CN110964718B - A rapid method for obtaining DNA condensates - Google Patents
A rapid method for obtaining DNA condensates Download PDFInfo
- Publication number
- CN110964718B CN110964718B CN201911349958.0A CN201911349958A CN110964718B CN 110964718 B CN110964718 B CN 110964718B CN 201911349958 A CN201911349958 A CN 201911349958A CN 110964718 B CN110964718 B CN 110964718B
- Authority
- CN
- China
- Prior art keywords
- dna
- ethanol
- solution
- mixed solution
- different
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 238000000034 method Methods 0.000 title claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 194
- JLVVSXFLKOJNIY-UHFFFAOYSA-N Magnesium ion Chemical compound [Mg+2] JLVVSXFLKOJNIY-UHFFFAOYSA-N 0.000 claims abstract description 26
- 229910001425 magnesium ion Inorganic materials 0.000 claims abstract description 26
- 239000011259 mixed solution Substances 0.000 claims abstract description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000007853 buffer solution Substances 0.000 claims abstract description 8
- 159000000003 magnesium salts Chemical class 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 31
- 239000007983 Tris buffer Substances 0.000 claims description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 2
- 238000009833 condensation Methods 0.000 abstract description 11
- 230000005494 condensation Effects 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 8
- 230000002378 acidificating effect Effects 0.000 abstract description 4
- 239000012445 acidic reagent Substances 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 description 155
- 230000037230 mobility Effects 0.000 description 21
- 238000002474 experimental method Methods 0.000 description 18
- 238000002296 dynamic light scattering Methods 0.000 description 17
- 230000002776 aggregation Effects 0.000 description 16
- 230000008859 change Effects 0.000 description 14
- 230000015271 coagulation Effects 0.000 description 14
- 238000005345 coagulation Methods 0.000 description 14
- 238000004220 aggregation Methods 0.000 description 12
- 150000002500 ions Chemical class 0.000 description 10
- 239000011777 magnesium Substances 0.000 description 10
- 230000007423 decrease Effects 0.000 description 9
- 239000010445 mica Substances 0.000 description 8
- 229910052618 mica group Inorganic materials 0.000 description 8
- 150000001768 cations Chemical class 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 238000005259 measurement Methods 0.000 description 7
- 230000008569 process Effects 0.000 description 7
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- 238000011160 research Methods 0.000 description 6
- 230000002195 synergetic effect Effects 0.000 description 6
- 239000011521 glass Substances 0.000 description 5
- 229920000867 polyelectrolyte Polymers 0.000 description 5
- 238000005054 agglomeration Methods 0.000 description 4
- 229940079593 drug Drugs 0.000 description 4
- 230000003993 interaction Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 230000009471 action Effects 0.000 description 3
- 239000000701 coagulant Substances 0.000 description 3
- 239000006059 cover glass Substances 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000001415 gene therapy Methods 0.000 description 3
- 229920002521 macromolecule Polymers 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 239000002245 particle Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 238000000089 atomic force micrograph Methods 0.000 description 2
- 125000002091 cationic group Chemical group 0.000 description 2
- 230000008602 contraction Effects 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 2
- -1 poly(ethylene glycol) Polymers 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- PFNFFQXMRSDOHW-UHFFFAOYSA-N spermine Chemical compound NCCCNCCCCNCCCN PFNFFQXMRSDOHW-UHFFFAOYSA-N 0.000 description 2
- 238000012876 topography Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 230000005653 Brownian motion process Effects 0.000 description 1
- LTMHDMANZUZIPE-AMTYYWEZSA-N Digoxin Natural products O([C@H]1[C@H](C)O[C@H](O[C@@H]2C[C@@H]3[C@@](C)([C@@H]4[C@H]([C@]5(O)[C@](C)([C@H](O)C4)[C@H](C4=CC(=O)OC4)CC5)CC3)CC2)C[C@@H]1O)[C@H]1O[C@H](C)[C@@H](O[C@H]2O[C@@H](C)[C@H](O)[C@@H](O)C2)[C@@H](O)C1 LTMHDMANZUZIPE-AMTYYWEZSA-N 0.000 description 1
- 241001089723 Metaphycus omega Species 0.000 description 1
