CN117165578A - Nucleic acid purification process - Google Patents
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- 238000000034 method Methods 0.000 title claims abstract description 26
- 230000008569 process Effects 0.000 title claims abstract description 21
- 238000001821 nucleic acid purification Methods 0.000 title claims abstract description 14
- 230000005291 magnetic effect Effects 0.000 claims abstract description 54
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 35
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 35
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 35
- 239000011324 bead Substances 0.000 claims abstract description 26
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000004005 microsphere Substances 0.000 claims abstract description 24
- 238000005406 washing Methods 0.000 claims abstract description 19
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 18
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- 230000009471 action Effects 0.000 claims abstract description 9
- 238000001914 filtration Methods 0.000 claims abstract description 9
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- 235000011114 ammonium hydroxide Nutrition 0.000 claims abstract description 8
- 229920001046 Nanocellulose Polymers 0.000 claims abstract description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 6
- 239000004202 carbamide Substances 0.000 claims abstract description 6
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims abstract description 6
- 239000003480 eluent Substances 0.000 claims abstract description 5
- 239000006166 lysate Substances 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims abstract description 4
- 238000001132 ultrasonic dispersion Methods 0.000 claims abstract description 4
- 239000012535 impurity Substances 0.000 claims abstract description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract 2
- 229920000734 polysilsesquioxane polymer Polymers 0.000 claims description 14
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 10
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims description 10
- 239000012153 distilled water Substances 0.000 claims description 10
- -1 iron ion salts Chemical class 0.000 claims description 7
- 229910052742 iron Inorganic materials 0.000 claims description 6
- SMZOUWXMTYCWNB-UHFFFAOYSA-N 2-(2-methoxy-5-methylphenyl)ethanamine Chemical compound COC1=CC=C(C)C=C1CCN SMZOUWXMTYCWNB-UHFFFAOYSA-N 0.000 claims description 5
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims description 5
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 5
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 5
- 238000007885 magnetic separation Methods 0.000 claims description 4
- 238000009210 therapy by ultrasound Methods 0.000 claims description 4
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 claims description 3
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 2
- WBHQBSYUUJJSRZ-UHFFFAOYSA-M sodium bisulfate Chemical compound [Na+].OS([O-])(=O)=O WBHQBSYUUJJSRZ-UHFFFAOYSA-M 0.000 claims description 2
- 229910000342 sodium bisulfate Inorganic materials 0.000 claims description 2
- 229910001429 cobalt ion Inorganic materials 0.000 claims 1
- XLJKHNWPARRRJB-UHFFFAOYSA-N cobalt(2+) Chemical compound [Co+2] XLJKHNWPARRRJB-UHFFFAOYSA-N 0.000 claims 1
- 150000002500 ions Chemical class 0.000 claims 1
- 229910001453 nickel ion Inorganic materials 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 239000004289 sodium hydrogen sulphite Substances 0.000 claims 1
- 238000000746 purification Methods 0.000 abstract description 4
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- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000002776 aggregation Effects 0.000 description 3
- 238000004220 aggregation Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
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- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 2
- 239000004810 polytetrafluoroethylene Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 229910001030 Iron–nickel alloy Inorganic materials 0.000 description 1
- 241000218378 Magnolia Species 0.000 description 1
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 description 1
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- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 239000011258 core-shell material Substances 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 230000026058 directional locomotion Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 239000012149 elution buffer Substances 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- ZJYYHGLJYGJLLN-UHFFFAOYSA-N guanidinium thiocyanate Chemical compound SC#N.NC(N)=N ZJYYHGLJYGJLLN-UHFFFAOYSA-N 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
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- 238000005259 measurement Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000002102 nanobead Substances 0.000 description 1
- 239000002121 nanofiber Substances 0.000 description 1
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- 229910052759 nickel Inorganic materials 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
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- 239000000047 product Substances 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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Abstract
The invention discloses a nucleic acid purification process, which belongs to the technical field of nucleic acid purification, and comprises the following steps: adding urea, cetyltrimethylammonium chloride and carboxylated POSS into acetic acid solution, stirring for 20min, adding nano cellulose, shaking uniformly, drying, washing, and removing impurities to obtain gel solution; adding the nano magnetic microspheres into absolute ethyl alcohol, performing ultrasonic dispersion, adding ammonia water and the gel solution, stirring for 6 hours, washing and drying for 12 hours to obtain magnetic beads; placing a nucleic acid sample in an EP tube, adding a lysate, standing, adding a magnetic bead reagent, uniformly mixing, and standing; secondly, under the action of a magnetic field, fixing magnetic beads, filtering and washing; thirdly, adding eluent, standing, fixing magnetic beads, and filtering to obtain purified nucleic acid. The nucleic acid purification process is mild in operation, simple in purification procedure, high in nucleic acid purity and simple and rapid to operate and elute.
