Matrix for microchannel screening and application thereof, sequencing gel for separating DNA and RNA as well as preparation method and application of sequencing gel

Abstract

The invention provides a matrix for microchannel screening and application thereof, a sequencing gel for separating DNA and RNA and a preparation method and application thereof, and the matrix comprises a copolymer of N, N-dimethylacrylamide and a hydrophilic monomer, and polyacrylamide; wherein the hydrophilic monomer is selected from any one or more than two of N-hydroxyethyl acrylamide, hydroxyethyl acrylate, hydroxyethyl methylacrylate, N-hydroxymethyl acrylamide and N-(2-hydroxyethyl)-2-methyl-2-acrylamide, and the hydrophilic monomer is selected from any one or more than two of N-hydroxyethyl acrylamide, hydroxyethyl acrylate, hydroxyethyl methylacrylate, N-hydroxymethyl acrylamide and N-(2-hydroxyethyl)-2-methyl-2-acrylamide. The sequencing gel comprises the matrix disclosed by the invention; the concentration of the matrix in the sequencing gel is 2%-6% (w / v), and the kinematic viscosity of the sequencing gel is 50-500 cP. The polymer in the matrix is good in compatibility, the matrix is good in stability and reproducibility, and the matrix can be applied to screening of biomacromolecules through capillary electrophoresis. The sequencing gel disclosed by the invention is relatively high in sequencing length in unit time, simple in preparation method and good in application prospect in the aspect of sequencing a base sequence.

Description

Technical Field

[0001] The present invention belongs to the technical field of screening matrix and nucleic acid sequencing, and specifically is a matrix for microchannel screening and application thereof, a sequencing gel for separating DNA and RNA and a preparation method and application thereof. Background Art

[0002] Capillary electrophoresis (CE) is a method of separating and analyzing components based on the different migration speeds of ions under the action of an electric field. It has the advantages of fast analysis speed, low sample consumption, high separation efficiency and low cost. Due to its high throughput and easy automation, it has become one of the most universal methods for separating biological macromolecules (such as DNA, RNA, polysaccharides and proteins). Since many biological macromolecules (such as DNA, RNA, etc.) have the same amount of charge per unit mass, they cannot be electrophoretically separated in free aqueous solution. Therefore, it is necessary to use a sieving matrix to distinguish charged macromolecules according to size. Traditional capillary gel electrophoresis (CGE) requires pumping a polymer monomer solution into a capillary for polymerization. This method has excellent particle size selectivity and sequencing read length, but the gel will change in volume due to heat and other reasons when it polymerizes in the capillary, which will then produce bubbles, uneven gel or even rupture, and will degrade under alkaline conditions and have a short lifespan.

[0003] Studies have shown that linear polymers can replace gels as sieving matrices, and linear polymers sieve DNA by forming a dynamic entangled network. Polymer materials such as polyacrylamide (PAM), poly N, N-dimethylacrylamide (PDMA), hydroxyethyl cellulose (HEC) and polyvinyl pyrrolidone (PVP) are often used as sieving matrices. Among them, PAM has a higher sequencing read length when used as a sieving matrix, but due to the existence of electroosmotic flow, when polyacrylamide alone is used as a sieving matrix for capillary electrophoresis, the capillary needs to be coated and modified to prevent the reduction of resolution. Other sieving matrices such as PDMA have problems such as low sequencing read length or long sequencing time, but studies have shown that polymers such as PDMA can effectively reduce electroosmotic flow in capillaries by self-coating capillaries. Therefore, by combining the advantages of the two polymers, it may be possible to prepare a sieving matrix with excellent sequencing performance.

[0004] Blending different polymers is a simple method for preparing a sieving matrix, but the compatibility problem between polymers makes it impossible to simply blend. Patent CN101351260B discloses a DNA separation matrix containing a high molecular weight poly N, N-dimethylacrylamide (PDMA) or its copolymer combined with a hydrophilic water-soluble polymer component poly N-hydroxyethylacrylamide (PHEA) to separate DNA sequencing fragments very quickly through microchannel electrophoresis; however, its sequencing read length can only reach more than 600 bases. Patent CN102220599B discloses a sieving matrix with a quasi-interpenetrating network prepared using acrylamide and N, N-dimethylacrylamide copolymer and poly N, N-dimethylacrylamide, and the sieving matrix has good sequencing ability and stability. However, the quasi-interpenetrating network does not remove unreacted monomers and initiators during the preparation process, which affects the final sequencing.

[0005] Through copolymerization, the sequencing ability and self-coating ability of different polymers can be effectively combined. Liguo Song et al. (Electrophoresis, 2001, 22: 729-736) studied the improvement of the sequencing performance of polymers by preparing random copolymers of acrylamide and N, N-dimethylacrylamide, and the separation efficiency of the copolymers was higher than that of polyacrylamide alone. US20150159009A1 discloses polyacrylamide polymers grafted with N, N-dimethylacrylamide, which can effectively inhibit electroosmotic flow and can effectively separate capillary fragments. However, both random copolymers and graft copolymers have problems such as uneven copolymerization, and the preparation method of graft copolymerization is relatively cumbersome.

