Production and preparation method of gel capable of remarkably improving capillary electrophoresis resolution

Abstract

The invention discloses a production and preparation method of gel capable of remarkably improving capillary electrophoresis resolution, the gel comprises a plurality of linear polymers, the plurality of linear polymers are mixed according to a certain proportion and then mixed with other reagents to prepare a polymer for gene capillary electrophoresis, and the other reagents comprise urea, an electrophoresis buffer solution and water; and the plurality of linear polymers are linear polyacrylamide, polyethylene glycol and hydroxyethyl cellulose.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gel production and preparation methods for significantly improving capillary electrophoresis resolution, in particular to a gel production and preparation method for significantly improving capillary electrophoresis resolution. BACKGROUND

[0002] At present, linear polymers play a key role in capillary electrophoresis (CE) of gene sequencing as a key screening medium. Its core function is to separate different lengths of DNA fragments through intermolecular interaction, thereby supporting the efficient operation of Sanger sequencing and other technologies. In the prior art, a single type of linear polymer separation gel is mostly used, and the separation capacity is weak, which reduces the efficiency. SUMMARY

[0003] In order to solve the technical problems existing in the prior art, the purpose of the present application is to provide a gel production and preparation method for significantly improving capillary electrophoresis resolution, which comprises several linear polymers. The several linear polymers are mixed according to a certain proportion and mixed with other reagents to prepare a polymer for gene capillary electrophoresis. The other reagents include urea, electrophoresis buffer and water. The several linear polymers are linear polyacrylamide, polyethylene glycol and hydroxyethyl cellulose.

[0004] Further, the synthesis of linear polyacrylamide is mainly realized by free radical polymerization of acrylamide monomers. The key is to avoid crosslinking between molecular chains to ensure that the product is linear structure. Linear polyacrylamide is generated by free radical homopolymerization of acrylamide monomers, and the reaction formula is as follows: ; The synthesis steps of linear polyacrylamide are as follows: Raw material preparation: acrylamide monomer (purity ≥ 99%), deionized water, free radical initiator (such as ammonium persulfate (APS), azobisisobutyronitrile (AIBN) or potassium persulfate), chain transfer agent (such as mercaptoethanol, used to control molecular weight); Reaction system configuration: add deionized water to a three-necked flask, and add acrylamide monomer under stirring to prepare a 5%-30% (mass fraction) aqueous solution; introduce nitrogen for 10-30 minutes to remove oxygen (to avoid inhibition of free radical polymerization by oxygen); Initiation of polymerization: add initiator (0.1%-1% of monomer mass), and if APS is used, a small amount of tetramethyl ethylenediamine (TEMED) is used as a catalyst (low-temperature initiation); control the reaction temperature (20-60℃, depending on the type of initiator), and stir for 2-24 hours until the solution viscosity increases significantly; Termination and purification: after the reaction is completed, a small amount of polymerization inhibitor (such as hydroquinone) is added to terminate the reaction; the product is precipitated into a large amount of acetone, filtered and vacuum dried to obtain white solid linear polyacrylamide.

[0005] Further, polyethylene glycol is a linear polymer generated by ring-opening polymerization of ethylene oxide (EO), and its general structure is HO-(CH2CH2O) n -H, the core of synthesis is to control the ring-opening polymerization process of ethylene oxide and the molecular weight distribution, the synthesis of polyethylene glycol is based on the cationic or anionic ring-opening polymerization of ethylene oxide, and the reaction formula is as follows: ; In the reaction, ethylene oxide (three-membered ring structure, large tension) undergoes ring-opening under the action of initiator, and the molecular chain is grown by repeated insertion of -CH2CH2O- units, and finally forms a linear polymer with hydroxyl groups at both ends; Initiation system: alcohol (such as methanol, ethylene glycol) as starter, alkali metal hydroxide (such as KOH, NaOH) or alkoxylate (such as NaOCH3) as catalyst, forming active alkoxide anion (RO⁻); Polymerization reaction: in a high-pressure reactor, the starter and catalyst are mixed, and ethylene oxide gas (excess) is introduced, and the reaction temperature (80-150℃) and pressure (0.5-3 MPa) are controlled; the active anion attacks the ring tension site of ethylene oxide, initiates ring-opening and continues to grow the chain segment; Termination reaction: when the target molecular weight is reached, the reaction is terminated by reducing the temperature and pressure, or adding an acidic substance (such as acetic acid) to neutralize the catalyst and deactivate the active chain end.