Description
Technical Field
The invention belongs to the technical field of nucleic acid purification, and particularly relates to a nucleic acid purification process.
Background
Nucleic acid is a carrier of genetic information, is the most important bioinformatic molecule, and is the main object of molecular biology research, so that purification of nucleic acid is the most important and fundamental technology in molecular biology experimental technology. At present, the magnetic bead method nucleic acid purification technology is most widely applied,Fe 3 0 4 the nano particles not only have the characteristics of high specific surface area and good conductivity of the nano material, but also have the advantages of the magnetic nano material and have good adsorptivity. However, the use of the polymer is limited because of the disadvantages of poor dispersibility and easy aggregation.
The existing biological magnetic beads for extracting nucleic acid have the problems of large particle size, small surface energy and low nucleic acid adsorption efficiency; in addition, the research shows that the magnetic microsphere prepared by the prior method has certain limitation, for example, the prepared magnetic microsphere has poor suspension property, the aggregation among microsphere materials is serious, the aggregation is easy to precipitate after standing, the hydrophilicity is poor, and the wall is easy to be stained; and a large amount of organic solvent is needed in the synthesis process, so that the waste liquid treatment cost is high.
Disclosure of Invention
The invention aims to provide a nucleic acid purification process for solving the problem of low nucleic acid adsorption efficiency.
The aim of the invention can be achieved by the following technical scheme:
a nucleic acid purification process comprising the steps of:
adding urea, cetyl trimethyl ammonium chloride and carboxylated cage polysilsesquioxane into an acetic acid solution, stirring for 20min, adding nanocellulose, shaking uniformly, drying, washing, and removing impurities to obtain a gel solution; adding the nano magnetic microspheres into absolute ethyl alcohol, performing ultrasonic dispersion, adding ammonia water and the gel solution, stirring for 6 hours, performing magnetic separation, washing, and drying for 12 hours to obtain magnetic beads; adding the lysate into the nucleic acid sample, standing for 3-5min, adding the magnetic beads, mixing, and standing for 3-5min;
secondly, under the action of a magnetic field, fixing magnetic beads, filtering and washing for 2-3 times;
thirdly, adding eluent, standing for 5-8min, fixing magnetic beads, and filtering to obtain purified nucleic acid.
Preferably, the dosage ratio of urea, cetyl trimethyl ammonium chloride, carboxylated cage polysilsesquioxane, acetic acid solution and nanocellulose is 5g:0.8g:5mL:15mL:3g; wherein the concentration of the acetic acid solution is 10%.
Preferably, the dosage ratio of the nano magnetic microsphere, the absolute ethyl alcohol, the ammonia water and the gel solution is 0.4g:45mL:5mL:0.6mL.
Preferably, the nano magnetic microsphere is prepared from any one or two of soluble iron ion salt and nickel-containing and cobalt-containing ion salt.
Preferably, the ionic valence of iron in the soluble iron ion salt is divalent or trivalent.
