CN111073886B - DNA extraction adsorption liquid based on superparamagnetism nano-particles, kit and DNA extraction method - Google Patents
DNA extraction adsorption liquid based on superparamagnetism nano-particles, kit and DNA extraction method Download PDFInfo
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- 238000001179 sorption measurement Methods 0.000 title claims abstract description 64
- 239000002105 nanoparticle Substances 0.000 title claims abstract description 56
- 238000007400 DNA extraction Methods 0.000 title claims abstract description 37
- 239000007788 liquid Substances 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 30
- 238000000605 extraction Methods 0.000 claims abstract description 51
- 239000000243 solution Substances 0.000 claims abstract description 48
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 32
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims abstract description 28
- 238000007885 magnetic separation Methods 0.000 claims abstract description 19
- 238000002156 mixing Methods 0.000 claims abstract description 19
- 239000012224 working solution Substances 0.000 claims abstract description 17
- 239000011780 sodium chloride Substances 0.000 claims abstract description 15
- 229910001629 magnesium chloride Inorganic materials 0.000 claims abstract description 14
- 239000007791 liquid phase Substances 0.000 claims abstract description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 22
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 14
- 239000012498 ultrapure water Substances 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 10
- 239000002245 particle Substances 0.000 claims description 9
- 238000010828 elution Methods 0.000 claims description 5
- 239000012530 fluid Substances 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 4
- 239000004353 Polyethylene glycol 8000 Substances 0.000 claims 1
- 229940085678 polyethylene glycol 8000 Drugs 0.000 claims 1
- 235000019446 polyethylene glycol 8000 Nutrition 0.000 claims 1
- 239000002202 Polyethylene glycol Substances 0.000 abstract description 19
- 229920001223 polyethylene glycol Polymers 0.000 abstract description 19
- 108020004414 DNA Proteins 0.000 description 65
- 239000006249 magnetic particle Substances 0.000 description 40
- 230000005291 magnetic effect Effects 0.000 description 12
- 239000003147 molecular marker Substances 0.000 description 10
- 238000000246 agarose gel electrophoresis Methods 0.000 description 9
- 238000012163 sequencing technique Methods 0.000 description 9
- 238000003860 storage Methods 0.000 description 9
- 238000002474 experimental method Methods 0.000 description 8
- 239000011550 stock solution Substances 0.000 description 8
- 238000004140 cleaning Methods 0.000 description 7
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- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
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- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- -1 salt ions Chemical class 0.000 description 3
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
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- 239000000047 product Substances 0.000 description 2
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- 238000000926 separation method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229920002594 Polyethylene Glycol 8000 Polymers 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 231100000481 chemical toxicant Toxicity 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- WTFXARWRTYJXII-UHFFFAOYSA-N iron(2+);iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+2].[Fe+3].[Fe+3] WTFXARWRTYJXII-UHFFFAOYSA-N 0.000 description 1
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Abstract
The invention provides a DNA extraction adsorption solution based on superparamagnetism nano-particles, a kit and a DNA extraction method, belonging to the technical field of DNA extraction, wherein the adsorption solution comprises polyethylene glycol, sodium chloride and magnesium chloride; the concentration of the polyethylene glycol is 100-250 mg/ml; the concentration of the sodium chloride is 1.25-2.75 mol/L; the concentration of the magnesium chloride is 10-20 mmol/L. The extraction method comprises the following steps: 1) Mixing superparamagnetic nano-particles with an adsorption solution; 2) Mixing a DNA sample to be extracted with a working solution, performing magnetic separation after adsorption extraction, and collecting superparamagnetic nanoparticles adsorbed with DNA; 3) Eluting the superparamagnetism nano particles adsorbed with DNA, magnetically separating, and collecting liquid phase components to obtain the extracted DNA. The adsorption liquid has simple components and high adsorption efficiency; the extraction method has the advantages of high extraction speed and low cost.
Description
Technical Field
The invention belongs to the technical field of DNA extraction, and particularly relates to a DNA extraction adsorption solution based on superparamagnetic nano particles, a kit and a DNA extraction method.
Background
DNA samples and DNA-based experiments are most common and fundamental in the life sciences field. High concentration, high purity DNA extraction is a key step in molecular biology experiments, and at the same time, it is the first step in the basis of many downstream experiments. Traditional methods of DNA extraction rely primarily on phenol chloroform or column purification. However, the use of toxic chemicals and the low extraction efficiency make both methods inadequate for the needs of modern scientific research.