[0006] Post-processing: remove unreacted ethylene oxide by distillation, decolorize and filter to obtain polyethylene glycol (liquid or solid) of different molecular weights.

[0007] Further, hydroxyethyl cellulose is a water-soluble cellulose derivative obtained by etherification of cellulose, and the core of its synthesis is to activate cellulose by alkalization, and then introduce hydroxyethyl (-CH2CH2OH) groups by etherification with ethylene oxide (EO); the following is the main synthesis method and key steps: The synthesis of hydroxyethyl cellulose consists of two steps of alkalization and etherification: Alkalization reaction: cellulose generates sodium cellulose salt (activated intermediate) under alkaline conditions, and the reaction formula is: ; Etherification reaction: the activated sodium cellulose salt undergoes nucleophilic substitution reaction with ethylene oxide to introduce hydroxyethyl groups, and the reaction formula is: ; By controlling the reaction conditions, the degree of substitution and the degree of polymerization of hydroxyethyl group can be adjusted, thus changing the water solubility and viscosity of hydroxyethyl cellulose.

[0008] Further, the obtained linear polymer is mixed in a certain proportion and mixed with other reagents to prepare a polymer for capillary electrophoresis of genes, as follows: Linear polymer (mixture): 4 g; Urea: 40 g; Electrophoresis buffer: 10 g; Add water to 100 g; Stir overnight, filter with 0.22 um membrane; The electrophoresis buffer is TE buffer (tris 100 mM, EDTA 10 mM, pH 8.0); DETAILED DESCRIPTION

[0009] Three linear polymer contents: linear polyacrylamide 2 g, polyethylene glycol 1 g, hydroxyethyl cellulose 1 g, three mixed into linear polymer (mixture): 4 g; Mix with urea, electrophoresis buffer and water, filter to obtain capillary filling medium for DNA separation; DETAILED DESCRIPTION

[0010] Three linear polymer contents: linear polyacrylamide 3 g, polyethylene glycol 0.5 g, hydroxyethyl cellulose 0.5 g, three mixed into linear polymer (mixture): 4 g; Mix with urea, electrophoresis buffer and water, filter to obtain capillary filling medium for DNA separation; DETAILED DESCRIPTION

[0011] Three linear polymer contents: linear polyacrylamide 3 g, polyethylene glycol 1 g, three mixed into linear polymer (mixture): 4 g; Mix with urea, electrophoresis buffer and water, filter to obtain capillary filling medium for DNA separation; DETAILED DESCRIPTION

[0012] Three linear polymer contents: linear polyacrylamide 3 g, hydroxyethyl cellulose 1 g, three mixed into linear polymer (mixture): 4 g; Mix with urea, electrophoresis buffer and water, filter to obtain capillary filling medium for DNA separation; DETAILED DESCRIPTION

[0013] Three linear polymer contents: linear polyacrylamide 4 g, mix with urea, electrophoresis buffer and water, filter to obtain capillary filling medium for DNA separation.

[0014] The beneficial effects of the present application compared to the prior art are that the mixing of multiple linear polymers can significantly improve the separation capacity of the gel. Example 1:

[0015] A production and preparation method of a gel for significantly improving the resolution of capillary electrophoresis, comprising mixing several linear polymers, mixing the several linear polymers according to a certain proportion with other reagents to prepare a polymer for gene capillary electrophoresis, the other reagents including urea, an electrophoresis buffer, and water; the several linear polymers are linear polyacrylamide, polyethylene glycol, and hydroxyethyl cellulose.