Preferably, the carboxylated cage polysilsesquioxane is prepared by the steps of:
adding octavinyl cage polysilsesquioxane and tetrahydrofuran into distilled water, carrying out ultrasonic treatment for 10min, heating to 70 ℃, adding ammonium persulfate and sodium bisulfate, stirring for 5min, adding acrylic acid, regulating the pH value to 4.5, reacting for 3h, and cooling to obtain carboxylated cage polysilsesquioxane.
Preferably, the octavinyl cage polysilsesquioxane, tetrahydrofuran, distilled water, ammonium persulfate, sodium bisulfite and acrylic acid are used in a ratio of 1.3g:2g:65mL:0.7g:0.2g:3g.
The invention has the beneficial effects that:
according to the invention, a netlike framework structure is formed between celluloses by utilizing carboxylated POSS, so that on one hand, the system contains more carboxyl and hydroxyl groups, the overall hydrophilicity is enhanced, the adsorption quantity of part of non-target nucleic acid is reduced, the specific recognition capability of the target nucleic acid is improved, the nucleic acid adsorption rate is greatly improved, and then the high-purity nucleic acid is obtained; and provides high active sites and good loading capacity. On the other hand, the integral tensile strength of the cellulose-carboxylated POSS gel is improved, the nanofiber forms tight combination when being subjected to the action of external force, the effects of transferring stress and resisting fracture and separation of adjacent areas are achieved, and the gel is endowed with excellent flexibility; coating the magnetic microsphere to form a core-shell magnetic microsphere, and exhibiting high stability in the processes of nucleic acid cleavage, separation and elution.
The magnetic beads obtained by the method have small particle size, reach the nanoscale, increase the surface energy of the magnetic beads, prolong the time for suspending the magnetic beads in the extracting solution, can be more efficiently contacted with the nucleic acid extract, and increase the extraction rate of the nucleic acid extract; the enrichment and separation of the nucleic acid extract are realized by the superparamagnetism and the magnetic field directional movement characteristic of the nano magnetic beads; the nano magnetic microsphere has the advantages of good corrosion resistance, high strength and high hardness.
The cellulose-carboxylated POSS gel prepared by the invention has smaller pore diameter to generate less gaseous heat transfer quantity, so that the gel has outstanding heat insulation performance, and the temperature range of the magnetic beads in storage and application is enlarged.
The nucleic acid purification process is mild in operation, simple in separation and purification procedure, free of a large device with high price, simple and rapid to operate, and high in product purity.
Detailed Description
The technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
The embodiment provides carboxylated cage polysilsesquioxane, which comprises the following specific steps:
1.3g of octavinyl cage polysilsesquioxane (model P815521, shanghai Michelia Biochemical technology Co., ltd.) and 2g of tetrahydrofuran are added into 65mL of distilled water, the mixture is subjected to ultrasonic treatment for 10min, the temperature is raised to 70 ℃, 0.7g of ammonium persulfate and 0.2g of sodium bisulfite are added, stirring is carried out for 5min, 3g of acrylic acid is added, the pH is regulated to 4.5, the reaction is carried out for 3h, and carboxylated POSS is obtained after cooling.
Example 2
The embodiment provides a nano magnetic microsphere, which comprises the following specific steps:
1.6g of Fe (NO) 3 ) 3 ·9H 2 0 and 0.58gNi (NO) 3 ) 2 ·6H 2 0 is added into 10mL of ethanol and 30mL of distilled water, stirred, placed in a polytetrafluoroethylene high-pressure reaction kettle for reaction for 10 hours at 200 ℃, cooled and filtered, and the absolute ethanol and the distilled water are respectively centrifugally washed for 3 timesDrying for 10h, grinding to obtain NiFe 2 O 4 A nano magnetic microsphere.
