CN1970752A - Method for recovery of DNA by using magnetic nanocomposite and reagent kit therefor - Google Patents
Method for recovery of DNA by using magnetic nanocomposite and reagent kit therefor Download PDFInfo
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- CN1970752A CN1970752A CN 200610131676 CN200610131676A CN1970752A CN 1970752 A CN1970752 A CN 1970752A CN 200610131676 CN200610131676 CN 200610131676 CN 200610131676 A CN200610131676 A CN 200610131676A CN 1970752 A CN1970752 A CN 1970752A
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Abstract
The invention discloses a recycling method of DNA and agent box through magnetic nanometer composite material in the biological technical domain, which comprises the following steps: (a) blending magnetic nanometer composite material and recycled sample; forming solid-liquid dispersing suspension; (b) adsorbing DNA to separate magnetic nanometer composite material from solid phase; obtaining magnetic nanometer composite material with adsorbed DNA; (c) washing impurity; evolving DNA from magnetic nanometer composite material. The magnetic nanometer composite agent box to recycle DNA possesses magnetic nanometer composite material solution, dissolving liquid, washing liquid I and washing liquid II and eluent.
Description
Technical field
The present invention relates to biological technical field.More specifically, the present invention relates to a kind ofly reclaim the method for DNA and be used to reclaim the test kit of DNA with magnetic nano-particle.
Background technology
The recovery of DNA is widely used, and for example reclaims high purity DNA and be used for genetic fingerprinting, gene sequencing, structure library, PCR check etc. from archaeal dna polymerase chain reaction (PCR) amplified production, polyacrylamide gel, sepharose, postdigestive biological specimen.The DNA recovery technology is a gordian technique in the molecular biology experiment, reclaim the efficient of DNA and the success or not that purity will be directly connected to subsequent experimental, therefore develop quick, efficient, simple and easy, the DNA recovery method is one of target of molecular biology worker always cheaply.
Main DNA recovery method is chemical method, glass bead method and post partition method at present, and step is comparatively loaded down with trivial details, and the DNA purity of recovery is not high.
The test kit that more external companies produce, the test kit as Promega company, Qiagen company, Gene company etc. produce costs an arm and a leg, and can't effectively promote.
Along with being gradually improved and progress of nanotechnology, magnetic nanometer composite material synthetic technology and the application prospect in biological field thereof have been subjected to people's attention day by day.Especially it has advantages such as separation method is easy and simple to handle, required equipment is cheap, and velocity of separation is fast, makes it become the requisite laboratory facilities of molecular biology research gradually, uses increasingly extensive.
The magnetic composite general requirement that is used for biotechnology has following characteristic: (1) possesses paramagnetism, is easy to absorption and wash-out.(2) has the bigger specific magnetising moment, to guarantee separation efficiency and to improve sensitivity.(3) composite material surface will be easy to carry out chemically modified, to be connected with drug molecule with different biologies.Require to have better biocompatibility when (4) being used in the body.Matrix material based on this purpose design is generally sandwich structure, kernel is generally the magnetic nano-particle of superparamagnetism, the centre is a coating layer, existing products adopts macromolecular material as coating layer mostly at present, outermost layer is then modified the functional group that specific effect is arranged with biological tissue, to satisfy the requirement of bioseparation.
The polymer microsphere that commercial magnetic composite adopts letex polymerization to obtain more has wherein coated the magnetic-particle of nano-scale.Yet the structure of high score bulbec is more loose usually, and the magnetic particle that wherein comprises breaks away from microballoon easily in long-time preservation process, thereby influences the performance of product.The polymer microsphere size of general letex polymerization gained is bigger, how at micrometer range, can not satisfy the requirement of the Application Areas of some needs smaller szie particles.The magnetic particle coating material of another kind of common employing is the polyose molecule, as dextran, chitosan etc., but the coating layer that this quasi-molecule forms is more unstable, a little less than the effect of itself and magnetic particle, exists magnetic particle easily to come off equally and causes the shortcoming of product failure.Therefore, this area presses for the new technology that reclaims DNA of exploitation simple and effectively.
