CN105524917A - Kit for extracting blood genome DNA based on magnetic bead method and use method for kit - Google Patents
Kit for extracting blood genome DNA based on magnetic bead method and use method for kit Download PDFInfo
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Abstract
The invention relates to a kit for extracting blood genome DNA based on a magnetic bead method. The kit comprises a cell lysis solution, a proteinase K solution, a magnetic bead dispersion solution, a first cleaning solution, a second cleaning solution and an elution solution, wherein the cell lysis solution comprises a guanidine compound, sodium chloride and a tween 20; a magnetic bead in the magnetic bead dispersion solution has the characteristics that a core is superparamagnetic ferroferric oxide, the surface is coated with silicon hydroxyl, and the particle size is 200-800nm; the first cleaning solution comprises Tris alkali, EDTA, isopropanol and water; the second cleaning solution is an ethanol solution; and the elution solution is Tris alkali or de-ionized water. The kit is simple to operate and can greatly improve working efficiency of blood genome DNA extraction; without pretreating erythrocytes, the cell lysis solution is directly adopted for pyrolyzing blood cells, and the magnetic bead dispersion solution can be added and combined with the genome DNA; and the kit is high in extraction volume and suitable for high-throughput experiments.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of test kit and the using method thereof of extracting poba gene group DNA based on paramagnetic particle method.
Background technology
Protocols in Molecular Biology develop rapidly in recent years, the research in genomic level has become the focus that numerous investigators study.Molecular biological a lot of normal experiment all needs to extract premised on genomic dna, no matter be gene sequencing, pcr amplification or structure BCA library etc., and the integrity of genomic dna concentration, purity, molecular weight ranges and the primary structure extracted all can have influence on downstream molecular biology experiment.In recent years precisely medical treatment and vitro diagnostic techniques flourish, need sample size to be processed is day by day huge, and therefore efficiently, conveniently, reliable, high-throughout DNA extraction method becomes the active demand of Related Research Domain.
The extracting method of DNA is a lot, as phenol/traditional extraction process such as chloroform extraction method, high salt precipitation method, because its extraction efficiency is low, is difficult to realize industrial applications, only still has a small amount of application at scientific research field.Commercial poba gene group DNA extraction method mainly centrifugal column method in the market, its principle be according on DNA and centrifugal column modify the reactive force such as hydrogen bond, electrostatic between chemical group, centrifugal column specific adsorption DNA in high level salt solution, after rinsing protein and other impurity, obtain genomic dna with low salt solutions wash-out.The more traditional DNA extraction method of centrifugal column type test kit is simple to operate, shortcoming operates in centrifuge tube for needing, DNA product can be obtained after needing eccentric cleaning repeatedly, operating process needs frequently to be produced by centrifugal column and proceed to centrifuge tube and coordinate a large amount of centrifugal work, cause the operating time to increase and needs are a large amount of artificial thereupon, can accept for its operation efficiency of one or several sample, but still undesirable for the work of extensive high-throughput DNA extraction.Current, the single projects of research field such as gene sequencing, precisely medical treatment need DNA sample quantity to be processed just can reach several ten thousand, hundreds of thousands of is even up to a million individual, investigators in the urgent need to develop one more efficiently, poba gene group DNA extraction method easily.
Paramagnetic particle method started to be applied in the Purification of different system DNA in recent years.Magnetic bead surfaces is modified with special chemical group, can form specific adsorption or desorption at different conditions, add and itself have paramagnetic characteristic DNA molecular, very convenient with mother liquor lock out operation after enrichment DNA.But method of the prior art needs after carrying out pre-treatment to red corpuscle, extracts the blood DNA sample of large volume.Though the method can carry out rapid extraction to the blood sample of large volume, can increase along with sample, due to erythrocytic pre-treatment step, then extraction time obviously increases, the workload also corresponding increase of experimenter.
Summary of the invention
For the deficiency that prior art exists, the object of the present invention is to provide a kind of test kit and using method thereof extracting poba gene group DNA based on paramagnetic particle method.This test kit can extract high purity genomic dna from the fresh or freezing anticoagulation of 200 μ L, and extracted amount can reach 8-12 μ g, and gained genomic dna product can directly apply to clinical vitro detection and use; And extraction step is simple and efficient, extraction efficiency is high.
