JP3940935B2 - Extraction and purification method of plasmid DNA - Google Patents

Description

[0001]
[Industrial application fields]
The present invention relates to a method for extracting plasmid DNA simply and with high purity from a microorganism or cell holding plasmid DNA using a nucleic acid-binding solid phase carrier, and a plasmid DNA extraction and purification reagent kit for use in the method. The reagent kit can also be applied to an automatic nucleic acid extraction apparatus.
[0002]
[Prior art]
Extraction and purification of nucleic acids from biological materials such as cells containing nucleic acids is an important step in the fields of genetic engineering and clinical diagnosis. For example, when trying to analyze a certain gene, it is necessary to first extract a nucleic acid such as DNA or RNA from a biological material such as a cell holding the gene. Further, in DNA / RNA diagnosis for detecting infectious agents such as bacteria and viruses, it is necessary to extract the nucleic acids of bacteria and viruses from biological materials such as blood and then detect them.
In general, nucleic acids such as DNA and RNA contained in biological materials do not exist in a free state, but exist in shells such as cell membranes and cell walls composed of proteins, lipids, and sugars. It itself forms a complex with the protein. Therefore, when extracting and purifying nucleic acid from biological material, first, nucleic acid is liberated by subjecting it to physical disruption treatment with ultrasound or heat, enzyme treatment with protease, treatment with surfactant or denaturing agent, It is necessary to purify the nucleic acid from the crushed material by extraction using an organic solvent such as phenol, ultracentrifugation, column chromatography using a carrier such as an ion exchanger, and the like. These techniques are combined and optimized according to the use of nucleic acids, starting materials, and extracted nucleic acids.
[0003]
Bacteria that retain plasmid DNA, especially plasmid DNA, are extracted from Escherichia coli by alkaline lysis and boil methods [Molecular cloning: A Laboratory manual, 2nd ed. (Cold Spring Harbor Laboratory Press, 1989)]. Has been done. However, these methods are troublesome operations because they involve complicated steps such as centrifugation. Furthermore, the DNA sample extracted by these methods contains a large amount of RNA, protein, and the like that are harmful to subsequent analysis. Therefore, in order to obtain plasmid DNA with high purity, after performing these extraction operations, there are complicated procedures such as ultracentrifugation using a density gradient by cesium chloride, ribonuclease digestion and phenol / chloroform extraction. In addition, it is necessary to perform RNA and protein removal operations that require a long time.
[0004]
In addition, as a method for easily extracting plasmid DNA without the need for genomic DNA and protein removal operation, a method using hydroxyapatite as a nucleic acid-binding solid phase [Beland, FA et al., J. Chromatografy, 174, 177-186 (1979)] is known. According to this method, it is possible to extract only the plasmid DNA in the lysate, and since RNA and protein hardly adsorb to hydroxyapatite, these contaminations can be almost prevented. However, in this method, a relatively high concentration buffer solution such as a 0.3 M phosphate buffer solution that inhibits an enzyme reaction such as restriction enzyme digestion is used as an eluate. Therefore, when the extracted plasmid DNA is used for analysis such as restriction enzyme digestion or sequencing, it is necessary to remove the buffer by dialysis, gel filtration, etc., which requires a long time. .
[0005]
On the other hand, as a simple nucleic acid extraction method, there is a method using silica as a nucleic acid-binding solid phase carrier [Japanese Patent Laid-Open No. 2-289596]. In this method, nucleic acid can be extracted from biological materials such as bacteria in one step, and since a low-concentration buffer such as water or TE buffer is used as the eluent, no special desalting and concentration operation is required. There is an advantage that the extracted nucleic acid can be used immediately for later analysis. However, when extraction of plasmid DNA from bacteria holding plasmid DNA by this method is attempted, genomic DNA is adsorbed onto silica in the same manner as plasmid DNA. Therefore, in order to extract only plasmid DNA with high purity, it is essential to further perform purification operations such as ultracentrifugation and column chromatography.