The prior art close with the present invention is Chinese patent ZL03111351.6, denomination of invention is " having magnetic core-shell particle of high-intensity magnetic field respective capabilities and preparation method thereof ", disclose with silicon-dioxide is the synthetic method of the magnetic nanometer composite material of coating layer, institute's synthetic magnetic nanometer composite material is suitable for separating and purifying of biomaterial and pharmaceutical products, can be as biomolecules and the directed delivery vehicles of drug molecule magnetic control.
At present, still do not utilize magnetic nanometer composite material to reclaim the technology of DNA.
Summary of the invention
The technical problem to be solved in the present invention is, uses with silicon-dioxide and reclaims DNA as the magnetic nanometer composite material of coating layer, reach easy, quick, efficiently, purpose cheaply.
The present inventor is on the basis of magnetic nanometer composite material of coating layer with silicon-dioxide having prepared, and finds that this magnetic nanometer composite material and dna molecular have the avidity of height, can be used as the effective medium that reclaims DNA.One object of the present invention is exactly to disclose a kind of method of utilizing magnetic nanometer composite material simple and effective ground to reclaim DNA.
Another purpose of the present invention provides a kind of test kit that utilizes magnetic nanometer composite material to reclaim DNA simple and effectively.
In a first aspect of the present invention, a kind of method of utilizing magnetic nanometer composite material to reclaim DNA is provided, said recovery DNA, be from lysate blended pcr amplification product, with lysate make adsorption liquid sample that DNA discharges from gelled specimen or the recovery samples such as biological specimen that discharged with lysate blended DNA reclaim DNA; Recycling step comprises:
(a) magnetic nanometer composite material is mixed with recovery sample, form solid-liquid homodisperse suspension liquid, the diameter of wherein said magnetic nanometer composite material is 0.2～10 μ m;
(b) magnetic nanometer composite material that will adsorb DNA with magnetic field is separated from solid-liquid homodisperse suspension liquid, thereby obtains being adsorbed with the magnetic nanometer composite material of DNA;
(c) with washings with the impurity flush away after, with elutriant DNA is parsed from magnetic nanometer composite material, thereby obtains pure dna solution; Said washings, its component are the salts solution of 1～8mol/l, the Tri(Hydroxymethyl) Amino Methane Hydrochloride of 0.5mol/l (Tris-HCl), pH=6.7, the ethylenediamine tetraacetic acid (EDTA) of 5～50mmol/l (EDTA) or be the sodium acetate of 0～2mol/l; Said elutriant is deionized water or 10 ± 2mmol/l Tris-HCl, pH=8.0.
But the inventive method simple and effective ground reclaims DNA from gel etc. contains the sample of DNA.
In another preference, described gelled specimen is that polyacrylamide gel, sepharose, dextrane gel etc. and other need carry out the sample that DNA reclaims.
In another preference, with lysate DNA is discharged from gelled specimen and obtain containing the adsorption liquid sample of DNA, the component of its lysate is the Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tris-HCl), ethylenediamine tetraacetic acid (EDTA) (EDTA) and 0.5～5% Triton X-100 (Triton) of 5～50mmol/l of pH=6.7 of salts solution, the 0.5mol/l of 1～8mol/l.
In another preference, described adsorption liquid sample prepares by following steps: the gelled specimen that will contain DNA is mixed with lysate, and 35 ℃～80 ℃ temperature were bathed 10～40 minutes.
In another preference, the kernel of described magnetic nanometer composite material is the magnetic ferrite nanoparticle, comprises Fe
3O
4Nanoparticle, γ-Fe
2O
3Nanoparticle or mix transition metal and the ferrite nano particles with superparamagnetism of compound, its shell is a silicon-dioxide.
In another preference, in step (c), also comprise step: divide and successively use washings I or cleaning solution II washing; Washing times is respectively 1～5 time, preferably is 2～3 times.The component of washings I is the salts solution of 1～8mol/l, the Tris-HCl of 0.5mol/l, pH=6.7, the EDTA of 5～50mmol/l; The component of cleaning solution II is the sodium acetate of 0～2mol/l.