Example 3
The embodiment provides a nano magnetic microsphere, which comprises the following specific steps:
1.6g FeCl 3 ·6H 2 0、0.54gCo(NO 3 ) 2 ·6H 2 O and 0.58gNiSO 4 ·6H 2 0 adding 30mL of ethanol and 100mL of distilled water, stirring, placing in a polytetrafluoroethylene high-pressure reaction kettle, reacting at 200 ℃ for 10h, cooling, filtering, respectively centrifugally washing with anhydrous ethanol and distilled water for 3 times, drying for 10h, and grinding to obtain Ni 0.5 Co 0.5 Fe 2 O 4 A nano magnetic microsphere.
Comparative example 1
Adding 1g FeO (OH) (CAS No. 1310-14-1, hubei Wankenkoku chemical Co., ltd.) into 200mL ammonia water with concentration of 6%, reacting at 200deg.C for 24h, cooling to room temperature, centrifuging, washing with distilled water until the pH of the supernatant is 7, washing with absolute ethanol, and drying to obtain Fe 3 0 4 A nano magnetic microsphere.
The magnetic nanobeads obtained in examples 2 to 3 and comparative example 1 were tested, and were magnetically tested using an MPMX-XL-7 type magnetic measurement system, and were characterized by SEM using a Quanta600FEG type field emission scanning electron microscope, and the test results are shown in Table 1:
TABLE 1
| Project | Saturation magnetization (emu/g) | Particle size (nm) |
| Example 2 | 78.69 | 40-45 |
| Example 3 | 79.55 | 40-45 |
| Comparative example 1 | 76.66 | 40-50 |
As can be seen from table 1, the nano magnetic microspheres in examples 2 to 3 have superparamagnetism, have higher paramagnetic properties than the magnetic nano microspheres in comparative example 1, and have more uniform particles and high dispersibility.
Example 4
The embodiment provides a nucleic acid purification process, which comprises the following specific steps:
adding 5g of urea, 0.8g of hexadecyl trimethyl ammonium chloride and 5mL of carboxylated POSS into 15mL of acetic acid solution, stirring for 20min, adding 3g of nanocellulose, shaking uniformly, drying at 80 ℃ and washing, and respectively soaking in absolute ethyl alcohol, ethyl acetate and n-hexane for 6h in sequence to obtain gel solution; adding 0.40g of the nano magnetic microsphere in the embodiment 2 into 45mL of absolute ethyl alcohol, carrying out ultrasonic treatment for 15min, adding 5mL of ammonia water and 0.6mL of the gel liquid, stirring for 6h, carrying out magnetic separation, washing, and drying for 12h to obtain magnetic beads; placing the nucleic acid sample in an EP tube, adding the lysate, standing for 5min, adding the magnetic beads, uniformly mixing, and standing for 5min at room temperature; fixing magnetic beads under the action of a magnetic field, filtering, adding washing liquid, and washing for 2-3 times; finally, adding eluent, standing for 5min, fixing magnetic beads, and filtering to obtain purified nucleic acid.
Example 5
The only difference from example 4 is that: the purified nucleic acid was obtained using the nano-magnetic microspheres of example 3, and the remaining raw materials and steps were the same as those of example 4.
Comparative example 4
The only difference from example 4 is that: the purified nucleic acid was obtained using the nano-magnetic microspheres of comparative example 1, and the remaining raw materials and steps were the same as in example 4.
Comparative example 5
The only difference from example 4 is that:
adding 60 anhydrous ethanol and 30 deionized water into 0.4g of the nano magnetic microsphere in the embodiment 2, performing ultrasonic dispersion for 3min, adding 0.24g of hexadecyl trimethyl ammonium bromide, stirring and reacting for 1h at 40 ℃, adding 2ml of ammonia water, stirring for 10min, adding 0.5g of tetraethoxysilane, reacting for 24h at 40 ℃, performing magnetic separation, washing with methanol for three times, and drying at 50 ℃ to obtain magnetic beads; the purified nucleic acid was obtained, and the other raw materials and steps were the same as those in example 4.