For achieving the above object, the present invention is achieved through the following technical solutions:
Extract a test kit of poba gene group DNA based on paramagnetic particle method, include the cell pyrolysis liquid of independently packing, proteolytic enzyme k solution, magnetic bead dispersion liquid, the first scavenging solution, the second scavenging solution and elutriant;
Wherein, described cell pyrolysis liquid includes guanidine compound, sodium-chlor and polysorbas20; Described guanidine compound is selected from Guanidinium hydrochloride, different sulfuric acid cyanoguanidine or its combination;
Magnetic bead in described magnetic bead dispersion liquid adopts kernel to be SPIO, and Surface coating has silicone hydroxyl, and particle diameter is the magnetic bead of 200-800nm;
Described first scavenging solution includes Tris alkali, EDTA, isopropyl alcohol and water;
Described second scavenging solution is ethanolic soln;
Described elutriant is Tris alkali or deionized water.
Preferably, the described test kit extracting poba gene group DNA based on paramagnetic particle method, wherein, in described cell pyrolysis liquid, the concentration of guanidine compound is 5-7mol/L, and the concentration of sodium-chlor is 0.2-0.5mol/L, and the volume fraction of polysorbas20 is 0.5%-2%.
Preferably, the described test kit extracting poba gene group DNA based on paramagnetic particle method, wherein, the deionized water solution of described magnetic bead dispersion liquid to be magnetic bead concentration be 20-40 μ g/ μ L.
Preferably, the described test kit extracting poba gene group DNA based on paramagnetic particle method, wherein, in described first scavenging solution, Tris paper mill wastewater is the concentration of 30-100mmol/L, EDTA is 6-20mmol/L, and the volume fraction of Virahol is 45%-65%.
Preferably, the described test kit extracting poba gene group DNA based on paramagnetic particle method, wherein, when described elutriant is Tris alkali, Tris paper mill wastewater is 0.9-1.1mmol/L.
Use the extracting method extracting the test kit of poba gene group DNA based on paramagnetic particle method according to any one of such scheme, comprising:
Step one: add blood sample to be measured, proteolytic enzyme k solution and cell pyrolysis liquid in reaction cup successively, is placed on turbula shaker and mixes fully, cracking 8-10 minute at 55-60 DEG C subsequently;
Step 2: add magnetic bead dispersion liquid and Virahol successively in reaction cup, is placed in mixing on turbula shaker and fully, reaction cup is placed in subsequently on magnetic sheet frame and leaves standstill until magnetic bead absorption is complete, removing clear liquid;
Step 3: use the first scavenging solution and the second scavenging solution to clean magnetic bead successively;
Step 4: magnetic bead is placed in 45 DEG C of vacuum drying oven vacuum-drying 3-5 minute, takes out, adds elutriant, be placed on turbula shaker and fully mix, and leaves standstill 5min with being placed in 55-60 DEG C;
Step 5: be separated with elutriant by magnetic bead, namely obtains the elutriant containing target dna.
Preferably, the described extracting method extracting the test kit of poba gene group DNA based on paramagnetic particle method, wherein, described reaction vessel is 48 orifice plates.
The invention has the beneficial effects as follows:
1, operation is succinct, and poba gene group DNA extraction working efficiency can be made to be improved significantly.This case coordinates the use of 48 orifice plates and magnetic sheet frame, complete genome DNA extraction work in blood, without the need to carrying out in centrifuge tube, directly being operated in 48 orifice plates, autospencer can be used to complete automatic liquid-feeding, do not need frequently to use whizzer.2 piece of 48 orifice plate can process 96 samples simultaneously, only needs the used time to be about 40-60min, and contrast the experiment of current centrifugal column method DNA extraction, the latter need coordinate the expensive device such as knockout plate whizzer, and still needing to be greater than 1h can complete; And use centrifugal column method, when operator carry out larger amt sample operation, great effort need be spent to be used for operation that centrifugal column proceeded to and produced centrifuge tube, operation is busy, easily produce misoperation.
2, without the need to carrying out pre-treatment to red corpuscle, after directly adopting cell pyrolysis liquid cracking blood cell, magnetic bead dispersion liquid can be added in conjunction with genomic dna.
3, DNA extraction output is up to 8-12 μ g.The method is developed based on paramagnetic particle method, and magnetic bead surfaces is modified with the chemical group that can carry out specific adsorption to DNA, and can realize quick adsorption under high salt, low ph value, can dissociate fast again under less salt, high ph-values, DNA extraction output is high.
4, without the need to using the organic solvent such as phenol, chloroform, operator safety is had no side effect.
5, working efficiency obtains and significantly promotes, and is applicable to high-throughput experiment.