The absorption that obtains by method steps of the present invention (b) magnetic nanometer composite material of DNA, can directly use, for example be used for the PCR reaction as template.Certainly, DNA desorb from the magnetic nanometer composite material can also be got off, form dna solution.Preferably, carry out step (c) again, add the elutriant contain Tris-HCl, stripping DNA from the magnetic nanometer composite material, thus obtain dna solution.
In a second aspect of the present invention, to pass through a kind of DNA and reclaimed test kit, it contains magnetic nanometer composite material, and described magnetic nanometer composite material has magnetic kernel and silica structure coating layer, and its diameter is 0.2～10 μ m.
Described test kit also contains:
Lysate, its component are the salts solution of 1～8mol/l, the Tris-HCl of 0.5mol/l, pH=6.7, the EDTA of 5～50mmol/l, 0.5～5%Triton;
Washings I, its component is the salts solution of 1～8mol/l, the Tris-HCl of 0.5mol/l, pH=6.7, the EDTA of 5～50mmol/l;
Cleaning solution II, its component are the sodium acetate (can be water) of 0～2mol/l;
Elutriant is 10 ± 2mmol/l Tris-HCl, pH=8.0 or deionized water.
In another preference, in lysate, described salts solution is NaCl or NaClO
4Solution or NaI solution, and concentration of salt solution is 5 ± 1mol/l; The concentration of described EDTA is 15 ± 5mmol/l;
In washings I, described salts solution is NaCl or NaClO
4Solution or NaI solution, and the concentration of salts solution is 4 ± 1mol/l; Described EDTA concentration is 20 ± 5mmol/l;
In cleaning solution II, the concentration of described sodium acetate is 1.5 ± 0.5mol/l.
Be used for lysate of the present invention, washings, elutriant and be not particularly limited, various lysates, washings, the elutriant that can adopt this area to adopt.In addition, the lysate in the test kit, washings, elutriant can be concentrated solution or diluent.
With the novel composite magnetic of the inventive method preparation, its size is adjustable between 0.2-10 μ m, and magnetic kernel is coated by the silica structural sheet of densification, can avoid coming off of magnetic particle fully.Simultaneously can slow down the oxidized speed of kernel, prolong its work-ing life.The outer silica structural sheet that coats has excellent biological compatibility.After utilizing different silylating reagents to carry out finishing, composite magnetic can combine with different pharmaceutical or biomolecules, thereby satisfies the special purposes such as directed transport of separation purification, detection, drug disposition or other biomolecules of external biological sample.
Major advantage of the present invention is:
1, utilize the magnetic nanometer composite material with superparamagnetism, a step is realized the recovery purifying of DNA.
2, method is simple, extracts fast, and operation can be finished in 1 hour usually.
3, can effectively remove inhibitor and pollutent, be applicable to Molecular Biology Lab.
4, according to the design of test kit, can estimate the extraction quantity of DNA, indicative function is played in later operation.
5, adopt the biological nano technology to carry out product development, significantly improve DNA extraction efficient, total extraction efficiency is more than 80%.
6, can with the supporting use of multiple biotechnology, as round pcr, enzyme incision technology, clone technology etc.
7, can realize the automatization of DNA reclaimer operation.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The preparation of magnetic nanometer composite material
Take by weighing the FeCl of 54.90g
36H
2The FeCl of O and 20.20g
24H
2O, the water dissolution of passing through letting nitrogen in and deoxidizing with 200ml obtains mixing solutions.Get 800ml water letting nitrogen in and deoxidizing in the round-bottomed flask of 1L, the mass percent concentration that adds 50ml is 25～28% strong aqua, rapidly to wherein pouring above-mentioned molysite mixing solutions into, reacts 1h down at 80 ℃ under vigorous stirring.After reaction finishes, isolate the solid of black with the permanent magnet of 0.1T from reaction soln, the gained solid obtains the Fe that particle diameter is 6～10nm 3～5 times with the high purity water cleaning
3O
4Nanoparticle.
Take by weighing 5.0g Na
2SiO
3(SiO
2Content is 45%) be dissolved in the 1000ml water, be 9.6 with the pH of resin cation (R.C.) regulator solution, to wherein adding the above-mentioned synthetic Fe of 2g
3O
4Nanoparticle.At room temperature, in the 1L round-bottomed flask, react 12h with the 150rpm stirring velocity.After reaction finishes, isolate the solid of black with the permanent magnet of 0.1T from reaction soln, the gained solid promptly gets the Fe that particle diameter is the coated with silica of 7～12nm 3～5 times with the high purity water cleaning
3O
4Nanoparticle.
