CN110964718A

CN110964718A - Method for rapidly obtaining DNA aggregate

Info

Publication number: CN110964718A
Application number: CN201911349958.0A
Authority: CN
Inventors: 王艳伟; 戴庭辉; 罗胜; 吕方怡; 许诗雨; 陈浩迪
Original assignee: Wenzhou University
Current assignee: Wenzhou University
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2020-04-07
Anticipated expiration: 2039-12-24
Also published as: CN110964718B

Abstract

The invention relates to a method for rapidly obtaining a DNA aggregate, which is characterized in that magnesium salt and DNA to be aggregated are added into a mixed solution containing water and ethanol, and the mixed solution containing water and ethanol is acidic or an acid reagent is added to make the mixed solution containing water and ethanol acidic. According to the invention, buffer solutions with different pH values are adopted to research the influence of the combined action of the pH value, the ethanol concentration and the magnesium ions on DNA condensation, and the lower the pH value and the higher the ethanol concentration are, the larger the DNA condensation effect is, the obviously accelerated the DNA size shortening time is, and especially under a system with the pH value less than or equal to 6, the influence of the ethanol concentration on the DNA condensation is obviously increased.

Description

Method for rapidly obtaining DNA aggregate

Technical Field

The invention relates to a method for rapidly obtaining a DNA aggregate.

Background

DNA is a carrier of genetic information, and a highly condensed and orderly arranged DNA structure keeps the stability of a genome, so that the replication is successfully completed. The gene therapy is to introduce the human health gene or the gene with therapeutic action into the pathogenic cells of human body through proper carrying medium to correct the pathogenic gene, so as to achieve the purpose of treating diseases. With the rise of gene therapy research, there is new interest in the study of DNA coagulation, which provides an important tool in gene delivery in gene therapy. DNA condensation is the process by which stretched DNA molecules collapse to form a compact ordered structure, typically comprising one or several molecules, and by forming a circular structure, the DNA is significantly smaller in size and can be incorporated into very small spaces within a cell or virus. There are many kinds of coagulants or ligands that cause DNA coagulation, such as multivalent cations, ethanol, proteins, neutral coagulation polymers, cationic liposomes, and some anticancer drugs, and DNA is coagulated into different forms by these coagulants.

Several theoretical models have been proposed to describe the principle of equilibrium ion distribution around polyelectrolytes. In solution, DNA condensation is highly dependent on the valency of the counter ion. Cations with valencies of 3 or greater are required to overcome the electrostatic repulsion inherent between nucleic acid fragments. Multivalent cations commonly found in cells are polyamines, such as spermidine and spermine, which play important roles in cell proliferation and growth. It is speculated that 89-90% of the phosphate charge of the DNA backbone must be neutralized to allow condensation of the DNA under various ionic conditions.

However, small counterions, such as Na, are more common in biological cells⁺，K⁺，Ca²⁺And Mg²⁺Metal cations with equivalent valency less than 3, which do not cause DNA aggregation, although they have a high affinity for the DNA backbone to neutralize most of the DNA charge. Divalent cation Mg widely distributed in biological systems²⁺And Ca²⁺Play an important role in many enzymatic activities associated with replication, transcription and recombination. We have found that even divalent counterions (e.g., Mg) can be found in a confined environment, e.g., inside the viral capsid²⁺) Also cause strong and non-monotonous effects^[1]. In another environment where DNA conformation is restricted, i.e.in a two-dimensional system^[2]Divalent counter ion introduction was also observedThe DNA is coagulated. Divalent counter ions, e.g. Mg²⁺It has a strong influence on the coagulation of DNA in a closed environment (e.g. the capsid inside the phage), similar to the higher valency counter ions. Intracellular pH regulation is important for controlling the cell cycle and proliferative capacity of cells, and pH-regulated DNA-based nanomaterials and nanodevices have roles for in vivo imaging, promising for clinical diagnosis and drug delivery. Therefore, investigation of Mg at various pH values²⁺The interaction with DNA is of great significance in medicine and life science.