Over the past few decades, magnetic beads have been widely used in nucleic acid extraction. The magnetic particle extracting reagent mainly comprises two parts of magnetic particles and adsorption liquid. The adsorption liquid mainly comprises polyethylene glycol (PEG) and salt ions. PEG and salt ions can phase change DNA molecules to adsorb to the magnetic particles. It is noted that the molecular weight and concentration of PEG and the type and concentration of salt ions used affect the effect of the adsorption solution. Therefore, under different extraction conditions, specific adjustment of the components and proportions of the adsorption solution is required according to the size of the target DNA fragment to be extracted and the nature of the magnetic particles to be used.
The magnetic particles are generally formed by wrapping ferroferric oxide by different modification groups, and the size, shape, surface group and magnetic response time of the different magnetic particles are different. There is also a large difference in concentration and purity obtained by nucleic acid extraction based on different magnetic particles. Among various magnetic particles, superparamagnetic nano particles (super paramagnetic iron oxide nanoparticles, SPION) have the fastest magnetic response time, have smaller average particle diameter, have no phenomenon of magnetic retention after the magnetic field is evacuated, and have more stable magnetic properties, so that the SPION has a larger application prospect in nucleic acid extraction. However, how to maximize the advantages of SPION has yet to be explored, and DNA extraction methods specific to SPION are still lacking. The exploration of the optimal use method of the SPION becomes an effective way for accelerating the popularization of the application of the technology and further improving the DNA extraction effect.
With the birth and rapid development of chip technology and second generation sequencing technology, a plurality of genome sequencing plans are proposed at home and abroad, and library construction and sequencing are becoming an indispensable link in scientific research. The enormous sequencing application requirements place higher demands on both sequencing costs and library construction costs. With the development of a technical platform, the sequencing cost gradually decreases, however, the high kit makes the library construction cost high, and the library construction cost of some samples is even higher than the sequencing cost of the samples. It is worth noting that the existing sequencing platform terminal is used for reading the DNA sequence, so that the operations such as extraction and purification of the DNA become the most basic and unavoidable links in the whole molecular biology experiment and library construction, and the research and development of the accurate, efficient and low-cost DNA extraction method is a key measure for reducing the construction cost of the whole library. This great need has prompted the creation of a variety of commercial magnetic particle extraction kits. However, the magnetic particles and adsorption liquid adopted by the existing extraction kit are different, so that the extraction effect is greatly different, and the price of the extraction kit is very high. The most widely used imported Beckman AMPure XP kit at present has the price of 216.67 yuan/ml; the extraction efficiency of other domestic magnetic particle extraction kits is lower, the scientific research requirements of high efficiency and accuracy are not met, and the price of the kit is about 100 yuan/ml. The high DNA extraction kit is very unreasonable, and the current situation also limits the development of basic biology and the popularization of sequencing technology in China to a certain extent.
Therefore, there is a need to develop a highly efficient and inexpensive DNA extraction method based on superparamagnetic nanoparticles.
Disclosure of Invention
In view of the above, the present invention aims to provide a superparamagnetic nanoparticle-based DNA extraction adsorption solution, a kit and a DNA extraction method; the adsorption liquid has simple components and high adsorption efficiency; the DNA extracted by the extraction method has high concentration and good purity; the extraction speed is high and the cost is low.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a DNA extraction adsorption solution based on superparamagnetism nano particles, which comprises polyethylene glycol, sodium chloride and magnesium chloride; the concentration of polyethylene glycol in the adsorption liquid is 100-250 mg/ml; the concentration of the sodium chloride is 1.25-2.75 mol/L; the concentration of magnesium chloride is 10-20 mmol/L.
Preferably, the concentration of polyethylene glycol in the adsorption liquid is 180-220 mg/ml; the concentration of the sodium chloride is 2.4-2.6 mol/L; the concentration of magnesium chloride is 15-18 mmol/L.
The invention provides a DNA extraction kit, which comprises the DNA extraction adsorption solution and superparamagnetic nano particles; the particle size of the superparamagnetic nano particles is 100 nm-1 mu m; the concentration of the superparamagnetic nano particles is 0.625-10 mg/ml.