[0006] In summary, the preparation technology of existing screening matrix mainly includes copolymerization and blending. Wherein copolymerization is divided into random copolymerization and graft copolymerization, and it is easy to have problems such as uneven copolymerization, and the preparation method of graft copolymerization is more complicated. Blending can be divided into direct blending and forming quasi-interpenetrating network, wherein direct blending can cause sequencing ability to decrease because of compatibility problems, and quasi-interpenetrating network can reduce the impact caused by incompatibility between polymers, but its preparation process can introduce impurities (such as unreacted monomers and initiator residues). Therefore, it is urgent to provide a screening matrix with simple preparation method and good polymer compatibility, especially a screening matrix with good sequencing ability. Summary of the invention

[0007] One of the objects of the present invention is to provide a matrix for microchannel screening and its application in screening biomacromolecules using capillary electrophoresis.

[0008] The second object of the present invention is to provide a sequencing gel for separating DNA and RNA, a preparation method thereof, and the use of the sequencing gel in sequencing base sequences.

[0009] The above objects of the present invention are achieved through the following technical solutions:

[0010] In a first aspect, the present invention provides a matrix for microchannel screening, the matrix comprising a copolymer of N,N-dimethylacrylamide and a hydrophilic monomer, polyacrylamide; wherein the hydrophilic monomer is selected from any one or more of N-hydroxyethylacrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, N-hydroxymethylacrylamide, N-(2-hydroxyethyl)-2-methyl-2-acrylamide, etc.

[0011] In some embodiments of the present invention, the copolymerization mass ratio of N,N-dimethylacrylamide to the hydrophilic monomer in the copolymer of N,N-dimethylacrylamide and the hydrophilic monomer is 9:1 to 1:9, preferably 9:1 to 5:5, and more preferably 6:4 to 8:2.

[0012] In some embodiments of the present invention, the mass ratio of the copolymer of N,N-dimethylacrylamide and a hydrophilic monomer to polyacrylamide in the matrix is ​​50:50 to 99:1, preferably 95:5 to 85:15.

[0013] In some embodiments of the present invention, the viscosity average molecular weight of the copolymer of N,N-dimethylacrylamide and a hydrophilic monomer is between 50 kDa and 300 kDa, preferably between 80 kDa and 250 kDa, and more preferably between 100 kDa and 200 kDa.

[0014] In some embodiments of the present invention, the viscosity average molecular weight of polyacrylamide is between 600 kDa and 2000 kDa, preferably between 800 kDa and 1600 kDa, and more preferably between 800 kDa and 1200 kDa.

[0015] In some embodiments of the present invention, the matrix is ​​obtained by blending a copolymer of N,N-dimethylacrylamide and a hydrophilic monomer, and polyacrylamide; preferably, the blending is carried out in water.

[0016] In some embodiments of the present invention, the copolymer of N,N-dimethylacrylamide and a hydrophilic monomer is prepared by redox radical polymerization of N,N-dimethylacrylamide and the hydrophilic monomer; preferably, during the polymerization process, the molecular weight (viscosity average molecular weight) of the copolymer is controlled by a chain transfer agent (amount or type, etc.).

[0017] In some embodiments of the present invention, the chain transfer agent is selected from any one or more of isopropyl alcohol, sodium bisulfite, thioglycolic acid, sodium formate and sodium hypophosphite.

[0018] In some embodiments of the present invention, the copolymer of N,N-dimethylacrylamide and a hydrophilic monomer is prepared by a method comprising the following steps:

[0019] (1) N,N-dimethylacrylamide is mixed with a hydrophilic monomer, and then a chain transfer agent, ammonium persulfate and water are added, and stirred for 20 to 40 minutes under the protection of an inert gas;

[0020] (2) adding tetramethylethylenediamine to the mixed solution obtained in step (1) and reacting for 2 to 8 hours;

[0021] (3) The reaction solution obtained in step (2) is dialyzed using a dialysis bag with a molecular weight cutoff of 14 kDa, and the dialyzed reaction solution is dried to obtain a copolymer.

[0022] In some embodiments of the present invention, in step (1), the mass ratio of the chain transfer agent, ammonium persulfate, water, N,N-dimethylacrylamide, and hydrophilic monomer is (1-6):(0.015-0.035):(150-250):(1-9):(1-9).

[0023] In some embodiments of the present invention, in step (1), the inert gas is nitrogen.

[0024] In some embodiments of the present invention, in step (2), the amount of tetramethylethylenediamine added is such that the ratio of ammonium persulfate to tetramethylethylenediamine is (15-35 mg):(20-45 μL).

[0025] In some embodiments of the present invention, in step (2), the drying method includes any one or two of freeze drying, vacuum drying, etc.

[0026] In a second aspect, the present invention provides a method for preparing a matrix for microchannel screening, the preparation method comprising the following steps:

[0027] ①. Prepare a copolymer of N,N-dimethylacrylamide and a hydrophilic monomer by redox free radical polymerization of N,N-dimethylacrylamide and a hydrophilic monomer; wherein the hydrophilic monomer is selected from any one or more of N-hydroxyethylacrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, N-hydroxymethylacrylamide, N-(2-hydroxyethyl)-2-methyl-2-acrylamide, etc.

[0028] ②. Blend polyacrylamide and the copolymer of N,N-dimethylacrylamide and hydrophilic monomer obtained in step ① at a mass ratio of 50:50 to 99:1.

[0029] In some embodiments of the present invention, the viscosity-average molecular weight of the copolymer of N,N-dimethylacrylamide and a hydrophilic monomer obtained in step ① is between 50 kDa and 300 kDa, preferably between 80 kDa and 250 kDa, and more preferably between 100 kDa and 200 kDa.

[0030] In some embodiments of the present invention, in step ①, the molecular weight of the copolymer is controlled by a chain transfer agent.

[0031] In some embodiments of the present invention, the chain transfer agent is selected from any one or more of isopropyl alcohol, sodium bisulfite, thioglycolic acid, sodium formate and sodium hypophosphite.