DNA, as a biological macromolecule, is a polyelectrolyte with different properties compared to neutral polymers and simple electrolytes. When dissolved in a polar solvent, DNA will ionize into highly charged polyions, and the surroundings are covered with many small counterions. Theoretical research results of a counter ion condensation theory, Monte Carlo simulation, Poisson-Boltzmann equation solution and the like show that the counter ions are condensed on the surface of the polyelectrolyte to form a thin-layer structure. From Manning's theory of counterion agglomeration, it is known that the counterion can neutralize most of the charges on DNA, but there are some charges on DNA that are not neutralized, and there are electrostatic repulsive forces between DNA molecules. How to overcome this residual electrostatic repulsive force, Shklovskii^[3]The research group proposed a new theoretical explanation. They believe that the counterions form a strongly associated fluid structure on the DNA surface similar to the Wigner crystal structure due to strong lateral repulsion effects. The counterions adsorbed on the surface of the DNA shield the charges of the DNA, so that the repulsive force between DNAs is reduced, and when the electrostatic repulsive force between DNAs is smaller than the electrostatic attractive force, the DNAs are aggregated. Since the electrostatic properties of DNA in solution are relatively good, electrophoretic mobility is often measured experimentally to measure the effective charge on DNA. There are many kinds of coagulants or ligands causing DNA coagulation, such as multivalent ions, ethanol, proteins, neutral coagulation polymers, cationic liposomes, and some anticancer drugs, and DNA will coagulate into different forms under the action of these drugs, which has attracted attention and research in different fields. The mechanism of condensation in poor solvents has been less studied, for whichWe know little. Recently, MacKin Tosh and colleagues predicted that disintegration of semi-flexible polymers in poor solvents occurs through a series of long-lived, metastable intermediates known as "tennis rackets". For experimental studies on this mechanism, ethanol can be used as a poor solvent for DNA and can be widely used in the fields of chemistry, biology and medicine, and thus ethanol can be a good choice. Fang et al^[4]The research finds that the ethanol concentration is from the full B type with 0 percent of ethanol concentration to the ethanol concentration>Full type a DNA conversion of 25%. Furthermore, divalent metal ions generally do not cause DNA aggregation under unspecific conditions^[5]But can cause DNA coagulation in an ethanol (about 15% by volume) -water mixed system^[6]. Alcohols and neutral or anionic polymers may also cause DNA agglomeration. High concentration ethanol is usually used to precipitate DNA, but the conditions are controlled so that DNA agglomeration occurs^[7]. Ethanol is a poor solvent for semi-flexible DNA, making DNA less interactive with the environment, and DNA tends to crowd into a more compact form. Thus, studies on the interaction of ethanol with DNA may provide insightful information to understand the second mechanism of condensation. And Mg²⁺The effect of drugs such as ethanol on DNA coagulation has been widely revealed, but studies on the synergistic effect of ethanol and pH on magnesium ions on DNA coagulation have not been intensively studied. Thus, different pH values, different volume fractions of ethanol and Mg are revealed²⁺Has great significance for the influence of DNA.

[1]Evilevitch,Alex,Fang,Tai L,Yoffe,Aron M,Castelnovo,Martin,DonaldC.Effects of Salt Concentrations and Bending Energy on the Extent of Ejectionof Phage Genomes.Biophysical Journal.2008；94:1110-20.

[2]Koltover I,Wagner K,Safinya CR.DNA Condensation in TwoDimensions.Proc Natl Acad Sci U S A.2000；97:14046.

[3]Nguyen T T,Grosberg A Y,Shklovskii B I.Screening of a chargedparticle by multivalent counterions in salty water:Giant charge inversion[J].The Journal of Chemical Physics,2000,113(3):1110-1125.

[4]Y.Fang,T.Spisz and J.Hoh,Nucleic Acids Res.,1999,27,1943.