The invention provides a DNA extraction method based on superparamagnetic nano particles, which comprises the following steps:
1) Mixing superparamagnetic nano particles with an adsorption solution to obtain a working solution;
2) Mixing a DNA sample to be extracted with the working solution in the step 1), performing magnetic separation after adsorption extraction, and collecting superparamagnetic nanoparticles adsorbed with DNA;
3) Eluting the superparamagnetism nano particles adsorbed with DNA, magnetically separating, and collecting liquid phase components to obtain the extracted DNA.
Preferably, the volume ratio of the superparamagnetic nanoparticle to the adsorption solution is 1 (1-16).
Preferably, the volume ratio of the DNA sample to be extracted to the working solution is 1 (0.5-2).
Preferably, the eluent used in the elution in step 3) is ultrapure water or TE solution.
Preferably, the magnetic separation time in step 2) and step 3) is independently 30s to 2min.
Preferably, after collecting the superparamagnetic nanoparticles adsorbed with DNA in step 2), a washing step is further included.
Preferably, the times of the cleaning are 1-3 times, and the cleaning is performed by ethanol with the volume concentration of 70% -80%.
The invention has the beneficial effects that: the DNA extraction adsorption liquid based on superparamagnetism nano particles provided by the invention comprises polyethylene glycol, sodium chloride and magnesium chloride; the components are simple, and the adsorption efficiency is high; the adsorption efficiency of the DNA fragment with the length of more than 100bp reaches more than 95 percent; the concentration of the magnetic nano particles needed by the adsorption liquid is low; the extraction method has short extraction time of only 12min, and the extraction cost of the method provided by the invention is as low as 4.38 yuan/ml; the DNA extracted by the extraction method has high concentration and high purity, and can be directly used for subsequent enzyme digestion, connection, PCR, sequencing and other reactions.
Drawings
FIG. 1 is a schematic diagram of a method for extracting DNA according to the present invention;
FIG. 2 is a schematic diagram of the method in example 1Prefabricated agarose gel electrophoresis systemAnalysis results of the extracted DNA molecular markers M1;
FIG. 3 is a schematic diagram of the embodiment 1 of the present inventionAnalysis results of the extracted DNA molecular markers M2 by a preformed agarose gel electrophoresis system.
FIG. 4 is a diagram of the embodiment 2 of the present inventionAnalysis results of the extracted DNA molecular markers M2 by a preformed agarose gel electrophoresis system.
Detailed Description
The invention provides a DNA extraction adsorption solution based on superparamagnetism nano particles, which comprises polyethylene glycol, sodium chloride and magnesium chloride; the concentration of polyethylene glycol in the adsorption liquid is 100-250 mg/ml; the concentration of the sodium chloride is 1.25-2.75 mol/L; the concentration of magnesium chloride is 10-20 mmol/L.
In the present invention, the concentration of polyethylene glycol in the adsorption solution is preferably 180 to 220mg/ml, more preferably 200mg/ml; the concentration of sodium chloride is preferably 2.4 to 2.6mol/L, more preferably 2.5mol/L; the concentration of the magnesium chloride is preferably 15 to 18mmol/L, more preferably 16.3mmol/L.
In the invention, the polyethylene glycol, sodium chloride and magnesium chloride are all commercially available products. In the present invention, the solvent of the adsorption liquid is preferably water; the method of disposing the adsorption liquid in the present invention is not particularly limited as long as three components can be realized within the above concentration ranges. In the present invention, the preparation method of the adsorption liquid preferably includes the following steps: s1) mixing and dissolving ultrapure water and sodium chloride, and filtering the mixture to obtain sodium chloride stock solution; s2) mixing and dissolving ultrapure water and magnesium chloride, and filtering the mixture to obtain magnesium chloride stock solution; s3) mixing and dissolving the sodium chloride stock solution, the magnesium chloride stock solution and the polyethylene glycol, and then filtering the mixture to obtain an adsorption solution. In the invention, the mixed dissolution is preferably carried out by adopting a room temperature rotator to turn over and dissolve; the pore size of the filter membrane is preferably 0.22. Mu.m. In the present invention, the adsorption solution is preferably stored at 3 to 5 ℃, more preferably at 4 ℃.
The invention provides a DNA extraction kit, which comprises the DNA extraction adsorption solution and superparamagnetic nano particles; the particle size of the superparamagnetic nano particles is 100 nm-1 mu m, more preferably 200-500 nm, most preferably 300nm; the concentration of the superparamagnetic nano-particles is 0.625-10 mg/ml, preferably 1.0-3.0 mg/ml, more preferably 1.25mg/ml. The source of the superparamagnetic nanoparticle is not particularly limited, and the superparamagnetic nanoparticle can be obtained by adopting a commercially available product which is conventional in the field.
The invention provides a DNA extraction method based on superparamagnetic nano particles, which comprises the following steps: 1) Mixing superparamagnetic nano particles with an adsorption solution to obtain a working solution; 2) Mixing a DNA sample to be extracted with the working solution in the step 1), performing magnetic separation after adsorption extraction, and collecting superparamagnetic nanoparticles adsorbed with DNA; 3) Eluting the superparamagnetism nano particles adsorbed with DNA, magnetically separating, and collecting liquid phase components to obtain the extracted DNA.
In the invention, superparamagnetic nano-particles are mixed with an adsorption solution to obtain a working solution. In the present invention, the volume ratio of the superparamagnetic nanoparticle to the adsorption solution is preferably 1 (1-16), more preferably 1 (6-10), and even more preferably 1:8. In the present invention, the mixing is achieved by vortex shaking. In the present invention, the superparamagnetic nanoparticle preferably further comprises a separation and washing process of the superparamagnetic nanoparticle before mixing with the adsorption solution. In the present invention, commercial superparamagnetic nanoparticles are preferably separated from the storage liquid by magnetic separation; then cleaning the superparamagnetic nano particles obtained by separation; the cleaning is preferably performed by using TE or ultrapure water, the number of times of cleaning is preferably 2, and the added volume of the TE or the ultrapure water is preferably 1-2 times of the volume of the superparamagnetic nano-particles. After the cleaning is finished, the ultrapure water is discarded, so that the residual liquid is fully volatilized. In the present invention, the working fluid may be used in batches for a short period of time, and the effective use time of the working fluid is within 30 days, preferably within 7 days, after completion of the preparation.
After working solution is obtained, mixing a DNA sample to be extracted with the working solution, performing magnetic separation after adsorption extraction, and collecting superparamagnetic nanoparticles adsorbed with DNA. In the present invention, the volume ratio of the DNA sample to be extracted to the working fluid is preferably 1 (0.5-2), more preferably 1:1.2. In the invention, the adsorption extraction is preferably standing incubation, and the time of the adsorption extraction is preferably 2min; the temperature of the adsorption extraction is preferably 25 ℃. In the present invention, the DNA sample to be extracted is not particularly limited, and all liquid samples containing DNA may be used. In the present invention, the mixing is preferably achieved by vortex oscillation. In the present invention, the time for the magnetic separation is preferably 30s to 2min. In the invention, after collecting the superparamagnetic nano-particles adsorbed with DNA, a washing step is further included; the number of times of the washing is preferably 1 to 3, more preferably 2, and the washing is preferably performed with ethanol having a volume concentration of 70% to 80%, more preferably with ethanol having a volume concentration of 75%. In the present invention, the added volume of the ethanol is preferably 1 to 2 times the volume of the superparamagnetic nanoparticle adsorbed with DNA. After the cleaning is finished, the ethanol is discarded, so that the residual liquid is fully volatilized. In the invention, after the working mixture is mixed with the DNA sample to be extracted, under the action of PEG, DNA molecules undergo a phase change process from spiral shape to sphere shape, and compressed sphere-shaped DNA molecules pass through Na + Is connected with carboxyl groups on the magnetic particles, thereby realizing the adsorption of DNA and the magnetic particles.