[0032] In some embodiments of the present invention, in step ①, the copolymerization mass ratio of N,N-dimethylacrylamide to the hydrophilic monomer is 9:1 to 1:9, preferably 9:1 to 5:5, and more preferably 6:4 to 8:2.

[0033] In some embodiments of the present invention, in step ②, the mass ratio of the copolymer of N,N-dimethylacrylamide and a hydrophilic monomer to polyacrylamide is 95:5 to 85:15.

[0034] In some embodiments of the present invention, in step ②, the viscosity-average molecular weight of the polyacrylamide is between 600 kDa and 2000 kDa, preferably between 800 kDa and 1600 kDa, and more preferably between 800 kDa and 1200 kDa.

[0035] In some embodiments of the present invention, in step ②, the blending is carried out in water.

[0036] In some embodiments of the present invention, step ② further comprises preparing polyacrylamide, which specifically comprises the following steps:

[0037] a) mixing acrylamide with a chain transfer agent, ammonium persulfate and water, and stirring for 20 to 40 minutes under the protection of an inert gas;

[0038] b) adding tetramethylethylenediamine to the mixed solution obtained in step a) and reacting for 2 to 8 hours;

[0039] c) dialyzing the reaction solution obtained in step b) using a dialysis bag with a molecular weight cutoff of 14 kDa, and drying the dialyzed reaction solution to obtain polyacrylamide.

[0040] In some embodiments of the present invention, in step a), the mass ratio of the chain transfer agent, ammonium persulfate, water and acrylamide is (1-6):(0.015-0.035):(150-250):(15-25).

[0041] In some embodiments of the present invention, in step a), the inert gas is nitrogen.

[0042] In some embodiments of the present invention, in step b), the amount of tetramethylethylenediamine added is such that the ratio of ammonium persulfate to tetramethylethylenediamine is (15-35 mg):(20-45 μL).

[0043] In some embodiments of the present invention, the reaction in step b) is carried out at room temperature.

[0044] In some embodiments of the present invention, in step b), the drying method includes any one or two of freeze drying, vacuum drying, etc.

[0045] In some embodiments of the present invention, the step ① specifically includes the following steps:

[0046] 1) Mix N,N-dimethylacrylamide and hydrophilic monomer, then add chain transfer agent, ammonium persulfate and water, and stir for 20 to 40 minutes under the protection of inert gas;

[0047] 2) Add tetramethylethylenediamine to the mixed solution obtained in step 1) and react for 2 to 8 hours;

[0048] 3) The reaction solution obtained in step (2) is dialyzed using a dialysis bag with a molecular weight cutoff of 14 kDa, and the dialyzed reaction solution is dried to obtain a copolymer.

[0049] In some embodiments of the present invention, in step 1), the mass ratio of chain transfer agent, ammonium persulfate, water, N,N-dimethylacrylamide and hydrophilic monomer is (1-6):(0.015-0.035):(150-250):(1-9):(1-9).

[0050] In some embodiments of the present invention, in step 1), the inert gas is nitrogen.

[0051] In some embodiments of the present invention, in step 2), the amount of tetramethylethylenediamine added is such that the ratio of ammonium persulfate to tetramethylethylenediamine is (15-35 mg):(20-45 μL).

[0052] In some embodiments of the present invention, in step 2), the drying method includes any one or two of freeze drying, vacuum drying, etc.

[0053] In a third aspect, the present invention provides use of the matrix described in the first aspect or the matrix prepared by the preparation method described in the second aspect in screening biomacromolecules using capillary electrophoresis.

[0054] In some embodiments of the present invention, the biomacromolecules include two or more biomacromolecules having the same charge per unit mass.

[0055] In some embodiments of the invention, the biomacromolecules include DNA and RNA.

[0056] In a fourth aspect, the present invention provides a sequencing gel for separating DNA and RNA, wherein the sequencing gel comprises the matrix described in the first aspect or the matrix prepared by the preparation method described in the second aspect.

[0057] In some embodiments of the present invention, the concentration of the matrix described in the first aspect or the matrix prepared by the preparation method described in the second aspect in the sequencing gel is 2% to 6% (w / v), preferably 2% to 4% (w / v).

[0058] In some embodiments of the present invention, the kinematic viscosity of the sequencing gel is 50 to 500 cP, preferably 200 to 400 cP.

[0059] In some embodiments of the present invention, the sequencing gel further comprises a buffer and a denaturant.

[0060] In some embodiments of the present invention, the buffer is selected from any one or more of tris(hydroxymethyl)aminomethane-acetate-EDTA buffer (TAE), tris(hydroxymethyl)methylaminopropanesulfonate sodium buffer (TAPS), tris(hydroxymethyl)aminomethane-tris(hydroxymethyl)methylaminopropanesulfonate sodium-EDTA buffer (TTE), etc., preferably TTE.

[0061] In some embodiments of the present invention, the concentration of the buffer in the sequencing gel is 50-150 mM, preferably 80-120 mM.

[0062] In some embodiments of the present invention, the denaturant is selected from any one or more of urea, 1,3-dimethylurea, dimethyl sulfoxide, formamide and 2-pyrrolidone, preferably urea.

[0063] In some embodiments of the present invention, the concentration of the denaturant in the sequencing gel is 5-8M, preferably 6-7M.

[0064] In a fifth aspect, the present invention provides a method for preparing the sequencing gel described in the fourth aspect, the preparation method comprising dissolving the matrix described in the first aspect or the matrix prepared by the preparation method described in the second aspect in a solvent.

[0065] In some embodiments of the present invention, the preparation method comprises mixing and dissolving a buffer, a denaturant, and the matrix described in the first aspect or the matrix prepared by the preparation method described in the second aspect in a solvent.

[0066] In some embodiments of the present invention, the solvent is water.

[0067] In some embodiments of the present invention, the method further comprises filtering the dissolved liquid to retain the filtrate; preferably, filtering is performed using a 0.22 μm filter membrane.

[0068] In a sixth aspect, the present invention provides use of the sequencing gel described in the fourth aspect or the sequencing gel prepared by the preparation method described in the fifth aspect in sequencing base sequences.

[0069] The beneficial effects of the present invention are as follows:

[0070] The present invention proposes a new matrix for microchannel screening. The matrix is ​​mainly polyacrylamide, and a copolymer obtained by copolymerizing N,N-dimethylacrylamide and a specific hydrophilic monomer is added for blending. The two polymers have good compatibility, which not only overcomes the problem of electroosmotic flow, but also further improves the sequencing ability of the matrix.

[0071] The present invention proposes to improve the hydrophilicity of N,N-dimethylacrylamide polymer by copolymerizing hydrophilic monomer with N,N-dimethylacrylamide, and then blending it with polyacrylamide to obtain a matrix for microchannel screening. The preparation method is simple to operate, the two polymers have good compatibility, the hydrogen bond content between the polymer molecules after mixing is high, and the prepared matrix has good stability and reproducibility, and can be used in the screening of biomacromolecules using capillary electrophoresis. In addition, the matrix preparation method of the present invention controls the molecular weight of the copolymer by a chain transfer agent during the copolymerization of N,N-dimethylacrylamide and the hydrophilic monomer, and can easily obtain a copolymer with a target molecular weight.

[0072] The sequencing gel of the present invention contains the matrix of the present invention. The two polymers in the matrix have good compatibility, good stability and good reproducibility. The preparation method is simple, the sequencing ability is good when sequencing base sequences, and the gel has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0073] Figure 1a The effect of isopropanol content on the molecular weight of polyacrylamide is demonstrated; the horizontal axis is the volume percentage of isopropanol to ultrapure water, and the vertical axis is the viscosity average molecular weight of polyacrylamide.

[0074] Figure 1b The effect of isopropanol content on the molecular weight of the copolymer of N,N-dimethylacrylamide and N-hydroxyethylacrylamide is shown; the horizontal axis is the volume percentage of isopropanol and ultrapure water, and the vertical axis is the viscosity average molecular weight of the copolymer.

[0075] Figure 2aThe hydrogen bond distribution between PAM and P(DMA-co-HEAA) in sequencing gel 1 is shown.

[0076] Figure 2b The distribution of hydrogen bonds between PAM and P(DMA-co-HEA) in sequencing gel 6 is shown.

[0077] Figure 2c The distribution of hydrogen bonds between PAM and P(DMA-co-HEMA) in sequencing gel 7 is shown.

[0078] Figure 2d The hydrogen bond distribution between PAM and P(DMA-co-NMA) in sequencing gel 8 is shown.

[0079] Figure 2e The hydrogen bond distribution between PAM and P(DMA-co-HEMAm) in sequencing gel 9 is shown.

[0080] Figure 3 The hydrogen bond distribution between PAM and PDMA in sequencing gel 1 is shown.

[0081] Figure 4a This is a graph showing the results of sequencing the 0-295 bp segment using sequencing gel 1.

[0082] Figure 4b This is the result of sequencing the 295-547 bp segment using sequencing gel 1.

[0083] Figure 4c This is the result of sequencing the 547-870 bp segment using sequencing gel 1. DETAILED DESCRIPTION

[0084] The following examples further illustrate the technology of the present invention. These examples are for illustration and example of the present invention and are not intended to limit the scope of the present invention in any form.

[0085] The polyacrylamide, poly N,N-dimethylacrylamide, poly N-hydroxyethylacrylamide, and copolymer of N,N-dimethylacrylamide and hydrophilic monomer used in the examples and comparative examples of the present invention were prepared in the following manner. All reagents used were purchased from Sigma-Aldrich; the dialysis bag was purchased from Biosharp and had a molecular weight cutoff of 14 kDa.

[0086] 1. Preparation of polyacrylamide (PAM)

[0087] Take 25g acrylamide and 2mL isopropanol in a three-necked flask, add 250mL ultrapure water (18.2MΩ·cm) and 35mg ammonium persulfate (APS), introduce nitrogen and stir for 30min at room temperature, add 40μL tetramethylethylenediamine (TEMED), and react for 6h at room temperature. The polymer solution obtained by the reaction is placed in a dialysis bag for dialysis, and the dialyzed polymer solution is freeze-dried to obtain a polymer solid. The viscosity average molecular weight of the polymer is measured by viscosity method to be 1000kDa.

[0088] In addition, referring to the above preparation method of PAM, the effect of the amount of isopropanol on the molecular weight of PAM was investigated by changing the amount of isopropanol. Figure 1a As shown, from Figure 1a It can be seen that the molecular weight of the polymer can be controlled by controlling the amount of isopropanol used.