In the 2L round-bottomed flask, add 900ml ethanol, 100ml water, 40ml mass percent concentration successively and be 25～28% strong aqua, 0.10g through the coated with silica synthetic Fe first time
3O
4Nanoparticle.At room temperature, drip the 0.6ml tetraethoxy, drip the 0.6ml tetraethoxy again behind the reaction 3h, continue reaction 12h with the 200rpm stirring velocity.Isolate the solid of black with the permanent magnet of 0.1T from reaction soln, the gained solid cleans with high purity water and promptly gets the multinuclear Fe that mean sizes is 0.4 μ m 3～5 times
3O
4-SiO
2Core-shell particle.
Embodiment 2
DNA reclaims test kit
Preparation utilizes the DNA of magnetic composite to reclaim test kit, comprises in it:
0.5ml concentration is magnetic nanometer composite material solution prepared among the embodiment 1 of 20mg/ml
100ml lysate, its component are 6mol/LNaI solution, the Tris-HCl of 0.5ml, pH=6.4, the EDTA of 25mmol/l;
100ml cleaning solution II, its component are the 1.5mol/l sodium acetate;
The 10ml elutriant is deionized water.
Embodiment 3
DNA reclaims test kit
Preparation utilizes the DNA of magnetic composite to reclaim test kit, and the magnetic nanometer composite material solution in it, cleaning solution II also comprise with embodiment 2:
100ml lysate, its component are 4mol/LNaCl solution, the Tris-HCl of 0.5ml, pH=6.4, the EDTA of 45mmol/l, 2.5%Triton;
100ml washings I, its component is the NaClO of 4mol/l
4Solution, the Tris-HCl of 0.5mol/l, pH=6.7, the EDTA of 25mmol/l;
10ml elutriant, its component are the Tris-HCl of 10mmol/l.
Embodiment 4
Reclaim sepharose DNA with magnetic nanometer composite material
1, (100mg～(gel fragment: the liquor capacity weight ratio) add in the lysate, the lysate component is with embodiment 2 500mg) to smash 1: 2 to 1: 4 by weight to pieces to cut the sepharose that contains dna fragmentation.
2,50 ℃ of dissolvings are dissolved fully until gel.
3, add magnetic nanometer composite material, room temperature absorption 20min.
4, be put into immediately behind the vortex vibration mixing test kit with magnet stand on, static 30s is adsorbed onto tube wall to the magnetic composite nano material.
5, after solution is abandoned in suction, with the washing of the cleaning solution II among the embodiment 23 times.
6, behind the drying at room temperature 10min, add elutriant, 65 ℃ of temperature are bathed 15min.
7, immediately centrifuge tube is put on the magnet stand, static 30s is adsorbed onto tube wall to magnetic nanometer composite material, and the sucking-off elutriant is stand-by to another centrifuge tube.
Embodiment 5
Reclaim DNA in the solution with magnetic nanometer composite material
1, in dna solution, adds 3 times of volume lysates, mixing.
All the other steps connect embodiment 4 steps 3～7.
Embodiment 6
Reclaim DNA in the polyacrylamide gel with magnetic nanometer composite material
1, cut the polyacrylamide gel that contains dna fragmentation and smash to pieces, soak with the lysate of the embodiment 2 of 2 times of volumes, 37 ℃ of temperature are bathed 2h.
2, the centrifugal 3min of 10000rpm draws the new centrifuge tube of supernatant solution to one.
3, all the other steps are with 3～7 of embodiment 4.
Embodiment 7
Reclaim DNA in the polyacrylamide gel with magnetic nanometer composite material
1, cut the polyacrylamide gel that contains dna fragmentation and smash to pieces, soak with 2 times of volume TNE, 37 ℃ of temperature are bathed 2h.
2, the centrifugal 3min of 10000rpm draws the new centrifuge tube of supernatant solution to one.