In the invention, the superparamagnetic nano-particles adsorbed with DNA are eluted, the magnetic separation is carried out, and the liquid phase component is collected, thus obtaining the extracted DNA. In the present invention, the eluent used for the elution is preferably ultrapure water or TE solution. In the present invention, the time for the magnetic separation is preferably 30s to 2min. In the present invention, the DNA obtained by the extraction is preferably stored at 3 to 5℃and more preferably at 4 ℃. In the invention, the elution utilizes the different solution properties of the eluent, and DNA molecules are recovered from spherical phase change to spiral phase change and are disconnected with magnetic particles, thereby realizing the elution of DNA;
the technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
The principle and steps of DNA extraction in this example are shown in FIG. 1; the method comprises the following specific steps:
preparation of adsorption liquid
The adsorption liquid in the example is composed of PEG, naCl, mgCl 2 The three components. The specific preparation method of the storage solution comprises the following steps:
storage solution A: 14.61g of NaCl is weighed, 40ml of ultrapure water is added, the mixture is placed in a room temperature rotary instrument for overturning and dissolving, after most of white particles are dissolved, the mixture is placed at room temperature for 1 to 6 hours, bubbles in a tube are fully dispersed, tube wall liquid drops are fully polymerized, the volume is conveniently and accurately fixed, the volume is fixed to 50ml, and then overturning and dissolving are carried out again. After complete dissolution, filtration was completed with a 0.22 μm microfiltration membrane filter and stored at 4℃for further use.
Storage solution B: weighing 4.066g of MgCl 2 ·6H 2 O is added with 4ml of ultrapure water, and is put into a room temperature rotator for overturning and dissolving, after the particles are dissolved, the particles are kept stand for longer than 1 to 6 hours at room temperature, the volume is fixed to 10ml, and then overturning and dissolving are carried out again. After complete dissolution, filtration was completed with a 0.22 μm microfiltration membrane filter and stored at 4℃for further use.
And preparing the adsorption liquid after the storage liquid is prepared. The specific method for preparing the adsorption liquid comprises the following steps: 4ml of stock solution A and 81.5. Mu.l of stock solution B were mixed, and 2g of PEG8000 was added. And (5) placing the mixture in a room temperature rotator for overturning and dissolving. After most of the PEG white tablet is dissolved, the solution is kept standing for longer than 1h at room temperature, then the volume is fixed to 10ml, and then the solution is turned over again. After complete dissolution, filtration was completed with a 0.22 μm microfiltration membrane filter and stored at 4℃for further use.
Preparation of magnetic particle working solution
The magnetic particle working solution consists of superparamagnetic nano particles and adsorption solution, and the carboxyl-modified coated magnetic particles adopted in the embodiment can be directly purchased from a biochemical reagent store, and the storage solution is 20% ethanol solution. The specific steps for preparing the magnetic particle working mixed liquid are as follows:
(1) Incubating the magnetic particle stock solution in advance. In order to ensure the accuracy of the volume, the magnetic particle storage liquid needs to be taken out from the temperature of 4 ℃ in advance, and the standing room temperature is longer than 30min. (it should be noted that this step is not necessary, as the temperature does not affect the properties of the superparamagnetic nanoparticle.
(2) 100 μl of the uniformly mixed magnetic particles were placed in an EP tube and magnetically separated. Under the action of external magnetic field such as magnetic frame, separating liquid phase from magnetic particles for 2min.
(3) The liquid phase was discarded and washed twice. After magnetic separation, the liquid phase was discarded by a pipette, and washed twice with 200. Mu.l of ultrapure water, and after washing, the residual liquid was sucked by a pipette.
(4) 800. Mu.l of the adsorption solution was added. The tube cap was closed and vortexed for 30s.
Extraction of DNA
In the invention, DNA molecules with different fragment sizes are taken as research objects, and in the embodiment, two different DNA molecules are adopted, namely a 50 bp-2500 bpDNA molecular marker M1 and a 100 bp-10 kbDNA molecular marker M2; the development of the subsequent experiments is supported by the following. The extraction process of different DNAs is consistent, and in this example, the molecular marker M1 is taken as an example to specifically describe the specific extraction process as follows:
(1) Incubating the magnetic particle working mixed solution in advance. In order to ensure the accuracy of the volume, the magnetic particle working mixed solution needs to be taken out from the temperature of 4 ℃ in advance and kept at the room temperature for longer than 30min. Mu.l of each M1 solution was placed in 2 different EP tubes, labeled s1, s2, respectively.
(2) s1 and s2 are added with 60 mu l of magnetic particle working solution. Respectively blowing and mixing, and standing at room temperature for 5min.
(3) Is arranged on a magnetic frame for magnetic separation. The magnetic separation time was 2min.