Accompanying drawing explanation
Fig. 1 is running gel figure (duct 1-2:2 part Omega centrifugal column method parallel laboratory test of extracting genomic dna product from blood in this case embodiment 1; Duct 3-5:3 part adopts the parallel laboratory test of this case isolation kit method).
Fig. 2 be in this case embodiment 2 from blood high-throughput extract complete genome DNA product running gel figure.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, can implement according to this with reference to specification sheets word to make those skilled in the art.
Embodiment 1
Extract a test kit of poba gene group DNA based on paramagnetic particle method, comprise independent packing:
The polysorbas20 mixing solutions of the Guanidinium hydrochloride of cell pyrolysis liquid: 6M, the sodium-chlor of 0.5M and volume fraction 1%.Concrete compound method: take 573g Guanidinium hydrochloride and 29g sodium-chlor, adds 800mL deionized water and stirring and dissolves, add 10mL polysorbas20, continue to add deionized water constant volume to 1L.
Proteolytic enzyme k solution: proteolytic enzyme k solution is a kind of commercial goods of maturation, it is the wider serine protease of a kind of nicking activity, and from woods Bai Shi Candida albicans, purifying obtains.Proteolytic enzyme k solution is exactly by obtained solution soluble in water for proteolytic enzyme k.
Magnetic bead dispersion liquid: concentration is the magnetic bead suspension of 20mg/mL, and magnetic bead kernel is Z 250 (Fe
3o
4), Surface coating SiO
2layer.Concrete compound method: take 2g magnetic bead, join in 100mL deionized water, ultrasonic disperse is even.
The isopropanol water solution of 50% (v/v) of the EDTA of Tris-HCl, 10mM of the first scavenging solution: 40mM.Concrete compound method: take 0.63gTris-HCl and 0.29gEDTA, the isopropanol water solution adding 80mL50% (v/v) fully dissolves, and continues to add the isopropanol water solution constant volume of 50% (v/v) to 100mL.
Second scavenging solution: the ethanolic soln of 80% (v/v).
The Tris alkali aqueous solution of elutriant: 1mM, pH value is 8.0.Concrete compound method: take 12mgTris alkali, adds 8 deionized waters and fully dissolves, and continues to add deionized water constant volume to 100mL, by concentrated hydrochloric acid adjust ph to 8.0.
Mentioned reagent box is used to extract poba gene group DNA product:
Step one: 4 DEG C or the freezing blood sample of thawed at room temperature; Water-bath is stabilized to 58 DEG C; The vibrational frequency of vortex mixed instrument is set as 1150rpm.
Step 2: add 200 μ L blood samples, 10 μ L proteolytic enzyme k solution and 200 μ L cell pyrolysis liquids in order with pipettor in 48 orifice plates, parallel 3 samples, vortex oscillation fully mixing in 30 seconds, is placed in water-bath cracking 10 minutes.
Step 3: take out 48 orifice plates, add 100 μ L magnetic bead dispersion liquids, 300 μ L Virahols respectively, 48 orifice plates are placed in after vortex mixed instrument vibrates 5 minutes, 48 orifice plates being loaded on 48 orifice plate dedicated magnetic grillages standing 20 seconds adsorbs completely to magnetic bead, 48 orifice plates are kept to be fixed on magnetic sheet frame, direct back-off abandoning supernatant, then back-off shape state is placed on thieving paper, thoroughly removes supernatant liquor and remains;
Step 4: 48 orifice plates are taken off from magnetic sheet base, 500 μ L first scavenging solutions are added respectively in sample well, 48 orifice plates are placed on vortex mixed instrument and vibrate 25 seconds, 48 orifice plates being reinstalled magnetic sheet frame adsorbs completely to magnetic bead in standing 20 seconds, 48 orifice plates are kept to be fixed on magnetic sheet frame, direct back-off abandoning supernatant, then back-off shape is placed on thieving paper, and thorough abandoning supernatant remains; Above cleaning operation twice is repeated subsequently with the second scavenging solution;
Step 5: 48 complete orifice plates of cleaning are placed in 45 DEG C of vacuum drying oven vacuum-dryings 3 minutes together with magnetic sheet frame, take out 48 orifice plates, in sample well, add 150 μ L elutriants respectively, 48 orifice plates are placed on vortex mixed instrument and vibrate 1 minute, 48 orifice plates are placed in water-bath and leave standstill 5 minutes;
48 orifice plates are positioned on magnetic sheet frame and leave standstill 20 seconds, keep 48 orifice plates to be fixed on magnetic sheet frame by step 6: taken off from vortex mixed instrument by 48 orifice plates, and transfer elutriant, in new centrifuge tube, namely obtains target dna product in the elutriant of clarification.