[0089] 2. Preparation of Poly (N,N-dimethylacrylamide) (PDMA)

[0090] Take 15mL N,N-dimethylacrylamide and 2mL isopropanol in a three-necked flask, add 150mL ultrapure water (18.2MΩ·cm) and 15mg ammonium persulfate (APS), introduce nitrogen and stir for 30min at room temperature, add 20μL tetramethylethylenediamine (TEMED), and react for 3h at room temperature. The polymer solution obtained by the reaction is placed in a dialysis bag for dialysis, and the dialyzed polymer solution is freeze-dried to obtain a polymer solid. The viscosity average molecular weight of the polymer is measured by viscosity method to be 210kDa.

[0091] 3. Preparation of poly (N-hydroxyethyl acrylamide) (PHEAA)

[0092] 15 mL of N-hydroxyethyl acrylamide and 4 mL of isopropanol were placed in a three-necked flask, 150 mL of ultrapure water (18.2 MΩ·cm) and 15 mg of ammonium persulfate (APS) were added, nitrogen was introduced and stirred at room temperature for 30 min, 20 μL of tetramethylethylenediamine (TEMED) was added, and the reaction was carried out at room temperature for 3 h. The polymer solution obtained by the reaction was placed in a dialysis bag for dialysis, and the dialyzed polymer solution was freeze-dried to obtain a polymer solid, and the viscosity average molecular weight of the polymer was measured by the viscosity method to be 250 kDa.

[0093] 4. Preparation of copolymer P(DMA-co-HEAA) with a mass ratio of 7:3 between N,N-dimethylacrylamide and N-hydroxyethylacrylamide

[0094] Take 14g N,N-dimethylacrylamide and 6g N-hydroxyethylacrylamide in a three-necked flask, add 3mL isopropanol and 35mg ammonium persulfate (APS), add 250mL ultrapure water (18.2MΩ·cm), introduce nitrogen and stir for 30min at room temperature, add 40μL tetramethylethylenediamine (TEMED), and react at room temperature for 6h. The copolymer solution obtained by the reaction is placed in a dialysis bag for dialysis, and the dialyzed polymer solution is freeze-dried to obtain a copolymer solid. The viscosity average molecular weight of the copolymer is measured by viscosity method to be 150kDa.

[0095] In addition, referring to the preparation method of the above copolymer, the effect of the amount of isopropanol on the molecular weight of the copolymer was investigated by changing the amount of isopropanol. Figure 1b As shown, from Figure 1b It can be seen that the molecular weight of the copolymer can be controlled by controlling the amount of isopropanol used.

[0096] 5. Preparation of copolymer P(DMA-co-HEAA) with a mass ratio of 5:5 between N,N-dimethylacrylamide and N-hydroxyethylacrylamide

[0097] Take 10g N,N-dimethylacrylamide and 10g N-hydroxyethylacrylamide in a three-necked flask, add 5mL isopropanol and 35mg ammonium persulfate (APS), add 250mL ultrapure water (18.2MΩ·cm), introduce nitrogen and stir for 30min at room temperature, add 40μL tetramethylethylenediamine (TEMED), and react at room temperature for 6h. The copolymer solution obtained by the reaction is placed in a dialysis bag for dialysis, and the dialyzed polymer solution is freeze-dried to obtain a copolymer solid. The viscosity average molecular weight of the copolymer is measured by viscosity method to be 100kDa.

[0098] VI. Preparation of copolymer P(DMA-co-HEAA) with a mass ratio of 9:1 between N,N-dimethylacrylamide and N-hydroxyethylacrylamide

[0099] Take 18g N,N-dimethylacrylamide and 2g N-hydroxyethylacrylamide in a three-necked flask, add 2mL isopropanol and 35mg ammonium persulfate (APS), add 250mL ultrapure water (18.2MΩ·cm), introduce nitrogen and stir for 30min at room temperature, add 40μL tetramethylethylenediamine (TEMED), and react at room temperature for 6h. The copolymer solution obtained by the reaction is placed in a dialysis bag for dialysis, and the dialyzed polymer solution is freeze-dried to obtain a copolymer solid. The viscosity average molecular weight of the copolymer is measured by viscosity method to be 120kDa.

[0100] VII. Preparation of copolymer P(DMA-co-HEA) with a mass ratio of 7:3 of N,N-dimethylacrylamide and hydroxyethyl acrylate

[0101] Take 14g N,N-dimethylacrylamide and 6g hydroxyethyl acrylate in a three-necked flask, add 3mL isopropanol and 35mg ammonium persulfate (APS), add 250mL ultrapure water (18.2MΩ·cm), introduce nitrogen and stir for 30min at room temperature, add 40μL tetramethylethylenediamine (TEMED), and react at room temperature for 6h. The copolymer solution obtained by the reaction is placed in a dialysis bag for dialysis, and the dialyzed polymer solution is freeze-dried to obtain a copolymer solid. The viscosity average molecular weight of the copolymer is measured by viscosity method to be 150kDa.

[0102] 8. Preparation of copolymer P(DMA-co-HEMA) with a mass ratio of 7:3 between N,N-dimethylacrylamide and hydroxyethyl methacrylate

[0103] Take 14g N,N-dimethylacrylamide and 6g hydroxyethyl methacrylate in a three-necked flask, add 3mL isopropanol and 35mg ammonium persulfate (APS), add 250mL ultrapure water (18.2MΩ·cm), introduce nitrogen and stir for 30min at room temperature, add 40μL tetramethylethylenediamine (TEMED), and react at room temperature for 6h. The copolymer solution obtained by the reaction is placed in a dialysis bag for dialysis, and the dialyzed polymer solution is freeze-dried to obtain a copolymer solid. The viscosity average molecular weight of the copolymer is measured by viscosity method to be 150kDa.