(4) The supernatant was discarded and washed twice with 80% (v/v) ethanol
(5) After all ethanol residues are completely volatilized, 20 μl of ultrapure water is added, and the DNA is eluted by blowing and mixing.
(6) After magnetic separation again, the liquid phase was transferred to a new EP tube.
(7) The obtained DNA solution was usedThe concentration was measured by the instrument and +.>The preformed agarose gel electrophoresis system detects the extent of the DNA bands.
In addition, beckman AMPure XP is taken as a comparison study in this example, and the extraction experiment of AMPure XP is performed according to the specification thereof, and the extracted samples are respectively marked as x1 and x2.
Analysis of results
The obtained DNA molecular marker M1 results, the DNA band range adoptsThe detection result of the prefabricated agarose gel electrophoresis system is shown in figure 2; the concentration is->The results of the instrumental measurements are shown in Table 1.
TABLE 1 extraction results of DNA molecular marker M1
The obtained DNA molecular marker M2 results, the DNA band range adoptsThe detection result of the prefabricated agarose gel electrophoresis system is shown in figure 3; the concentration is->The results of the instrument measurements are shown in Table 2.
TABLE 2 extraction results of DNA molecular marker M2
From this, it can be seen that the extraction efficiencies obtained by the two methods in tables 1 and 2 are almost identical, and that the present invention has an even more stable effect. While the slaveAs can be seen from the detection results of the prefabricated agarose gel electrophoresis system in FIG. 2 and FIG. 3, the ranges of the DNA bands obtained by the two methods are almost consistent, and the invention has recovery efficiency of nearly 100% for the DNA fragments of 150 bp-10 kp. Therefore, the invention can completely replace the existing AmPure XP kit in terms of extraction effect, and is even better. Meanwhile, the extraction cost of the invention is as low as 4.38 yuan/ml, and the cost is only 1/50 of that of an AMPure XP kit and 1/25 of that of a domestic kit.
Example 2
The principle and steps of DNA extraction in this example are shown in FIG. 1; the method comprises the following specific steps:
preparation of adsorption liquid
The adsorption solution in this example consisted of PEG, naCl, mgCl2 three components, and was prepared as in example 1.
Preparation of magnetic particle working solution
The magnetic particle working solution consists of superparamagnetic nano particles and adsorption solution, and the carboxyl-modified coated magnetic particles adopted in the embodiment can be directly purchased from a biochemical reagent store, and the storage solution is 20% ethanol solution. The specific steps for preparing the magnetic particle working mixed liquid are as follows:
incubating the magnetic particle stock solution in advance. In order to ensure the accuracy of the volume, the magnetic particle storage liquid needs to be taken out from the temperature of 4 ℃ in advance, and the standing room temperature is longer than 30min. (it should be noted that this step is not necessary, as the temperature does not affect the properties of the superparamagnetic nanoparticle.
100 μl of the uniformly mixed magnetic particles were placed in an EP tube and magnetically separated. Under the action of external magnetic field such as magnetic frame, separating liquid phase from magnetic particles for 2min.
The liquid phase was discarded and washed twice. After magnetic separation, the liquid phase was discarded by a pipette, and washed twice with 200. Mu.l of ultrapure water, and after washing, the residual liquid was sucked by a pipette.
100. Mu.l, 400. Mu.l, 800. Mu.l and 1600. Mu.l of the adsorption solution were added to give final concentrations of the magnetic particles of 10mg/ml,2.5mg/ml,1.25mg/ml and 0.625mg/ml, respectively, and these were designated as magnetic particle working solutions Mwb (Mag working buffer 10), mwb2.5, mwb1.25 and Mwb0.625, respectively. The tube cap was closed and vortexed for 30s.
Extraction of DNA
In the invention, DNA molecules with different fragment sizes are taken as research objects, and in the embodiment, the DNA molecular marker M1 with the fragment of 50 bp-2500 bpis adopted. The specific extraction process is as follows:
incubating the magnetic particle working mixed solution in advance. In order to ensure the accuracy of the volume, the magnetic particle working mixed solution needs to be taken out from the temperature of 4 ℃ in advance and kept at the room temperature for longer than 30min. Mu.l of each M1 solution was placed in 8 different EP tubes, labeled 10A,10B,2.5A,2.5B,1.25A,1.25B, 0.245A, 0.245B, respectively; 60. Mu.l of the magnetic particle working fluid Mwb, mwb2.5, mwb1.25 and Mwb0.625 were added in this order. Respectively blowing and mixing, and standing at room temperature for 5min.