[0016] Further, the synthesis of linear polyacrylamide is mainly realized by radical polymerization of acrylamide monomers, the key is to avoid crosslinking between molecular chains to ensure that the product is linear structure. Linear polyacrylamide is generated by radical homopolymerization of acrylamide monomers, and the reaction formula is as follows: ; The synthesis steps of linear polyacrylamide are as follows: Raw material preparation: acrylamide monomer (purity ≥ 99%), deionized water, free radical initiator (such as ammonium persulfate (APS), azobisisobutyronitrile (AIBN) or potassium persulfate), chain transfer agent (such as mercaptoethanol, used to control molecular weight); Reaction system configuration: add deionized water to a three-necked flask, add acrylamide monomer under stirring, and prepare a 5%-30% (mass fraction) aqueous solution; introduce nitrogen for 10-30 minutes to remove oxygen (to avoid inhibition of free radical polymerization by oxygen); Initiation of polymerization: add initiator (0.1%-1% of the mass of monomers), if APS is used, a small amount of tetramethyl ethylenediamine (TEMED) is used as a catalyst (low-temperature initiation); control the reaction temperature (20-60°C, depending on the type of initiator), and stir for 2-24 hours until the viscosity of the solution increases significantly; Termination and purification: after the reaction is completed, a small amount of polymerization inhibitor (such as hydroquinone) is added to terminate the reaction; pour the product into a large amount of acetone to precipitate, filter and vacuum dry to obtain white solid linear polyacrylamide.

[0017] Further, polyethylene glycol is a linear polymer generated by ring-opening polymerization of ethylene oxide (EO), and its general structure is HO-(CH2CH2O) n -H, the core of the synthesis is to control the ring-opening polymerization process and molecular weight distribution of ethylene oxide, and the synthesis of polyethylene glycol is based on cationic or anionic ring-opening polymerization of ethylene oxide, and the reaction formula is as follows: ; In the reaction, ethylene oxide (three-membered ring structure, large tension) undergoes ring-opening under the action of initiator, and the molecular chain is continuously grown by repeated insertion of -CH2CH2O- units, and finally a linear polymer with hydroxyl groups at both ends is formed; Initiation system: alcohol (such as methanol, ethylene glycol) as starter, alkali metal hydroxide (such as KOH, NaOH) or alkoxylate (such as NaOCH3) as catalyst, forming active alkoxide anion (RO⁻); Polymerization reaction: in a high-pressure reaction kettle, the starter and catalyst are mixed, and ethylene oxide gas (excess) is introduced, and the reaction temperature (80-150℃) and pressure (0.5-3 MPa) are controlled; the active anion attacks the ring tension site of ethylene oxide, initiates ring-opening and continuously grows the chain segment; Termination reaction: when the target molecular weight is reached, the reaction is terminated by reducing the temperature and pressure, or adding acidic substances (such as acetic acid) to neutralize the catalyst and deactivate the active chain end.

[0018] Post-processing: remove unreacted ethylene oxide by distillation, and obtain polyethylene glycol (liquid or solid) of different molecular weights by decolorization and filtration.

[0019] Further, hydroxyethyl cellulose is a water-soluble cellulose derivative obtained by etherification of cellulose. The synthesis core is to activate cellulose by alkalization, and then introduce hydroxyethyl (-CH2CH2OH) groups by etherification with ethylene oxide (EO). The following are the main synthesis methods and key steps: The synthesis of hydroxyethyl cellulose consists of two steps of alkalization and etherification: Alkalization reaction: cellulose generates sodium cellulose salt (activated intermediate) under alkaline conditions, and the reaction formula is: ; Etherification reaction: the activated sodium cellulose salt undergoes nucleophilic substitution reaction with ethylene oxide to introduce hydroxyethyl groups, and the reaction formula is: ; By controlling the reaction conditions, the degree of substitution of hydroxyethyl and the degree of polymerization can be adjusted, so as to change the water solubility and viscosity of hydroxyethyl cellulose.

[0020] Further, the obtained linear polymer is mixed according to a certain proportion and mixed with other reagents to prepare a polymer for capillary electrophoresis of genes, as follows: Linear polymer (mixture): 4g; Urea: 40g; Electrophoresis buffer: 10g; Add water to 100g; Stir overnight and filter through a 0.22um filter membrane; The electrophoresis buffer is TE buffer (tris 100mM, EDTA 10mM, PH 8.0); DETAILED DESCRIPTION

[0021] The content of the three linear polymers is: 2g of linear polyacrylamide, 1g of polyethylene glycol, and 1g of hydroxyethyl cellulose, which are mixed into a linear polymer (mixture) of 4g; The mixture is stirred with urea, electrophoresis buffer and water, and filtered to obtain a capillary filling medium for DNA separation; DETAILED DESCRIPTION

