CN1257180C - Fast DNA extracting and purifying process with shell magnetic nanoscale particle of aminated silica - Google Patents
Fast DNA extracting and purifying process with shell magnetic nanoscale particle of aminated silica Download PDFInfo
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- CN1257180C CN1257180C CNB021398186A CN02139818A CN1257180C CN 1257180 C CN1257180 C CN 1257180C CN B021398186 A CNB021398186 A CN B021398186A CN 02139818 A CN02139818 A CN 02139818A CN 1257180 C CN1257180 C CN 1257180C
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Abstract
The present invention relates to a method for rapidly extracting and purifying DNA by aminated silicon oxide shell magnetic nanometer particles. The aminated silicon oxide shell magnetic nanometer particles with positive charges are used as a carrier to directly react with a tissue cracking solution or a cell cracking solution, the aminated silicon oxide shell magnetic nanometer particles with positive charges and the DNA with negative charges in the tissue cracking solution or the cell cracking solution form nanometer particle-DNA compounds by electrostatic action, and the nanometer particle-DNA compounds are separated from the tissue cracking solution or the cell cracking solution under the action of an external magnetic field. The DNA combined on the surface of the nanometer particles is separated by a dissociation solution, and thereby, the rapid extraction and purification of the DNA is realized. The method does not need a phenol/ chloroform extraction method to remove protein in a ribonucleic acid solution, the steps are simplified and the efficiency is enhanced. Meanwhile, the pollution of phenol/chloroform on the environment and the toxic action on a human body are relieved. The method provides a new means for researches of gene transduction, mutation detection, gene diagnosis and treatment, etc. and DNA purification in application.
Description
Technical field: the present invention relates to the Application Areas and the nanobiology field of nano material, refer more particularly to nano particle application and development to the quick extracting and purifying of DNA in molecular biology.
Background technology: along with finishing of the Human Genome Project, contemporary biology, medical science etc. have been deep into molecular level, and the demand of the DNA biomacromolecule that carries out high efficiency separation, enrichment, extraction, reprinting that utilized is also improved day by day.Yet DNA extraction purification process commonly used at present all gos deep into experimental requirements because of existing self-defect can't satisfy people.Phenol/chloroform extraction method once was an extraction DNA method classical in the molecular biology, but since phenol/chloroform to the huge toxic action of human body with to the strong pollution of environment, in the range of application that will limit to it today greatly of advocating environmental protection.DNA extraction agent box on the current market is because operation steps more complicated, raw material need pre-treatment and raw material self physicochemical property not to cause productive rate high and can't satisfy people's requirement by force.
Summary of the invention: at the above-mentioned defective of prior art, technical problem to be solved by this invention provide a kind of adopt cheap and easy to manufacture, in neutral solution, be method on the occasion of the fast succinct extracting and purifying DNA of the amination silicon-dioxide shell magnetic nanoparticles of electrokinetic potential, tool physical and chemical stability.
Technical scheme of the present invention is to be carrier with the positively charged amination silicon-dioxide shell magnetic nanoparticles with magnetic, directly interact with organizing lysate or cell pyrolysis liquid, positively charged amination silicon-dioxide shell magnetic nanoparticles forms amination silicon-dioxide shell magnetic nanoparticles-DNA mixture with organizing DNA electronegative in lysate or the cell pyrolysis liquid by electrostatic interaction, the effect of outside magnetic field realizes amination silicon-dioxide shell magnetic nanoparticles-DNA mixture down and organizes separating of lysate or cell pyrolysis liquid, the DNA that will be combined in amination silicon-dioxide shell magnetic nanoparticles surface by dissociation solution disintegrates down again, thereby realize fast purifying, set up a kind of new DNA rapid extraction purification process based on the silicon-dioxide shell magnetic nanoparticles to DNA.
This method does not need to adopt phenol/chloroform extraction method to remove protein in the nucleic acid solution, has simplified experimental procedure, shortened the time, improved efficient, has removed phenol/chloroform simultaneously to the pollution of environment with to the toxic action of human body; Compare with the DNA extraction agent box extracting and purifying DNA on the current market, it does not need amination silicon-dioxide shell magnetic nanoparticles is carried out pre-treatment; DNA compares with the hydroxyapatite column chromatography purification, and amination silicon-dioxide shell magnetic nanoparticles has huge specific surface area, can improve the efficient of purify DNA.This method be established as gene transfer, sudden change research such as detection, gene diagnosis and gene therapy and use in the purifying of DNA a kind of new means and fast succinct extracting and purifying DNA method soon are provided.