[0104] IX. Preparation of copolymer P(DMA-co-NMA) with a mass ratio of 7:3 between N,N-dimethylacrylamide and N-hydroxymethylacrylamide

[0105] Take 14g N,N-dimethylacrylamide and 6g N-hydroxymethylacrylamide in a three-necked flask, add 3mL isopropanol and 35mg ammonium persulfate (APS), add 250mL ultrapure water (18.2MΩ·cm), introduce nitrogen and stir for 30min at room temperature, add 40μL tetramethylethylenediamine (TEMED), and react at room temperature for 6h. The copolymer solution obtained by the reaction is placed in a dialysis bag for dialysis, and the dialyzed polymer solution is freeze-dried to obtain a copolymer solid. The viscosity average molecular weight of the copolymer is measured by viscosity method to be 150kDa.

[0106] 10. Preparation of copolymer P(DMA-co-HEMAm) with a mass ratio of 7:3 of N,N-dimethylacrylamide and N-(2-hydroxyethyl)-2-methyl-2-acrylamide

[0107] Take 14g N,N-dimethylacrylamide and 6g N-(2-hydroxyethyl)-2-methyl-2-acrylamide in a three-necked flask, add 3mL isopropanol and 35mg ammonium persulfate (APS), add 250mL ultrapure water (18.2MΩ·cm), introduce nitrogen and stir for 30min at room temperature, add 40μL tetramethylethylenediamine (TEMED), and react at room temperature for 6h. The copolymer solution obtained by the reaction is placed in a dialysis bag for dialysis, and the dialyzed polymer solution is freeze-dried to obtain a copolymer solid. The viscosity average molecular weight of the copolymer is measured by viscosity method to be 150kDa.

[0108] Example 1

[0109] A matrix for microchannel screening is obtained by mixing PAM with a viscosity average molecular weight of 1000 kDa and a copolymer with a viscosity average molecular weight of 150 kDa and a mass ratio of N,N-dimethylacrylamide to N-hydroxyethylacrylamide of 7:3 at a mass ratio of 90:10.

[0110] Example 2

[0111] A matrix for microchannel screening is obtained by mixing PAM with a viscosity average molecular weight of 1000 kDa and a copolymer with a viscosity average molecular weight of 100 kDa and a mass ratio of 5:5 of N,N-dimethylacrylamide and N-hydroxyethylacrylamide at a mass ratio of 90:10.

[0112] Example 3

[0113] A matrix for microchannel screening is obtained by mixing PAM with a viscosity average molecular weight of 1000 kDa and a copolymer with a viscosity average molecular weight of 120 kDa and a mass ratio of N,N-dimethylacrylamide to N-hydroxyethylacrylamide of 9:1 at a mass ratio of 90:10.

[0114] Example 4

[0115] A method for preparing a sequencing gel for separating DNA and RNA, comprising adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.3 g of a copolymer with a viscosity average molecular weight of 150 kDa and a mass ratio of N,N-dimethylacrylamide to N-hydroxyethylacrylamide of 7:3, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, filtering the polymer solution with a 0.22 μm filter membrane, and obtaining a sequencing gel 1. The hydrogen bond distribution between PAM and P(DMA-co-HEAA) in the sequencing gel 1 is as follows: Figure 2a shown.

[0116] Example 5

[0117] A method for preparing a sequencing gel for separating DNA and RNA comprises the following steps: adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.3 g of a copolymer with a viscosity average molecular weight of 100 kDa and a mass ratio of N,N-dimethylacrylamide to N-hydroxyethylacrylamide of 5:5, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, and filtering the polymer solution with a 0.22 μm filter membrane to obtain a sequencing gel 2.

[0118] Example 6

[0119] A method for preparing a sequencing gel for separating DNA and RNA comprises the following steps: adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.3 g of a copolymer with a viscosity average molecular weight of 120 kDa and a mass ratio of N,N-dimethylacrylamide to N-hydroxyethylacrylamide of 9:1, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, and filtering the polymer solution with a 0.22 μm filter membrane to obtain sequencing gel 3.

[0120] Example 7

[0121] A method for preparing a sequencing gel for separating DNA and RNA comprises the following steps: adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.06 g of a copolymer with a viscosity average molecular weight of 150 kDa and a mass ratio of N,N-dimethylacrylamide to N-hydroxyethylacrylamide of 7:3, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, and filtering the polymer solution with a 0.22 μm filter membrane to obtain sequencing gel 4.

[0122] Example 8

[0123] A method for preparing a sequencing gel for separating DNA and RNA comprises the following steps: adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 3 g of a copolymer with a viscosity average molecular weight of 150 kDa and a mass ratio of N,N-dimethylacrylamide to N-hydroxyethylacrylamide of 7:3, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, and filtering the polymer solution with a 0.22 μm filter membrane to obtain sequencing gel 5.

[0124] Example 9

[0125] A method for preparing a sequencing gel for separating DNA and RNA, wherein 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.3 g of a copolymer with a viscosity average molecular weight of 150 kDa and a mass ratio of N,N-dimethylacrylamide to hydroxyethyl acrylate of 7:3, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water are added to a polytetrafluoroethylene beaker, and the polymer is slowly stirred to completely dissolve, and the polymer solution is filtered with a 0.22 μm filter membrane to obtain sequencing gel 6. The hydrogen bond distribution between PAM and P(DMA-co-HEA) in sequencing gel 6 is as follows: Figure 2b shown.