[0022] The content of the three linear polymers is: 3g of linear polyacrylamide, 0.5g of polyethylene glycol, and 0.5g of hydroxyethyl cellulose, which are mixed into a linear polymer (mixture) of 4g; The mixture is stirred with urea, electrophoresis buffer and water, and filtered to obtain a capillary filling medium for DNA separation; DETAILED DESCRIPTION

[0023] The content of the three linear polymers is: 3g of linear polyacrylamide, 1g of polyethylene glycol, and 1g of hydroxyethyl cellulose, which are mixed into a linear polymer (mixture) of 4g; The mixture is stirred with urea, electrophoresis buffer and water, and filtered to obtain a capillary filling medium for DNA separation; DETAILED DESCRIPTION

[0024] The content of the three linear polymers is: 3g of linear polyacrylamide, 1g of polyethylene glycol, and 1g of hydroxyethyl cellulose, which are mixed into a linear polymer (mixture) of 4g; The mixture is stirred with urea, electrophoresis buffer and water, and filtered to obtain a capillary filling medium for DNA separation; DETAILED DESCRIPTION

[0025] The content of the three linear polymers is: 3g of linear polyacrylamide, 1g of polyethylene glycol, and 1g of hydroxyethyl cellulose, which are mixed into a linear polymer (mixture) of 4g;

[0026] The above embodiments are only preferred embodiments of the present application, and cannot be used to limit the scope of protection of the present application. Any non-essential changes and substitutions made by those skilled in the art based on the present application are within the scope of the present application.

Claims

1. A method for producing and preparing a gel that significantly improves the resolution of capillary electrophoresis, characterized in that: It includes several linear polymers, which are mixed in a certain proportion and then mixed with other reagents to prepare a polymer for gene capillary electrophoresis. Other reagents include urea, electrophoresis buffer, and water; the linear polymers are linear polyacrylamide, polyethylene glycol, and hydroxyethyl cellulose.

2. The method for producing and preparing a gel that significantly improves the resolution of capillary electrophoresis according to claim 1, characterized in that, The synthesis of linear polyacrylamide is mainly achieved through the free radical polymerization of acrylamide monomers. The key is to avoid cross-linking between molecular chains to ensure that the product has a linear structure. Linear polyacrylamide is generated from acrylamide monomers through a free radical homopolymerization reaction, as shown in the following reaction formula: ； The synthesis steps of linear polyacrylamide are as follows: Raw material preparation: Acrylamide monomer (purity ≥99%), deionized water, free radical initiator (such as ammonium persulfate (APS), azobisisobutyronitrile (AIBN) or potassium persulfate), chain transfer agent (such as mercaptoethanol, used to control molecular weight); Reaction system preparation: Add deionized water to a three-necked flask, add acrylamide monomer while stirring, and prepare a 5%-30% (mass fraction) aqueous solution; purge with nitrogen for 10-30 minutes to remove oxygen (to avoid oxygen inhibiting free radical polymerization). Initiate polymerization: Add an initiator (0.1%-1% of the monomer mass). If using APS, a small amount of tetramethylethylenediamine (TEMED) is required as a catalyst (low-temperature initiation). Control the reaction temperature (20-60℃, depending on the type of initiator) and stir the reaction for 2-24 hours until the solution viscosity increases significantly. Termination and purification: After the reaction is completed, a small amount of polymerization inhibitor (such as hydroquinone) is added to terminate the reaction; the product is poured into a large amount of acetone to precipitate, filtered, and then vacuum dried to obtain a white solid linear polyacrylamide.