- 238000000342 Monte Carlo simulation Methods 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229920006318 anionic polymer Polymers 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000005537 brownian motion Methods 0.000 description 1
- 210000000234 capsid Anatomy 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000004568 cement Substances 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 239000010941 cobalt Substances 0.000 description 1
- 229910017052 cobalt Inorganic materials 0.000 description 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- LTMHDMANZUZIPE-PUGKRICDSA-N digoxin Chemical compound C1[C@H](O)[C@H](O)[C@@H](C)O[C@H]1O[C@@H]1[C@@H](C)O[C@@H](O[C@@H]2[C@H](O[C@@H](O[C@@H]3C[C@@H]4[C@]([C@@H]5[C@H]([C@]6(CC[C@@H]([C@@]6(C)[C@H](O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)C[C@@H]2O)C)C[C@@H]1O LTMHDMANZUZIPE-PUGKRICDSA-N 0.000 description 1
- 229960005156 digoxin Drugs 0.000 description 1
- LTMHDMANZUZIPE-UHFFFAOYSA-N digoxine Natural products C1C(O)C(O)C(C)OC1OC1C(C)OC(OC2C(OC(OC3CC4C(C5C(C6(CCC(C6(C)C(O)C5)C=5COC(=O)C=5)O)CC4)(C)CC3)CC2O)C)CC1O LTMHDMANZUZIPE-UHFFFAOYSA-N 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 230000005684 electric field Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 229920005570 flexible polymer Polymers 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000001298 force spectroscopy Methods 0.000 description 1
- 238000001476 gene delivery Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000011503 in vivo imaging Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 229920000831 ionic polymer Polymers 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229910021645 metal ion Inorganic materials 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 150000007523 nucleic acids Chemical group 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000002798 polar solvent Substances 0.000 description 1
- 229920000768 polyamine Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000020988 regulation of intracellular pH Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000010079 rubber tapping Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 229940063675 spermine Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000007704 transition Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
Landscapes
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及一种快速获取DNA凝聚物的方法，在包含水和乙醇的混合溶液中加入镁盐、待凝聚的DNA，所述包含水和乙醇的混合溶液为酸性或加入酸试剂使包含水和乙醇的混合溶液变为酸性。本发明采用不同PH值的缓冲溶液进行研究PH值、乙醇浓度、镁离子三者共同作用对DNA凝聚的影响，发现PH越低、乙醇浓度越高，DNA凝聚作用越大，DNA尺寸缩短时间明显加快，尤其是在PH≤6的体系下，乙醇浓度对DNA凝聚的影响明显增大。
The present invention relates to a method for rapidly obtaining DNA condensates. Magnesium salts and DNA to be condensed are added to a mixed solution containing water and ethanol. The mixed solution containing water and ethanol is acidic or an acid reagent is added to make the mixed solution containing water and ethanol. The mixed solution of ethanol becomes acidic. In the present invention, buffer solutions with different pH values are used to study the effect of pH value, ethanol concentration and magnesium ion on DNA cohesion. Accelerated, especially in the system of PH≤6, the effect of ethanol concentration on DNA condensation increased significantly.
Description
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]DNA aggregation by divalent counterions was also observed. 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 canMost of the charges on the DNA are neutralized, but part of the charges on the DNA are not neutralized, and electrostatic repulsive force exists 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. Little is known about the mechanism of condensation in poor solvents. 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, the study of the interaction of ethanol with DNA may provide insightsInformation 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,Donald C.Effects of Salt Concentrations and Bending Energy on the Extent of Ejection of Phage Genomes.Biophysical Journal.2008；94:1110-20.
[2]Koltover I,Wagner K,Safinya CR.DNA Condensation in Two Dimensions.Proc Natl Acad Sci U S A.2000；97:14046.
[3]Nguyen T T,Grosberg A Y,Shklovskii B I.Screening of a charged particle 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):3mMMg2+10% ethanol, pH 8.0, 6.0, 5.0; (g) - (i) 3mMMg2+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 Mg2+，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. Changes in the DNA aggregation morphology, critical aggregation force and electrophoretic mobility in this environment were investigated by AFM, MT and DLS, respectively.