[5]Yoshikawa,V.V.Vasilevskaya A.R.Khokhlov Y.Matsuzawa and K.,Collapse of single DNA molecule in poly(ethylene glycol)solutions.J.Chem.Phys.,1995.102:16.

[6] Wang Yangwei, Hugao Ming, forest yoga, DNA coagulation caused by the synergistic effect of ethanol and cobalt trichlorohexamine [ J ]. proceedings of Zhejiang university (science edition), 2012(04):56-61.

[7] Zhang Wen science, AFM imaging of single molecule level polymer interaction and study of single molecule force spectroscopy, published in macromolecules 2011.9: 913-.

Disclosure of Invention

The object of the present invention is to overcome the disadvantages and shortcomings of the prior art and to provide a method for rapidly obtaining DNA aggregates.

The technical scheme adopted by the invention is as follows: a method for rapidly obtaining DNA aggregates, which comprises adding magnesium salt and DNA to be aggregated to a mixed solution containing water and ethanol, wherein the mixed solution containing water and ethanol is acidic or adding an acid reagent to make the mixed solution containing water and ethanol acidic.

The mixed solution containing water and ethanol is a mixed solution of a buffer solution and ethanol.

The volume fraction of the ethanol in the mixed solution of the buffer solution and the ethanol is greater than or equal to 10%.

The pH of the mixed solution containing water and ethanol is less than or equal to 6.

The invention has the following beneficial effects: according to the invention, buffer solutions with different pH values are adopted to research the influence of the combined action of the pH value, the ethanol concentration and magnesium ions on DNA condensation, the lower the pH value, the higher the ethanol concentration and the larger the DNA condensation effect are found, the time for shortening the DNA size is obviously accelerated, and especially under a system with the pH value less than or equal to 6, the influence of the ethanol concentration on the DNA condensation is obviously increased.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is within the scope of the present invention for those skilled in the art to obtain other drawings based on the drawings without inventive exercise.

Fig. 1 is a schematic view of magnetic tweezers;

FIG. 2 alcohol with 3mM Mg at different pH values and different concentrations²⁺AFM images of environmentally induced DNA. (a) - (c) 3mM MG²⁺,pH＝8.0、6.0、5.0；(d)-(f):3mMMg ²⁺10% ethanol, pH 8.0, 6.0, 5.0; (g) - (i) 3mMMg ²⁺20% ethanol, pH 8.0, 6.0, 5.0; (j) - (l) 3mM MG ²⁺30% ethanol, pH 8.0, 6.0, 5.0;

FIG. 3 shows the relationship between the electrophoretic mobility of DNA and pH at different concentrations of ethanol;

FIG. 4 is a graph showing the relationship between the electrophoretic mobility of DNA and the concentration of ethanol in different pH solution environments;

FIG. 5 shows a graph at 3mMMg²⁺And extension time profile of DNA measured with MT during DNA stretching in 5mM Tris at different pH values and different volume fractions. (a) (b), (c):3mM Mg^2+，pH＝8.0，6.0，5.0。(d)，(e)，(f)：3mM Mg ²⁺10% ethanol, pH 8.0, 6.0, 5.0.(g), (h), (i):3mM Mg ²⁺20% ethanol, pH 8.0, 6.0, 5.0.(j, (k), (l):3mM Mg ²⁺30% ethanol, pH 8.0, 6.0, 5.0;

FIG. 6 is a graph of the DNA extension time measured by MT during the contraction of DNA in different solution environments.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the accompanying drawings.

The following three experimental means, Atomic Force Microscope (AFM), single molecular Magnetic Tweezers (MT) and Dynamic Light Scattering (DLS), were used to analyze DNA molecules at different pH values and different volume fractions of ethanol and 3mM Mg²⁺Experimental studies were performed in solution. By usingAFM, MT and DLS were used to examine changes in DNA aggregation morphology, critical aggregation force and electrophoretic mobility in this environment.