Is arranged on a magnetic frame for magnetic separation. The magnetic separation time was 2min.
The supernatant was discarded and washed twice with 80% (v/v) ethanol
After all ethanol residues are completely volatilized, 20 μl of ultrapure water is added, and the DNA is eluted by blowing and mixing.
After magnetic separation again, the liquid phase was transferred to a new EP tube.
The obtained DNA solution was usedThe concentration was measured by the instrument and +.>The preformed agarose gel electrophoresis system detects the extent of the DNA bands.
In addition, beckman AMPure XP is taken as a comparison study in this example, and an extraction experiment of AMPure XP is performed according to the specification thereof, and the extracted samples are respectively marked as xA and xB.
Analysis of results
The obtained DNA molecular marker M1 results, the DNA band range adoptsThe detection result of the prefabricated agarose gel electrophoresis system is shown in fig. 4; the concentration is->The results of the instrument measurements are shown in Table 3.
TABLE 3 extraction results of DNA molecular marker M1
From the results, it can be seen that the final concentration of different superparamagnetic particles can extract DNA with high efficiency, and the DNA extraction efficiency is from 85% to 97% when the final concentration of superparamagnetic particles is between 0.625 and 10mg/mlAs can be seen from FIG. 4, the DNA bands obtained by the different methods are almost identical. Therefore, the invention can completely replace the existing AmPure XP kit in terms of extraction effect, and is even better. Meanwhile, in a specific experiment, different magnetic particle concentrations can be used according to different requirements, and the extraction cost of a preferable method for the final concentration of the magnetic particles of 1.25mg/ml is as low as 4.38 yuan/ml, and the cost is only 1/50 of an AMPure XP kit and 1/25 of a domestic kit; if more DNA samples are used, the final concentration of 0.625mg/ml can be adopted, so that the extraction efficiency of more than 85% is ensured, and the cost is further reduced.
According to the embodiment, the invention specifically aims at the superparamagnetic nano-particles, the simplest optimized adsorption solution is adopted, the DNA extraction process is simple and rapid, the effect is equivalent or even better, and the cost is far lower than that of the existing kit; these all make the present invention irreplaceable advantages over existing DNA extraction methods.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. A DNA extraction method based on superparamagnetic nano particles, which is characterized by comprising the following steps:
1) Mixing the superparamagnetic nano particles with the DNA extraction adsorption solution to obtain a working solution;
2) Mixing a DNA sample to be extracted with the working solution in the step 1), performing magnetic separation after adsorption extraction, and collecting superparamagnetic nanoparticles adsorbed with DNA;
the adsorption extraction is carried out, and the standing time is 5min;
3) Eluting the superparamagnetism nano-particles adsorbed with DNA, magnetically separating, and collecting liquid phase components to obtain the extracted DNA;
the DNA extraction adsorption liquid is DNA extraction adsorption liquid based on superparamagnetic nano particles;
the concentration of polyethylene glycol 8000 in the extracting and adsorbing solution is 200mg/ml; the concentration of sodium chloride is 2mol/L; the concentration of magnesium chloride is 16.3mmol/L;
the particle size of the superparamagnetic nano particles is 300nm; the concentration of the superparamagnetic nano-particles is 1.25mg/ml.
2. The extraction method according to claim 1, wherein the volume ratio of the superparamagnetic nanoparticles to the adsorption solution in step 1) is 1 (1-16).
3. The extraction method according to claim 1, wherein the volume ratio of the DNA sample to be extracted in the step 2) to the working fluid is 1 (0.5-2).
4. The method according to claim 1, wherein the eluent used in the elution in step 3) is ultrapure water or TE solution.
5. The extraction method according to claim 1, wherein the magnetic separation time in step 2) and step 3) is independently 30s to 2min.
6. The method of claim 1, further comprising a washing step after collecting the DNA-adsorbed superparamagnetic nanoparticles in step 2).
7. The extraction method according to claim 6, wherein the number of times of washing is 1 to 3, and the washing is performed with ethanol having a volume concentration of 70 to 80%.
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