Experiment, simultaneously to same DNA sample, uses Omega column method poba gene group DNA extraction kit to carry out 2 parts of parallel control experiment.
Agarose gel electrophoresis detects: reclaim product to gained 5 parts of DNA, get 2 μ L loadings respectively, carry out agarose gel electrophoresis, glue figure (see Fig. 1) display obtain DNA product band complete display, not degraded, every part of DNA band brightness of extracting is suitable with Omega column method result.
OD value detects: use ultramicron ultraviolet-visible spectrophotometer to carry out OD value to 5 parts of DNA recovery samples and detect (see table 1).Detected result display uses present method from blood, extract that the genomic dna product assay obtained is high and purity is good.
In table 1 embodiment 1 from blood the OD value detected result of extracting genomic dna product
Note: sample 1-2:Omega column method experimental result; Sample 3-5: this test kit and methods experiment result.
Embodiment 2
The test kit of embodiment 1 is used to carry out high-throughput extracting experiment to 16 parts of poba gene group DNA:
Step one: 4 DEG C or the freezing blood sample of thawed at room temperature; Water-bath is stabilized to 58 DEG C; The vibrational frequency of vortex mixed instrument is set as 1150rpm.
Step 2: be sequentially added into 200 μ L blood samples, 10 μ L proteolytic enzyme k solution, 200 μ L cell pyrolysis liquids with hyperchannel autospencer in 48 orifice plates, parallel 16 increment product, vortex oscillation fully mixing in 30 seconds, is placed in water-bath cracking 10 minutes.
Step 3: take out 48 orifice plates, after adding 100 μ L magnetic bead dispersion liquids, 300 μ L Virahols respectively with hyperchannel autospencer in 16 sample wells, 48 orifice plates are placed in vortex mixed instrument to vibrate 5 minutes, 48 orifice plates being placed on 48 orifice plate magnetic sheet framves standing 20 seconds adsorbs completely to magnetic bead, 48 orifice plates are kept to be fixed on magnetic sheet frame, direct back-off abandoning supernatant, then back-off shape state is placed on thieving paper, thoroughly removes supernatant liquor and remains;
Step 4: 48 orifice plates are taken off from magnetic sheet base, in sample well, 500 μ L first scavenging solutions are added respectively with hyperchannel autospencer, afterwards 48 orifice plates are placed on vortex mixed instrument and vibrate 25 seconds, 48 orifice plates being reinstalled on magnetic sheet frame standing 20 seconds adsorbs completely to magnetic bead, 48 orifice plates are kept to be fixed on magnetic sheet frame, direct back-off abandoning supernatant, then back-off shape is placed on thieving paper, thorough abandoning supernatant; Above cleaning operation twice is repeated with the second scavenging solution;
Step 5: 48 complete orifice plates of cleaning are placed in 45 DEG C of vacuum drying oven vacuum-dryings 3 minutes together with magnetic sheet frame, take out 48 orifice plates, in sample well, 150 μ L elutriants are added respectively with hyperchannel autospencer, 48 orifice plates are placed in after vortex mixed instrument vibrates 1 minute, 48 orifice plates are put in water-bath and leave standstill 5 minutes;
Step 6: 48 orifice plates are taken off from vortex mixed instrument, 48 orifice plates are positioned on magnetic sheet frame and leave standstill 20 seconds, keep 48 orifice plates to be fixed on magnetic sheet frame, with hyperchannel autospencer transfer elutriant in PCR plate, obtain target dna product in the elutriant of clarification.
Agarose gel electrophoresis detects: reclaim product to gained 16 parts of DNA, get 2 μ L loadings respectively, carry out agarose gel electrophoresis, glue figure (see Fig. 2) display to obtain complete genome DNA product collimation good, band is clear, without hangover and assorted band, show that the genomic dna integrity extracted is good, and without other DNA pollution.
OD value detects: use ultramicron ultraviolet-visible spectrophotometer to reclaim product to 16 parts of genomic dnas and carried out OD value detection (see table 2).Detected result display uses present method to extract that the DNA product assay obtained is high to poba gene group DNA and purity is good.
In table 2 embodiment 2 from blood the OD detected value of high-throughput extracting genomic dna product
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend described.