Description of drawings: now in conjunction with the accompanying drawings the present invention is explained.
Fig. 1 is the electrokinetic potential ζ of the used amination silicon-dioxide shell magnetic nanoparticles of the present invention and the graphic representation of Ph value.
Among the figure:
The ferroferric oxide gel nano particle
The iso-electric point of the magnetic nanoparticle that this method adopted is all about 9.6, in pH neutral normal temperature medium, the amino protonated of nano grain surface can produce clean positive electricity effectively, and electro kinetic potential is up to 35mv, can be directly and tissue or cell pyrolysis liquid interact, DNA electronegative in positively charged amination silicon-dioxide shell magnetic nanoparticles and tissue or the cell pyrolysis liquid forms amination silicon-dioxide shell magnetic nanoparticles-DNA mixture by electrostatic interaction with the ion pair form, by utilizing the magnetic action of kernel, can realize under the effect of outside magnetic field that amination silicon-dioxide shell magnetic nanoparticles-DNA mixture and tissue or cell pyrolysis liquid are fast soon, succinctly, separate efficiently.
Fig. 2 is the gel electrophoresis figure of the hepatic tissue DNA of mouse, is the DNA of amination silicon-dioxide shell magnetic nanoparticles extraction of the present invention and the comparison between conventional method (phenol/chloroform extraction method) the extraction DNA.
(1) uses the extractive DNA of amination silicon-dioxide shell magnetic nanoparticles among the figure
(2) extract DNA (, therefore can not extract) with nano SiO 2 particle owing to do not have positive charge
(3) DNA that extracts with ordinary method
Obviously, common nano SiO 2 particle positively charged not under neutral environment, can not extract DNA, and positively charged amination silicon-dioxide shell magnetic nanoparticles method can successfully be extracted DNA under neutral environment, extracting DNA with ordinary method compares, simplified extraction steps, shortened the extracting time, improved extraction efficiency, removed phenol/chloroform simultaneously to the pollution of environment with to the toxic action of human body.
Embodiment:
1. cracking tissue or cell: get an amount of tissue or cell, add an amount of extracting lysate after being milled to tissue juice or enchylema fast on ice cracking 3-5 hour.
2. form the mixture of amination silicon-dioxide shell magnetic nanoparticles-DNA: utilize amination silicon-dioxide shell magnetic nanoparticles in pH neutral normal temperature medium, the amino protonated of its surface can produce clean positive electricity effectively, and electro kinetic potential is up to this characteristic of 35mv, add an amount of amination silicon-dioxide shell magnetic nanoparticles in tissue after cracking or the enchylema through high-temperature sterilization, the vibration mixing, fully in conjunction with 5-30 minute, form the mixture of amination silicon-dioxide shell magnetic nanoparticles-DNA in room temperature (15-25 ℃).
3. separated and collected amination silicon-dioxide shell magnetic nanoparticles-DNA mixture: utilize the kernel of amination silicon-dioxide shell magnetic nanoparticles to have the characteristics of magnetic, separate adding under the action of a magnetic field, collect amination silicon-dioxide shell magnetic nanoparticles-DNA mixture.
4. the DNA on the amination of dissociating silicon-dioxide shell magnetic nanoparticles-DNA mixture: in amination silicon-dioxide shell magnetic nanoparticles-DNA mixture, add dissociation solution (sodium chloride solution of 2-3mol/L or the buffered soln of pH10-12), placed 5-30 minute under the room temperature, allow DNA fully elute.Left standstill under the outside magnetic field 5-20 minute, amination silicon-dioxide shell magnetic nanoparticles is fully precipitated, and DNA is distributed in the settled solution of upper strata, collects the upper strata settled solution, repeat this step 2-3 time, guarantee that the DNA on amination silicon-dioxide shell magnetic nanoparticles surface all disintegrates down.
5. concentration of DNA product: the characteristic of utilizing DNA can sedimentation in Virahol to separate out, in the upper strata settled solution of collecting that includes DNA, add an amount of Virahol, allow its thorough mixing, placed 10-15 minute down in 0 ℃ to-20 ℃, in 4 ℃ of centrifugal 15-30 of following 12000-15000g minutes, DNA is precipitated out from solution then.
6. collect DNA: outwell the upper strata settled solution, with washing with alcohol DNA precipitation, recentrifuge, outwell the upper strata ethanolic soln, allow residual ethanol volatilize naturally totally, add 50 microlitre pH and be 8.0 TE damping fluid dissolving DNA precipitation, obtain being fit to the dna solution of concentration, preserve down in 4 ℃.
Embodiment (hepatic tissue with the extraction small white mouse is an example): the method that adopts amination silicon-dioxide shell magnetic nanoparticles quick extracting and purifying DNA from the hepatic tissue of small white mouse
1. get 1.302 gram small white mouse hepatic tissues, add 18ml extracting lysate, after being milled to homogenate fast on ice, became to organize lysate in 4 hours in 60 ℃ of following cracking.
2. get 500ul and organize lysate, add the amination silicon-dioxide shell magnetic nanoparticles of 1 milliliter of 20mg/mL through high-temperature sterilization, vibration at room temperature fully in conjunction with 10-15 minute, forms amination silicon-dioxide shell magnetic nanoparticles-DNA mixture.
3. will leave standstill 5 minutes under amination silicon-dioxide shell magnetic nanoparticles-DNA mixture outside magnetic field, amination silicon-dioxide shell magnetic nanoparticles-DNA mixture is fully precipitated, the upper strata settled solution is outwelled, with ultrapure water washing 1 time, to leave standstill 5 minutes under amination silicon-dioxide shell magnetic nanoparticles-DNA mixture outside magnetic field once more, the upper strata settled solution is outwelled, collected amination silicon-dioxide shell magnetic nanoparticles-DNA mixture.
4. the sodium chloride solution that adds 500uL 2mol/L in the amination silicon-dioxide shell magnetic nanoparticles-DNA mixture amination silicon-dioxide shell magnetic nanoparticles-DNA mixture that suspends again, vibration, under room temperature, placed 5 minutes, allow DNA fully elute.Fully precipitate amination silicon-dioxide shell magnetic nanoparticles under the outside magnetic field, collect the upper strata settled solution.Repeat this step 3 time, guarantee that the DNA on amination silicon-dioxide shell magnetic nanoparticles surface all disintegrates down.
5. the Virahol that adds 1mL in the upper strata settled solution of collecting allows its thorough mixing, in 0 ℃ to-20 ℃ placement 10 minutes down, centrifugal then 15 minutes, makes DNA and solution layering.
6. outwell the upper strata settled solution, obtain the DNA product, with washing with alcohol DNA precipitation, recentrifuge is outwelled the upper strata ethanolic soln, allows residual ethanol volatilize naturally totally, add 50 microlitre pH and be 8.0 TE damping fluid dissolving DNA precipitation, obtain dna solution, preserve down in 4 ℃.
Among the present invention, described amination silicon-dioxide shell magnetic nanoparticles can make with following method:
The first step at first prepares the nano ferriferrous oxide colloid, being about to 20mL 1mol/L ferric chloride aqueous solutions (now joining) mixes with the ferrous chloride solution (with the preparation of 2mol/L hydrochloric acid) of 5mL 2mol/L, the ammoniacal liquor that adds 250mL0.7mol/L, after stirring half an hour under the room temperature, separate with magnet, outwell supernatant liquor.The gained precipitation adds redistilled water to 250mL then with 30mL1mol/L Tetramethylammonium hydroxide (TMA) dissolving.With the aperture is the sand core funnel filtration of 40-80 μ m, removes the accumulative macrobead.