3, add 3 times of volume lysates, obtain recovery sample.
4, magnetic nanometer composite material is mixed with recovery sample, the magnetic nanometer composite material that will adsorb DNA with magnet is separated from suspension liquid again, thereby obtains being adsorbed with the magnetic nanometer composite material of DNA.
5, the magnetic nanometer composite material that will be adsorbed with DNA is used the cleaning solution II washed twice again with washings I washed twice, with elutriant DNA is parsed from magnetic nanometer composite material at last, thereby obtains pure dna solution.
Embodiment 8
Reclaim DNA in the PCR product with magnetic nanometer composite material
In pcr amplification product solution, add 3 times of volume lysates, mixing.
All the other steps are pressed embodiment 4 steps 3～7.
Embodiment 9
Reclaim the quality evalution of DNA
The dna direct that reclaims among the embodiment 4,7,8 is carried out electrophoresis.One of them swimming lane is that L is DNA standard substance ψ * 174/HaeIII (the allelotrope Ladder of pcr amplified fragment), so that compare the DNA recovering effect of recovery sample.
The result shows that all sample standard deviations have obtained ideal and reclaimed the result.But the DNA organic efficiency in agarose and polyacrylamide gel is lower than the dna fragmentation that reclaims slightly from pcr amplification product.
Claims (6)
1, a kind of method that reclaims DNA with magnetic nanometer composite material, it is characterized in that, said recovery DNA, be from lysate blended pcr amplification product, with lysate make adsorption liquid sample that DNA discharges from gelled specimen or the recovery samples such as biological specimen that discharged with lysate blended DNA reclaim DNA; Recycling step comprises:
(a) magnetic nanometer composite material is mixed with recovery sample, form solid-liquid homodisperse suspension liquid, the diameter of wherein said magnetic nanometer composite material is 0.2～10 μ m;
(b) magnetic nanometer composite material that will adsorb DNA with magnetic field is separated from solid-liquid homodisperse suspension liquid, thereby obtains being adsorbed with the magnetic nanometer composite material of DNA;
(c) with washings with the impurity flush away after, with elutriant DNA is parsed from magnetic nanometer composite material, thereby obtains pure dna solution; Said washings, its component are the salts solution of 1～8mol/l, the Tri(Hydroxymethyl) Amino Methane Hydrochloride of 0.5mol/l, pH=6.7, the ethylenediamine tetraacetic acid (EDTA) of 5～50mmol/l or be the sodium acetate of 0～2mol/l; Said elutriant is deionized water or 10 ± 2mmol/l Tris-HCl, pH=8.0.
2, according to the described method that reclaims DNA with magnetic nanometer composite material of claim 1, it is characterized in that, said adsorption liquid sample prepares by following steps: the gelled specimen that will contain DNA is mixed with lysate, and 35 ℃～80 ℃ temperature were bathed 10～40 minutes; Said gelled specimen is polyacrylamide gel, sepharose, dextrane gel.
According to claim 1 or 2 described methods, it is characterized in that 3, in (c) step, said washing divides and successively uses washings I, cleaning solution II washing with magnetic nanometer composite material recovery DNA; Washing times is respectively 1～5 time; The component of washings I is the salts solution of 1～8mol/l, the Tri(Hydroxymethyl) Amino Methane Hydrochloride of 0.5mol/l, pH=6.7, the ethylenediamine tetraacetic acid (EDTA) of 5～50mmol/l; The component of cleaning solution II is the sodium acetate of 0～2mol/l.
4, according to claim 1 or 2 described methods, it is characterized in that the kernel of said magnetic nanometer composite material is the magnetic ferrite nanoparticle, comprises Fe with magnetic nanometer composite material recovery DNA
3O
4Nanoparticle, γ-Fe
2O
3Nanoparticle or mix transition metal and the ferrite nano particles with superparamagnetism of compound, its shell is a silicon-dioxide.
