KR100486179B1 - Cell lysis composition, method and kit for isolating and purifing nucleic acids - Google Patents

Abstract

Chaotropic salts selected from the group consisting of 2M to 6M guanidine HCl, guanidine SCN and NaCl; 0.5% to 10% by weight of nonionic surfactant; PIPES of 10 mM to 50 mM; 0.1 mM to 5 mM EDTA; And a cell lysis composition comprising 0.2% to 5% sodium lauryl salcosylate, a method for separating and purifying nucleic acids using the same, and a kit comprising the same.

Description

CELL LYSIS COMPOSITION, METHOD AND KIT FOR ISOLATING AND PURIFING NUCLEIC ACIDS

(Technology)

The present invention relates to cell lysis compositions, methods and kits for isolating nucleic acids present in cells. More specifically, the present invention is directed to cytolytic compositions, methods, and kits for lysing cells and isolating and purifying DNA from cultures of cells containing DNA. The present invention relates in particular to improved cytolysis compositions, methods and kits for separating and purifying plasmid DNA from microbial cells using DNA, more particularly improved cytolysis buffer.

(Background)

In the art currently performed in the field of biotechnology, nucleic acid fragments are routinely isolated and purified from prokaryotic or eukaryotic or viral cultures. These isolated fragments can then be used for a variety of industrial applications, such as recombinant engineering, sequencing or diagnostic probes, and other genes for the analysis of genes of the entire protein or peptide to which the fragment belongs. Traditionally, nucleic acids have been isolated and purified from proteins and other intracellular materials by lysing in the presence of a surfactant such as sodium dodecyl sulfate and an aqueous solution of a salt such as potassium acetate, followed by extraction with phenol or chloroform or mixtures thereof.

In recombinant DNA technology, DNA molecules are generally isolated from bacterial cell cultures or bacteriophage cultures. For example, double stranded plasmid DNA is produced and isolated in bacterial cells, such as Escherichia coli , which are cultured in liquid nutrient broth medium.

Methods commonly used for isolation of plasmid DNA from bacterial cultures are detailed in HC Birnboim and J. Doly, "A Rapid Alkaline Extraction Procedure for Screening Recombinant Plasmid DNA", Nucleic Acids Res., 7: 1513-1523 (1979). It is described.

Small-scale experiments to isolate plasmid DNA, better known as miniprep, are routinely used in molecular biology experiments. Ausubel, F. M. et al. (1989), Current protocols in Molecular Biology, Vol. 2, John Wiley & Sons, NY.

This method has the advantage of being able to prepare plasmid DNA from a large number of samples in a short time, but it is generally lower in purity and requires a lower degree of purity than larger plasmid DNA purification methods, including ultracentrifugation using a CsCl gradient. It can be used only in a few limited applications and has the disadvantage that there is a need for purification again if subsequent experiments require the production of higher purity plasmid DNA.

Therefore, the traditional small scale method of separating and purifying plasmid DNA by ethanol precipitation method (Lis and Schleif, Nucleic Acids Res., 2: 383-389 (1975)) after using three buffers represented by dilution, disruption and neutralization of cells. Methods that are more effective in terms of time and purity of the target DNA than the plasmid DNA separation method have been devised. An example of such methods is plasmid DNA extraction and purification, which is pure DNA separation through a column developed to improve purity and overcome the inconvenience of using ethanol precipitation in the step of separating DNA from cell lysate. . Horn et al., Human Gene Ther., 6: 565-573 (1995); US Pat. No. 5,707,812; Chandra et al., Anal. Biochem., 203: 169-172 (1992); Marquet et al., BioPharm.:26-37 (1995), etc.)

In recent years, extraction and purification kits including columns using this technique have become the basis for the plasmid DNA purification method. The principle of plasmid DNA extraction using such a column uses the principle of structural interaction between water molecules and DNA using glass fiber or silica membrane that specifically binds to DNA, and then the DNA molecules are separated from proteins and other intracellular materials. To refine.

The currently available commercially available plasmid DNA purification system including the use of such a column is essentially composed of three steps of buffer treatment and purification using a column, similarly to the cell lysis process in the conventional method described above. The disadvantage is that it is cumbersome and time consuming to process a large number of samples.

On the other hand, the DNA separation method using such a column is currently highly developed, and considering that the time required for the final step of using the column during the entire DNA separation and purification process is very short and the required degree of mastery is very low, column centrifugation or The necessity of simplifying the process and time of the step of preparing the cell lysate prior to filtration is increasing.