First, the experimental process

1. Material

Ethanol was purchased from Korea company (Shanghai, China), and chemical reagents such as magnesium chloride were purchased from Sigma-Aldrich company, and used in a 5 mmol. L state before use^－1And (5) diluting with a Tris buffer solution. Ultrapure water was deionized and purified by a purification system having a resistivity of 18.2 M.OMEGA.cm, which was purified by a Milli-Q system (Millipore corporation, USA). Double-stranded phage lambda-DNA (48502bp) was purchased from New England Biolabs, Inc., at a primary concentration of 500 ng. mu.L DNA^-1. Phosphate Buffered Saline (PBS) was used in our MT sample preparation with 10mM phosphate, 140mM NaCl, pH 8.0.

2. Detection method

Dynamic light scattering

Dynamic Light Scattering (DLS) is mainly used to measure the properties of a sample according to the change of laser Light, and is also called Photon Correlation Spectroscopy (PCS), because Light irradiates on a measured substance to generate scattered Light, and DLS is used to detect the size and potential of the substance by detecting the change of the intensity and frequency of the scattered Light with time, and can also measure the relationship between the change of Light intensity and time. DLS technology is widely used for researching the interaction between DNA and counter ions and can also be used for researching the physicochemical properties of proteins and other macromolecules. In this experiment, a DLS apparatus (Malvern Zetasizer nano ZS90) was mainly used, and He — Ne laser (l 633nm) and avalanche photodiode (for detecting scattered light) were applied to measure the zeta potential and electrophoretic mobility of the substance. Under the action of an external electric field, the polyelectrolyte moves towards an electrode with opposite charges, and moves relative to a counter ion in a medium, which is called electrophoresis. The lambda-DNA in our experiments can be seen as a linear strongly charged polyelectrolyte, the electrophoretic mobility μ of DNA is given by:

where f (κ r) is a function of κ r, called the Henry function, where r is the particle radius, 1/k is the Debye length, η is the viscosity coefficient of the medium,

is the zeta potential and ε is the dielectric constant of the medium. The zeta potential of the particles to be measured can be directly measured by the electrophoresis method by utilizing the DLS technology, and the electrophoretic mobility of the particles to be measured can be obtained by the formula.

In the experiment, 1000. mu.l of the test solution was pipetted into a centrifuge tube and the DNA was diluted to 1 ng.. mu.l in the solution^-1The concentration of (c). Incubation at room temperature for 5 minutes was required prior to measurement. And finally, taking out 1000 mu l of mixed liquid to be detected in the centrifugal tube by using a liquid transfer device, transferring the mixed liquid to a zeta capillary sample cell, and then putting the mixed liquid in a dynamic light scattering sample groove for experiment. During the measurement, the sample cell was kept at a constant temperature of 25 °.

Atomic Force Microscope (AFM) imaging

The experimental instrument used a multimode atomic force microscope (JPK Instruments AG, Berlin Germany) with tapping mode. The experiment was performed with a scan rate of 1 Hz and the image pixels were 512 x 512. The information of height, width, etc. of the DNA image was analyzed using JPK Data Processing analysis software version 4.2. The AFM works by using the extremely small tip at one end of the internal cantilever and the extremely weak interatomic force between the samples to be measured, so as to obtain the surface topography information of the samples.

In the experimental process, a mica sheet with the size of 1 centimeter multiplied by 1 centimeter is flatly pasted on a glass slide with the specification of 25.4 multiplied by 76.2mm by a double faced adhesive tape, and the mica sheet is carefully dissociated by a frosted tape until the surface of the mica sheet is flat and clean and is used as a substrate for DNA adsorption. For each sample, a defined amount of lambda DNA and a different volume fraction of ethanol were added to the MgCl2 solution and adjusted to the desired pH. The final concentration of lambda-DNA was set to 1ng. mu.L^-1The final concentration of MgCl2 was 3 mmol.L^－1. 50 μ L of the previously prepared mixture was pipetted, deposited onto freshly dissociated mica plates and incubated at room temperatureFor 5 minutes. After incubation, the residual solution on the surface of the mica sheet was sucked off, and the sample was rinsed with 30 μ L of water about 10 times and then lightly blown dry with nitrogen. And placing the film in the device box for about 0.5h to be scanned.