[0006]
[Problems to be solved by the invention]
An object of the present invention is to solve the above-mentioned problems of the existing technology, and in a short time and with high purity without requiring complicated operations from microorganisms or cells holding plasmid DNA. It is to provide a method for extraction and purification.
[0007]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors have easily extracted plasmid DNA from microorganisms or cells holding plasmid DNA using an appropriate cell lysate, organic solvent and nucleic acid-binding solid phase carrier. The inventors have found that it can be purified and have reached the present invention.
[0008]
That is, the present invention is a method for extracting and purifying plasmid DNA, comprising the following steps (a) to (c).
(A) adding a cell lysate containing a chaotropic substance, a pH 3-6 cell lysate, an organic solvent extract and a nucleic acid-binding solid phase carrier to a microorganism or cells holding plasmid DNA, and mixing or contacting them; Adsorb plasmid DNA on a solid support,
(B) washing the solid phase carrier adsorbed with the plasmid DNA in the step (a) with a washing solution; and
(C) The plasmid DNA is eluted from the solid phase carrier washed in the step (b) with an eluent.
[0009]
Further, the present invention is a plasmid DNA extraction / purification reagent kit comprising a pH 3-6 lysis solution containing a chaotropic substance, an organic solvent extract, a nucleic acid binding solid phase carrier, a washing solution and an eluate.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The method for extracting and purifying plasmid DNA according to the present invention can be roughly divided into three stages: (a) dissolution / adsorption process, (b) washing process, and (c) elution process.
[0011]
(A) In the lysis / adsorption step, a microorganism or cell is lysed by adding, mixing or contacting a cell lysate, an organic solvent, or a nucleic acid binding solid phase carrier to the microorganism or cell holding the plasmid DNA, and plasmid DNA Is adsorbed to a nucleic acid-binding solid phase.
[0012]
As a microorganism or cell holding the plasmid DNA used in the present invention, for example, a transformant of Escherichia coli is representative. In the case of this Escherichia coli transformant, usually, the culture is performed overnight using an appropriate selective medium according to a known conventional method, and collected by an operation such as centrifugation, and used as a starting material. The plasmid DNA to be extracted here is used as a vector as is well known. Accordingly, the plasmid DNA as used herein naturally includes cosmid DNA.
[0013]
The cell lysate used in the present invention contains a buffer and has a pH of 3-6. This may be contained in the cell lysate in advance, or may be added as a buffer after lysing the cells. The buffering agent is not particularly limited as long as it is generally used, but more preferably has a buffering ability at any pH within the range of pH 3-6. For example, sodium acetate-acetic acid, sodium acetate-hydrochloric acid and the like can be mentioned, and the use concentration is preferably 1 to 500 nm and the pH is preferably in the range of 3 to 6.
[0014]
The cell lysate used in the present invention contains a chaotropic substance. The chaotropic substance is particularly limited as long as it has an action of increasing the water solubility of a hydrophobic molecule, which is generally known as a chaotropic substance, and further contributes to the binding of plasmid DNA to a solid phase. Not. Specific examples include guanidine thiocyanate, guanidine hydrochloride, sodium iodide, potassium iodide, sodium perchlorate and the like. Of these, guanidine thiocyanate is preferably used. The concentration of these chaotropic substances used varies depending on the chaotropic substance used. For example, when guanidine thiocyanate is used, it is preferably used in a range of 3 to 5.5M.
