JPS61187793A - Plasmid DNA separation and purification method - Google Patents

Abstract

PURPOSE:A cyclic double-stranded DNA containing RNA and other DNA is subjected to a specific gel filtration chromatography to effect short-time and economical isolation and purification of the titled DNA. CONSTITUTION:A cyclic double-stranded DNA containing other components is previously treated with ribonuclease to digest a part of RNA, then subjected to gel filtration chromatography of spherical, hydrophilic semirigid vinyl polymer gel which has hydroxyl groups and ether groups on its side chains and swells in aqueous solutions, more than 1,000Angstrom average pore diameter and less than 100mum average particle size using, as an eluate, an aqueous solution of 7.0-8.5pH, containing 2-500mM of tris hydrochloride, 1-50mM of EDTA and 10-1,000mM of NaCl to separate and purify a plasmid DNA of 3-12Kbp.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method for separating and purifying plasmid DNA. This invention relates to a method for separating and purifying plasmid DNA from DNA.

[Conventional technology]

Plasmid DNA is an important material in the field of genetic engineering, and is usually obtained by separating and purifying a solution obtained by lysing the cells of a microorganism.

The method that has been commonly used in the past to separate and purify plasmid DNA, particularly to separate plasmid DNA from RNA and chromosomal DNA, utilizes the difference in density resulting from the difference in affinity between plasmid DNA and ethidium bromide. Ethidium bromide-cesium chloride equilibrium density gradient ultracentrifugation was performed.

However, this method requires a long time to operate the ultracentrifuge and remove ethidium bromide and cesium chloride, and cesium chloride is expensive and difficult to reuse. It has drawbacks such as non-negligible operating costs and risks, and the use of ethidium bromide, a known carcinogen, in high concentrations, which may pose no harm to the human body.

Recently, there has been progress in developing carriers for column chromidography for fractionating high molecular weight substances based on the principle of molecular sieves, and there have been several reports of their use in purifying cilasmid DNA. The methods used therein include, for example, (1) Tris-HCl-EDTA-N containing agarose dal filtration carrier and sodium azide;
Combination with aCL aqueous eluate [Lillis et al.
is at *1m), Analytical Bioch@m1stry
), vol. 120, pp. 52-58 (1982)'l, (2
) Agarose dal filtration carrier and Tris-HCl-EDTA
- combination with NaCl aqueous eluate [Holmus (D, S.

Holmea), Analytical Biochemistry
(Analytical Biochemistry
), vol. 127, pp. 428-433 (1982)), (3) polarization with a large network structure? Combination of Limar Dal and Tris phosphate eluate containing Na('t) [Byvater et al.
), F7 /L/ Masha Japan Co., Ltd. Product Catalog, March 1983] Ri(4) Allyldextran-N
, a method using N'-methylenebisacrylamide crosslinked resin [♂Ross et al. (Boro@, I at at, )
, Gone, Vol. 22, pp. 191-201 (1983)].

[Problem that the invention seeks to solve]

In these known methods, the separation of circular double-stranded DNA from chromosomal DNA, etc. is not always sufficient, and further improvements have been desired.

[Means and effects for solving the problem] The present invention has solved this problem as a result of intensive study on a method of combining a specific carrier and a specific eluent. That is, the present invention
Circular double-stranded DNA containing RNA and other DNA as coexisting components is treated with Tris hydrochloride, EDTA and salt) IJ
Hydrophilic semi-rigid cross-linked vinyl having hydroxyl groups and ether bonds in side chains and swells in aqueous solution, N
It is characterized by being subjected to r/L/filtration chromatography on IJ Mama Dal! A method for separating and purifying lasmid DNA is provided.

The hydrophilic semi-rigid vinyl polymer gel used is approximately 50
01. Preferably, it is a spherical colloid having a giant average pore diameter of about 1,000 Å or more and an average particle diameter of about 1004 m or less. Although the upper limit of the average pore diameter is not limited, it is approximately 1×10 4 ^. It is preferable that the pore diameter of each particle be as uniform as possible. There is no particular lower limit to the average particle diameter, but if it is too small, chromatography will take a long time (or high pressure will need to be used), so it is practically about 35 mm. It is also preferable that the particle diameter of each particle is as uniform as possible.

Hydrophilic hard vinyl cross-linked like this? As a refugel,
For example, Toyop*arl (registered trademark, manufactured by Toyo Soda Kogyo Co., Ltd.) HW-758 can be mentioned.

In the present invention, the hydrophilic hard vinyl polymer gel can be packed into a column and used by ordinary liquid chromatography techniques. Including voice part 7
．． 8.5 aqueous buffer solution is used.

The concentration of Tris hydrochloride in this solution is about 2 to about 500
On the order of mM, preferably about 10 to about Zo.

It is about mM. The concentration of EDTA is about 1 to about 50 mM, preferably about 2 to about 20 mM. The concentration of sodium chloride is about 10 to about 1004mM or less, preferably about 50 to about 500mM.

The circular double-stranded DNA that can be separated and purified by the present invention has a size of about 3 to about 12 kb.

The circular double-stranded DNA used in the present invention can be combined with RNA and other D
The sample containing NA is obtained by treating microbial cells with such circular double-stranded DNA with lysozyme, etc. to lyse them, and then boiling the resulting clarified lysate.
lmes, D, S-), Analytical Chemistry (
Analytical Ch@m1stry), 12
7, pp. 428-433], by separating it from proteins, lipids, etc. by a phenol extraction method, an ethanol precipitation method, an ethanol precipitation method, etc., and dissolving it in an appropriate buffer.

In the present invention, such a sample is treated with ribonuclease A in advance to digest a considerable portion of the RNA, and then subjected to dyf-perchromatography. Separation and purification of lasmid DNA can be carried out more effectively and efficiently.

〔Example〕

Example 1 The sample containing the pna 322 plasmid obtained in Sample Preparation Example 1 was dissolved in p) 17.5 buffer solution IR1 containing 20 nM Tris-hydrochloride, 10 molar EDTA, and 100 mM NaCl, and dissolved in Toyopearl ( (Registered Trademark: Previous) Relfiltration chromatography was performed using HW-758 as a dull carrier. The conditions were as follows. Detection was carried out by absorbance measurement at 254 nm in both Examples and Comparative Examples Column: 1.5 mm x 30 m x 2 Jl of each fraction: 60 drops Elution pressure: about 5.0 kg! /α2 Elution rate: 36ffi//hour The obtained chromatogram is shown in FIG. In Figure 1, 1 is chromosomal DNA, 2 is plasmid DNA, and 3 is RN.
Each of A indicates one. FIG. 2 shows electrophoretic images of each fraxin on an agarose gel.

10 μL of each eluate fraction was subjected to 0.747 I low flow electrophoresis. Figure 2 shows its migration and turns. Fraction numbers 26, 28 and 30 contained chromosomal DNA, and fraction numbers 34, 36, 38 and 40 contained plasmid DNA.

Furthermore, it is shown that fraction numbers 52, 54, 56, and 58 contain RNA as a main component. In addition, H of λ-phage DNA is used as a molecular weight marker (1).
lnd m digest and molecular marker (2) (standard)
The pBR322 plasmid containing the dimorphism was used as a pBR322 plasmid.

Example 2 The sample containing the ptyc 8 plasmid obtained in Sample Preparation Example 2 was dissolved in -8.0 solution (hereinafter referred to as TI buffer) 1d containing 10rrM Tris hydrochloride and 1mM EDTA.

Immobilized ribonuclease A (manufactured by Sigma, Ribonu
cleas@As R-4001s Insoluble enzyme derived from bovine pancreas supported on agarose beads) 5 units were washed several times with water, and then Sephadex swollen with water (
5ephadsx trademark) G-251,7mj was added and packed into the column.

The column was equilibrated with TI buffer, and the sample solution was passed through it and washed with TE buffer. Precipitate the DNA component from the passthrough 12-, and add the precipitate to the same elution buffer 1d as in Example 1.
Dissolved in Toyopearl (registered trademark: supra) H
Relfiltration chromatography was performed using W-758 as a gel carrier. The conditions were as follows: O Column: 1.5 cIgIX46clR Amount of each fraction: 40 drops Elution pressure: Crab hydrostatic pressure: 90 Elution rate: 12 WLl/hr.

The obtained chromatogram is shown in Figure 3. In Figure 3, 1
．． 2 and 3 have the same meaning as in FIG.

Example 3 The same immobilized ribonuclease A column used in Example 2 and the Toyopearl (registered trademark) column were connected so that the eluate from the former would flow into the latter. After equilibrating the Ryoriki Ram with the same eluate used in Example 1, a sample containing the pUC8 plasmid obtained in the same manner as in Sample Preparation Example 2 was dissolved in this eluate by 1 dK, and the immobilized reconuclease A was dissolved. The eluate was used for digestion with ribonuclease AK and Toyopearl.
(registered trademark: supra) Dull filtration chromatography using HW-75S was performed at the same time. The elution conditions were as follows.

Amount of each fraction: 40 drops Elution pressure Hydrostatic pressure 90 cIn Elution rate = 12 g/hr The resulting chromatogram is shown in FIG. 1 in Figure 4,
2 and 3 have the same meaning as in FIG.