[0126] Example 10

[0127] A method for preparing a sequencing gel for separating DNA and RNA, comprising adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.3 g of a copolymer with a viscosity average molecular weight of 150 kDa and a mass ratio of N,N-dimethylacrylamide to hydroxyethyl methacrylate of 7:3, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, filtering the polymer solution with a 0.22 μm filter membrane, and obtaining sequencing gel 7. The hydrogen bond distribution between PAM and P(DMA-co-HEMA) in sequencing gel 7 is as follows: Figure 2c shown.

[0128] Embodiment 11

[0129] A method for preparing a sequencing gel for separating DNA and RNA, comprising adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.3 g of a copolymer with a viscosity average molecular weight of 150 kDa and a mass ratio of N,N-dimethylacrylamide to N-hydroxymethylacrylamide of 7:3, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, filtering the polymer solution with a 0.22 μm filter membrane, and obtaining sequencing gel 8. The hydrogen bond distribution between PAM and P(DMA-co-NMA) in sequencing gel 8 is as follows: Figure 2d shown.

[0130] Example 12

[0131] A method for preparing a sequencing gel for separating DNA and RNA, comprising adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.3 g of a copolymer with a viscosity average molecular weight of 150 kDa and a mass ratio of N,N-dimethylacrylamide to N-(2-hydroxyethyl)-2-methyl-2-acrylamide of 7:3, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, filtering the polymer solution with a 0.22 μm filter membrane, and obtaining sequencing gel 9. The hydrogen bond distribution between PAM and P(DMA-co-HEMAm) in sequencing gel 9 is as follows: Figure 2e shown.

[0132] Comparative Example 1

[0133] A method for preparing a sequencing gel for separating DNA and RNA, comprising adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.3 g of PDMA with a viscosity average molecular weight of 210 kDa, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, and filtering the polymer solution with a 0.22 μm filter membrane to obtain a comparative sequencing gel 1. The hydrogen bond distribution between PAM and PDMA in the comparative sequencing gel 1 is as follows: Figure 3 shown.

[0134] Comparative Example 2

[0135] A method for preparing a sequencing gel for separating DNA and RNA comprises the following steps: adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.3 g of PHEAA with a viscosity average molecular weight of 250 kDa, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, and filtering the polymer solution with a 0.22 μm filter membrane to obtain a comparative sequencing gel 2.

[0136] Comparative Example 3

[0137] A method for preparing a sequencing gel for separating DNA and RNA comprises the following steps: adding 3 g of PAM with a viscosity average molecular weight of 1000 kDa, 0.21 g of PDMA with a viscosity average molecular weight of 210 kDa, 0.09 g of PHEAA with a viscosity average molecular weight of 250 kDa, 48 g of urea, 10 mL of 1M TTE buffer and 45 mL of water into a polytetrafluoroethylene beaker, slowly stirring to completely dissolve the polymer, filtering the polymer solution with a 0.22 μm filter membrane, and obtaining a comparative sequencing gel 3.

[0138] Test Case

[0139] DNA sequencing was performed using the PAM with a viscosity average molecular weight of 1000 kDa, PDMA with a viscosity average molecular weight of 210 kDa, PHEAA with a viscosity average molecular weight of 250 kDa, comparative sequencing gel 1, comparative sequencing gel 2, comparative sequencing gel 3, and sequencing gels 1 to 6 prepared above as sequencing gels. The sequencing instrument was an ABI 3730XL genetic analyzer, and the standard product was Z-BigDyeV3; sequencing conditions: temperature 60°C, injection voltage 1.5 kV, injection time 25 s, operating voltage 7.3 kV, and operating time 170 min.

[0140] Use warm water to clean the gel pump and capillary, pump the sequencing gel into the sequencer according to the operating procedures, and after spatial positioning, inject the standard into the capillary for sequencing. The sequencing results are shown in Table 1. The sequencing results of sequencing gel 1 are shown in Table 1. Figure 4a -c.

[0141] Table 1 Sequencing lengths of different sequencing gels

[0142]

[0143]

[0144] As can be seen from Table 1, when PAM is used alone to prepare sequencing gel, the sequencing length can only reach 631bp due to the poor self-coating performance of PAM on the capillary. When PDMA or PHEAA is used alone to prepare sequencing gel, the sequencing capacity at the same concentration is poor, and can only reach 320bp or 317bp. When a blend of PAM and any one of PDMA, PHEAA, P(DMA-co-HEAA), or P(DMA-co-HEA) is used to prepare sequencing gel, the sequencing capacity is improved, and at the same blending mass ratio, the use of a blend of PAM and P(DMA-co-HEAA) or P(DMA-co-HEA) is more improved than the use of a blend of PAM and PDMA or PHEAA. When a blend of three polymers, PAM, PDMA, and PHEAA, is used to prepare sequencing gel, the sequencing capacity cannot be improved compared to using PAM alone to prepare sequencing gel.

[0145] In addition, from Figure 2a -e, Figure 3 It can be seen from the comparison that there are more hydrogen bonds between the two polymers in the sequencing gel of the present invention, the compatibility in the sequencing gel is better, and it is more conducive to the sequencing gel to remain stable for a long time.

[0146] In summary, the good compatibility of the polymers in the sequencing gel of the present invention is beneficial to improving the stability of the sequencing gel, and the sequencing gel of the present invention has better sequencing ability than the existing sequencing gel.

[0147] It should be noted that the embodiments described above are only used to explain the present invention and do not constitute any limitation of the present invention. The present invention has been described with reference to the embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than restrictive words. The present invention may be modified as specified within the scope of the claims of the present invention, and the present invention may be revised without departing from the scope and spirit of the present invention. Although the present invention described therein relates to specific methods, materials and embodiments, it does not mean that the present invention is limited to the specific examples disclosed therein, on the contrary, the present invention can be extended to all other methods and applications with the same functions.