Second step, with hexanaphthene (7.5ml), tensio-active agent triton x-100 (TritonX-100) (1.8ml) and n-hexyl alcohol (1.8ml) (volume ratio is about 4.2/1/1), mix, add an amount of water (360 μ L) as disperse phase, form water in oil microcapsule after stirring 5min, add 200 μ L nano ferriferrous oxide colloids, stir.Again 100 μ L tetraethoxys (TEOS) and 100 μ L N-(β-aminoethyl)-γ-An Bingjisanyiyangjiguiwan (AEAPS) are mixed (1/1, volume ratio) joins after in the microcapsule system, after stirring, their the same one-step hydrolysis in microcapsule of ammonia-catalyzed that add 100 μ L, the entire reaction time is 24 hours, react complete back and use the acetone breakdown of emulsion, collecting granules under the outside magnetic field.Can prepare amination silicon-dioxide shell magnetic nanoparticles.
The present invention has following advantage:
One: the isoelectric point of the amination silica shell magnetic nanoparticles that the method adopts is about 9.6, at pH In the neutral normal temperature medium, the amino protonated of amination silica shell magnetic nanoparticles surface can just produce only effectively Electricity, and electro kinetic potential does not need amination silica shell magnetic nanoparticles is modified again up to 35mv.
Its two: amination silica shell magnetic nanoparticles directly interacts with organizing lysate or cell pyrolysis liquid, and is logical Cross electrostatic interaction and form amination silica shell magnetic nanoparticles-DNA compound, outside magnetic field with the ion pair form Effect is lower can realize amination silica shell magnetic nanoparticles-DNA compound and tissue or cell pyrolysis liquid quick, Succinctly, separate efficiently.
Its three: this method shortened the extracting time, improved extraction efficiency, removed phenol/chloroform simultaneously to the pollution of environment with to the toxic action of human body.
Its four: compare with hydroxyapatite column chromatography purification DNA method with genome DNA extraction test kit extracting and purifying DNA method, it does not need again amination silicon-dioxide shell magnetic nanoparticles to be carried out pre-treatment, and amination silicon-dioxide shell magnetic nanoparticles has huge specific surface area, can improve the productive rate of extract product.The purifying that is established as DNA in gene transfer, sudden change research such as detection, gene diagnosis and gene therapy and the application of this method provides a kind of new means and method.
Claims (2)
1, a kind of method that adopts the quick extracting and purifying DNA of amination silicon-dioxide shell magnetic nanoparticles, it is characterized in that: the amination silicon-dioxide shell magnetic nanoparticles with positively charged and tool magnetic is a carrier, directly interact with organizing lysate or cell pyrolysis liquid, positively charged amination silicon-dioxide shell magnetic nanoparticles with organize DNA electronegative in lysate or the cell pyrolysis liquid to form nano particle-DNA mixture by electrostatic interaction, the effect of outside magnetic field realizes nano particle-DNA mixture down and organizes separating of lysate or cell pyrolysis liquid, the DNA that will be combined in nano grain surface by dissociation solution disintegrates down again, thereby realizes the extracting and purifying to DNA; Described amination silicon-dioxide shell magnetic nanoparticles makes in order to the below method:
The first step at first prepares the nano ferriferrous oxide colloid, the 20mL 1mol/L ferric chloride aqueous solutions of soon now joining mixes with the ferrous chloride solution of 5mL 2mol/L, wherein the ferrous chloride solution of 2mol/L is prepared with 2mol/L hydrochloric acid, the ammoniacal liquor that adds 250mL 0.7mol/L, after stirring half an hour under the room temperature, separate with magnet, outwell supernatant liquor, the gained precipitation is dissolved with 30mL lmol/L Tetramethylammonium hydroxide, add redistilled water then to 250mL, with the aperture is the sand core funnel filtration of 40-80 μ m, removes the accumulative macrobead;
Second step: with volume ratio is that 4.2: 1: 1 hexanaphthene, tensio-active agent triton x-100 and n-hexyl alcohol mixes, add 360 μ L water as disperse phase, form water in oil microcapsule after stirring 5min, add 200 μ L nano ferriferrous oxide colloids, stir; The 100 μ L tetraethoxys and 100 that with volume ratio are 1: 1 again join in the microcapsule system after μ L N-(β-aminoethyl)-γ-An Bingjisanyiyangjiguiwan mixes, after stirring, their the same one-step hydrolysis in microcapsule of ammonia-catalyzed that add 100 μ L, the entire reaction time is 24 hours, react complete back acetone breakdown of emulsion, collecting granules under the outside magnetic field can prepare amination silicon-dioxide shell magnetic nanoparticles.