3. The method for producing and preparing a gel that significantly improves capillary electrophoresis resolution according to claim 2, characterized in that, Polyethylene glycol is a linear polymer produced by the ring-opening polymerization of ethylene oxide (EO), with the general structural formula HO-(CH2CH2O). n -H, the core of the synthesis is controlling the ring-opening polymerization process and molecular weight distribution of ethylene oxide. The synthesis of polyethylene glycol is based on the cationic or anionic ring-opening polymerization of ethylene oxide, and the reaction formula is as follows: ； In the reaction, ethylene oxide (a three-membered ring structure with high strain) undergoes ring opening under the action of an initiator, and the molecular chain grows by repeatedly inserting -CH2CH2O- units, eventually forming a linear polymer with hydroxyl groups at both ends; Initiation system: Using alcohols (such as methanol and ethylene glycol) as initiators and alkali metal hydroxides (such as KOH and NaOH) or alkoxy compounds (such as NaOCH3) as catalysts, active alkoxide anions (RO⁻) are formed. Polymerization reaction: In a high-pressure reactor, the initiator and catalyst are mixed, and ethylene oxide gas (in excess) is introduced. The reaction temperature (80-150℃) and pressure (0.5-3 MPa) are controlled. Active negative ions attack the ring strain sites of ethylene oxide, initiating ring opening and continuous chain segment growth. Termination of reaction: Once the target molecular weight is reached, the reaction is terminated by cooling or depressurizing, or by adding an acidic substance (such as acetic acid) to neutralize the catalyst and deactivate the active chain ends. Post-processing: Unreacted ethylene oxide is removed by distillation, and polyethylene glycol (liquid or solid) of different molecular weights is obtained by decolorization and filtration.

4. The method for producing and preparing a gel that significantly improves the resolution of capillary electrophoresis according to claim 3, characterized in that: Hydroxyethyl cellulose is a water-soluble cellulose derivative obtained by etherification of cellulose. Its synthesis primarily involves activating cellulose through alkalization followed by etherification with ethylene oxide (EO) to introduce a hydroxyethyl (-CH2CH2OH) group. The main synthesis methods and key steps are as follows: The synthesis of hydroxyethyl cellulose involves two steps: alkalization and etherification. Alkalinization reaction: Cellulose reacts under alkaline conditions to form sodium cellulose (an activation intermediate). The reaction equation is as follows: ; Etherification reaction: Activated sodium cellulose undergoes a nucleophilic substitution reaction with ethylene oxide, introducing a hydroxyethyl group through ring-opening. The reaction formula is as follows: ； By controlling the reaction conditions, the degree of substitution and polymerization of hydroxyethyl can be adjusted, thereby changing the water solubility and viscosity of hydroxyethyl cellulose.

5. The method for producing and preparing a gel that significantly improves the resolution of capillary electrophoresis according to claim 4, characterized in that: The obtained linear polymer was mixed in a certain proportion and then combined with other reagents to prepare a polymer for gene capillary electrophoresis, as follows: Linear polymer (mixture): 4g; Urea: 40g; Electrophoresis buffer: 10g; Replenish moisture to 100g; Stir overnight, then filter through a 0.22µm membrane. The electrophoresis buffer was TE buffer (tris 100mM, EDTA 10mM, pH 8.0). Specific implementation method 1: Contents of three linear polymers: linear polyacrylamide 2g, polyethylene glycol 1g, hydroxyethyl cellulose 1g, the three mixed into a linear polymer (mixture): 4g; The mixture was stirred with urea, electrophoresis buffer and water, and then filtered to obtain a capillary packing medium for DNA separation. Specific implementation method 2: Contents of the three linear polymers: linear polyacrylamide 3g, polyethylene glycol 0.5g, hydroxyethyl cellulose 0.5g, and the three mixed into a linear polymer (mixture): 4g; The mixture was stirred with urea, electrophoresis buffer and water, and then filtered to obtain a capillary packing medium for DNA separation. Specific implementation method 3: Contents of the three linear polymers: linear polyacrylamide 3g, polyethylene glycol 1g, and the three mixed into a linear polymer (mixture): 4g; The mixture was stirred with urea, electrophoresis buffer and water, and then filtered to obtain a capillary packing medium for DNA separation. Specific implementation method 4: Content of the three linear polymers: linear polyacrylamide 3g, hydroxyethyl cellulose 1g, and the three mixed into a linear polymer (mixture): 4g; The mixture was stirred with urea, electrophoresis buffer and water, and then filtered to obtain a capillary packing medium for DNA separation. Specific implementation method 5: The three linear polymer contents were as follows: 4g of linear polyacrylamide was mixed with urea, electrophoresis buffer and water, stirred and filtered to obtain capillary filling medium for DNA separation.

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