Claims

1. A matrix for microchannel screening, characterized in that: The matrix includes a copolymer of N,N-dimethylacrylamide and a hydrophilic monomer, and polyacrylamide; wherein the hydrophilic monomer is selected from any one or more of N-hydroxyethylacrylamide, hydroxyethyl acrylate, hydroxyethyl methacrylate, N-hydroxymethylacrylamide, and N-(2-hydroxyethyl)-2-methyl-2-acrylamide.

2. The matrix according to claim 1, characterized in that The copolymerization mass ratio of N,N-dimethylacrylamide to the hydrophilic monomer in the copolymer of N,N-dimethylacrylamide and the hydrophilic monomer is 9:1 to 1:9, preferably 9:1 to 5:5, and more preferably 8:2 to 6:

4.

3. The matrix according to claim 1 or 2, characterized in that The mass ratio of the copolymer of N,N-dimethylacrylamide and hydrophilic monomer to polyacrylamide in the matrix is ​​50:50 to 99:1, preferably 95:5 to 85:15; and / or, the viscosity average molecular weight of the copolymer of N,N-dimethylacrylamide and a hydrophilic monomer is between 50 kDa and 300 kDa, preferably between 80 kDa and 250 kDa, and more preferably between 100 kDa and 200 kDa; And / or, the viscosity average molecular weight of the polyacrylamide is between 600 kDa and 2000 kDa, preferably between 800 kDa and 1600 kDa, and more preferably between 800 kDa and 1200 kDa.

4. The matrix according to any one of claims 1 to 3, characterized in that The copolymer of N,N-dimethylacrylamide and a hydrophilic monomer is prepared by redox free radical polymerization of N,N-dimethylacrylamide and the hydrophilic monomer; preferably, the molecular weight of the copolymer is controlled by a chain transfer agent during the polymerization process; more preferably, the chain transfer agent is selected from any one or more of isopropanol, sodium bisulfite, thioglycolic acid, sodium formate and sodium hypophosphite.

5. The matrix according to any one of claims 1 to 4, characterized in that The copolymer of N,N-dimethylacrylamide and a hydrophilic monomer is prepared by a method comprising the following steps: (1) N,N-dimethylacrylamide is mixed with a hydrophilic monomer, and then a chain transfer agent, ammonium persulfate and water are added, and stirred for 20 to 40 minutes under the protection of an inert gas; (2) adding tetramethylethylenediamine to the mixed solution obtained in step (1) and reacting for 2 to 8 hours; (3) dialyzing the reaction solution obtained in step (2) using a dialysis bag with a molecular weight cutoff of 14 kDa, and drying the dialyzed reaction solution to obtain a copolymer; Preferably, in step (1), the mass ratio of the chain transfer agent, ammonium persulfate, water, N,N-dimethylacrylamide, and hydrophilic monomer is (1-6):(0.015-0.035):(150-250):(1-9):(1-9); And / or, in step (1), the inert gas is nitrogen; And / or, in the step (2), the amount of tetramethylethylenediamine added is such that the ratio of ammonium persulfate to tetramethylethylenediamine is (15-35 mg):(20-45 μL); And / or, in step (2), the drying method includes any one or both of freeze drying and vacuum drying.

6. Use of the matrix according to any one of claims 1 to 5 in screening biomacromolecules using capillary electrophoresis; preferably, the biomacromolecules include two or more biomacromolecules with the same charge per unit mass; more preferably, the biomacromolecules include DNA and RNA.

7. A sequencing gel for separating DNA and RNA, characterized in that: The sequencing gel comprises the matrix described in any one of claims 1 to 5; preferably, the concentration of the matrix described in any one of claims 1 to 5 in the sequencing gel is 2% to 6% (w / v), preferably 2% to 4% (w / v); more preferably, the kinematic viscosity of the sequencing gel is 50 to 500 cP, preferably 200 to 400 cP.

8. The sequencing gel according to claim 7, characterized in that: The sequencing gel further comprises a buffer and a denaturant; preferably, The buffer is selected from any one or more of tris(hydroxymethyl)aminomethane-acetate-EDTA buffer, tris(hydroxymethyl)methylaminopropanesulfonate sodium buffer, and tris(hydroxymethyl)aminomethane-tris(hydroxymethyl)methylaminopropanesulfonate sodium-EDTA buffer, preferably tris(hydroxymethyl)aminomethane-tris(hydroxymethyl)methylaminopropanesulfonate sodium-EDTA buffer; and / or, the concentration of the buffer in the sequencing gel is 50 to 150 mM, preferably 80 to 120 mM; and / or, the denaturant is selected from any one or more of urea, 1,3-dimethylurea, dimethyl sulfoxide, formamide and 2-pyrrolidone, preferably urea; And / or, the concentration of the denaturant in the sequencing gel is 5-8M, preferably 6-7M.

9. The method for preparing a sequencing gel according to claim 7 or 8, characterized in that: The preparation method comprises dissolving the matrix according to any one of claims 1 to 5 in a solvent; preferably, the method comprises mixing and dissolving a buffer, a denaturant and the matrix according to any one of claims 1 to 5 in a solvent; more preferably, the solvent is water.

10. Use of the sequencing gel according to claim 7 or 8 or the sequencing gel prepared by the preparation method according to claim 9 in sequencing base sequences.

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