、某一长度的DNA或者某一序列特异的DNA进行提取。
目前实验室常用的DNA分离纯化方法包括硅胶膜离心吸附柱法以及磁珠吸附法。两种方法的核心原理类似，均是通过硅胶膜或者磁珠表面键合的基团，在一定的缓冲液条件下，同DNA表面所带的电荷进行吸附，从而将目的DNA分离纯化。这两种方法都已经被开发为成熟的商业试剂盒，在市场上能够买到。以TIANGEN公司的通用型DNA纯化回收试剂盒为例，该试剂盒采用独特的缓冲体系和离心吸附柱，既可从TAE或TBE琼脂糖凝胶中回收DNA片段，又可用于直接纯化PCR产物，同时除去蛋白质、其它有机化合物、无机盐离子及寡核苷酸引物等杂质，能够满足多种实验需要。溶胶液中含有pH指示剂，可根据颜色来判断溶胶状态。凝胶回收试剂盒的原理是：在凝胶融化液中凝胶块被迅速融化并释放出DNA，加入高离液序列溶液后DNA片断被选择性吸附到硅胶膜上，经漂洗液洗涤去除残留在硅胶膜上的杂质和高浓度盐离子后，吸附到硅胶膜上的DNA片断经洗脱缓冲液或水洗脱下来，即可用于各种分子生物学实验。磁珠吸附法核酸提取为运用纳米技术对超顺磁性纳米颗粒的表面进行改良和表面修饰后，制备成超顺磁性氧化硅纳米磁珠。该磁珠能在微观界面上与核酸分子特异性地识别和高效结合。利用氧化硅纳米微球的超顺磁性，在Chaotropic盐(盐酸胍、异硫氰酸胍等)和外加磁场的作用下，能从血液、动物组织、食品、病原微生物等样本中将DNA和RNA分离出来，可应用于临床疾病诊断、输血安全、法医学鉴定、环境微生物检测、食品安全检测、分子生物学研究等多种领域。
实验室常用的DNA回收方法，成本相对较高，操作流程也比较繁琐和复杂，无法对特异DNA序列进行纯化，并且需要使用含有有毒化学试剂的缓冲液。
发明内容
本发明提供一种核酸合成装置和纯化装置、其用途及核酸合成方法和纯化方法，核酸合成装置不仅可以用于寡核苷酸序列的合成，还可以利用固定在CPG上的寡核苷酸序列进行酶切产物、PCR产物的纯化等，具有结构简单、体积小、重量轻、效率高、成本低、功能多样的优点；核酸纯化装置能够对特异核酸序列进行纯化，具有高效、快速、方便、安全无毒的优点。
根据第一方面，一种实施例中提供一种核酸合成装置，包括固相载体，该固相载体包括可控孔径玻璃球(CPG)，CPG是未被修饰的裸CPG，其表面具有羟基，羟基可通过共价键合连接亚磷酰胺保护的核苷酸单体或多聚体以进行核酸合成。
本领域普通技术人员熟知，目前寡核苷酸的合成普遍采用第二代CPG Frits合成柱，其中CPG为二氧化硅材质，并且CPG表面通常带有化学基团的通用连接臂(Universal Linker)修饰，例如
Figure PCTCN2018121092-appb-000003
Figure PCTCN2018121092-appb-000004
等等。而在本申请中，CPG是未被修饰的裸CPG，是指CPG表面富含硅羟基，并且CPG表面未带有任何化学基团修饰，如连接臂(Universal Linker)修饰等。
在优选实施例中，上述核酸是DNA，相应地，上述核酸合成装置是DNA合成装置。
在优选实施例中，上述CPG的孔径为
Figure PCTCN2018121092-appb-000005
(埃)至
Figure PCTCN2018121092-appb-000006
在优选实施例中，上述核酸合成装置还包括筛板，上述筛板包括上筛板和下筛板，上述CPG置于上述上筛板和下筛板之间。
在优选实施例中，上述上筛板经过亲水处理或选自亲水材料，上述下筛板选自疏水材料。
在优选实施例中，上述固相载体还包括热塑性聚合物树脂，上述热塑性聚合物树脂与CPG烧结形成含可控孔径玻璃球的滤芯(CPG Frits)。
在优选实施例中，上述热塑性聚合物树脂选自聚乙烯，优选超高分子量聚乙烯(UHMW-PE)或高密度聚乙烯(HDPE)。
在优选实施例中，上述烧结的温度是160℃至250℃，优选180℃。
在优选实施例中，上述CPG Frits的直径≤3.5mm，厚度≥1.5mm。
在优选实施例中，上述CPG Frits的直径为3mm，厚度为4mm。
在优选实施例中，每个CPG Frits含有0.1-10mg CPG，优选4mg CPG。
在优选实施例中，上述CPG Frits是亲水性的CPG Frits。
在优选实施例中，上述亲水性的CPG Frits是通过亲水处理获得的CPG Frits。
在优选实施例中，上述亲水处理是先用醇类溶剂润湿，再键合上表面活性剂。
在优选实施例中，上述醇类溶剂为含C1-10直链或支链的醇类溶剂，优选为甲醇、乙醇、丙醇、正丁醇、乙二醇、甘油、及它们的含水溶剂或它们的组合，更优选为95％(v/v)的乙醇；
上述表面活性剂为阴离子表面活性剂、阳离子表面活性剂、两性离子表面活性剂或非离子表面活性剂，优选为十二烷基硫酸钠、苯扎溴铵、聚山梨酯、脂肪酸山梨坦、季铵化物、卵磷脂、氨基酸或它们的组合。
在优选实施例中，上述核酸合成装置还包括容纳装置，上述固相载体置于上述容纳装置中。
在优选实施例中，上述容纳装置是空柱管。
在优选实施例中，上述空柱管内预留至少一个装配位，上述装配位是圆柱体，上述固相载体呈圆柱状，使上述固相载体与上述装配位完全切合，上述装配位的高度大于上述固相载体的厚度。
在优选实施例中，上述空柱管的柱长为70mm，空柱管上部内径为4.52mm，到上述装配位上部逐渐收口至3mm，上述装配位的内径为2.95mm，上述装配位的高度为5mm。
根据第二方面，一种实施例中提供一种核酸合成设备，包括如第一方面的核酸合成装置。
根据第三方面，一种实施例中提供一种第一方面的核酸合成装置在核酸合成中的用途。
在优选实施例中，使用固相亚磷酰胺三酯法进行上述核酸合成。
根据第四方面，一种实施例中提供一种核酸合成方法，包括使用第一方面的核酸合成装置或第二方面的核酸合成设备进行核酸合成，上述核酸合成装置中CPG表面的羟基用作待合成核酸的合成起始点，通过共价键合连接亚磷酰胺保护的核苷酸单体或多聚体。
在优选实施例中，上述方法是固相亚磷酰胺三酯法。
根据第五方面，一种实施例中提供一种核酸纯化装置，包括固相载体，固相载体包括可控孔径玻璃球(CPG)，CPG是未被修饰的裸CPG，其表面具有羟基，羟基通过共价键合连接一段寡核苷酸序列，寡核苷酸序列与待纯化核酸全部或部分特异性互补配对或随机配对。
在优选实施例中，上述寡核苷酸序列与待纯化核酸的3’端或/和5’端的一段区域全部或部 分特异性互补配对或随机配对。
在优选实施例中，上述寡核苷酸序列的长度是6至12个碱基长度。
在优选实施例中，上述待纯化核酸是DNA，相应地，上述核酸纯化装置是DNA纯化装置。
在优选实施例中，上述待纯化核酸是生物学反应产物，优选酶切产物或PCR产物。
在优选实施例中，上述CPG的孔径为
Figure PCTCN2018121092-appb-000007
(埃)至
Figure PCTCN2018121092-appb-000008
在优选实施例中，上述核酸纯化装置还包括筛板，上述筛板包括上筛板和下筛板，上述CPG置于上述上筛板和下筛板之间。
在优选实施例中，上述上筛板经过亲水处理或选自亲水材料，上述下筛板选自疏水材料。
在优选实施例中，上述固相载体还包括热塑性聚合物树脂，上述热塑性聚合物树脂与CPG烧结形成含可控孔径玻璃球的滤芯(CPG Frits)。
在优选实施例中，上述热塑性聚合物树脂选自聚乙烯，优选超高分子量聚乙烯(UHMW-PE)或高密度聚乙烯(HDPE)。
在优选实施例中，上述CPG Frits的直径≤3.5mm，厚度≥1.5mm。
在优选实施例中，上述核酸纯化装置还包括容纳装置，上述固相载体置于上述容纳装置中。
在优选实施例中，上述容纳装置是空柱管。
在优选实施例中，上述空柱管内预留至少一个装配位，上述装配位是圆柱体，上述固相载体呈圆柱状，使上述固相载体与上述装配位完全切合，上述装配位的高度大于上述固相载体的厚度。
根据第六方面，一种实施例中提供一种核酸纯化设备，包括如第五方面的核酸纯化装置。
根据第七方面，一种实施例中提供一种核酸纯化方法，包括将含有待纯化核酸的溶液加入如第五方面的核酸纯化装置中孵育，使上述待纯化核酸与上述核酸纯化装置中的上述寡核苷酸序列全部或部分特异性互补配对或随机配对结合；然后将上述待纯化核酸从上述核酸纯化装置上洗脱下来。
在优选实施例中，上述待纯化核酸为单链核酸。
在优选实施例中，上述核酸纯化装置在与上述溶液孵育之前，先进行氨解脱保护处理。 在申请中，氨解脱保护处理是指，在氨解条件下脱去合成寡核苷酸所用单体或多聚体的杂环碱基氨基上的保护基团，以及磷酯羟基上的氰乙基保护基，使其恢复寡核苷酸本身的结构。
在优选实施例中，上述氨解脱保护处理在90℃下进行2小时；然后，使用乙腈/水溶液和超纯水为流动相进行洗涤，离心，得到连接有上述寡核苷酸序列的核酸纯化装置后再与上述溶液孵育。
在优选实施例中，上述孵育之后，以乙醇/水溶液为流动相进行洗涤，除去未结合的核酸和/或酶和/或缓冲液，之后再将上述待纯化核酸从上述核酸纯化装置上洗脱下来。
在优选实施例中，上述将上述待纯化核酸从上述核酸纯化装置上洗脱下来，具体包括：在加热条件下，使配对结合的待纯化核酸和寡核苷酸序列变性解链，用加热超纯水将解开的单链待纯化核酸洗脱下来。
在优选实施例中，上述待纯化核酸从上述核酸纯化装置上洗脱下来后再进行退火，使互补单链恢复双螺旋结构。
根据第八方面，一种实施例中提供一种第五方面的核酸纯化装置在核酸纯化中的用途。
本发明的核酸合成装置中，CPG是未被修饰的裸CPG，不带通用连接臂(Universal Linker)，表面暴露有一定数量的羟基，寡核苷酸引物等核酸合成完毕经过氨解后处理，连接在CPG上的寡核苷酸引物等核酸不会被切下来，仍然连接在固相载体上。因此，本发明的核酸合成装置能够同时实现合成核酸和固定核酸的目的，不仅可以用于寡核苷酸引物等核酸的合成，还可以利用固定在CPG上的寡核苷酸引物等核酸进行酶切产物、PCR产物的纯化等，具有结构简单、体积小、重量轻、效率高、成本低、功能多样的优点。
本发明的核酸纯化装置，能够对特异核酸序列进行纯化，具有高效、快速、方便、安全无毒等优点，克服了现有的核酸分离纯化方法无法纯化特异DNA序列、操作复杂、效率低下、分离速度慢、经济成本高以及需要使用有毒化学试剂的缺陷。
附图说明
图1为本发明实施例中核酸合成柱或核酸纯化柱设计示意图，其中，1-空柱管；2-装配在空柱管内的CPG Frits；
图2为本发明实施例中核酸合成柱或核酸纯化柱的CPG Frits横截面示意图，其中，3-PE颗粒之间搭桥形成的刚性网状结构；4-不带通用连接臂(Universal Linker)、未被修饰的裸CPG 颗粒；
图3为本发明实施例中核酸合成柱的CPG结构设计原理图，其中，未被修饰的裸CPG颗粒表面具有羟基(-OH)；
图4为本发明实施例中核酸纯化柱的CPG结构设计原理图，其中，未被修饰的裸CPG颗粒表面连接有一段寡核苷酸序列；
图5为本发明实施例中核酸纯化方法流程示意图；
图6为本发明实施例中纯化获得的DNA片段在1.2％琼脂糖凝胶上的电泳结果图，其中，1-使用连接有6nt简并寡核苷酸序列的DNA纯化柱回收PCR产物；2-使用连接有8nt简并寡核苷酸序列的DNA纯化柱回收PCR产物；3-使用连接有10nt简并寡核苷酸序列的DNA纯化柱回收PCR产物；4-使用连接有12nt简并寡核苷酸序列的DNA纯化柱回收PCR产物；M-DL2000 DNA Marker。
具体实施方式
下面通过具体实施方式结合附图对本发明作进一步详细说明。在以下的实施方式中，很多细节描述是为了使得本发明能被更好的理解。然而，本领域技术人员可以毫不费力的认识到，其中部分特征在不同情况下是可以省略的，或者可以由其他元件、材料、方法所替代。
另外，说明书中所描述的特点、操作或者特征可以以任意适当的方式结合形成各种实施方式。同时，方法描述中的各步骤或者动作也可以按照本领域技术人员所能显而易见的方式进行顺序调换或调整。因此，说明书和附图中的各种顺序只是为了清楚描述某一个实施例，并不意味着是必须的顺序，除非另有说明其中某个顺序是必须遵循的。
本发明的一个实施例中提供一种核酸合成装置，该装置包括固相载体，该固相载体包括可控孔径玻璃球(CPG)，该CPG是未被修饰的裸CPG，其表面不携带化学基团的通用连接臂(Universal Linker)，其表面具有羟基，羟基用作核酸合成的起始点，用于通过共价键合连接亚磷酰胺保护的核苷酸单体或多聚体以进行核酸合成。
本发明实施例中，CPG表面的羟基与核苷酸的共价键合连接可以通过经典的固相亚磷酰胺三酯法实现，与现有技术不同的是，本发明实施例中采用的CPG是未被修饰的裸CPG，其表面的羟基充当固相亚磷酰胺三酯法反应中5’-羟基的作用与加入的亚磷酰胺保护的核苷酸单体或多聚体发生键合反应。
本发明的核酸合成装置适用于任何合适的核酸的合成，例如包括但不限于DNA和RNA等，优选DNA的合成。因此，在优选实施例中，本发明的核酸合成装置是DNA合成装置。
本发明实施例中，所采用的可控孔径玻璃球(CPG)球体内部有很多不规则的孔道，孔隙网络庞大，孔道的大小称为孔径，孔径稳定，通常选择孔径
Figure PCTCN2018121092-appb-000009
(埃)至
Figure PCTCN2018121092-appb-000010
(优选
Figure PCTCN2018121092-appb-000011
)的CPG用于本发明实施例的核酸合成装置的制备，也可以选择其它孔径。在具体应用中，可以根据具体情况，来确定所采用的可控孔径玻璃球(CPG)的孔径。
本发明实施例中，核酸合成装置可以设计成各种合适的形式。本发明提供两种示例性的形式。
在第一种形式中，核酸合成装置包括可控孔径玻璃球(CPG)和筛板，筛板包括上筛板和下筛板，可控孔径玻璃球(CPG)置于上筛板和下筛板之间，筛板可以采用超高分子量聚乙烯(UHMW-PE)或高密度聚乙烯(HDPE)等热塑性聚合物树脂粉末烧结而成。筛板的作用是阻挡CPG不漏下来而合成试剂能够通过。上筛板可以经过亲水处理或使用亲水材料，下筛板可以使用疏水材料。此外，该核酸合成装置还包括容纳装置，可控孔径玻璃球(CPG)等固相载体以及任选的筛板置于容纳装置中，该容纳装置可以是空柱管，该空柱管可以采用聚丙烯注塑而成。
在第二种形式中，核酸合成装置包括可控孔径玻璃球(CPG)和热塑性聚合物树脂烧结形成的含可控孔径玻璃球的滤芯(CPG Frits)作为固相载体。烧结的温度可以是160℃至250℃，优选180℃。热塑性聚合物树脂可以选自聚乙烯或聚丙烯等，优选超高分子量聚乙烯(UHMW-PE)或高密度聚乙烯(HDPE)。聚乙烯(PE)颗粒包裹并固定CPG颗粒，PE颗粒之间搭桥形成一定的孔径，CPG颗粒均匀地嵌入PE搭桥而成的刚性网状结构中，形成具有确定孔径和孔隙率的CPG Frits。在寡核苷酸序列合成过程中，合成试剂流经CPG内部孔道与PE搭桥形成的孔道，使试剂流道足够长，从而增加化学反应的时间以及反应物与CPG上的反应位点接触的机会。聚合物的包裹作用使CPG颗粒固定，不会由于试剂的流动而发生转动，进一步延长了试剂和CPG上的反应位点的接触时间，提高了合成效率和产物纯度。在该种形式中，CPG Frits呈圆柱状，其直径可以是任何合适的直径，例如直径≤3.5mm；厚度可以是任何合适的厚度，例如厚度≥1.5mm。优选地，CPG Frits的直径为3mm，厚度为4mm。每个CPG Frits含有0.1-10mg CPG，优选4mg CPG。
由于核酸合成所使用的PE材料等具有超强的疏水性，而分子生物学反应如PCR反应等大多在水溶液中进行，这种疏水性材质限制了其进一步的应用，通过亲水处理，能够实现这种材质对水溶液的吸收，这将拓展其在合成生物学领域的广泛应用。因此，为提高CPG Frits 的亲水性，CPG Frits可以采用经亲水处理的CPG Frits。亲水处理可以是先用醇类润湿，再键合上表面活性剂。
其中，醇类溶剂可以是含C1-10直链或支链的醇类溶剂，优选甲醇、乙醇、丙醇、正丁醇、乙二醇、甘油、及它们的含水溶剂或它们的组合，更优选为95％(v/v)的乙醇。表面活性剂可以是阴离子表面活性剂、阳离子表面活性剂、两性离子表面活性剂或非离子表面活性剂，优选十二烷基硫酸钠、苯扎溴铵、聚山梨酯、脂肪酸山梨坦、季铵化物、卵磷脂、氨基酸或它们的组合。
在一些优选实施例中，本发明的核酸合成装置还包括容纳装置，CPG Frits等固相载体置于容纳装置中，该容纳装置可以是空柱管，该空柱管可以采用聚丙烯注塑而成。
在一个优选实施例中，容纳装置是空柱管，固相载体CPG Frits制成圆柱状置于空柱管中，空柱管内预留至少一个装配位，装配位是圆柱体，没有斜度，固相载体呈圆柱状与空柱管完全切合，装配位的高度大于固相载体的厚度。
在一个优选实施例中，空柱管为聚丙烯注塑而成，采用梯度直口设计，预留的CPG Frits装配位是圆柱体，没有斜度。空柱管的柱长为70mm，空柱管上部内径为4.52mm，到装配位上部逐渐收口至3mm，装配位的内径为2.95mm，装配位的高度为5mm。
此外，本发明的核酸合成装置中，容纳装置除了使用空柱管以外，还可以使用其他合适形式，例如多孔板，如24孔板、96孔板、384孔板等，每个CPG Frits、或CPG和筛板分别置于多孔板的每个孔中，形成一种多孔板形式的核酸合成装置，能够批量化进行核酸合成。
如图1、图2和图3所示，在本发明的一个示例性的实施例中，核酸合成装置是核酸合成柱，该核酸合成柱包括：空柱管1和装配在空柱管内的CPG Frits 2。其中，CPG为不带通用连接臂(Universal Linker)、未被修饰的裸CPG载体，表面暴露有一定数量的羟基，CPG的孔径为
Figure PCTCN2018121092-appb-000012
CPG Frits 2由未被修饰的裸CPG颗粒4和热塑性聚合物树脂(例如PE)烧结形成，PE颗粒之间搭桥形成刚性网状结构3。烧结温度可以是160℃至250℃，优选180℃。烧结时间可以是5分钟至10小时等，具体可以根据需要确定。图3示出，裸CPG载体的表面暴露有一定数量的羟基(-OH)，通过该羟基可以与待加入的亚磷酰胺保护的核苷酸单体或多聚体之间形成共价键合，以进行核酸合成。
本发明的一种实施例中提供一种核酸合成设备，设备包括本发明实施例的核酸合成装置。本发明的核酸合成设备，除了包括本发明的核酸合成装置以外，其他部分例如液路系统、气路系统、控制系统、传动系统等可以按照现有技术实施。例如，将Dr.Oligo 192核酸合成仪 中使用的核酸合成装置替换为本发明的核酸合成装置，即可作为本发明的核酸合成设备的一个例子。
本发明的一种实施例中提供本发明的核酸合成装置在合成核酸的方法中的用途。合成的核酸可以是任何合适的核酸，例如包括但不限于DNA和RNA等，优选DNA。
在优选实施例中，采用固相亚磷酰胺三酯法进行核酸的合成。例如，将本发明的核酸合成装置作为Dr.Oligo 192核酸合成仪中的合成柱，并在该合成仪上采用经典的固相亚磷酰胺三酯法合成一段寡核苷酸引物序列，即可实现本发明的目的。
本发明的一种实施例中提供一种核酸合成方法，该方法使用本发明的核酸合成装置或核酸合成设备进行核酸合成，核酸合成装置中CPG表面的羟基用作待合成核酸的合成起始点，通过共价键合连接亚磷酰胺保护的核苷酸单体或多聚体。在优选实施例中，核酸合成方法是固相亚磷酰胺三酯法。
本发明的一个实施例中提供一种核酸纯化装置，该装置包括固相载体，该固相载体包括可控孔径玻璃球(CPG)，该CPG是未被修饰的裸CPG，不带通用连接臂(Universal Linker)，其表面具有羟基，这些羟基通过共价键合连接一段寡核苷酸序列，该寡核苷酸序列与待纯化核酸全部或部分特异性互补配对或随机配对。
本发明实施例中，CPG表面的羟基与寡核苷酸序列的共价键合连接可以通过经典的固相亚磷酰胺三酯法实现，与现有技术不同的是，本发明实施例中采用的CPG是未被修饰的裸CPG，其表面的羟基充当固相亚磷酰胺三酯法反应中5’-羟基的作用与加入的亚磷酰胺保护的核苷酸单体或多聚体发生键合反应。