Moreover, in recent years, with the development of genetic engineering, a large number of samples have to be processed at the same time in a short time. Also, since the results of the experiment can be trusted only when reproducible results can be obtained even in repetitive experiments, the method is performed with a minimum of steps. Increasingly, there is a growing need to simplify processing. Furthermore, such a simplified process is required to ensure the purity and amount of plasmid DNA to the extent that subsequent application of the experiment is possible. Another advantage of process simplification is that it increases the reproducibility and success probability of the result by the experimenter's skill.

Thus, the present inventors used a simpler method by using only one newly provided cytolysis buffer instead of three different buffers conventionally used for the purpose of dilution, crushing and neutralization for extracting DNA from cells. We found the surprising fact that we can isolate large amounts of high purity DNA at a fraction of the cost in less time. In addition, a kit for plasmid DNA separation and purification comprising the novel cell lysis buffer was prepared, and the plasmid DNA can be separated at a higher yield with a higher reproducibility in a shorter step and time than the conventional method using the buffer from various cells. The present invention has been completed by revealing that there is.

It is an object of the present invention to provide an improved cytolytic composition for isolating and purifying plasmid DNA contained in cells.

It is an object of the present invention to provide a simplified method for isolating and purifying plasmid DNA contained in cells.

It is another object of the present invention to provide a kit for use in a simplified process for isolating and purifying plasmid DNA contained in cells.

To achieve this object, a chaotropic salt selected from the group consisting of 2M to 6M guanidine HCl, guanidine SCN and NaCl, 0.5% to 10% by weight of nonionic surfactant, 10mM to 50mM of PIPES, 0.1 mM to 5 mM EDTA; And 0.2% to 5% sodium lauryl salcosylate It provides a cell lysis composition comprising.

In another aspect of the invention,

(a) collecting the cells from the cell culture;

(b) a catotropic salt selected from the group consisting of 2M to 6M guanidine HCl, guanidine SCN and NaCl, 0.5% to 10% by weight of nonionic surfactant, 10mM to 50mM PIPES, 0.1mM to 5mM EDTA, And suspending the cell collection of step (a) in a cell lysis composition comprising 0.2 wt% to 5 wt% sodium lauryl salcosylate to incubate for 1 to 10 minutes to provide lysate.

(c) removing the insoluble material in the cell lysate of step (b) to obtain a cell lysate;

(d) isolating and collecting DNA from the cell lysate of step (c), thereby providing a method for separating intracellular plasmid DNA.

The cell is preferably a microbial cell, more preferably a bacterial cell. In addition, step c) preferably provides a solution containing the plasmid DNA without centrifugation of insoluble impurities from the plasmid DNA in the cell lysate.

In another aspect, the present invention provides a kit for use in the above plasmid DNA separation method.

Simple and rapid plasmid DNA purification methods using the cell lysis compositions provided herein produce high purity plasmids with very high yields. This method introduces a cell lysis method using a simplified cell lysis composition which is completely different from the basic lysis based method of performing three steps of buffer treatment to obtain cell lysate in the conventional method. Employs a column purification method conventionally used. This method typically produces 30 μg to 60 μg of plasmid DNA per 3 ml of bacterial culture, with a purity _ranging from 1.8 to 2.0 (OD _260/280 ). Plasmids prepared according to this method were superior in other linkage experiments, including higher DNA sequencing success rates compared to plasmids isolated by conventional three-step lysis methods based on basic lysis.

Since the plasmids are purified to higher purity than conventional miniprep methods, the isolated plasmid DNA can be used as a sample for a variety of genetic engineering follow-up experiments, the process is simplified to increase reproducibility, and is more efficient in terms of time and cost. Provide a method. The described method allows for larger scale-ups as needed.

Hereinafter, the present invention will be described in detail.

Justice

As used herein, "plasmid" refers to a circular gene having a gene that can be artificially combined to replicate in E. coli bacteria and yeast.

As used herein, "cell lysis" refers to the outflow of cytosolic solution by breaking down the cell membrane of a single cell organism having a cell membrane such as bacteria.

As used herein, "DNA adherent column" refers to a column packed with cationic metal beads, or cationic adsorption fibers.

Specific method of carrying out the present invention

The present invention provides a cytolytic composition.

The lysis composition of the present invention comprises a catotropic salt selected from the group consisting of 2M to 6M guanidine HCl, guanidine SCN and NaCl, 0.5% to 10% by weight of nonionic surfactant, 10mM to 50mM of PIPES and 0.1mM to A cell lysis composition is provided comprising 5 mM EDTA.

The cytolytic composition is prepared from an aqueous solution in which a chaotropic salt, a nonionic surfactant, PIPES and EDTA is dissolved, selected from the group consisting of guanidine HCl, guanidine SCN and NaCl. More preferably, the first treatment solution contains RNase A at a concentration of 100 ug / ml.