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 for 5 minutes at room temperature. 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, the DNA adsorbed on the surface of mica is highly compressed and the size in the transverse direction is not changed, so that AFM only flattens the morphology of DNA in the system, but the structure is not changed. 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 middle part consisting ofThe cross of one or more DNA molecules forms a core 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 results of DLS measurements with changes in pH of the solution or Mg after addition of 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%); phase change of ethanol volume fraction in low concentration range (0-10%) at pH less than 6More pronounced 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). In the low volume fraction ethanol in fig. 5(d), 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 ethanol at pH 8.0Volume fraction of 10% even if very small pulling force is applied: (<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 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)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911349958.0A CN110964718B (en) | 2019-12-24 | 2019-12-24 | A rapid method for obtaining DNA condensates |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911349958.0A CN110964718B (en) | 2019-12-24 | 2019-12-24 | A rapid method for obtaining DNA condensates |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110964718A CN110964718A (en) | 2020-04-07 |
| CN110964718B true CN110964718B (en) | 2021-08-13 |
Family
ID=70036392
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911349958.0A Expired - Fee Related CN110964718B (en) | 2019-12-24 | 2019-12-24 | A rapid method for obtaining DNA condensates |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110964718B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114891780B (en) * | 2022-04-15 | 2023-10-13 | 温州大学 | A way to increase DNA flexibility |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107033219B (en) * | 2017-04-07 | 2020-07-28 | 中国石油大学(华东) | A kind of self-assembled peptide and its application as DNA coagulation reagent |
-
2019
- 2019-12-24 CN CN201911349958.0A patent/CN110964718B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN110964718A (en) | 2020-04-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| von Maltzahn et al. | Positively charged surfactant-like peptides self-assemble into nanostructures | |
| Atkin et al. | Adsorption of 12-s-12 gemini surfactants at the silica− aqueous solution interface | |
| Zhao | Self-assembly of DNA segments on graphene and carbon nanotube arrays in aqueous solution: A molecular simulation study | |
| Pal et al. | Water-induced physical gelation of organic solvents by N-(n-alkylcarbamoyl)-l-alanine amphiphiles | |
| Shi et al. | Experimental determination of particle size-dependent surface charge density for silica nanospheres | |
| Sajja et al. | Dynamic behavior of RNA nanoparticles analyzed by AFM on a mica/air interface | |
| Pal et al. | Helical nanobots as mechanical probes of intra-and extracellular environments | |
| Zaldivar et al. | Self-assembled nanostructures of peptide amphiphiles: Charge regulation by size regulation | |
| Fauquignon et al. | Large and giant unilamellar vesicle (s) obtained by self-assembly of poly (dimethylsiloxane)-b-poly (ethylene oxide) diblock copolymers, membrane properties and preliminary investigation of their ability to form hybrid polymer/lipid vesicles | |
| Trotsenko et al. | Conformational transitions of flexible hydrophobic polyelectrolytes in solutions of monovalent and multivalent salts and their mixtures | |
| Hsieh et al. | Correlative light-electron microscopy of lipid-encapsulated fluorescent nanodiamonds for nanometric localization of cell surface antigens | |
| Jiang et al. | Serum-induced degradation of 3D DNA box origami observed with high-speed atomic force microscopy | |
| Sek et al. | Molecular resolution imaging of an antibiotic peptide in a lipid matrix | |
| CN110964718B (en) | A rapid method for obtaining DNA condensates | |
| Dhindsa et al. | Enhanced dynamics of hydrated trna on nanodiamond surfaces: A combined neutron scattering and md simulation study | |
| El Hamoui et al. | Interactions between DNA and the Hfq amyloid-like region trigger a viscoelastic response | |
| Yu et al. | Carbon Nanotube/Biocompatible Bola‐Amphiphile Supramolecular Biohybrid Materials: Preparation and Their Application in Bacterial Cell Agglutination | |
| Judah et al. | Influence of buffers, ionic strength, and pH on the volume phase transition behavior of acrylamide-based nanogels | |
| Tian et al. | pH-regulated reversible transition between polyion complexes (PIC) and hydrogen-bonding complexes (HBC) with tunable aggregation-induced emission | |
| Arango et al. | Nanostructured Surface Functionalization of Polyacrylamide Hydrogels Below the Length Scale of Hydrogel Heterogeneity | |
| Vodolazkaya et al. | Estimation of Nanoparticle’s Surface Electrostatic Potential in Solution Using Acid–Base Molecular Probes. III. Experimental Hydrophobicity/Hydrophilicity and Charge Distribution of MS2 Virus Surface | |
| Crisci et al. | Bioconjugation of a PNA probe to zinc oxide nanowires for label-free sensing | |
| Alfei et al. | Synthesis, characterization and broad-spectrum bactericidal effects of ammonium methyl and ammonium ethyl styrene-based nanoparticles | |
| Chrysostomou et al. | Hydrophilic random cationic copolymers as polyplex-formation vectors for DNA | |
| Ono et al. | Dynamics of DNA condensates at the solid− liquid interface by atomic force microscopy |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20210813 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |