Nucleic acid synthesis device and purification device, use thereof, and nucleic acid synthesis method and purification method

Abstract

A nucleic acid synthesis device and a purification device, uses thereof, and a nucleic acid synthesis method and a purification method, wherein the nucleic acid synthesis device comprises a solid phase carrier, the solid phase carrier comprises a controlled pore glass ball (CPG), the CPG is an unmodified naked CPG, and the surface of the CPG has a hydroxyl group, which is used as a starting point for nucleic acid synthesis and is used to connect phosphoramidite-protected nucleotide monomers or polymers through covalent bonding to perform nucleic acid synthesis. The nucleic acid synthesis device of the present invention can not only be used for the synthesis of oligonucleotide primers, but also can use the oligonucleotide primers fixed on the CPG for enzyme cutting, purification of PCR products, etc., and has the advantages of simple structure, small size, light weight, high efficiency, low cost, and diverse functions.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid synthesis, and in particular to a nucleic acid synthesis device and a purification device, uses thereof, and a nucleic acid synthesis method and a nucleic acid purification method. Background Art

[0002] The classic DNA synthesis column includes a solid phase carrier, a sieve plate, and an empty column tube. The solid phase carrier is usually a controlled pore glass ball (CPG). The CPG ball has many irregular channels inside, and the pore network is huge. The size of the channel is called the pore size. The pore size is stable and the pore size is usually selected. and CPG is used for DNA synthesis, and other pore sizes can also be selected. The sieve plate is usually sintered with ultra-high molecular weight polyethylene (UHMW-PE) or high-density polyethylene (HDPE) powder. In the DNA synthesis column, the function of the sieve plate is to prevent CPG from leaking out while allowing the synthesis reagents to pass through. The empty column tube is made of polypropylene injection molding.

[0003] The second-generation universal DNA synthesis column consists of a filter element containing controlled pore glass balls (Controlled Pore Glass Frits, CPG Frits) and an empty column tube. CPG Frits are sintered from CPG and UHMW-PE or HDPE powders. PE particles wrap and fix CPG particles, and bridges between PE particles to form a certain pore size. CPG particles are evenly embedded in the rigid mesh structure formed by PE bridges to form a sieve plate with a certain pore size and porosity. During the primer synthesis process, the synthesis reagent flows through the internal pores of CPG and the pores formed by PE bridges, making the reagent flow channel long enough, thereby increasing the time of the chemical reaction and the chance of the reactants contacting the reaction sites on CPG. The encapsulation of the polymer fixes the CPG particles and does not rotate due to the flow of the reagents, further prolonging the contact time between the reagents and the reaction sites on CPG, thereby improving the synthesis efficiency and product purity.

[0004] At present, the synthesis of oligonucleotides such as primers generally adopts 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. It is usually modified with a universal linker with a chemical group. The initial step of the synthesis is to use trichloroacetic acid (TCA) to remove the DMT protecting group on the linker connected to CPG to obtain a free 5'-hydroxyl for the next step of condensation reaction with phosphoramidite monomers. After the synthesis is completed, the oligonucleotide connected to CPG will be cut off after aminolysis treatment and can be used for subsequent experiments after purification, but it cannot achieve the effect of fixing oligonucleotides. Because the PE material used has super hydrophobicity, CPG is wrapped in it, so this synthesis column cannot absorb reaction solutions such as enzyme cutting and PCR.

[0005] The separation and purification of DNA is a key step in molecular biology operations. The separated DNA can be used for downstream gene cloning, probe preparation and other experiments. The separation and purification of DNA usually includes two types: 1) Total DNA extraction. This separation and purification only requires the removal of other impurities in tissues, cells or in vitro DNA synthesis reaction products, and then all the required DNA genetic material is separated by physical or chemical methods. 2) Specific DNA extraction. This separation and purification usually requires the extraction of specific DNA in tissues, cells or in vitro DNA synthesis amplification products, including a certain functional type of DNA (such as plasmid DNA), a certain length of DNA or a certain sequence-specific DNA, according to the purpose of the experiment.

[0006] At present, the commonly used DNA separation and purification methods in laboratories include the silica membrane centrifugal adsorption column method and the magnetic bead adsorption method. The core principles of the two methods are similar. Both methods use the groups bonded to the surface of the silica membrane or magnetic beads to adsorb the charges on the surface of the DNA under certain buffer conditions, thereby separating and purifying the target DNA. Both methods have been developed into mature commercial kits and are available on the market. Taking TIANGEN's universal DNA purification and recovery kit as an example, the kit uses a unique buffer system and centrifugal adsorption column, which can not only recover DNA fragments from TAE or TBE agarose gels, but also can be used to directly purify PCR products, while removing impurities such as proteins, other organic compounds, inorganic salt ions and oligonucleotide primers, which can meet a variety of experimental needs. The sol contains a pH indicator, and the sol state can be judged by color. The principle of the gel recovery kit is that the gel blocks are quickly melted in the gel melting solution and the DNA is released. After adding the high-chaotropic sequence solution, the DNA fragments are selectively adsorbed onto the silica gel membrane. After washing with the rinse solution to remove the impurities and high-concentration salt ions remaining on the silica gel membrane, the DNA fragments adsorbed onto the silica gel membrane are eluted with the elution buffer or water and can be used in various molecular biology experiments. The magnetic bead adsorption method for nucleic acid extraction is to use nanotechnology to improve and modify the surface of superparamagnetic nanoparticles to prepare superparamagnetic silica nanoparticles. The magnetic beads can specifically recognize and efficiently bind to nucleic acid molecules at the microscopic interface. Using the superparamagnetism of silica nanoparticles, under the action of Chaotropic salts (guanidine hydrochloride, guanidine isothiocyanate, etc.) and an external magnetic field, DNA and RNA can be separated from samples such as blood, animal tissues, food, and pathogenic microorganisms. It can be applied to clinical disease diagnosis, blood transfusion safety, forensic identification, environmental microbiology detection, food safety detection, molecular biology research and many other fields.