Claims (7)
1. extract a test kit of poba gene group DNA based on paramagnetic particle method, it is characterized in that, include the cell pyrolysis liquid of independently packing, proteolytic enzyme k solution, magnetic bead dispersion liquid, the first scavenging solution, the second scavenging solution and elutriant;
Wherein, described cell pyrolysis liquid includes guanidine compound, sodium-chlor and polysorbas20; Described guanidine compound is selected from Guanidinium hydrochloride, different sulfuric acid cyanoguanidine or its combination;
Magnetic bead in described magnetic bead dispersion liquid adopts kernel to be SPIO, and Surface coating has silicone hydroxyl, and particle diameter is the magnetic bead of 200-800nm;
Described first scavenging solution includes Tris alkali, EDTA, isopropyl alcohol and water;
Described second scavenging solution is ethanolic soln;
Described elutriant is Tris alkali or deionized water.
2. the test kit of poba gene group DNA is extracted as claimed in claim 1 based on paramagnetic particle method, it is characterized in that, in described cell pyrolysis liquid, the concentration of guanidine compound is 5-7mol/L, and the concentration of sodium-chlor is 0.2-0.5mol/L, and the volume fraction of polysorbas20 is 0.5%-2%.
3. extract as claimed in claim 1 the test kit of poba gene group DNA based on paramagnetic particle method, it is characterized in that, the deionized water solution of described magnetic bead dispersion liquid to be magnetic bead concentration be 20-40 μ g/ μ L.
4. the test kit of poba gene group DNA is extracted as claimed in claim 1 based on paramagnetic particle method, it is characterized in that, in described first scavenging solution, Tris paper mill wastewater is the concentration of 30-100mmol/L, EDTA is 6-20mmol/L, and the volume fraction of Virahol is 45%-65%.
5. extract the test kit of poba gene group DNA as claimed in claim 1 based on paramagnetic particle method, it is characterized in that, when described elutriant is Tris alkali, Tris paper mill wastewater is 0.9-1.1mmol/L.
6. use the extracting method extracting the test kit of poba gene group DNA based on paramagnetic particle method according to any one of claim 1-5, it is characterized in that, comprising:
Step one: add blood sample to be measured, proteolytic enzyme k solution and cell pyrolysis liquid in reaction vessel successively, is placed on turbula shaker and mixes fully, cracking 8-10 minute at 55-60 DEG C subsequently;
Step 2: add magnetic bead dispersion liquid and Virahol successively in reaction vessel, is placed in mixing on turbula shaker and fully, is placed in by reaction vessel subsequently on magnetic sheet frame and leaves standstill until magnetic bead absorption is complete, removing clear liquid;
Step 3: use the first scavenging solution and the second scavenging solution to clean magnetic bead successively;
Step 4: magnetic bead is placed in 45 DEG C of vacuum drying oven vacuum-drying 3-5 minute, takes out, adds elutriant, be placed on turbula shaker and fully mix, and leaves standstill 5min with being placed in 55-60 DEG C;
Step 5: be separated with elutriant by magnetic bead, namely obtains the elutriant containing target dna.
7. extract the extracting method of the test kit of poba gene group DNA as claimed in claim 6 based on paramagnetic particle method, it is characterized in that, described reaction vessel is 48 orifice plates.
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| CN108048454A (en) * | 2018-02-08 | 2018-05-18 | 天津威高分子诊断科技有限公司 | A kind of paramagnetic particle method bone genome DNA extracting reagent kit |
| CN109207474A (en) * | 2018-10-10 | 2019-01-15 | 南通中科基因医学检验所有限公司 | A kind of peripheral blood genome DNA rapid extraction method |
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| CN111019940A (en) * | 2019-12-25 | 2020-04-17 | 上海派森诺生物科技股份有限公司 | Extracting solution for directly extracting whole blood genome DNA and extracting method thereof |
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| CN112592919A (en) * | 2020-12-22 | 2021-04-02 | 哈尔滨赛信生物科技开发有限公司 | Kit for extracting blood genome DNA by using paramagnetic particle method and application thereof |
| CN112553193A (en) * | 2020-12-23 | 2021-03-26 | 苏州中科先进技术研究院有限公司 | Kit for extracting whole blood DNA by paramagnetic particle method and use method thereof |
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| WO2023050156A1 (en) * | 2021-09-29 | 2023-04-06 | 京东方科技集团股份有限公司 | Nucleic acid extraction composition, nucleic acid extraction device and nucleic acid extraction method |
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Application publication date: 20160427 |