Examples 4-5 and comparative examples 4-5 were applied:
the lysis buffer was 4M guanidine thiocyanate, 20% Triton-X100 and 50mM Tris-HCl mixture, pH 6; wherein the washing liquid is 80% absolute ethanol; the eluent is TE elution buffer with pH of 8.
Placing a nucleic acid sample into an EP tube containing magnetic beads, and performing cracking, washing and eluting under the action of a magnetic field to obtain purified nucleic acid; the nucleic acid sample was taken at 12.5. Mu.L, the beads at 2mg, the lysis buffer at 50. Mu.L, and the eluate at 20. Mu.L.
The purified nucleic acid was subjected to fluorescent quantitative PCR test, and the analysis results are shown in Table 2:
TABLE 2
| Project | OD260/280 | OD260/230 | Concentration (ng/. Mu.L) | Extraction yield (%) |
| Example 4 | 1.86 | 2.10 | 5.9 | 95 |
| Example 5 | 1.88 | 2.11 | 6.1 | 96 |
| Comparative example 4 | 1.84 | 2.01 | 5.8 | 93 |
| Comparative example 5 | 1.80 | 1.83 | 4.3 | 85 |
As can be seen from Table 2, the extraction yield and concentration in examples 4 to 5 are higher than those in comparative examples 4 to 5, indicating that the nucleic acid purification process of the present invention has a higher purification yield in terms of adsorption efficiency.
It is noted that relational terms such as first and second, and the like are used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (7)
1. A nucleic acid purification process comprising the steps of:
adding urea, cetyl trimethyl ammonium chloride and carboxylated cage polysilsesquioxane into an acetic acid solution, stirring for 20min, adding nanocellulose, shaking uniformly, drying, washing, and removing impurities to obtain a gel solution; adding the nano magnetic microspheres into absolute ethyl alcohol, performing ultrasonic dispersion, adding ammonia water and the gel solution, stirring for 6 hours, performing magnetic separation, washing, and drying for 12 hours to obtain magnetic beads; adding the lysate into the nucleic acid sample, standing for 3-5min, adding the magnetic bead reagent, mixing, and standing for 3-5min;
secondly, under the action of a magnetic field, fixing magnetic beads, filtering and washing for 2-3 times;
thirdly, adding eluent, standing for 5-8min, fixing magnetic beads, and filtering to obtain purified nucleic acid.
2. The process for purifying nucleic acid according to claim 1, wherein the ratio of urea, cetyltrimethylammonium chloride, carboxylated cage polysilsesquioxane, acetic acid solution and nanocellulose is 5g:0.8g:5mL:15mL:3g; wherein the concentration of the acetic acid solution is 10%.
3. The process for purifying nucleic acid according to claim 1, wherein the ratio of the amount of the nano magnetic microspheres, absolute ethyl alcohol, ammonia water and gel solution is 0.4g:45mL:5mL:0.6mL.
4. The process for purifying nucleic acid according to claim 1, wherein the nano-magnetic microspheres are prepared from one or both of soluble iron ion salts and salts containing nickel and cobalt ions.
5. The process for purifying a nucleic acid according to claim 4, wherein the ion valence of iron in the soluble iron ion salt is divalent or trivalent.
6. The process for purifying a nucleic acid according to claim 1, wherein the carboxylated cage polysilsesquioxane is prepared by:
adding octavinyl cage polysilsesquioxane and tetrahydrofuran into distilled water, carrying out ultrasonic treatment for 10min, heating to 70 ℃, adding ammonium persulfate and sodium bisulfate, stirring for 5min, adding acrylic acid, regulating the pH value to 4.5, reacting for 3h, and cooling to obtain carboxylated cage polysilsesquioxane.
7. The process for purifying a nucleic acid according to claim 6, wherein the octavinyl cage polysilsesquioxane, tetrahydrofuran, distilled water, ammonium persulfate, sodium bisulphite and acrylic acid are used in an amount ratio of 1.3g:2g:65mL:0.7g:0.2g:3g.
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