As the nonionic surfactant in the cytolytic composition, both polyethyleneglycol type nonionic surfactants and polyhydric alcohol type nonionic surfactants may be used. Preferably, Triton X-100, Tween (; sorbitan) is used. Ethylene oxide adducts of esters) or 2-mercaptoethanol, most preferably Triton X-100.

The chaotropic salt in the cytolytic composition component can be used with any chaotropic salt used in the art for ionic purposes that serves to bind nucleic acids to substances such as silica gel charged to the column with an affinity bond. Preferably, guanidine HCl, guanidine SCN (thiocyanate), NaCl are used, most preferably guanidine HCl.

The concentration of the chaotropic salt selected from the group consisting of guanidine HCl, guanidine SCN and NaCl in the cell lysis composition component is preferably 2M to 6M, more preferably 2.5M to 4M. The concentration of PIPES is preferably 10 mM to 50 mM, more preferably 10 mM to 30 mM. The concentration of the nonionic surfactant is preferably 0.5% by weight to 10% by weight, more preferably 1% by weight to 5% by weight. The concentration of EDTA is preferably 0.1 mM to 5 mM, more preferably 1 mM to 3 mM.

In another aspect the invention provides methods for plasmid DNA isolation and purification.

Plasmid DNA isolation and purification methods of the present invention comprises the steps of (a) collecting cells from the cell culture;

(b) from 2M to 6M guanidine HCl, guanidine SCN and NaCl, a chaotropic salt selected from the group consisting of 0.5 wt% to 10 wt% nonionic surfactant, 10 mM to 50 mM PIPES and 0.1 mM to 5 mM EDTA. Suspending the cell collection of step (a) in a cell lysis composition comprising incubating for 1 minute to 10 minutes to provide a cell lysate;

(c) removing the insoluble material in the cell lysate of step (b) to obtain a cell lysate;

(d) isolating and collecting DNA from the cell lysate of step (c), thereby providing a method for separating intracellular plasmid DNA.

The incubation time of the lysis step using the lysis composition is from 3 minutes to 10 minutes, preferably 3 minutes, most preferably 5 minutes. If the most optimal 10 minutes were used, about 95% of the DNA could be obtained, and a satisfactory result could be obtained even with a short time of 5 minutes.

Plasmid sizes that can be separated by the present invention are 0.07 kb to 100 kb, preferably 2 kb to 100 kb, more preferably 1000 kb or less. If the size of the plasmid is too large, efficiency can be reduced when separating from the column.

Step (d) may be used in any conventional method for DNA purification. Preferably ethanol precipitation, other nucleic acid precipitation methods or centrifugation or filtration using DNA affinity columns may be used, more preferably centrifugation or filtration using DNA affinity columns.

Step (d) comprises the steps of (i) loading the liquid phase of step (c) into a DNA adhesion column; And (ii) eluting the attached DNA after column washing. The DNA adhesion column may be any column conventionally used for DNA separation, but a column consisting essentially of anion exchange resin, glass fiber or silica membrane may be used, and silica membrane is particularly preferable. Preferably the wash buffer is 50-80% ethanol and 100 mM to 1500 mM NaCl aqueous solution and the elution buffer is 5 mM to 20 mM Tris-HCl aqueous solution.

Plasmid DNA separation method of the present invention has the advantage of reducing the total amount of the step and the treatment buffer and the time required to separate the DNA in the separation and purification of the plasmid from the cells. In addition, by using a combination of surfactant and guanidine HCl from the cell disruption stage, it is possible to minimize the possibility of intracellular proteins affecting DNA, thereby minimizing the possibility of DNA damage caused by nuclease, a problem of the existing DNA prep. It is possible at the same time and allows the provision of higher purity plasmid products.

The present invention also provides a kit used to carry out the cell lysis and DNA separation and purification methods.

Plasmid DNA separation kit of the present invention is a catotropic salt selected from the group consisting of 2M to 6M guanidine HCl, guanidine SCN and NaCl, 0.5% to 10% by weight of nonionic surfactant, 10mM to 50mM of PIPES, 0.1mM Cell lysis compositions comprising from 5 mM EDTA and from 1 to 500 μg / ml RNase A.

Preferably the kit comprises (b) a DNA adhesion column; (c) column wash buffer; And (d) column elution buffer, wherein the DNA attachment column may be any affinity DNA attachment column used conventionally and the wash buffer and elution buffer used may be any known conventionally appropriate for each column. It may be a buffer of.

In one preferred embodiment, the DNA adhesion column is a spin column, and elution is performed by centrifugation or filtration.

Using such methods and kits has the advantage of reducing the time required to prepare plasmid DNA with higher purity and better application of other linked experiments and saving reagents by working with a simplified process than conventional methods. In addition, by allowing the cell lysis and DNA separation step that is divided into three steps in the conventional one step, it is possible to obtain a more reproducible results by reducing the chance of mistakes and errors that the operator can make.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples, since the gist of the present invention is limited only by the claims. Will be evident to those of ordinary knowledge in Esau.

Example

Example 1: Preparation of lysis buffer of the present invention

(Manufacture example 1)

We preferred 3M guanidine-HCl, 5 wt% Triton X-100, 25 mM PIPES, 2 mM EDTA, and 1 wt% sodium lauryl as one preferred embodiment of the cytolytic composition for separating intracellular plasmid DNA. An aqueous solution consisting of salcosylate was prepared.

(Manufacture example 2)

As one preferred embodiment of the cell lysis composition for isolating intracellular plasmid DNA, the inventors have described 2M guanidine-HCl, 10 wt% Triton X-100, 50 mM PIPES, 1 mM EDTA and 1 wt% sodium lauryl salil. An aqueous solution consisting of cosylate was prepared.

Example 2: Plasmid Separation Using Cytolytic Buffer of the Invention in E. coli

The present inventors isolated and purified the plasmid DNA according to the method of the present invention using the cell lysis composition of the present invention prepared in Example 1 and the preferred kit of the present invention.

1) Inoculated with E. coli (XL1 Blue) single colony containing plasmid DNA pBluescript II (commercially available from Stratagene) at 37 ° C. in 3 ml LB broth containing 50 μg / ml of ampicillin and incubated for 16 hours. Then, the cells were precipitated and recovered at 13000 rpm for 2 minutes.

2) The recovered cells were treated with 300 μl of 3M guanidine-HCl, 5 wt% Triton X-100, 25 mM PIPES and 2 mM EDTA, 1 wt% sodium lauryl salcosylate and 100 μg / ml RNase A. Resuspended in the composed aqueous solution and incubated for 5 minutes at room temperature. The mixture was occasionally stirred during incubation.

3) The whole incubated cell lysate was loaded into a spin column (purchased from Canada BBI) and centrifuged at 12000 rpm for 30 seconds, followed by 500 μl of 50% ethanol, 1 M potassium acetate, 0.1 M NaCl, 1 mM EDTA and Wash buffer containing 10 mM Tris Cl (pH 7.5) was added and centrifuged for 15 seconds at 12000 rpm. After the flow was discarded and the washing step was repeated once, centrifugation was performed for 15 seconds at 12000 rpm with the addition of a wash buffer containing 500 μl of 50% ethanol, 0.1 M NaCl, 1 mM EDTA, and 10 mM Tris Cl (pH 7.5). Centrifugation was performed at 12000 rpm for 10 seconds to remove any remaining wash solution.

4) The column was placed in a collection tube and 50 μl of 10 mM Tris HCl (pH 8.0) aqueous solution was added to the column, followed by centrifugation at 13000 rpm for 1 minute.

5) 0.2, 0.5 and 1 μl of the collected 50 μl plasmid DNA aqueous solution was taken and loaded onto a 1% agarose gel, followed by electrophoresis at 110 V for 15 minutes.

The total time for separation and purification is 17 minutes.

Agarose gel photos using 0.5 μl in 50 μl of isolated and purified pBluescript II plasmid DNA aqueous solution are shown in FIG. 2 (lane 1).

Comparative Example 1 Plasmid Separation Using Commercial Miniprep Kit Available Commercially in E. coli

Plasmid pBluescript II DNA contained in the cells collected according to step 1) of the method of Example 2 was carried out by Qiagen's Qiagen Plasmid Mini and three foreign companies (Omega's "Plasmid Mini Kit I" and Qiagen's "Plasmid Mini Kit" Qbiogen). "Plasmid Mini Kit") Three domestic companies (Bionia's "AccuPrep Plasmid Extraction Kit", General BioSystem's "Plasmid DNA mini prep kit" and Atman Bioscience's "DNA purification kit") Separated. Isolation plasmid pBluescript II DNA was electrophoresed on agarose gel.

The total time for separation and purification is 27 minutes-35 minutes.

Agarose gel photos using 0.5 μl in 50 μl of isolated and purified pBluescript II plasmid DNA aqueous solution are shown in FIG. 2 (lanes 2 to 7 respectively for A to F).

The concentrations and total amounts of pBluescript II plasmid DNA isolated and purified using the kit of the present invention including the cell lysis composition of the present invention in Example 2 and Comparative Example 1, and a conventional commercially available kit are shown in Table 1 below.

lane manufacturer Concentration (μg / μl) Total separation amount (㎍) One Kit of the invention 1,200 60 2 Omega Corp 300 15 3 Qbiogen 300 15 4 Qiagen 800 40 5 Bioneer 700 35 6 General Biosystems Corporation 300 15 7 Atman Bioscience Corp 700 35

Example 3 Restriction Enzyme Cleavage Experiment Using DNA Isolated by the Method of the Present Invention

DNA isolated in Example 2 was digested with restriction enzyme (HindIII / EcoRI) according to the manufacturer's instructions and then electrophoresed on 1% agarose gel, and the result is shown in FIG. 3. Each lane in Figure 3 shows the results by restriction enzyme cleavage of 13 miniprep products using the kit of the present invention.

Example 4 DNA Sequencing Using DNA Isolated by the Method of the Invention

DNA sequencing was performed using DNA isolated by the method of Example 2. PCR was performed by mixing 200 ng of sample with 1 pmol of primer and 2 μl of Bio-dye terminator from Applied Biosystem, and PCR products were analyzed using 3700 model of Applied Biosystem after ethanol purification.

The results are shown in FIG.

The methods and kits provided by the present invention can be used to prepare bacterial lysates and to isolate and purify plasmid DNA more efficiently in terms of time and cost, as well as to isolate and purify the plasmid miniprep from conventionally used products. Higher plasmids and higher DNA purity than the product of the method allows for more diverse and improved follow-up experiments.

1 is a flow chart comparing the plasmid DNA extraction method according to the method (A) and the conventional method (B) of the present invention.

Figure 2 is a comparative agarose gel photograph of plasmid DNA isolated and purified using the kit of the invention and a conventional commercially available kit.

Figure 3 is an agarose gel photograph showing the restriction enzyme cleavage results of plasmid DNA isolated and purified using the method of the present invention.

4 is a comparative photograph of the results of DNA sequencing assays performed using the kit of the present invention and a conventional commercially available kit.

Claims (10)

A chaotropic salt selected from the group consisting of 2M to 6M guanidine HCl, guanidine SCN and NaCl; 0.5% to 10% by weight of nonionic surfactant; PIPES of 10 mM to 50 mM; 0.1 mM to 5 mM EDTA; And A cytolytic composition comprising 0.2 wt% to 5 wt% sodium lauryl salcosylate. The cytolytic composition of claim 1, wherein the nonionic surfactant is 0.5% to 10% by weight of Triton X-100. The lysis composition according to claim 1, wherein the composition further comprises 1 to 500 g / ml of RNase A. (a) collecting the cells from the prokaryotic culture; (b) a catotropic salt selected from the group consisting of 2M to 6M guanidine HCl, guanidine SCN and NaCl, 0.5% to 10% by weight of nonionic surfactant, 10mM to 50mM PIPES, 0.1mM to 5mM EDTA, Suspending the cell collection of step (a) in a cell lysis composition comprising 0.2% to 5% by weight sodium lauryl salcosylate and 1 to 500 μg / ml of RNase A and incubating the cells for 1 minute to 10 minutes Providing a lysate; (c) removing the insoluble material in the cell lysate of step (b) to obtain a cell lysate; (d) isolating and collecting DNA from the cell lysate of step (c). The method of claim 3 or 4, wherein the nonionic surfactant is Triton X-100. The process of claim 3 or 4, wherein step (d) (i) loading the liquid phase of step (c) into a DNA adherent column; And (ii) eluting the attached DNA after column washing. 7. The method of claim 6, wherein said DNA adherent column is a spin column and eluting is by centrifugation. 2M to 6M guanidine HCl, 0.5% to 10% by weight nonionic surfactant, 10mM to 50mM PIPES, 0.1mM to 5mM EDTA, 0.2% to 5% sodium lauryl salcosyl and 1 to 500 A kit for isolating intracellular plasmid DNA comprising a cell lysis composition comprising μg / ml of RNase A. The kit of claim 6 wherein the kit is (b) a DNA adhesion column; (c) a first wash buffer and a second wash buffer; And (d) A kit for separating intracellular plasmid DNA further comprising column elution buffer. The method according to claim 8 or 9, wherein the DNA adhesion column is a spin column and the first wash buffer is 50% ethanol, 1M potassium acetate, 0.1M NaCl, 1 mM EDTA and 10mM Tris Cl, the second wash buffer is 50% Ethanol, 0.1M NaCl, 1 mM EDTA and 10 mM Tris Cl, and the elution buffer is 10 mM Tris HCl.