[0007] The DNA recovery methods commonly used in laboratories are relatively costly, with cumbersome and complicated procedures. They are unable to purify specific DNA sequences and require the use of buffer solutions containing toxic chemical reagents. Summary of the invention

[0008] The present invention provides a nucleic acid synthesis device and a purification device, uses thereof, and a nucleic acid synthesis method and a purification method. The nucleic acid synthesis device can not only be used for the synthesis of oligonucleotide sequences, but also can utilize the oligonucleotide sequences fixed on CPG to purify enzyme cleavage products and PCR products, and 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, and has the advantages of high efficiency, rapidity, convenience, safety and non-toxicity.

[0009] According to the first aspect, an embodiment provides a nucleic acid synthesis device, including a solid phase carrier, which includes a controlled pore glass ball (CPG), and the CPG is an unmodified naked CPG with hydroxyl groups on its surface. The hydroxyl groups can be covalently bonded to phosphoramidite-protected nucleotide monomers or polymers for nucleic acid synthesis.

[0010] It is well known to those skilled in the art that the synthesis of oligonucleotides currently generally adopts the second-generation CPG Frits synthesis column, in which CPG is made of silica and the surface of CPG is usually modified with a universal linker with chemical groups, such as In the present application, CPG is unmodified naked CPG, which means that the surface of CPG is rich in silanol groups, and the surface of CPG is not modified with any chemical groups, such as universal linker modification.

[0011] In a preferred embodiment, the nucleic acid is DNA, and accordingly, the nucleic acid synthesis device is a DNA synthesis device.

[0012] In a preferred embodiment, the pore size of the CPG is (Angstrom) to

[0013] In a preferred embodiment, the nucleic acid synthesis device further comprises a sieve plate, the sieve plate comprises an upper sieve plate and a lower sieve plate, and the CPG is placed between the upper sieve plate and the lower sieve plate.

[0014] In a preferred embodiment, the upper sieve plate is hydrophilically treated or selected from hydrophilic materials, and the lower sieve plate is selected from hydrophobic materials.

[0015] In a preferred embodiment, the solid phase carrier further comprises a thermoplastic polymer resin, and the thermoplastic polymer resin is sintered with CPG to form a filter element (CPG Frits) containing controlled pore size glass balls.

[0016] In a preferred embodiment, the thermoplastic polymer resin is selected from polyethylene, preferably ultra-high molecular weight polyethylene (UHMW-PE) or high-density polyethylene (HDPE).

[0017] In a preferred embodiment, the sintering temperature is 160°C to 250°C, preferably 180°C.

[0018] In a preferred embodiment, the diameter of the CPG Frits is ≤3.5 mm and the thickness is ≥1.5 mm.

[0019] In a preferred embodiment, the CPG Frits have a diameter of 3 mm and a thickness of 4 mm.

[0020] In a preferred embodiment, each CPG Frits contains 0.1-10 mg CPG, preferably 4 mg CPG.

[0021] In a preferred embodiment, the CPG Frits are hydrophilic CPG Frits.

[0022] In a preferred embodiment, the hydrophilic CPG Frits are CPG Frits obtained by hydrophilic treatment.

[0023] In a preferred embodiment, the hydrophilic treatment is performed by first wetting with an alcohol solvent and then bonding with a surfactant.

[0024] In a preferred embodiment, the alcohol solvent is a C1-10 straight-chain or branched alcohol solvent, preferably methanol, ethanol, propanol, n-butanol, ethylene glycol, glycerol, and aqueous solvents thereof or combinations thereof, more preferably 95% (v / v) ethanol;

[0025] The surfactant is an anionic surfactant, a cationic surfactant, a zwitterionic surfactant or a nonionic surfactant, preferably sodium lauryl sulfate, benzalkonium bromide, polysorbate, sorbitan fatty acid, quaternary ammonium compound, lecithin, amino acid or a combination thereof.

[0026] In a preferred embodiment, the nucleic acid synthesis device further comprises a containing device, and the solid phase carrier is placed in the containing device.

[0027] In a preferred embodiment, the above-mentioned receiving device is an empty column tube.

[0028] In a preferred embodiment, at least one assembly position is reserved in the above-mentioned empty column tube, the above-mentioned assembly position is a cylinder, and the above-mentioned solid phase carrier is cylindrical, so that the above-mentioned solid phase carrier is completely fitted with the above-mentioned assembly position, and the height of the above-mentioned assembly position is greater than the thickness of the above-mentioned solid phase carrier.

[0029] In a preferred embodiment, the column length of the above-mentioned empty column tube is 70mm, the inner diameter of the upper part of the empty column tube is 4.52mm, and it gradually narrows to 3mm at the upper part of the above-mentioned assembly position. The inner diameter of the above-mentioned assembly position is 2.95mm, and the height of the above-mentioned assembly position is 5mm.

[0030] According to the second aspect, an embodiment provides a nucleic acid synthesis device, comprising the nucleic acid synthesis apparatus according to the first aspect.

[0031] According to the third aspect, an embodiment provides use of the nucleic acid synthesis device of the first aspect in nucleic acid synthesis.

[0032] In a preferred embodiment, the nucleic acid synthesis is performed using a solid phase phosphoramidite triester method.

[0033] According to the fourth aspect, a method for nucleic acid synthesis is provided in one embodiment, comprising using the nucleic acid synthesis device of the first aspect or the nucleic acid synthesis equipment of the second aspect to perform nucleic acid synthesis, wherein the hydroxyl group on the CPG surface of the above-mentioned nucleic acid synthesis device is used as the synthesis starting point of the nucleic acid to be synthesized, and phosphoramidite-protected nucleotide monomers or polymers are connected by covalent bonding.

[0034] In a preferred embodiment, the above method is a solid phase phosphoramidite triester method.

[0035] According to the fifth aspect, an embodiment provides a nucleic acid purification device, including a solid phase carrier, the solid phase carrier includes a controlled pore glass ball (CPG), the CPG is an unmodified naked CPG, and its surface has hydroxyl groups, and the hydroxyl groups are covalently bonded to an oligonucleotide sequence, and the oligonucleotide sequence is specifically complementary or randomly paired with all or part of the nucleic acid to be purified.

[0036] In a preferred embodiment, the oligonucleotide sequence is specifically complementary or randomly paired with all or part of a region at the 3' end and / or 5' end of the nucleic acid to be purified.

[0037] In a preferred embodiment, the length of the above oligonucleotide sequence is 6 to 12 bases.

[0038] In a preferred embodiment, the nucleic acid to be purified is DNA, and accordingly, the nucleic acid purification device is a DNA purification device.

[0039] In a preferred embodiment, the nucleic acid to be purified is a biological reaction product, preferably an enzyme cleavage product or a PCR product.

[0040] In a preferred embodiment, the pore size of the CPG is (Angstrom) to

[0041] In a preferred embodiment, the nucleic acid purification device further comprises a sieve plate, the sieve plate comprises an upper sieve plate and a lower sieve plate, and the CPG is placed between the upper sieve plate and the lower sieve plate.

[0042] In a preferred embodiment, the upper sieve plate is hydrophilically treated or selected from hydrophilic materials, and the lower sieve plate is selected from hydrophobic materials.

[0043] In a preferred embodiment, the solid phase carrier further comprises a thermoplastic polymer resin, and the thermoplastic polymer resin is sintered with CPG to form a filter element (CPG Frits) containing controlled pore size glass balls.

[0044] In a preferred embodiment, the thermoplastic polymer resin is selected from polyethylene, preferably ultra-high molecular weight polyethylene (UHMW-PE) or high-density polyethylene (HDPE).

[0045] In a preferred embodiment, the diameter of the CPG Frits is ≤3.5 mm and the thickness is ≥1.5 mm.

[0046] In a preferred embodiment, the nucleic acid purification device further comprises a containing device, and the solid phase carrier is placed in the containing device.

[0047] In a preferred embodiment, the above-mentioned receiving device is an empty column tube.

[0048] In a preferred embodiment, at least one assembly position is reserved in the above-mentioned empty column tube, the above-mentioned assembly position is a cylinder, and the above-mentioned solid phase carrier is cylindrical, so that the above-mentioned solid phase carrier is completely fitted with the above-mentioned assembly position, and the height of the above-mentioned assembly position is greater than the thickness of the above-mentioned solid phase carrier.

[0049] According to the sixth aspect, an embodiment provides a nucleic acid purification device, including the nucleic acid purification device of the fifth aspect.

[0050] According to the seventh aspect, an embodiment provides a method for purifying nucleic acid, comprising adding a solution containing the nucleic acid to be purified into a nucleic acid purification device as in the fifth aspect for incubation, so that the nucleic acid to be purified is specifically complementary paired or randomly paired with all or part of the oligonucleotide sequence in the nucleic acid purification device; and then eluting the nucleic acid to be purified from the nucleic acid purification device.

[0051] In a preferred embodiment, the nucleic acid to be purified is a single-stranded nucleic acid.

[0052] In a preferred embodiment, the nucleic acid purification device is subjected to an amino deprotection treatment before being incubated with the solution. In the application, the amino deprotection treatment refers to removing the protecting groups on the amino groups of the heterocyclic bases of the monomers or polymers used to synthesize the oligonucleotides and the cyanoethyl protecting groups on the phosphoester hydroxyl groups under aminolysis conditions to restore the structure of the oligonucleotides themselves.

[0053] In a preferred embodiment, the above-mentioned amino deprotection treatment is carried out at 90°C for 2 hours; then, acetonitrile / water solution and ultrapure water are used as mobile phases for washing and centrifugation to obtain a nucleic acid purification device connected with the above-mentioned oligonucleotide sequence, which is then incubated with the above-mentioned solution.

[0054] In a preferred embodiment, after the incubation, washing is performed using an ethanol / water solution as a mobile phase to remove unbound nucleic acids and / or enzymes and / or buffers, and then the nucleic acids to be purified are eluted from the nucleic acid purification device.

[0055] In a preferred embodiment, the eluting of the nucleic acid to be purified from the nucleic acid purification device specifically comprises: denaturing and melting the paired nucleic acid to be purified and the oligonucleotide sequence under heating conditions, and eluting the unzipped single-stranded nucleic acid to be purified with heated ultrapure water.

[0056] In a preferred embodiment, the nucleic acid to be purified is eluted from the nucleic acid purification device and then annealed to restore the double helix structure of the complementary single strands.

[0057] According to the eighth aspect, one embodiment provides use of the nucleic acid purification device of the fifth aspect in nucleic acid purification.

[0058] In the nucleic acid synthesis device of the present invention, CPG is an unmodified naked CPG without a universal linker, and a certain number of hydroxyl groups are exposed on the surface. After the synthesis of nucleic acids such as oligonucleotide primers is completed and subjected to aminolysis, nucleic acids such as oligonucleotide primers connected to CPG will not be cut off and remain connected to the solid phase carrier. Therefore, the nucleic acid synthesis device of the present invention can simultaneously achieve the purpose of synthesizing nucleic acids and fixing nucleic acids, and can not only be used for the synthesis of nucleic acids such as oligonucleotide primers, but also can use nucleic acids such as oligonucleotide primers fixed on CPG to purify enzyme cleavage products and PCR products, etc., and has the advantages of simple structure, small size, light weight, high efficiency, low cost, and diverse functions.