Experiment with magnetic tweezers

Single molecule Magnetic Tweezers (MT)

The magnetic tweezers device consists of an inverted microscope, a Charge Coupled Device (CCD) controlled by a personal computer and the like and is used for obtaining a force spectrum of DNA in a counter ion solution. In the experiment, a homemade experiment sample groove is used for carrying out the experiment, the sample groove is formed by placing a cover glass with the thickness of 0.17mm between two glass slides, the side cover glass is not aligned with the two glass slides, two glass microtubes are respectively inserted into two ends of the side cover glass, and finally, the middle gap is solidified and sealed by glass cement to form a microtank. Before the experiment, after the sample tank is silanized, a digoxin resistant medicine (1%) is introduced, and the sample tank is vertically kept for about 6 hours; then introducing bovine serum albumin solution (BSA, 5%), and standing for about 30 min; and finally, mixing the modified DNA, the streptavidin and the magnetic ball, introducing the mixture into a sample tank, standing for 1h, and searching for a single DNA molecule in the sample tank under an inverted microscope (as shown in figure 1). The magnet is used for exerting tension on the magnetic ball on the DNA, thereby achieving the purpose of stretching the DNA. In the experiment, a micro-injection pump is used for injecting prepared medicines into a sample groove to enable the medicines to react with single DNA, then the force is slowly reduced, the change process of the length and the time of the DNA is recorded by a CCD, and the movement of a magnetic ball is analyzed by using an image processing program to obtain a force spectrum curve graph. And the cohesion is obtained according to the principle of measuring the force along the Brownian motion of the magnetic ball in the direction vertical to the DNA. The cohesive force described below is a critical cohesive force and corresponds to the first time the DNA is coagulated.

Second, experimental results

Morphology of DNA under AFM

The AFM can change the actual form of DNA in ethanol solutions with different pH values and different concentrations. But treatment of DNA adsorbed on a surface with a multivalent cation solution produces a circle that is similar in size (i.e., diameter) to a circle in solution, but has a smaller height (axial). That is, DNA adsorbed on the surface of mica is highly compressed only, laterallyDoes not change in size, so AFM simply flattens the morphology of the DNA in the system, but does not change its structure. Therefore, in order to directly observe the change of DNA morphology, we observed the DNA with different pH values and different volume fractions of ethanol and Mg²⁺Topography under environment. Figure 2 shows ethanol and Mg at different pH values (pH 8.0-5.0) and different volume fractions (0, 10%, 20%, 30%)²⁺AFM images of DNA in the environment. In FIG. 2(a-c), we see that the DNA on mica plates is in a free and loose linear form and does not change with the decrease of pH value, and we can not find the aggregated DNA. But to Mg at different pH values (pH 8.0, 6.0, 5.0)²⁺The DNA solution is added with 10% ethanol and the morphology of the DNA changes from a free, loose state to a flower-like structure consisting of a number of networks, which is formed by the crossing of one or more DNA molecules, as shown in FIG. 2 (d-f). FIG. 2(g-i) shows that the morphology of DNA changes from kinked to compact flower shape when the ethanol volume fraction is increased to 20%. FIG. 2(j-l) shows that the DNA morphology D was a compact flower when the ethanol volume fraction was increased to 30%. The flower-like structure has a core in the middle formed by the intersection of one or more DNA molecules, with some loose threads on either side. From this we can see Mg at pH 6²⁺Addition of ethanol (10% by volume) to the DNA solution also caused DNA aggregation when its concentration and pH were below the critical values, indicating that ethanol and pH had a synergistic effect on the aggregation of DNA by magnesium ions. We can also see that DNA condensation with ethanol volume fraction increases and pH value decreases and increases. As can be seen, the DNA coagulation effect is obviously enhanced with the increase of the volume fraction of the ethanol and the decrease of the pH value.