[0015]
The cell lysate may contain a surfactant for the purpose of disrupting the cell membrane or denaturing proteins contained in the cells. The surfactant is not particularly limited as long as it is generally used for nucleic acid extraction from cells and the like. Specifically, polyoxyethylene octylphenyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene Nonionic surfactants such as sorbitan monooleate, cationic surfactants such as dodecyltrimethylammonium bromide, dodecyltrimethylammonium chloride, cetyltrimethylammonium bromide, negative ions such as sodium dodecyl sulfate, sodium N-lauroyl sarcosine, sodium cholate Examples include amphoteric surfactants such as ionic surfactants and phosphatidylethanolamines. Of these, nonionic surfactants such as polyoxyethylene octyl phenyl ether and polyoxyethylene sorbitan monolaurate are preferably used. The concentration of these surfactants varies depending on the surfactant used. For example, when polyoxyethylene octylphenyl ether is used, it is preferably used in a range of 0.1 to 3%. .
[0016]
The organic solvent used in the present invention is not particularly limited as long as it does not interfere with the binding of plasmid DNA to the solid phase and prevents the binding of genomic DNA to the solid phase. The details of this principle are unknown, but the polarity of the liquid phase is moderately lowered by adding an organic solvent to the liquid phase, so that plasmid DNA and genomic DNA having different molecular surface polarities can be combined with the solid phase. It is considered that the bond selectivity is given.
Specific examples of the organic solvent used in the present invention include water saturated phenol, buffer saturated phenol, chloroform, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 3-methyl-1-propanol, acetone and the like. Is mentioned. Of these, water-saturated phenol or buffer-saturated phenol, or a mixture of these saturated phenol and chloroform in an appropriate ratio is preferable.
[0017]
The nucleic acid-binding solid phase carrier used in the present invention is not particularly limited as long as it has a hydrophilic surface capable of adsorbing nucleic acid in the presence of chaotropic ions, that is, holding it by reversible binding. Specifically, silicon dioxide, that is, silica is preferably used. In addition, other materials composed of silica, such as glass, diatomaceous earth, or those subjected to surface treatment by chemical modification, unless they prevent reversible binding to nucleic acids as described above In addition, complexes with other substances such as superparamagnetic metal oxides are also included. Specific examples of the form of the nucleic acid-binding solid phase carrier include particles, filters, reaction vessels, and the like, but are not particularly limited. Among these, considering the efficiency of adsorption and elution, the particle form is more preferable, and the particle size is more preferably 0.05 to 500 μm.
[0018]
In the present invention, the cell lysate, organic solvent, and nucleic acid-binding solid phase may be added separately or simultaneously.
[0019]
(B) The washing step is a step in which only the nucleic acid-binding solid phase carrier adsorbed with plasmid DNA is separated as much as possible from the mixture of cell disrupted material, cell lysate, organic solvent, and nucleic acid-binding solid phase carrier. At this time, it is preferable to repeatedly wash about 1 to 3 times using a cleaning liquid.
[0020]
Specific means for separating the nucleic acid-binding solid phase carrier in the present invention varies depending on the form of the solid phase carrier to be used. For example, when the nucleic acid-binding solid phase is in the form of particles, centrifugation, filtration, column operation and the like are preferable. Furthermore, if a particle in which superparamagnetic metal oxide is contained in the particles is used as a solid phase carrier, a simple magnetic separation method using a magnet or the like is possible, which is more preferable.
[0021]
The washing solution used in the present invention is not particularly limited as long as it does not promote elution of plasmid DNA from the solid phase and prevents binding of genomic DNA or protein to the solid phase. Specifically, a 3 to 5.5 M guanidine thiocyanic acid solution or 40% to 100% ethanol is preferable, and it is more preferable to use these cleaning solutions in combination. That is, first, it is preferable to wash with 40% to 100% ethanol after washing with a guanidine thiocyanic acid solution.
In addition, when the cell lysate and organic solvent used in the lysis / adsorption process at the beginning are used as the washing solution, it is more effective for removing genomic DNA and proteins. At this time, it is preferable to subsequently wash with 40% to 100% ethanol.