[0059] The nucleic acid purification device of the present invention can purify specific nucleic acid sequences and has the advantages of high efficiency, rapidity, convenience, safety and non-toxicity. It overcomes the defects of existing nucleic acid separation and purification methods that are unable to purify specific DNA sequences, have complex operations, low efficiency, slow separation speed, high economic cost and require the use of toxic chemical reagents. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Schematic diagram of the design of a nucleic acid synthesis column or a nucleic acid purification column in an embodiment of the present invention, wherein 1-empty column tube; 2-CPG Frits assembled in the empty column tube;

[0061] Figure 2 Schematic diagram of the cross section of the CPG Frits of the nucleic acid synthesis column or nucleic acid purification column in the embodiment of the present invention, wherein: 3- a rigid network structure formed by bridging between PE particles; 4- a bare CPG particle without a universal linker and not modified;

[0062] Figure 3 The schematic diagram of the CPG structure design of the nucleic acid synthesis column in the embodiment of the present invention, wherein the surface of the unmodified bare CPG particles has hydroxyl groups (-OH);

[0063] Figure 4 The schematic diagram of the CPG structure design of the nucleic acid purification column in the embodiment of the present invention, wherein an oligonucleotide sequence is connected to the surface of the unmodified bare CPG particles;

[0064] Figure 5 This is a schematic diagram of the nucleic acid purification method in an embodiment of the present invention;

[0065] Figure 6 The figure is the electrophoresis result of the DNA fragment purified in the embodiment of the present invention on 1.2% agarose gel, wherein 1-the PCR product is recovered using a DNA purification column connected with a 6nt degenerate oligonucleotide sequence; 2-the PCR product is recovered using a DNA purification column connected with an 8nt degenerate oligonucleotide sequence; 3-the PCR product is recovered using a DNA purification column connected with a 10nt degenerate oligonucleotide sequence; 4-the PCR product is recovered using a DNA purification column connected with a 12nt degenerate oligonucleotide sequence; M-DL2000 DNA Marker. DETAILED DESCRIPTION

[0066] The present invention is further described in detail below by specific embodiments in conjunction with the accompanying drawings. In the following embodiments, many details are described to enable the present invention to be better understood. However, those skilled in the art can easily recognize that some of the features can be omitted in different situations, or can be replaced by other elements, materials, methods.

[0067] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementations. At the same time, the steps or actions in the method description can also be interchanged or adjusted in a manner that is obvious to those skilled in the art. Therefore, the various sequences in the specification and the drawings are only for the purpose of clearly describing a certain embodiment and are not meant to be a required sequence, unless otherwise specified that a certain sequence must be followed.

[0068] In one embodiment of the present invention, a nucleic acid synthesis device is provided, which includes a solid phase carrier, which includes a controlled pore glass ball (CPG), and the CPG is an unmodified naked CPG, and a universal linker (Universal Linker) that does not carry a chemical group on its surface, and has a hydroxyl group on its surface. The hydroxyl group is used as a starting point for nucleic acid synthesis and is used to connect phosphoramidite-protected nucleotide monomers or polymers through covalent bonding to perform nucleic acid synthesis.

[0069] In the embodiment of the present invention, the covalent bonding connection between the hydroxyl groups on the surface of CPG and the nucleotide can be achieved by the classic solid-phase phosphoramidite triester method. Different from the prior art, the CPG used in the embodiment of the present invention is an unmodified naked CPG, and the hydroxyl groups on its surface act as the 5'-hydroxyl groups in the solid-phase phosphoramidite triester reaction to react with the added phosphoramidite-protected nucleotide monomers or polymers.

[0070] The nucleic acid synthesis device of the present invention is suitable for the synthesis of any suitable nucleic acid, for example including but not limited to DNA and RNA, etc., preferably the synthesis of DNA. Therefore, in a preferred embodiment, the nucleic acid synthesis device of the present invention is a DNA synthesis device.

[0071] In the embodiment of the present invention, the controlled pore glass sphere (CPG) used has many irregular channels inside the sphere, and the pore network is huge. The size of the channel is called the pore size, and the pore size is stable. The pore size is usually selected. (Angstrom) to (Preferred ) 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. In specific applications, the pore size of the controlled pore glass ball (CPG) used can be determined according to specific circumstances.

[0072] In the embodiments of the present invention, the nucleic acid synthesis device can be designed in various suitable forms. The present invention provides two exemplary forms.

[0073] In the first form, 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 ball (CPG) is placed between the upper sieve plate and the lower sieve plate, and the sieve plate can be sintered with thermoplastic polymer resin powders such as ultra-high molecular weight polyethylene (UHMW-PE) or high-density polyethylene (HDPE). The function of the sieve plate is to prevent CPG from leaking down and allow the synthetic reagent to pass through. The upper sieve plate can be hydrophilically treated or use hydrophilic materials, and the lower sieve plate can use hydrophobic materials. In addition, the nucleic acid synthesis device also includes a containing device, and a solid phase carrier such as a controlled pore glass ball (CPG) and an optional sieve plate are placed in the containing device, and the containing device can be an empty column tube, which can be formed by injection molding of polypropylene.

[0074] In the second form, the nucleic acid synthesis device includes a filter element (CPG Frits) containing controlled pore glass balls (CPG) formed by sintering a thermoplastic polymer resin as a solid phase carrier. The sintering temperature can be 160°C to 250°C, preferably 180°C. The thermoplastic polymer resin can 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 CPG particles, and bridges between PE particles to form a certain pore size. CPG particles are evenly embedded in the rigid mesh structure formed by PE bridges to form CPG Frits with a certain pore size and porosity. During the synthesis of oligonucleotide sequences, the synthesis reagent flows through the internal pores of CPG and the pores formed by PE bridges, so that the reagent flow channel is long enough, thereby increasing 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 fixes the CPG particles and prevents them from rotating due to the flow of reagents, further prolonging the contact time between the reagents and the reaction sites on the CPG, thereby improving the synthesis efficiency and product purity. In this form, the CPG Frits are cylindrical, and their diameter can be any suitable diameter, such as a diameter ≤3.5 mm; the thickness can be any suitable thickness, such as a thickness ≥1.5 mm. Preferably, 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.

[0075] Since the PE materials used in nucleic acid synthesis have super hydrophobicity, and molecular biological reactions such as PCR reactions are mostly carried out in aqueous solution, this hydrophobic material limits its further application. Through hydrophilic treatment, this material can absorb aqueous solution, which will expand its wide application in the field of synthetic biology. Therefore, in order to improve the hydrophilicity of CPG Frits, CPG Frits can use CPG Frits that have been hydrophilically treated. The hydrophilic treatment can be first wetted with alcohols and then bonded with surfactants.

[0076] Wherein, the alcohol solvent can be a C1-10 straight chain or branched alcohol solvent, preferably methanol, ethanol, propanol, n-butanol, ethylene glycol, glycerol, and their aqueous solvents or their combinations, more preferably 95% (v / v) ethanol. The surfactant can be an anionic surfactant, a cationic surfactant, a zwitterionic surfactant or a nonionic surfactant, preferably sodium lauryl sulfate, benzalkonium bromide, polysorbate, fatty acid sorbitan, quaternary ammonium compound, lecithin, amino acid or their combinations.

[0077] In some preferred embodiments, the nucleic acid synthesis device of the present invention further comprises a containing device, in which a solid phase carrier such as CPG Frits is placed. The containing device can be an empty column tube, which can be made of polypropylene injection molding.

[0078] In a preferred embodiment, the containing device is an empty column tube, and 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 with no slope. The solid phase carrier is cylindrical and fits perfectly with the empty column tube. The height of the assembly position is greater than the thickness of the solid phase carrier.

[0079] In a preferred embodiment, the hollow column tube is made of polypropylene injection molding, adopts a gradient straight mouth design, and the reserved CPG Frits assembly position is a cylinder without a slope. The column length of the hollow column tube is 70mm, the inner diameter of the upper part of the hollow column tube is 4.52mm, and the upper part of the assembly position gradually narrows to 3mm. The inner diameter of the assembly position is 2.95mm, and the height of the assembly position is 5mm.

[0080] In addition, in the nucleic acid synthesis device of the present invention, the containing device may use other suitable forms besides empty column tubes, such as a multi-well plate, such as a 24-well plate, a 96-well plate, a 384-well plate, etc. Each CPG Frits, or CPG and sieve plate are respectively placed in each hole of the multi-well plate, forming a multi-well plate-type nucleic acid synthesis device capable of performing nucleic acid synthesis in batches.

[0081] like Figure 1 , Figure 2 and Figure 3 As shown, in an exemplary embodiment of the present invention, the nucleic acid synthesis device is a nucleic acid synthesis column, which includes: an empty column tube 1 and CPG Frits 2 assembled in the empty column tube. The CPG is a bare CPG carrier without a universal linker and unmodified, with a certain number of hydroxyl groups exposed on the surface. The pore size of the CPG is CPG Frits 2 are formed by sintering unmodified bare CPG particles 4 and thermoplastic polymer resin (such as PE), and the PE particles are bridged to form a rigid network structure 3. The sintering temperature can be 160° C. to 250° C., preferably 180° C. The sintering time can be 5 minutes to 10 hours, etc., which can be determined according to needs. Figure 3 It is shown that a certain number of hydroxyl groups (-OH) are exposed on the surface of the naked CPG carrier, through which covalent bonds can be formed with the phosphoramidite-protected nucleotide monomers or polymers to be added to perform nucleic acid synthesis.

[0082] In one embodiment of the present invention, a nucleic acid synthesis device is provided, and the device includes the nucleic acid synthesis device of the embodiment of the present invention. In addition to including the nucleic acid synthesis device of the present invention, the other parts of the nucleic acid synthesis device of the present invention, such as the liquid system, gas system, control system, transmission system, etc., can be implemented according to the prior art. For example, replacing the nucleic acid synthesis device used in Dr. Oligo 192 nucleic acid synthesizer with the nucleic acid synthesis device of the present invention can be used as an example of the nucleic acid synthesis device of the present invention.

[0083] In one embodiment of the present invention, the use of the nucleic acid synthesis device of the present invention in a method for synthesizing nucleic acid is provided. The synthesized nucleic acid can be any suitable nucleic acid, for example including but not limited to DNA and RNA, etc., preferably DNA.

[0084] In a preferred embodiment, the nucleic acid is synthesized by solid phase phosphoramidite triester method. For example, the nucleic acid synthesis device of the present invention is used as a synthesis column in Dr. Oligo 192 nucleic acid synthesizer, and a section of oligonucleotide primer sequence is synthesized by the classic solid phase phosphoramidite triester method on the synthesizer, thereby achieving the purpose of the present invention.

[0085] In one embodiment of the present invention, a method for nucleic acid synthesis is provided, wherein the method uses the nucleic acid synthesis device or nucleic acid synthesis equipment of the present invention to synthesize nucleic acid, wherein the hydroxyl group on the CPG surface of the nucleic acid synthesis device is used as the synthesis starting point of the nucleic acid to be synthesized, and phosphoramidite-protected nucleotide monomers or polymers are connected by covalent bonding. In a preferred embodiment, the nucleic acid synthesis method is a solid phase phosphoramidite triester method.

[0086] In one embodiment of the present invention, a nucleic acid purification device is provided, which includes a solid phase carrier, which includes a controlled pore glass ball (CPG). The CPG is an unmodified naked CPG without a universal linker (UniversalLinker), and has hydroxyl groups on its surface. These hydroxyl groups are covalently bonded to an oligonucleotide sequence, and the oligonucleotide sequence is specifically complementary or randomly paired with all or part of the nucleic acid to be purified.

[0087] In the embodiment of the present invention, the covalent bonding connection between the hydroxyl groups on the surface of CPG and the oligonucleotide sequence can be achieved by the classic solid-phase phosphoramidite triester method. Different from the prior art, the CPG used in the embodiment of the present invention is unmodified naked CPG, and the hydroxyl groups on its surface act as the 5'-hydroxyl groups in the solid-phase phosphoramidite triester reaction to react with the added phosphoramidite-protected nucleotide monomers or polymers.

[0088] In the embodiment of the present invention, the oligonucleotide sequence is specifically complementary or randomly paired with all or part of the nucleic acid to be purified, so that the specific nucleic acid sequence can be purified purposefully. 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. Generally speaking, the pairing position of the oligonucleotide sequence on the nucleic acid to be purified is selected from a region at the 3' end or / and the 5' end of the nucleic acid to be purified. Specifically, it can be a region at the 3' end of the nucleic acid to be purified, it can also be a region at the 5' end of the nucleic acid to be purified, or it can be a region at each of the 3' end and the 5' end of the nucleic acid to be purified.

[0089] Generally speaking, there is no particular restriction on the length of the oligonucleotide sequence, and any length can be designed according to specific needs. However, based on experimental results, it is determined that an oligonucleotide sequence with a length of 6 to 12 bases can achieve excellent results, such as 6 bases, 8 bases, 10 bases or 12 bases.

[0090] In the embodiments of the present invention, the term "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. In a preferred embodiment of the present invention, the nucleic acid to be purified is DNA, and therefore, the nucleic acid purification device of the present invention is a DNA purification device. In a more preferred embodiment, the nucleic acid to be purified is a biological reaction product, which can be a product of any biological, biochemical or other reaction, and the product needs to be specifically purified to a specific fragment, and typical but non-limiting examples of such biological reaction products are enzyme cleavage products or PCR products.

[0091] In the embodiment of the present invention, the CPG sphere used has many irregular channels inside, and the pore network is huge. The size of the channel is called the pore size. The pore size is stable, and the pore size is usually selected. (Angstrom) to (Preferred ) is used in the preparation of the nucleic acid purification device of the embodiment of the present invention, and other pore sizes can also be selected. In specific applications, the pore size of the CPG used can be determined according to the specific conditions of the nucleic acid to be purified.

[0092] In the embodiments of the present invention, the nucleic acid purification device can be designed in various suitable forms. The present invention provides two exemplary forms.

[0093] In the first form, the nucleic acid purification device includes 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, and the sieve plate can be sintered with thermoplastic polymer resin powder such as ultra-high molecular weight polyethylene (UHMW-PE) or high-density polyethylene (HDPE). The function of the sieve plate is to prevent CPG from leaking down while various reagents can pass through. The upper sieve plate can be hydrophilically treated or use hydrophilic materials, and the lower sieve plate can use hydrophobic materials. In addition, the nucleic acid purification device also includes a containing device, and a solid phase carrier such as CPG and an optional sieve plate are placed in the containing device. The containing device can be an empty column tube, and the empty column tube can be made of polypropylene injection molding.

[0094] In the second form, the nucleic acid purification device includes a filter element (CPG Frits) containing controlled pore glass balls formed by sintering CPG and a thermoplastic polymer resin as a solid phase carrier. The thermoplastic polymer resin can be selected from polyethylene or polypropylene, preferably ultra-high molecular weight polyethylene (UHMW-PE) or high-density polyethylene (HDPE). Polyethylene (PE) particles wrap and fix CPG particles, and bridges between PE particles to form a certain pore size. CPG particles are uniformly embedded in the rigid mesh structure formed by PE bridges to form CPG Frits with a certain pore size and porosity. In this form, CPG Frits can be cylindrical, and its diameter can be any suitable diameter, such as a diameter of ≤3.5mm; the thickness can be any suitable thickness, such as a thickness of ≥1.5mm. Preferably, the diameter of CPG Frits is 3mm and the thickness is 4mm. In addition, the nucleic acid purification device can also include a containing device, and the solid phase carriers such as CPG Frits are placed in the containing device. The containing device can be an empty column tube, and the empty column tube can be made of polypropylene injection molding. In a preferred embodiment, the containing device is an empty column tube, and 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 with no slope. The solid phase carrier is cylindrical and fits perfectly with the empty column tube. The height of the assembly position is greater than the thickness of the solid phase carrier.

[0095] In addition, in the nucleic acid purification device of the present invention, in addition to using empty column tubes, the containing device 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 CPG and sieve plates are respectively placed in each hole of the multi-well plate, forming a multi-well plate-type nucleic acid purification device that can perform nucleic acid purification in batches.

[0096] like Figure 1 , Figure 2 and Figure 4As shown, in an exemplary embodiment of the present invention, the nucleic acid purification device is a nucleic acid purification column, which includes: an empty column tube 1 and CPG Frits 2 assembled in the empty column tube. The CPG is a bare CPG carrier without a universal linker and unmodified, with a certain number of hydroxyl groups exposed on the surface. The pore size of the CPG is CPG Frits 2 are formed by sintering unmodified bare CPG particles 4 and thermoplastic polymer resin (such as PE), and the PE particles are bridged to form a rigid network structure 3. The sintering temperature can be 160° C. to 250° C., preferably 180° C. The sintering time can be 5 minutes to 10 hours, etc., which can be determined according to needs. Figure 4 It is shown that a certain number of hydroxyl groups (-OH) are exposed on the surface of the naked CPG carrier, and the hydroxyl groups are connected to an oligonucleotide sequence through covalent bonding. The oligonucleotide sequence is specifically complementary or randomly paired with all or part of the nucleic acid to be purified.

[0097] In a preferred embodiment, CPG Frits are made by mixing CPG and UHMW-PE powders uniformly and sintering at 180°C. The diameter of CPG Frits is 3mm and the thickness of CPG Frits is 4mm. CPG Frits are hydrophilic treated, and the content of CPG in each CPG Frits is 4mg. The hydrophilic treatment process of CPG Frits can be first wetting with alcohols and then bonding with surfactants. The alcohol solvent can be a C1-10 straight chain or branched alcohol solvent, preferably methanol, ethanol, propanol, n-butanol, ethylene glycol, glycerol, and their aqueous solvents or combinations thereof, more preferably 95% (v / v) ethanol. The surfactant can 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 hollow column tube can be made of polypropylene injection molding, with a gradient straight mouth design, and the reserved CPG Frits assembly position is a cylinder without a slope. The column length of the hollow column tube is 70mm, the inner diameter of the upper part of the hollow column tube is 4.52mm, and it gradually narrows to 3mm at the upper part of the assembly position. The inner diameter of the assembly position is 2.95mm, and the height of the assembly position is 5mm.

[0098] In one embodiment of the present invention, a nucleic acid purification device is provided, including the nucleic acid purification apparatus according to the embodiment of the present invention.

[0099] In one embodiment of the present invention, a method for purifying nucleic acid is provided, which comprises adding a solution containing the nucleic acid to be purified into the nucleic acid purification device of the present invention for incubation, so that the nucleic acid to be purified is specifically complementary paired or randomly paired with the oligonucleotide sequence in the nucleic acid purification device; and then eluting the nucleic acid to be purified from the nucleic acid purification device.

[0100] In the embodiments of the present invention, the nucleic acid to be purified may be any suitable nucleic acid, including but not limited to DNA and RNA. In a preferred embodiment of the present invention, the nucleic acid to be purified is DNA. In a more preferred embodiment, the nucleic acid to be purified is a biological reaction product, which may be a product of any biological, biochemical or other reaction, and the product requires specific purification of a specific fragment, and typical but non-limiting examples of such biological reaction products are enzyme cleavage products or PCR products.

[0101] In the embodiments of the present invention, the nucleic acid to be purified may be a nucleic acid containing at least a portion of a single strand, including but not limited to a nucleic acid to be purified having a protruding single strand at the end and a double strand in the middle, or a nucleic acid to be purified having a double strand at the end and a single strand in the middle, or a nucleic acid to be purified having a single strand throughout. In a preferred embodiment of the present invention, the nucleic acid to be purified is a single-stranded nucleic acid.

[0102] In a preferred embodiment of the present invention, the nucleic acid purification device is first subjected to an ammonium deprotection treatment before being incubated with the solution to be purified. The ammonium deprotection treatment can be performed, for example, at 90° C. for 2 hours; then, the nucleic acid purification device is washed with an acetonitrile / water solution and ultrapure water as mobile phases, centrifuged, and a pure nucleic acid purification device connected with an oligonucleotide sequence is obtained, which is then incubated with the solution to be purified.

[0103] like Figure 5 As shown, the nucleic acid to be purified is a PCR product, which is denatured and unwound at high temperature (e.g., 95° C.) to form a single strand, wherein both ends of the single strand carry a sequence complementary to the oligonucleotide sequence connected to the CPG surface in the nucleic acid purification device; the denatured and unwound PCR product is loaded into the nucleic acid purification device for incubation, and the oligonucleotide sequence connected to the CPG surface is complementary to the sequence segments at both ends of the single strand; washing (e.g., washing with ethanol / water solution as the mobile phase) is performed to remove unbound DNA and impurities such as enzymes and buffers in the reaction system; then the nucleic acid to be purified is eluted from the nucleic acid purification device, for example, under heating conditions, the paired nucleic acid to be purified and the oligonucleotide sequence are denatured and unwound, and the unwound single strand of the nucleic acid to be purified is eluted with heated ultrapure water; finally, the eluted nucleic acid is denatured and annealed again to restore the double helix structure of the complementary single strands to obtain a purified DNA product.

[0104] In one embodiment of the present invention, a use of the nucleic acid purification device of the present invention in nucleic acid purification is provided, wherein an oligonucleotide sequence is connected to the hydroxyl group on the CPG surface of the nucleic acid purification device, and the oligonucleotide sequence is specifically complementary or randomly paired with all or part of the nucleic acid to be purified.

[0105] The technical solutions of the present invention are described in detail below through examples. It should be understood that the examples are merely exemplary and cannot be construed as limiting the scope of protection of the present invention.

[0106] Example

[0107] In this embodiment, the specific experimental process and steps of the nucleic acid purification method are as follows:

[0108] (1) Using the classic solid phase phosphoramidite triester method on a Dr.Oligo 192 nucleic acid synthesizer, a hydrophilic DNA synthesis column (the synthesis column has CPG Frits built in it, which includes a naked CPG carrier, and a certain number of hydroxyl groups are exposed on the surface of the naked CPG carrier) is used to synthesize a purification "bait" sequence (i.e., a degenerate oligonucleotide sequence) that is randomly paired with the DNA to be purified. The sequence lengths of the synthesized degenerate oligonucleotides are 6 nt, 8 nt, 10 nt, and 12 nt, respectively, and are connected to the CPG Frits to obtain the desired DNA purification column;

[0109] (2) The obtained DNA purification column was subjected to an ammonium deprotection treatment at 90° C. for 2 hours, and then washed with 400 μL of acetonitrile / water (volume ratio 90:10) and 300 μL of ultrapure water as mobile phases, and centrifuged to obtain a DNA purification column connected with a pure degenerate oligonucleotide sequence;

[0110] (3) After PCR amplification, 25 μL of a DNA product solution containing a target fragment of 350 bp was obtained, which was heated at 95°C for 5 minutes to denature and unwind into a single strand;

[0111] (4) The denatured DNA aqueous solution obtained in step (3) was quickly loaded into a DNA purification column, absorbed by CPG Frits, and incubated at 25° C. for 5 minutes to bind to the oligonucleotides on the column;

[0112] (5) Wash twice with 400 μL ethanol / water (volume ratio 80:20) as the mobile phase and centrifuge to remove impurities such as unbound DNA;

[0113] (6) Heat at 95°C for 5 minutes to denature and melt the DNA, elute the unwound single-stranded DNA with 20 μL of 95°C ultrapure water, centrifuge and collect;

[0114] (7) Denaturing the DNA aqueous solution obtained in step (6) by heating it again at 95° C. for 5 minutes;

[0115] (8) Cool to 60°C and anneal for 10 minutes to obtain pure PCR products.

[0116] The purified PCR product was detected using a NanoDrop UV spectrophotometer. 260 / OD 280 The ratio is 1.8-2.0.

[0117] Table 1 below shows the comparison results of the purification effects of DNA purification columns connected with oligonucleotides of different lengths (all of which are random sequences of degenerate base N) for purifying PCR products under the same conditions.

[0118] Table 1

[0119]

[0120] Figure 6 The figure is a graph showing the electrophoresis result of the DNA fragments purified in the above example on a 1.2% agarose gel, wherein lane 1 represents the recovery of PCR products using a DNA purification column connected to a 6nt degenerate oligonucleotide sequence; lane 2 represents the recovery of PCR products using a DNA purification column connected to an 8nt degenerate oligonucleotide sequence; lane 3 represents the recovery of PCR products using a DNA purification column connected to a 10nt degenerate oligonucleotide sequence; lane 4 represents the recovery of PCR products using a DNA purification column connected to a 12nt degenerate oligonucleotide sequence; and M represents DL2000 DNA Marker.

[0121] from Figure 6 As shown in Table 1, the nucleic acid synthesis device of the present invention is used to connect the degenerate oligonucleotide sequence. Based on the complementary pairing relationship between the oligonucleotide sequence and the nucleic acid to be purified, the purification of enzyme cutting, PCR products, etc. can be completed quickly and effectively, greatly shortening the purification time. The obtained DNA fragments are detected by ultraviolet spectrophotometer, and OD 260 / OD 280 The ratio reaches 1.8-2.0, the purified DNA product has high purity, and the purification recovery rate is about 50%.

[0122] The above specific examples are used to illustrate the present invention, which are only used to help understand the present invention and are not used to limit the present invention. For those skilled in the art to which the present invention belongs, some simple deductions, modifications or substitutions can be made according to the idea of ​​the present invention.

Claims

1. A nucleic acid purification device, It is characterized in that The device includes a solid phase carrier, which includes a controlled pore glass ball (CPG). The CPG is an unmodified naked CPG, whose surface is rich in silanol groups and is not modified with any chemical groups. The silanol groups are connected to an oligonucleotide sequence through covalent bonding. The oligonucleotide sequence is specifically complementary or randomly paired with all or part of the nucleic acid to be purified, and the length of the oligonucleotide sequence is 6 to 12 bases.

2. The nucleic acid purification device according to claim 1, It is characterized in that The oligonucleotide sequence is specifically complementary to all or part of a region at the 3' end and / or the 5' end of the nucleic acid to be purified.

3. The nucleic acid purification device according to claim 1, It is characterized in that The nucleic acid to be purified is DNA, and accordingly, the nucleic acid purification device is a DNA purification device.

4. The nucleic acid purification device according to claim 3, It is characterized in that The nucleic acid to be purified is a biological reaction product.

5. The nucleic acid purification device according to claim 4, It is characterized in that The nucleic acid to be purified is an enzyme cleavage product or a PCR product.

6. The nucleic acid purification device according to claim 1, It is characterized in that The pore size of the CPG is 500Å (angstroms) to 2000Å.

7. The nucleic acid purification device according to claim 1, It is characterized in that The nucleic acid purification device further comprises a sieve plate, wherein the sieve plate comprises an upper sieve plate and a lower sieve plate, and the CPG is placed between the upper sieve plate and the lower sieve plate.

8. The nucleic acid purification device according to claim 7, It is characterized in that The upper sieve plate is treated with hydrophilicity or selected from hydrophilic materials, and the lower sieve plate is selected from hydrophobic materials.

9. The nucleic acid purification device according to claim 1, It is characterized in that The solid phase carrier also 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 controlled pore sizes.

10. The nucleic acid purification device according to claim 9, It is characterized in that The thermoplastic polymer resin is selected from polyethylene.

11. The nucleic acid purification device according to claim 9, It is characterized in that The thermoplastic polymer resin is ultra-high molecular weight polyethylene (UHMW-PE) or high-density polyethylene (HDPE).

12. The nucleic acid purification device according to claim 9, It is characterized in that The diameter of the CPG Frits is ≤3.5 mm and the thickness is ≥1.5 mm.

13. The nucleic acid purification device according to claim 1, It is characterized in that The nucleic acid purification device also includes a containing device, and the solid phase carrier is placed in the containing device.

14. The nucleic acid purification device according to claim 13, It is characterized in that The containing device is an empty column tube.

15. The nucleic acid purification device according to claim 14, It is characterized in that At least one assembly position is reserved in the empty column tube, the assembly position is a cylinder, the solid phase carrier is cylindrical, the solid phase carrier is completely fitted with the assembly position, and the height of the assembly position is greater than the thickness of the solid phase carrier.

16. A nucleic acid purification device, It is characterized in that The device comprises the nucleic acid purification device according to any one of claims 1 to 15.

17. A method for purifying nucleic acid, It is characterized in that The method comprises adding a solution containing the nucleic acid to be purified into the nucleic acid purification device as described in any one of claims 1 to 15 for incubation, so that the nucleic acid to be purified is specifically complementary paired or randomly paired with all or part of the oligonucleotide sequence in the nucleic acid purification device; and then eluting the nucleic acid to be purified from the nucleic acid purification device.

18. The method for purifying nucleic acid according to claim 17, It is characterized in that The nucleic acid to be purified is a single-stranded nucleic acid.

19. The method for purifying nucleic acid according to claim 17, It is characterized in that The nucleic acid purification device is first subjected to an amino deprotection treatment before being incubated with the solution.

20. The method for purifying nucleic acid according to claim 19, It is characterized in that The amino deprotection treatment is carried out at 90° C. for 2 hours; then, acetonitrile / water solution and ultrapure water are used as mobile phases for washing and centrifugation to obtain a nucleic acid purification device connected with the oligonucleotide sequence and then incubated with the solution.

21. The method for purifying nucleic acid according to claim 17, It is characterized in that After the incubation, washing is performed using ethanol / water solution as the 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.

22. The method for purifying nucleic acid according to claim 17, It is characterized in that The eluting of the nucleic acid to be purified from the nucleic acid purification device specifically includes: denaturing and melting the paired nucleic acid to be purified and the oligonucleotide sequence under heating conditions, and eluting the unwound single-stranded nucleic acid to be purified with heated ultrapure water.

23. The method for purifying nucleic acid according to claim 22, It is characterized in that The nucleic acid to be purified is eluted from the nucleic acid purification device and then annealed to restore the double helix structure of the complementary single strands.

24. Use of the nucleic acid purification device according to any one of claims 1 to 15 in nucleic acid purification.

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