2. Measurement of electrophoretic mobility

The effective charge of DNA changes with its morphology, i.e., the electrophoretic mobility of DNA changes depending not only on the surface charge of DNA, but also on the coagulation of DNA. Thus, the electrophoretic mobility of DNA was investigated by DLS experiments at different pH values (pH 5-8), different volume fractions (0, 10%, 20%, 30%, 40%) of ethanol and Mg²⁺Changing relationships in the environment. Table 1 and FIGS. 3 and 4 show the experimental results of DLS measurementFruit, changing the pH of the solution or adding Mg after ethanol²⁺The resulting tendency of electrophoretic mobility of DNA changes. When the pH of the solution was 8.0 by changing the pH alone, the electrophoretic mobility of DNA was-0.69X 10^-4(cm2V^-1s^-1) The electrophoretic mobility of the DNA is increased along with the reduction of the pH value, and the change trend of the electrophoretic mobility of the DNA is gradual and smooth along with the reduction of the pH value. When ethanol is added to the solution, the electrophoretic mobility of the DNA increases as a whole and increases as the volume fraction of ethanol increases. Each data point is the average of three consecutive measurements with the corresponding standard deviation as the error. It can be obtained that the electrophoretic mobility of DNA decreases gradually but is always negative as the volume fraction of ethanol increases and the pH value decreases. When the pH value is more than 6, the influence of the change of the pH value on the electrophoretic mobility of the DNA is extremely small; at a pH of less than 6, the effect of the change in pH on the electrophoretic mobility of DNA is large. At pH above 6, the volume fraction of ethanol is less variable at low concentration ranges (0-10%) than at higher concentration ranges (10% -40%); at a pH of less than 6, the change in the volume fraction of ethanol is more pronounced in the low concentration range (0-10%) than in the higher concentration range (10% -40%).

Table 1 electrophoretic mobilities of DNA at different pH values (pH 5-8), different volume fractions (0, 10%, 20%, 30%, 40%) of ethanol and Mg²⁺Changing relationships in an environment

3. Measurement of cohesion

The magnitude of the critical cohesive force of DNA is related to the effective charge of DNA, and as the positive charge in solution increases, the more tightly the bonds between the phosphate groups of DNA and between the phosphate groups and the mica surface become. FIGS. 5(a) - (c) show that as force is reduced, DNA extension changes linearly with time and no step-wise jumps are seen. This procedure demonstrates that DNA does not coagulate. When 10% ethanol is added to Mg²⁺DNA extension time curve jumps when pH goes from 8.0 to 5.0 in DNA solution, except pH 8.0 in fig. 5(d) -5 (f). Low volume fraction B in FIG. 5(d)In alcohol, no DNA coagulation occurred at pH 8.0. In fig. 5(e) -5 (f), the curves show that as the amount of ethanol friction increases, a longer time from 13 μm to 6 μm is required.

In FIGS. 5(g) -5 (i), when 20% ethanol is Mg²⁺The time for the DNA extension time curve to jump to and shorten by the same length when the pH in the DNA solution is added from 8.0 to 5.0 decreases with increasing ethanol volume fraction. The time was changed from 430s to 5s, and the force increased as the volume fraction of ethanol increased. Thus, this phenomenon demonstrates that the cohesive strength increases with decreasing pH and increasing ethanol. In FIGS. 5(j) -5 (l), when 30% ethanol is added to mg²⁺In DNA solutions, the DNA extension time curve has a greater jump at pH values from 8.0 to 5.0, while the time for decreasing the same length decreases rapidly. The time was changed from 400s to 2s, and the force increased as the volume fraction of ethanol increased.