[0022]
(C) The elution step is a step of eluting the DNA from the nucleic acid binding solid phase carrier to which the plasmid DNA is adsorbed. Therefore, the eluent used in the present invention is not particularly limited as long as it promotes the elution of plasmid DNA from the solid phase. Specifically, water or TE buffer [10 mM Tris-HCl buffer, 1 mM EDTA, pH 8.0] is preferable. The plasmid DNA recovered at this time can be directly used for an enzyme reaction using a restriction enzyme, a DNA polymerase, or the like without performing desalting and concentration operations such as dialysis and ethanol precipitation.
[0023]
As described above, the method for extracting and purifying plasmid DNA according to the present invention consists of simple steps, so it can be easily applied to plasmid DNA extraction and purification kits and nucleic acid extraction apparatuses that automate solid phase separation and reagent dispensing operations. It is clear that it can be applied to.
[0024]
【Example】
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to these examples.
Example 1 Extraction of Plasmid DNA from E. coli pBR322 / HB101 (1) Preparation of E. coli pBR322 / HB101 100 μg / ml of E. coli HB101 transformant (pBR322 / HB101) transformed with plasmid pBR322 (manufactured by Toyobo Co., Ltd.) Inoculated into 100 ml of LB medium [10 g / l tryptone, 5 g / l yeast extract, 5 g / l sodium chloride (pH 7.5)] containing ampicillin, and cultured at a culture temperature of 37 ° C. and a shaking speed of 180 rpm for 15 hours. After culturing, 1.5 ml of the culture solution is dispensed into 1.5 ml microtubes, centrifuged at 12,000 rpm for 1 minute in a high-speed microcooled centrifuge (MR-150: manufactured by Tommy Seiko Co., Ltd.), and the supernatant The microbial cells obtained by removing were used as extraction materials.
[0025]
(2) Extraction and purification of plasmid DNA 500 μl of cell lysate [4.7 M guanidine thiocyanic acid, 92 mM sodium acetate-hydrochloric acid (pH 4.0), 1.2% polyoxyethylene octyl phenyl ether] , 20 mM EDTA] was added to lyse, followed by 500 μl TE buffer saturated phenol / chloroform (1: 1) and mixed vigorously. To this, 40 μl of 0.5 g / ml magnetic silica (particle size 1-10 μm, containing 30% iron tetroxide particles, specific surface area 280 m ² / g, pore volume 0.025 ml / g, surface pore diameter 2-6 nm : Suzuki Oil) suspension was added and mixed at room temperature for 10 minutes.
Next, the microtube was placed on a magnetic stand (MPC-M: manufactured by Dynal) to collect magnetic silica particles, and the supernatant was removed. Remove the microtube from the magnetic stand, add 1 ml of washing solution [5.3 M guanidine thiocyanic acid, 52 mM Tris-hydrochloric acid (pH 6.4)] and mix well. The particles were washed by removing.
Similarly, the particles were washed again with 1 ml of washing solution, and then the particles were washed twice with 1 ml of 70% ethanol and once with 100% ethanol. After removing the supernatant, the microtube was placed on a heat block set at 55 ° C. and left for 20 minutes to evaporate and remove ethanol in the tube, thereby drying the particles. 100 μl of sterilized water was added thereto and mixed at room temperature for 10 minutes, and then placed on a magnetic stand to collect magnetic silica particles, and the supernatant was collected. The recovered liquid volume was approximately 80 μl.
[0026]
10 μl of the collected solution is subjected to agarose gel electrophoresis, and the result of photography after ethidium bromide staining is shown in FIG. 1 (lane 3). As is clear from FIG. 1 (lane 3), almost no contamination with E. coli genome-derived DNA or RNA was observed, confirming that plasmid DNA was extracted and purified with high purity.
In FIG. 1, lane 1 is a size marker comprising a PstI digest of lambda phage DNA, lane 2 is a migration pattern of commercially available pBR322 DNA, lane 3 is a migration pattern of pBR322 DNA extracted and purified by the method shown in Example 1, lane 4 shows the migration pattern of EcoRI digest of commercially available pBR322 DNA, and Lane 5 shows the migration pattern of EcoRI digest of pBR322 DNA extracted and purified by the method shown in Example 1.