2, the method for the quick extracting and purifying DNA of employing amination silicon-dioxide shell magnetic nanoparticles according to claim 1 is characterized in that its concrete steps are:
(1) cracking tissue or cell: take by weighing an amount of tissue or cell, add an amount of extracting lysate after being milled to tissue juice or enchylema fast on ice cracking 3-5 hour;
(2) mixture of formation amination silicon-dioxide shell magnetic nanoparticles-DNA: utilize amination silicon-dioxide shell magnetic nanoparticles in pH neutral normal temperature medium, the amino protonated of its surface can produce clean positive electricity effectively, and electro kinetic potential is up to this characteristic of 30mv, add an amount of amination silicon-dioxide shell magnetic nanoparticles in tissue after cracking or the enchylema through high-temperature sterilization, the vibration mixing, fully in conjunction with 5-30 minute, form the mixture of amination silicon-dioxide shell magnetic nanoparticles-DNA in 15-25 ℃ room temperature;
(3) collect amination silicon-dioxide shell magnetic nanoparticles-DNA mixture: utilize the kernel of amination silicon-dioxide shell magnetic nanoparticles to have the characteristics of magnetic, under the effect of externally-applied magnetic field, separate, collect amination silicon-dioxide shell magnetic nanoparticles-DNA mixture;
(4) dissociate DNA on amination silicon-dioxide shell magnetic nanoparticles-DNA mixture: in amination silicon-dioxide shell magnetic nanoparticles-DNA mixture, add dissociation solution, described dissociation solution is the sodium chloride solution of 2-3mol/L or the buffered soln of pH10-12, placed 5-30 minute under the room temperature, allow DNA fully elute.Left standstill under the outside magnetic field 5-20 minute, amination silicon-dioxide shell magnetic nanoparticles is fully precipitated, and DNA is distributed in the settled solution of upper strata, collects the upper strata settled solution, repeat this step 2-3 time, guarantee that the DNA on amination silicon-dioxide shell magnetic nanoparticles surface all disintegrates down;
(5) concentration of DNA product: the characteristic of utilizing DNA can sedimentation in Virahol to separate out, in the upper strata settled solution of collecting that includes DNA, add an amount of Virahol, allow its thorough mixing, placed 10-15 minute down in 0 ℃ to-20 ℃, in 4 ℃ of centrifugal 15-30 of following 12000-15000g minutes, DNA is precipitated out from solution then;
(6) collect DNA: outwell the upper strata settled solution, with washing with alcohol DNA precipitation, recentrifuge, outwell the upper strata ethanolic soln, allow residual ethanol volatilize naturally totally, add 50 microlitre pH and be 8.0 TE damping fluid dissolving DNA precipitation, obtain being fit to the dna solution of concentration, preserve down in 4 ℃.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101851617A (en) * | 2010-04-28 | 2010-10-06 | 长春市博坤生物科技有限公司 | Extracting method of viral nucleic acid capable of being used for high flux full automation extraction by magnetic bead method |
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| CN101612541B (en) * | 2009-07-17 | 2011-09-07 | 江南大学 | Preparation of polyacrylic acid coated ferroferric oxide magnetic nano-particles and application thereof |
| CN104759260B (en) * | 2015-04-14 | 2018-04-20 | 河海大学 | A kind of amino functional magnetic silica ferroso-ferric oxide composite nano materials and preparation method thereof |
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| CN109761240A (en) * | 2019-03-19 | 2019-05-17 | 大连大学 | A kind of preparation method of amination silica dioxide granule |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101851617A (en) * | 2010-04-28 | 2010-10-06 | 长春市博坤生物科技有限公司 | Extracting method of viral nucleic acid capable of being used for high flux full automation extraction by magnetic bead method |
| CN101851617B (en) * | 2010-04-28 | 2012-11-21 | 长春市博坤生物科技有限公司 | Extracting method of viral nucleic acid capable of being used for high flux full automation extraction by magnetic bead method |
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