CN1243010C - Fast DNA extracting and purifying process with nanoscale particle of aminated silica - Google Patents
Fast DNA extracting and purifying process with nanoscale particle of aminated silica Download PDFInfo
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- CN1243010C CN1243010C CNB021398178A CN02139817A CN1243010C CN 1243010 C CN1243010 C CN 1243010C CN B021398178 A CNB021398178 A CN B021398178A CN 02139817 A CN02139817 A CN 02139817A CN 1243010 C CN1243010 C CN 1243010C
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Abstract
The present invention relates to a method for rapidly extracting and purifying DNA by aminated silicon oxide nanometer particles. The aminated silicon oxide nanometer particles with positive charges are used as a carrier and directly react with a tissue cracking solution or a cell cracking solution, the aminated silicon oxide 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, and the nanometer particle-DNA compounds are separated from the tissue cracking solution or the cell cracking solution by centrifugal action, and furthermore, the DNA combined on the surfaces of the nanometer particles is separated by a dissociation solution. Thereby, the rapid purification of the DNA is realized. The method does not need to adopt a phenol/ chloroform extraction method to remove protein in a ribonucleic acid solution, thereby, the time is shortened 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, treatment, etc. and extraction and purification of DNA 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 a DNA extraction 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 nano SiO 2 particle material of electrokinetic potential, tool physical and chemical stability.
Technical scheme of the present invention is to be carrier with positively charged amination nano SiO 2 particle, directly interact with organizing lysate or cell pyrolysis liquid, positively charged amination nano SiO 2 particle forms amination nano SiO 2 particle-DNA mixture with organizing DNA electronegative in lysate or the cell pyrolysis liquid by electrostatic interaction, realize amination nano SiO 2 particle-DNA mixture and organize separating of lysate or cell pyrolysis liquid by centrifugation, the DNA that will be combined in amination nano SiO 2 particle 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 amination nano SiO 2 particle to DNA.
This method does not need to adopt phenol/chloroform extraction method to remove protein in the nucleic acid solution, 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 the amination nano SiO 2 particle is carried out pre-treatment; DNA compares with the hydroxyapatite column chromatography purification, and the amination nano SiO 2 particle 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 nano SiO 2 particle of the present invention and the graphic representation of Ph value.
Among the figure:
The iso-electric point of the amination nano SiO 2 particle that this method adopted is about 9.6, in pH neutral normal temperature medium, the amino proton on amination nano SiO 2 particle 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 nano SiO 2 particle and tissue or the cell pyrolysis liquid forms amination nano SiO 2 particle-DNA mixture by electrostatic interaction with the ion pair form, and is fast soon by realizing amination nano SiO 2 particle-DNA mixture and tissue or cell pyrolysis liquid under the centrifugation effect, succinctly, separate efficiently.
Fig. 2 is the gel electrophoresis figure of the hepatic tissue DNA of mouse, is the DNA of amination nano SiO 2 particle extraction of the present invention and the comparison between conventional method (phenol/chloroform extraction method) the extraction DNA.
(1) DNA MARKER among the figure (marker contrast)
(2) DNA that extracts with ordinary method
(3) with the extractive DNA of amination nano SiO 2 particle
Obviously, the DNA product basically identical of Ti Quing between the two, but amination nano SiO 2 particle method has successfully been simplified extraction steps, has shortened the extracting time, improved extraction efficiency, has 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 nano SiO 2 particle-DNA: utilize the amination nano SiO 2 particle in pH neutral normal temperature medium, the amino protonated of amination nano SiO 2 particle surface can produce clean positive electricity effectively, and electro kinetic potential is up to this characteristic of 30mv, add an amount of amination nano SiO 2 particle 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 nano SiO 2 particle-DNA in room temperature (15-25 ℃).
3. separated and collected amination nano SiO 2 particle-DNA mixture: utilize the density variation between amination nano SiO 2 particle-DNA mixture and the cracking tissue juice, the amination nano SiO 2 particle is carried out centrifugal treating, amination nano SiO 2 particle-DNA mixture is fully precipitated, collect amination nano SiO 2 particle-DNA mixture.
4. the DNA on the amination of dissociating nano SiO 2 particle-DNA mixture: in amination nano SiO 2 particle-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.Then to the amination nano SiO 2 particle in 4 ℃ of centrifugal 10-15 minutes, the amination nano SiO 2 particle is fully precipitated, DNA is distributed in the settled solution of upper strata, collect the upper strata settled solution in another centrifuge tube, repeat this step 2-3 time, guarantee that the DNA on amination nano SiO 2 particle 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 and 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 dna solution, preserve down in 4 ℃.
Embodiment (hepatic tissue with the extraction small white mouse is an example): the method that adopts amination nano SiO 2 particle 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 nano SiO 2 particle of 1 milliliter of 15mg/mL through high-temperature sterilization, vibration fully in conjunction with 10-15 minute, forms amination nano SiO 2 particle-DNA mixture under room temperature.
3. amination nano SiO 2 particle-DNA mixture is carried out centrifugal treating, under 4 ℃ temperature, centrifugal 3 minutes of 5000g, amination nano SiO 2 particle-DNA mixture is fully precipitated, with ultrapure water washing 1 time, in centrifugal 3 minutes of 4 ℃ of 5000g, outwell the upper strata settled solution once more, collect amination nano SiO 2 particle-DNA mixture.