5, a kind ofly reclaim the test kit of the method for DNA, it is characterized in that, contain in the test kit with magnetic nanometer composite material:
Magnetic nanometer composite material, said magnetic nanometer composite material have magnetic kernel and silica structure coating layer, and its diameter is 0.2～10 μ m;
Lysate, its component are the salts solution of 1～8mol/l, the Tri(Hydroxymethyl) Amino Methane Hydrochloride of 0.5mol/l, pH=6.7, the ethylenediamine tetraacetic acid (EDTA) of 5～50mmol/l, 0.5～5% Triton X-100;
Washings I, its component is the salts solution of 1～8mol/l, the Tri(Hydroxymethyl) Amino Methane Hydrochloride of 0.5mol/l, pH=6.7, the ethylenediamine tetraacetic acid (EDTA) of 5～50mmol/l;
Cleaning solution II, its component are the sodium acetate of 0～2mol/l;
Elutriant is 10 ± 2 mmol/l Tris-HCl, pH=8.0 or deionized waters.
According to the described test kit of claim 5, it is characterized in that 6, said salts solution is NaCl or NaClO
4Solution or NaI solution, and concentration of salt solution is 5 ± 1mol/l; The concentration of the ethylenediamine tetraacetic acid (EDTA) of being washed is 15 ± 5mmol/l; In washings I, said salts solution is NaCl or NaClO
4Solution or NaI solution, and the concentration of salts solution is 4 ± 1mol/l; Said ethylenediamine tetraacetic acid (EDTA) concentration is 20 ± 5mmol/l; In cleaning solution II, the concentration of said sodium acetate is 1.5 ± 0.5mol/l.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101818143A (en) * | 2010-04-28 | 2010-09-01 | 长春市博坤生物科技有限公司 | Ultra-low amount DNA extraction kit and method based on silica magnetic compound particles |
| CN102146369A (en) * | 2010-12-24 | 2011-08-10 | 吉林大学 | Coordination based method for extracting DNA (Deoxyribonucleic Acid) with solid-phase medium |
| CN102834518A (en) * | 2010-01-08 | 2012-12-19 | 霍夫曼-拉罗奇有限公司 | Improved recovery of nucleic acids from magnetic glass particles |
| CN105562132A (en) * | 2016-01-04 | 2016-05-11 | 上海医脉赛科技有限公司 | Device for extracting and detecting biological sample |
| CN105734044A (en) * | 2014-12-08 | 2016-07-06 | 苏州新波生物技术有限公司 | Rinsing liquid for nucleic acid extraction purification |
| CN112159806A (en) * | 2020-12-01 | 2021-01-01 | 广东和信健康科技有限公司 | Nucleic acid dissociation liquid and application thereof in nucleic acid extraction |
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2006
- 2006-11-29 CN CN 200610131676 patent/CN1970752A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102834518A (en) * | 2010-01-08 | 2012-12-19 | 霍夫曼-拉罗奇有限公司 | Improved recovery of nucleic acids from magnetic glass particles |
| CN102834518B (en) * | 2010-01-08 | 2015-04-01 | 霍夫曼-拉罗奇有限公司 | Improved recovery of nucleic acids from magnetic glass particles |
| CN101818143A (en) * | 2010-04-28 | 2010-09-01 | 长春市博坤生物科技有限公司 | Ultra-low amount DNA extraction kit and method based on silica magnetic compound particles |
| CN102146369A (en) * | 2010-12-24 | 2011-08-10 | 吉林大学 | Coordination based method for extracting DNA (Deoxyribonucleic Acid) with solid-phase medium |
| CN105734044A (en) * | 2014-12-08 | 2016-07-06 | 苏州新波生物技术有限公司 | Rinsing liquid for nucleic acid extraction purification |
| CN105562132A (en) * | 2016-01-04 | 2016-05-11 | 上海医脉赛科技有限公司 | Device for extracting and detecting biological sample |
| CN105562132B (en) * | 2016-01-04 | 2018-06-26 | 上海医脉赛科技有限公司 | A kind of device extracted and detect biological sample |
| CN112159806A (en) * | 2020-12-01 | 2021-01-01 | 广东和信健康科技有限公司 | Nucleic acid dissociation liquid and application thereof in nucleic acid extraction |
| CN112159806B (en) * | 2020-12-01 | 2021-03-30 | 广东和信健康科技有限公司 | Application of nucleic acid dissociation liquid in nucleic acid extraction and purification |
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