[0027]
(3) Restriction enzyme digestion of plasmid DNA 1 μl of 10 × H buffer [500 mM Tris-hydrochloric acid (pH 7.5), 1 M sodium chloride, 100 mM magnesium chloride, 10 mM in 8 μl of the recovered solution obtained in (2) above. Dithiothreitol] and 1 μl of 1 U / μl EcoRI (manufactured by Toyobo Co., Ltd.) were added and mixed, and allowed to stand at 37 ° C. for 3 hours.
[0028]
The reaction solution was subjected to agarose gel electrophoresis, and the result of photography after ethidium bromide staining is shown in FIG. 1 (lane 5). As is clear from FIG. 1 (lane 5), the plasmid DNA extracted and purified by the method of the present invention is completely cleaved by the restriction enzyme EcoRI, and it can be immediately confirmed that it can be used for restriction enzyme digestion.
[0029]
Example 2 Extraction of Plasmid DNA from E. coli pUC19 / JM109 (1) Preparation of E. coli pUC19 / JM109 100 μg / ml of E. coli JM109 transformant (pUC19 / JM109) transformed with plasmid pUC19 (manufactured by Toyobo) Was inoculated into 100 ml of SB medium [32 g / l tryptone, 20 g / l yeast extract, 5 g / l sodium chloride, 5 ml 1N NaOH] and cultured for 15 hours at a culture temperature of 37 ° C. and a shaking speed of 180 rpm. After culturing, 1.5 ml of the culture solution is dispensed into 1.5 ml microtubes, centrifuged at 12,000 rpm for 1 minute in a high-speed microcooled centrifuge (MR-150: manufactured by Tommy Seiko Co., Ltd.), and the supernatant The microbial cells obtained by removing were used as extraction materials.
[0030]
(2) Extraction and purification of plasmid DNA In the same manner as in Example 1, plasmid pUC19 DNA was extracted and purified from the cells prepared in (1) above.
The cells were lysed by adding 600 μl of cell lysate [5M guanidine thiocyanate, 100 mM sodium acetate-hydrochloric acid (pH 4.0)], and then 300 μl of TE buffer saturated phenol was added and mixed vigorously. 40 μl of 0.5 g / ml magnetic silica (particle size 1-10 μm, containing 30% iron tetroxide particles, specific surface area 280 m ² / g, pore volume 0.025 ml / g, surface pore diameter 2-6 nm (Suzuki Yushi Co., Ltd.) suspension was added and mixed at room temperature for 10 minutes.
Next, the microtube was placed on a magnetic stand (MPC-M: manufactured by Dynal) to collect magnetic silica particles, and the supernatant was removed. Further, the microtube was removed from the magnetic stand, and 750 μl of cell lysate [5M guanidine thiocyanic acid, 100 mM sodium acetate-hydrochloric acid (pH 4.0)] and 150 μl of TE buffer saturated phenol were added and mixed well as a washing solution. Similarly, the particles were washed by placing on a magnetic stand and removing the supernatant.
Similarly, the particles were washed again, followed by washing the particles twice with 1 ml of 70% ethanol. After removing the supernatant, 100 μl of sterilized water was added thereto, mixed at room temperature for 10 minutes, and then placed on a magnetic stand to collect magnetic silica particles, and the supernatant was collected. The recovered liquid volume was approximately 100 μl.
[0031]
3 μl of the recovered solution is subjected to agarose gel electrophoresis, and after photography with ethidium bromide, the photographed result is shown in FIG. 2 (lane 3). As is clear from FIG. 2 (lane 3), almost no contamination with E. coli genome-derived DNA or RNA was observed, confirming that plasmid DNA was extracted and purified with high purity.
[0032]
(3) Restriction enzyme digestion of plasmid DNA As in Example 1, 8 μl of 1 μl of 10 × L buffer [100 mM Tris-HCl (pH 7.5), 100 mM magnesium chloride, 10 mM dithiothreitol], and 1 μl of 1 U / μl of KpnI (manufactured by Toyobo Co., Ltd.) were added and mixed, and allowed to stand at 37 ° C. for 3 hours.
The reaction solution was subjected to agarose gel electrophoresis, and after photography with ethidium bromide, the photographed result is shown in FIG. 2 (lane 5). As is clear from FIG. 2 (lane 5), it can be confirmed that the plasmid DNA extracted and purified by the method according to the present invention is completely cleaved by the restriction enzyme KpnI and can be immediately used for restriction enzyme digestion. In FIG. 2, lane 1 is a molecular weight marker composed of PstI digest of lambda phage DNA, lane 2 is a migration pattern of commercially available pUC19 DNA, lane 3 is a migration pattern of pUC19 DNA extracted and purified by the method shown in Example 2, lane 4 shows the migration pattern of KpnI digest of commercially available pUC19 DNA, and lane 5 shows the migration pattern of KpnI digest of pUC19 DNA extracted and purified by the method shown in Example 2.
[0033]
【The invention's effect】
According to the present invention, by using a lysate containing a chaotropic substance having a pH of 3 to 6 and a solid phase carrier for nucleic acid binding, a highly pure plasmid DNA is specifically detected from microorganisms or cells holding the plasmid DNA. Further, the plasmid DNA can be extracted and purified in a short time and in a simple manner without the need for complicated post-treatment operations.
[Brief description of the drawings]
FIG. 1 is a photograph replacing a drawing showing an agarose gel electrophoresis pattern of plasmid pBR322 DNA extracted and purified by the method of the present invention and its restriction enzyme digest.
FIG. 2 is a photograph replacing a drawing showing an agarose gel electrophoresis pattern of plasmid pUC19DNA extracted and purified by the method of the present invention and its restriction enzyme digest.

Claims (8)

A method for extracting and purifying plasmid DNA, comprising the following steps (a) to (c):
(A) A cell lysate having a pH of 3 to 6 containing a chaotropic substance, a TE buffer-saturated phenol / chloroform (1: 1) equivalent to the cell lysate, or a cell lysate By adding, mixing or contacting a nucleic acid-binding solid phase carrier containing an extract containing half amount of TE buffer saturated phenol and silica, the plasmid DNA is adsorbed on the solid phase carrier,
(B) washing the solid phase carrier adsorbed with the plasmid DNA in the step (a) with a washing solution; and
(C) The plasmid DNA is eluted from the solid phase carrier washed in the step (b) with an eluent.   The method for extracting and purifying plasmid DNA according to claim 1, wherein the microorganism or cell holding the plasmid DNA is a bacterium.   The method for extracting and purifying plasmid DNA according to claim 1, wherein the cell lysate having a chaotropic substance and having a pH of 3 to 6 comprises guanidine thiocyanate and sodium acetate-hydrochloric acid (pH 4.0).   The method for extracting and purifying plasmid DNA according to claim 1, wherein the nucleic acid-binding solid phase carrier is a particle.   The method for extracting and purifying plasmid DNA according to claim 1, wherein the nucleic acid-binding solid phase carrier is a particle containing a superparamagnetic metal oxide.   The method for extracting and purifying plasmid DNA according to claim 1, wherein the eluate is water or TE buffer. Nucleic acid-binding solid phase carrier a charge of material comprising superparamagnetic metal oxide, further plasmid DNA extracted and purified according to claim 1, wherein by using magnetic comprising the step of separating the nucleic acid binding solid carrier and liquid Law. An extract composed of a chaotropic substance-containing pH 3-6 solution, a cell lysate equivalent to TE buffer saturated phenol / chloroform (1: 1), or a cell lysate half of TE buffer saturated phenol , silica A plasmid DNA extraction and purification reagent kit comprising a solid phase carrier for nucleic acid binding, a washing solution and an eluate.

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