本发明实施例中，寡核苷酸序列与待纯化核酸全部或部分特异性互补配对或随机配对，因此，能够有目的地对特异核酸序列进行纯化，在已知待纯化核酸的序列的情况下，容易根据碱基互补配对原则设计上述寡核苷酸序列。一般而言，寡核苷酸序列在待纯化核酸上的配对位置选在待纯化核酸的3’端或/和5’端的一段区域，具体而言，可以是待纯化核酸的3’端的一段区域，也可以是待纯化核酸的5’端的一段区域，还可以是在待纯化核酸的3’端和5’端各有一段区域。
一般而言，寡核苷酸序列的长度没有特别限制，可以根据具体需要设计任意长度，但是根据试验效果确定寡核苷酸序列的长度在6至12个碱基长度范围内能取得优异效果，例如6个碱基、8个碱基、10个碱基或12个碱基。
本发明实施例中，称为“核酸纯化装置”，意味着本发明的装置可以纯化任何合适的核酸， 包括但不限于DNA和RNA等。在本发明的优选实施例中，待纯化核酸是DNA，因此，本发明的核酸纯化装置是一种DNA纯化装置。在更优选的实施例中，待纯化核酸是生物学反应产物，可以是任何生物学、生物化学等反应的产物，而该产物需要特异性纯化特定片段，这样的生物学反应产物典型但非限定性的例子是酶切产物或PCR产物等。
本发明实施例中，所采用的CPG球体内部有很多不规则的孔道，孔隙网络庞大，孔道的大小称为孔径，孔径稳定，通常选择孔径
Figure PCTCN2018121092-appb-000013
(埃)至
Figure PCTCN2018121092-appb-000014
(优选
Figure PCTCN2018121092-appb-000015
)的CPG用于本发明实施例的核酸纯化装置的制备，也可以选择其它孔径。在具体应用中，可以根据待纯化核酸的具体情况，来确定所采用的CPG的孔径。
本发明实施例中，核酸纯化装置可以设计成各种合适的形式。本发明提供两种示例性的形式。
在第一种形式中，核酸纯化装置包括CPG和筛板，筛板包括上筛板和下筛板，CPG置于上筛板和下筛板之间，筛板可以采用超高分子量聚乙烯(UHMW-PE)或高密度聚乙烯(HDPE)等热塑性聚合物树脂粉末烧结而成。筛板的作用是阻挡CPG不漏下来而各种试剂能够通过。上筛板可以经过亲水处理或使用亲水材料，下筛板可以使用疏水材料。此外，该核酸纯化装置还包括容纳装置，CPG等固相载体以及任选的筛板置于容纳装置中，该容纳装置可以是空柱管，该空柱管可以采用聚丙烯注塑而成。
在第二种形式中，核酸纯化装置包括CPG和热塑性聚合物树脂烧结形成的含可控孔径玻璃球的滤芯(CPG Frits)作为固相载体。热塑性聚合物树脂可以选自聚乙烯或聚丙烯等，优选超高分子量聚乙烯(UHMW-PE)或高密度聚乙烯(HDPE)。聚乙烯(PE)颗粒包裹并固定CPG颗粒，PE颗粒之间搭桥形成一定的孔径，CPG颗粒均匀地嵌入PE搭桥而成的刚性网状结构中，形成具有确定孔径和孔隙率的CPG Frits。在该种形式中，CPG Frits可以呈圆柱状，其直径可以是任何合适的直径，例如直径≤3.5mm；厚度可以是任何合适的厚度，例如厚度≥1.5mm。优选地，CPG Frits的直径为3mm，厚度为4mm。此外，该核酸纯化装置还可以包括容纳装置，CPG Frits等固相载体置于容纳装置中，该容纳装置可以是空柱管，该空柱管可以采用聚丙烯注塑而成。在一个优选实施例中，容纳装置是空柱管，固相载体CPG Frits制成圆柱状置于空柱管中，空柱管内预留至少一个装配位，装配位是圆柱体，没有斜度，固相载体呈圆柱状与空柱管完全切合，装配位的高度大于固相载体的厚度。
此外，本发明的核酸纯化装置中，容纳装置除了使用空柱管以外，还可以使用其他合适形式，例如多孔板，如24孔板、96孔板、384孔板等，每个CPG Frits、或CPG和筛板分别置于多孔板的每个孔中，形成一种多孔板形式的核酸纯化装置，能够批量化进行核酸纯化。
如图1、图2和图4所示，在本发明的一个示例性的实施例中，核酸纯化装置是核酸纯化柱，该核酸纯化柱包括：空柱管1和装配在空柱管内的CPG Frits 2。其中，CPG为不带通用连接臂(Universal Linker)、未被修饰的裸CPG载体，表面暴露有一定数量的羟基，CPG的孔径为
Figure PCTCN2018121092-appb-000016
CPG Frits 2由未被修饰的裸CPG颗粒4和热塑性聚合物树脂(例如PE)烧结形成，PE颗粒之间搭桥形成刚性网状结构3。烧结温度可以是160℃至250℃，优选180℃。烧结时间可以是5分钟至10小时等，具体可以根据需要确定。图4示出，裸CPG载体的表面暴露有一定数量的羟基(-OH)，该羟基通过共价键合连接一段寡核苷酸序列，上述寡核苷酸序列与待纯化核酸全部或部分特异性互补配对或随机配对。
在一个优选实施例中，CPG Frits是CPG和UHMW-PE粉末混合均匀后于180℃烧结而成，CPG Frits的直径为3mm，CPG Frits的厚度为4mm。CPG Frits经过亲水处理，每颗CPG Frits中CPG的含量为4mg。其中，CPG Frits的亲水处理过程可以是先用醇类润湿，再键合上表面活性剂。其中，醇类溶剂可以是含C1-10直链或支链的醇类溶剂，优选甲醇、乙醇、丙醇、正丁醇、乙二醇、甘油、及它们的含水溶剂或它们的组合，更优选为95％(v/v)的乙醇。表面活性剂可以是阴离子表面活性剂、阳离子表面活性剂、两性离子表面活性剂或非离子表面活性剂，优选十二烷基硫酸钠、苯扎溴铵、聚山梨酯、脂肪酸山梨坦、季铵化物、卵磷脂、氨基酸或它们的组合。空柱管可以是聚丙烯注塑而成，采用梯度直口设计，预留的CPG Frits装配位是圆柱体，没有斜度。空柱管的柱长为70mm，空柱管上部内径为4.52mm，到装配位上部逐渐收口至3mm，装配位的内径为2.95mm，装配位的高度为5mm。
本发明的一个实施例中提供一种核酸纯化设备，包括如本发明实施例的核酸纯化装置。
本发明的一个实施例中提供一种核酸纯化方法，该方法包括将含有待纯化核酸的溶液加入本发明的核酸纯化装置中孵育，使待纯化核酸与核酸纯化装置中的寡核苷酸序列特异性互补配对或随机配对结合；然后将待纯化核酸从核酸纯化装置上洗脱下来。
本发明实施例中，待纯化核酸可以是任何合适的核酸，包括但不限于DNA和RNA等。在本发明的优选实施例中，待纯化核酸是DNA。在更优选的实施例中，待纯化核酸是生物学反应产物，可以是任何生物学、生物化学等反应的产物，而该产物需要特异性纯化特定片段，这样的生物学反应产物典型但非限定性的例子是酶切产物或PCR产物等。
本发明实施例中，待纯化核酸可以是含有至少一部分单链的核酸，包括但不限于待纯化核酸末端为突出的单链而中间为双链，或者待纯化核酸末端为双链，中间为单链，或者待纯化核酸整条链均为单链。在本发明的优选实施例中，待纯化核酸为单链核酸。
在本发明的一个优选实施例中，核酸纯化装置在与待纯化的溶液孵育之前，先进行氨解脱保护处理。氨解脱保护处理，例如，可以在90℃下进行2小时；然后，使用乙腈/水溶液和超纯水为流动相进行洗涤，离心，得到连接有寡核苷酸序列的纯净的核酸纯化装置，之后再与待纯化的溶液孵育。
如图5所示，待纯化核酸是PCR产物，该PCR产物高温(例如95℃)变性解旋形成单链，其中两端分别带有一段与核酸纯化装置中CPG表面连接的寡核苷酸序列互补的序列；将变性解旋的PCR产物上样至核酸纯化装置中孵育，CPG表面连接的寡核苷酸序列与单链两端的序列区段互补；洗涤(例如，以乙醇/水溶液为流动相进行洗涤)，除去未结合的DNA以及反应体系中的酶、缓冲液等杂质；然后将待纯化核酸从核酸纯化装置上洗脱下来，例如，在加热条件下，使配对结合的待纯化核酸和寡核苷酸序列变性解链，用加热超纯水将解开的单链待纯化核酸洗脱下来；最后将洗脱下来的核酸再次变性、退火，使互补单链恢复双螺旋结构，得到纯化的DNA产物。
本发明的一种实施例中提供本发明的核酸纯化装置在核酸纯化中的用途，其中，核酸纯化装置的CPG表面的羟基上连接一段寡核苷酸序列，该寡核苷酸序列与待纯化核酸全部或部分特异性互补配对或随机配对。
以下通过实施例详细说明本发明的技术方案，应当理解，实施例仅是示例性的，不能理解为对本发明保护范围的限制。
实施例
本实施例中，核酸纯化方法的具体实验过程和步骤如下：
(1)在Dr.Oligo 192核酸合成仪上采用经典的固相亚磷酰胺三酯法，利用亲水DNA合成柱(该合成柱内置CPG Frits，其包括裸CPG载体，该裸CPG载体的表面暴露有一定数量的羟基)合成一段与待纯化DNA随机配对的纯化“钓饵”序列(即简并寡核苷酸序列)，合成的简并寡核苷酸的序列长度分别为6nt、8nt、10nt、12nt，并且连接在CPG Frits上，即获得所需的DNA纯化柱；
(2)对获得的DNA纯化柱在90℃下进行2小时氨解脱保护处理后，依次以400μL乙腈/水(体积比90:10)和300μL超纯水为流动相进行洗涤，离心，得到连接有纯净简并寡核苷酸序列的DNA纯化柱；
(3)经PCR扩增获得含有大小为350bp目的片段的DNA产物溶液25μL，于95℃加热5分钟变性解旋为单链；
(4)将步骤(3)中获得的变性DNA水溶液，快速上样至DNA纯化柱内，被CPG Frits吸收，于25℃孵育5分钟与柱上寡核苷酸结合；
(5)以400μL乙醇/水(体积比80:20)为流动相洗涤2次，离心，除去未结合DNA等杂质；
(6)于95℃加热5分钟变性解链，用20μL 95℃超纯水将解开的单链DNA洗脱下来，离心并收集；
(7)将步骤(6)中获得的DNA水溶液再次于95℃加热5分钟变性；
(8)冷却至60℃退火10分钟，获得纯净的PCR产物。
使用NanoDrop紫外分光光度计对纯化所得PCR产物进行检测，OD 260/OD 280比值为1.8-2.0。
如下表1为连接有不同长度寡核苷酸(均为简并碱基N的随机序列)的DNA纯化柱在相同条件下纯化PCR产物的纯化效果比较结果。
表1
Figure PCTCN2018121092-appb-000017
图6为上述实施例中纯化获得的DNA片段在1.2％琼脂糖凝胶上的电泳结果图，其中，泳道1表示使用连接有6nt简并寡核苷酸序列的DNA纯化柱回收PCR产物；泳道2表示使用连接有8nt简并寡核苷酸序列的DNA纯化柱回收PCR产物；泳道3表示使用连接有10nt简并寡核苷酸序列的DNA纯化柱回收PCR产物；泳道4表示使用连接有12nt简并寡核苷酸序列的DNA纯化柱回收PCR产物；M表示DL2000 DNA Marker。
从图6与表1结果可知，使用本发明的核酸合成装置连接简并寡核苷酸序列，基于该寡核苷酸序列与待纯化核酸的互补配对关系，可快速有效完成酶切、PCR产物等的纯化工作，大大缩短纯化时间；所得到的DNA片段用紫外分光光度计检测，OD 260/OD 280比值达1.8-2.0，纯化得到的DNA产物纯度高，纯化回收率约为50％。
以上应用了具体个例对本发明进行阐述，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。

Claims (47)

  1. 一种核酸合成装置，其特征在于，所述装置包括固相载体，所述固相载体包括可控孔径玻璃球(CPG)，所述CPG是未被修饰的裸CPG，其表面具有羟基，所述羟基可通过共价键合连接亚磷酰胺保护的核苷酸单体或多聚体以进行核酸合成。
  2. 根据权利要求1所述的核酸合成装置，其特征在于，所述核酸是DNA，相应地，所述核酸合成装置是DNA合成装置。
  3. 根据权利要求1所述的核酸合成装置，其特征在于，所述CPG的孔径为
    Figure PCTCN2018121092-appb-100001
    (埃)至
    Figure PCTCN2018121092-appb-100002
  4. 根据权利要求1所述的核酸合成装置，其特征在于，所述核酸合成装置还包括筛板，所述筛板包括上筛板和下筛板，所述CPG置于所述上筛板和下筛板之间。
  5. 根据权利要求4所述的核酸合成装置，其特征在于，所述上筛板经过亲水处理或选自亲水材料，所述下筛板选自疏水材料。
  6. 根据权利要求1所述的核酸合成装置，其特征在于，所述固相载体还包括热塑性聚合物树脂，所述热塑性聚合物树脂与CPG烧结形成含可控孔径玻璃球的滤芯(CPG Frits)。
  7. 根据权利要求6所述的核酸合成装置，其特征在于，所述热塑性聚合物树脂选自聚乙烯，优选超高分子量聚乙烯(UHMW-PE)或高密度聚乙烯(HDPE)。
  8. 根据权利要求6所述的核酸合成装置，其特征在于，所述烧结的温度是160℃至250℃，优选180℃。
  9. 根据权利要求6所述的核酸合成装置，其特征在于，所述CPG Frits的直径≤3.5mm，厚度≥1.5mm。
  10. 根据权利要求9所述的核酸合成装置，其特征在于，所述CPG Frits的直径为3mm，厚度为4mm。
  11. 根据权利要求6所述的核酸合成装置，其特征在于，每个所述CPG Frits含有0.1-10mg CPG，优选4mg CPG。
  12. 根据权利要求6所述的核酸合成装置，其特征在于，所述CPG Frits是亲水性的CPG Frits。
  13. 根据权利要求12所述的核酸合成装置，其特征在于，所述亲水性的CPG Frits是通过亲水处理获得的CPG Frits。
  14. 根据权利要求13所述的核酸合成装置，其特征在于，所述亲水处理是先用醇类溶剂润湿，再键合上表面活性剂。
  15. 根据权利要求14所述的核酸合成装置，其特征在于，所述醇类溶剂为含C1-10直链或支链的醇类溶剂，优选为甲醇、乙醇、丙醇、正丁醇、乙二醇、甘油、及它们的含水溶剂或它们的组合，更优选为95％(v/v)的乙醇；
    所述表面活性剂为阴离子表面活性剂、阳离子表面活性剂、两性离子表面活性剂或非离子表面活性剂，优选为十二烷基硫酸钠、苯扎溴铵、聚山梨酯、脂肪酸山梨坦、季铵化物、卵磷脂、氨基酸或它们的组合。
  16. 根据权利要求1所述的核酸合成装置，其特征在于，所述核酸合成装置还包括容纳装置，所述固相载体置于所述容纳装置中。
  17. 根据权利要求16所述的核酸合成装置，其特征在于，所述容纳装置是空柱管。
  18. 根据权利要求17所述的核酸合成装置，其特征在于，所述空柱管内预留至少一个装配位，所述装配位是圆柱体，所述固相载体呈圆柱状，使所述固相载体与所述装配位完全切合，所述装配位的高度大于所述固相载体的厚度。
  19. 根据权利要求18所述的核酸合成装置，其特征在于，所述空柱管的柱长为70mm，空柱管上部内径为4.52mm，到所述装配位上部逐渐收口至3mm，所述装配位的内径为2.95mm，所述装配位的高度为5mm。
  20. 一种核酸合成设备，其特征在于，所述设备包括如权利要求1-19任一项所述的核酸合成装置。
  21. 权利要求1-19任一项所述的核酸合成装置在核酸合成中的用途。
  22. 根据权利要求21所述的用途，其特征在于，使用固相亚磷酰胺三酯法进行所述核酸合成。
  23. 一种核酸合成方法，其特征在于，所述方法包括使用权利要求1-19任一项所述的核酸合成装置或权利要求20所述的核酸合成设备进行核酸合成，所述核酸合成装置中CPG表面的羟基用作待合成核酸的合成起始点，通过共价键合连接亚磷酰胺保护的核苷酸单体或多聚体。
  24. 根据权利要求23所述的核酸合成方法，其特征在于，所述方法是固相亚磷酰胺三酯法。
  25. 一种核酸纯化装置，其特征在于，所述装置包括固相载体，所述固相载体包括可控孔径玻璃球(CPG)，所述CPG是未被修饰的裸CPG，其表面具有羟基，所述羟基通过共价键合连接一段寡核苷酸序列，所述寡核苷酸序列与待纯化核酸全部或部分特异性互补配对或随机配对。
  26. 根据权利要求25所述的核酸纯化装置，其特征在于，所述寡核苷酸序列与待纯化核酸的3’端或/和5’端的一段区域全部或部分特异性互补配对或随机配对。
  27. 根据权利要求25所述的核酸纯化装置，其特征在于，所述寡核苷酸序列的长度是6至12个碱基长度。
  28. 根据权利要求25所述的核酸纯化装置，其特征在于，所述待纯化核酸是DNA，相应地，所述核酸纯化装置是DNA纯化装置。
  29. 根据权利要求28所述的核酸纯化装置，其特征在于，所述待纯化核酸是生物学反应产物，优选酶切产物或PCR产物。
  30. 根据权利要求25所述的核酸纯化装置，其特征在于，所述CPG的孔径为
    Figure PCTCN2018121092-appb-100003
    (埃)至
    Figure PCTCN2018121092-appb-100004
  31. 根据权利要求25所述的核酸纯化装置，其特征在于，所述核酸纯化装置还包括筛板，所述筛板包括上筛板和下筛板，所述CPG置于所述上筛板和下筛板之间。
  32. 根据权利要求31所述的核酸纯化装置，其特征在于，所述上筛板经过亲水处理或选自亲水材料，所述下筛板选自疏水材料。
  33. 根据权利要求25所述的核酸纯化装置，其特征在于，所述固相载体还包括热塑性聚合物树脂，所述热塑性聚合物树脂与CPG烧结形成含可控孔径玻璃球的滤芯(CPG Frits)。
  34. 根据权利要求33所述的核酸纯化装置，其特征在于，所述热塑性聚合物树脂选自聚乙烯，优选超高分子量聚乙烯(UHMW-PE)或高密度聚乙烯(HDPE)。
  35. 根据权利要求34所述的核酸纯化装置，其特征在于，所述CPG Frits的直径≤3.5mm，厚度≥1.5mm。
  36. 根据权利要求25所述的核酸纯化装置，其特征在于，所述核酸纯化装置还包括容纳装置，所述固相载体置于所述容纳装置中。
  37. 根据权利要求36所述的核酸纯化装置，其特征在于，所述容纳装置是空柱管。
  38. 根据权利要求37所述的核酸纯化装置，其特征在于，所述空柱管内预留至少一个装 配位，所述装配位是圆柱体，所述固相载体呈圆柱状，使所述固相载体与所述装配位完全切合，所述装配位的高度大于所述固相载体的厚度。
  39. 一种核酸纯化设备，其特征在于，所述设备包括如权利要求25-38任一项所述的核酸纯化装置。
  40. 一种核酸纯化方法，其特征在于，所述方法包括将含有待纯化核酸的溶液加入如权利要求25-38任一项所述的核酸纯化装置中孵育，使所述待纯化核酸与所述核酸纯化装置中的所述寡核苷酸序列全部或部分特异性互补配对或随机配对结合；然后将所述待纯化核酸从所述核酸纯化装置上洗脱下来。
  41. 根据权利要求40所述的核酸纯化方法，其特征在于，所述待纯化核酸为单链核酸。
  42. 根据权利要求40所述的核酸纯化方法，其特征在于，所述核酸纯化装置在与所述溶液孵育之前，先进行氨解脱保护处理。
  43. 根据权利要求42所述的核酸纯化方法，其特征在于，所述氨解脱保护处理在90℃下进行2小时；然后，使用乙腈/水溶液和超纯水为流动相进行洗涤，离心，得到连接有所述寡核苷酸序列的核酸纯化装置后再与所述溶液孵育。
  44. 根据权利要求40所述的核酸纯化方法，其特征在于，所述孵育之后，以乙醇/水溶液为流动相进行洗涤，除去未结合的核酸和/或酶和/或缓冲液，之后再将所述待纯化核酸从所述核酸纯化装置上洗脱下来。
  45. 根据权利要求40所述的核酸纯化方法，其特征在于，所述将所述待纯化核酸从所述核酸纯化装置上洗脱下来，具体包括：在加热条件下，使配对结合的待纯化核酸和寡核苷酸序列变性解链，用加热超纯水将解开的单链待纯化核酸洗脱下来。
  46. 根据权利要求40所述的核酸纯化方法，其特征在于，所述待纯化核酸从所述核酸纯化装置上洗脱下来后再进行退火，使互补单链恢复双螺旋结构。
  47. 权利要求25-38任一项所述的核酸纯化装置在核酸纯化中的用途。
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