FIG. 6(b) shows a transition between Mg²⁺And (3) the process of DNA shrinkage in a solution with pH of 6.0 and ethanol volume fraction of 20%. In fig. 6(a), we can see that even if very small pulling force is applied under the environment of pH 8.0 and ethanol volume fraction of 10%, (<0.5pN), the extension of the DNA will also maintain an almost constant length over time. This indicates that the DNA did not aggregate in this environment. However in FIG. 6(b) we can see that in Mg²⁺pH 6.0, ethanol volume fraction 20%, in which a significant DNA contraction process occurs, we can see a sudden change in the DNA extension time curve. This indicates that DNA is aggregated in this environment. And its cohesion force was measured to be 1.7 pN. From this, we can also obtain ethanol, pH and Mg²⁺Has synergistic effect on DNA aggregation. When the force is greater than 10pN, the extension of DNA varies discontinuously and stepwise with time, and the extension of DNA cannot stretch the contour length in the final extension process. This may be accompanied by DNA aggregation that changes the persistence length of DNA. To quantify the observations, we measured DNA at different pH values, different ethanol volume fractions and Mg²⁺The cohesion force under the environment is shown in table 1 below. In single molecule experiments, the displacement of the magnetic sphere is in the micrometer range, corresponding to a change of less than 0.03pN in applied magnetic force. In thatIn most cases, the measurement error is less than 5%. Thus, the force can be considered constant and no further adjustment is required during the experiment. Table 2 lists DNA at different pH values (pH 8.0, 6.0, 5.0) and different volume fractions (0, 10%, 20%) of ethanol and Mg²⁺Cohesion Fc under ambient conditions. As can be seen from the table, when ethanol was not added to solutions at different pH values (pH 8.0, 6.0, 5.0), Fc was 0PN and when 10% ethanol was added, Fc was changed to 0PN, 1.3PN, and 1.5PN, respectively. When ethanol increased to 20%, Fc became 1.1PN, 1.8PN, 2.1 PN. From this, we know that DNA cohesive force increases with the increase of ethanol volume fraction and the decrease of pH. In contrast to the case of multivalent cations, we cannot observe the compactness of DNA at very low forces when the ethanol concentration is as high as 65%. However, when Mg²⁺When added to the solution, DNA aggregation was induced by ethanol volume fraction of 20% at pH 8.0. And when the pH value is reduced, the critical volume fraction of DNA agglomeration caused by ethanol is less than or equal to 10 percent. Thus, ethanol and pH have a synergistic effect on the magnesium ions causing DNA aggregation.

TABLE 2 DNA cohesion (Fc) as a function of ethanol volume fraction and pH

Adopting AFM, MT and DLS technology to treat Mg with different pH values and different ethanol volume fractions²⁺The DNA molecules in solution were studied experimentally. It was found by AFM experiments that the coagulation of DNA increases with increasing volume fraction of ethanol and decreasing pH. The increase of DNA cohesion with the increase of ethanol volume fraction and the decrease of pH value can be measured by MT experiment. And the change of the electrophoretic mobility of the DNA under the same environment is measured by using a DLS experiment, and the electrophoretic mobility of the DNA is gradually reduced but is always negative along with the increase of the alcohol concentration and the reduction of the pH value. From the above three experiments, it can be obtained that ethanol and pH have a synergistic effect on DNA aggregation caused by magnesium ions.

The above disclosure is only for the purpose of illustrating the preferred embodiments of the present invention, and it is therefore to be understood that the invention is not limited by the scope of the appended claims.

Claims

1. A method for rapidly obtaining a DNA aggregate, comprising: adding a magnesium salt, DNA to be coagulated, to a mixed solution containing water and ethanol which is acidic or adding an acid agent to make the mixed solution containing water and ethanol acidic.

2. The method for rapidly obtaining a DNA aggregate according to claim 1, wherein: the mixed solution containing water and ethanol is a mixed solution of a buffer solution and ethanol.

3. The method for rapidly obtaining a DNA aggregate according to claim 2, wherein: the volume fraction of the ethanol in the mixed solution of the buffer solution and the ethanol is greater than or equal to 10%.

4. The method for rapidly obtaining a DNA aggregate according to claim 3, wherein: the pH of the mixed solution containing water and ethanol is less than or equal to 6.

5. The method for rapidly obtaining a DNA aggregate according to claim 4, wherein: the pH of the mixed solution containing water and ethanol is less than 6.