4. the sodium chloride solution that adds 500uL 2mol/L in amination nano SiO 2 particle-DNA mixture of collecting, the amination that suspends again nano SiO 2 particle, vibration was placed 5 minutes under room temperature, allowed DNA fully elute.To centrifugal 2 minutes of amination nano SiO 2 particle 5000g under 4 ℃ temperature, collect the upper strata settled solution in another centrifuge tube then, repeat this step 3 time, guarantee that the DNA on amination nano SiO 2 particle 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, in centrifugal 15 minutes of 4 ℃ of following 12000-15000g, makes DNA and solution layering then.
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 nano SiO 2 particle can make with the following method:
With hexanaphthene (7.5ml), Triton TX-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, stir and form water in oil microcapsule after 5 minutes, again 100 μ L tetraethoxys (TEOS) and 100 μ LN-(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 use acetone breakdown of emulsion, centrifugation collecting granules.Can prepare the amination nano SiO 2 particle.
The present invention has following advantage:
One: the amination nano SiO 2 particle isoelectric point that the method adopts about 9.6, normal in pH neutrality In the temperature medium, the amino protonated of amination nano SiO 2 particle surface can produce clean positive electricity effectively, and electro kinetic potential Up to 30mv, do not need the amination nano SiO 2 particle is modified again.
Its two: the amination nano SiO 2 particle directly interacts with organizing lysate or cell pyrolysis liquid, passes through static Work forms amination nano SiO 2 particle-DNA compound in order to the ion pair form, can realize under centrifugal effect Amination nano SiO 2 particle-DNA compound and tissue or cell pyrolysis liquid are fast, succinctly, separate efficiently.
Its three: this method has been simplified extraction steps, shortened the extracting time, improved extraction efficiency, has 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 the amination nano SiO 2 particle to be carried out pre-treatment, and the amination nano SiO 2 particle 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 nano SiO 2 particle, with positively charged amination nano SiO 2 particle is carrier, directly interact with organizing lysate or cell pyrolysis liquid, positively charged amination nano SiO 2 particle with organize DNA electronegative in lysate or the cell pyrolysis liquid to form nano particle-DNA mixture by electrostatic interaction, realize nano particle-DNA mixture and organize separating of lysate or cell pyrolysis liquid by centrifugation, the DNA that will be combined in nano grain surface by dissociation solution disintegrates down again, thereby realizes the fast purifying to DNA; It is characterized in that: concrete processing step is:
(1) cracking tissue or cell are got 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 nano SiO 2 particle-DNA: utilize the amination nano SiO 2 particle in pH neutral normal temperature medium, the amino protonated of amination nano SiO 2 particle surface can produce clean positive electricity effectively, and electro kinetic potential is up to this characteristic of 30mv, add an amount of amination nano SiO 2 particle in tissue after cracking or the enchylema through high-temperature sterilization, the vibration mixing, under 15-25 ℃ room temperature,, form the mixture of amination nano SiO 2 particle-DNA fully in conjunction with 5-30 minute;
(3) separated and collected amination nano SiO 2 particle-DNA mixture: utilize the density variation between amination nano SiO 2 particle-DNA mixture and the cracking tissue juice, the amination nano SiO 2 particle is carried out centrifugal treating, amination nano SiO 2 particle-DNA mixture is fully precipitated, collect amination nano SiO 2 particle-DNA mixture;
(4) dissociate DNA. on amination nano SiO 2 particle-DNA mixture: in amination nano SiO 2 particle-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; Then to the amination nano SiO 2 particle in 4 ℃ of centrifugal 10-15 minutes, the amination nano SiO 2 particle is fully precipitated, DNA is distributed in the settled solution of upper strata, collect the upper strata settled solution in another centrifuge tube, repeat this step 2-3 time, guarantee that the DNA on amination nano SiO 2 particle 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 ℃.
2, the method for the quick extracting and purifying DNA of employing amination nano SiO 2 particle according to claim 1 is characterized in that described amination nano SiO 2 particle makes with the following method:
It with volume ratio 4.2: 1: 1 hexanaphthene, tensio-active agent Triton TX-100 and n-hexyl alcohol, mix, the water that adds 360 μ L is as disperse phase, stir and form water in oil microcapsule after 5 minutes, the 100 μ L tetraethoxy TEOS 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, the centrifugation collecting granules can prepare the amination nano SiO 2 particle.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021398178A CN1243010C (en) | 2002-12-11 | 2002-12-11 | Fast DNA extracting and purifying process with nanoscale particle of aminated silica |
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| CNB021398178A CN1243010C (en) | 2002-12-11 | 2002-12-11 | Fast DNA extracting and purifying process with nanoscale particle of aminated silica |
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| CN1243010C true CN1243010C (en) | 2006-02-22 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100439918C (en) * | 2006-06-29 | 2008-12-03 | 复旦大学 | Method for enriching, desalting protein or polypeptide in minute quantities, and carrying out analysis directly |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100374858C (en) * | 2006-03-30 | 2008-03-12 | 复旦大学 | Method for simultanuously enriching desalting and appraising micro protein or polypeptide solution |
| CN102091575B (en) * | 2009-12-14 | 2013-02-13 | 深圳先进技术研究院 | Ferroferric oxide magnetic nanoparticles and preparation method thereof |
| CN103837519B (en) * | 2014-04-10 | 2016-11-16 | 中国科学院合肥物质科学研究院 | Surface enhanced raman spectroscopy measures the method for Polychlorinated Biphenyls |
| CN106520522A (en) * | 2016-12-06 | 2017-03-22 | 厦门华厦学院 | DNA extraction device and method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN100439918C (en) * | 2006-06-29 | 2008-12-03 | 复旦大学 | Method for enriching, desalting protein or polypeptide in minute quantities, and carrying out analysis directly |
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