Example 4 Digestion with ribonuclease A and Toy were carried out in the same manner as in Example 3 except that half of the sample obtained in Sample Preparation Example 3 was used.
Gel filtration was performed using opearl (registered trademark: supra). The obtained chromatogram is shown in FIG. 1, 2 and 3 in FIG. 5 have the same meanings as in FIG.

Examples 5 and 6 Ribonuclease digestion and Delp filtration were performed in the same manner as in Example 4, except that the sodium chloride concentration of the eluate was changed to 200 mM (Example 5) and 300 mM (Example 6). The obtained romatodalum is shown in FIG. 6 (Example 5) and FIG. 7 (Example 6). 1, 2 and 3 in both figures represent the same meanings as those in FIG.

Example 7 20mM) Liss hydrochloride, 200mM NaCt,
0.7 m of anion exchange cellulose (DEAE cellulose (Whatman DI52, trademark)) equilibrated with 1 mM EDTA, pH 7.4 buffer was applied to the column, and the plasmid DNA obtained in Examples 4 to 6 was added to the column. A total amount of 80+++j) of the fractions was passed through the tube to adsorb DNA.

After washing the column with 40t of the same buffer, 20mM)
IJS hydrochloride, 1mM NaCt, 1mM ED
Elution was performed with TA, pH 7.4 buffer 5d. The eluate was precipitated with ethanol (twice), rinsed with ethanol, dried under vacuum, and dissolved in TE buffer (500 fitf).The yield calculated from the absorbance at 260 nm was about 420 μ9.

Sample Preparation Example 1 Escherichia coli carrying plasmid pBR322, B. coli HBIOI strain (B, coli HB 101/p
BR322) was precultured in LB medium containing 25 μm1/IrLl of ampicillin, and this was cultured in the same medium 1! ! was inoculated at 0.5 inch, and cultured with shaking at 37°C for 9 hours. After completion of the culture, the bacterial cells were collected by centrifugation, and added with 20mM Liss hydrochloride, 100mM NaCl, 5mM EDTA.
8M buffer containing pH 7.5 was added and mixed.

Add to this 8-group sucrose, 5% Triton X-100゜50 m
MEDTA, 50 mM) p containing lithium hydrochloride
) After adding and mixing 70rnl of 18.0 buffer solution, add lysozyme solution (so that the concentration is 10Tn9/lut,
10 mM lysozyme) lys hydrochloride and 1 mM
PI(s, ) containing EDTA.

(dissolved in the buffer solution) 5d was added. The resulting solution was heat-treated in boiling water for 75 seconds while being gently stirred. After quenching this, centrifuge it and add isopro-9 to the supernatant.
Nol 50d was added, mixed and kept at -20°C for 30 minutes. Collect the precipitate by centrifugation and add 0.3 M
Add sodium acetate solution 7,5 rILt and mix;
Insoluble matter was removed by centrifugation.

Add 20 rILt of ethanol to this, and after mixing -20
Leave overnight at 9°C.

The precipitate was collected by centrifugation, rinsed with ethanol, and vacuum dried to obtain a starting sample for the present invention.

Sample preparation examples 2 and 3! Nijieri shakori HB1 with lasmid pBR322
Plasmid pUC8 (Sample Preparation Example 2) in place of the 01 strain
and pUC9 (Sample Preparation Example 3), E. coli strain JM83 (E, coil JM83/p
A starting sample was prepared in the same manner as in Sample Preparation Example 1 using UC8 and E. coli JM83/pUC9).
Compatible samples were obtained for each. These cilasmids have been described by Gene (Vieira, J* et al., Gon
e), Vol. 19, p. 259 (1982).

〔Effect of the invention〕

According to the present invention, cilasmid D, which has been difficult to obtain with conventional technology,
Contamination of chromosomal DNA into the NA fraction can be almost completely prevented.

Furthermore, according to the present invention, highly purified plasmid DNA can be obtained in a shorter time and more economically than with conventional techniques.

[Brief explanation of drawings]

FIG. 1 and FIGS. 3 to 7 are diagrams showing chromatograms obtained when plasmid DNA was separated and purified according to the present invention, and FIG. 2 shows agarose electrophoresis/ It is a figure showing a 4' tongue. ""- Shinbe Koji 1, C 1st ward, Figure 3, Line 4, Figure 5, Figure 6, 1n, Doda 456 Hatchi

Claims (4)

[Claims] (1) Cyclic double-stranded DNA containing RNA and other DNA as coexisting components is prepared using an aqueous developing solution containing Tris hydrochloride, EDTA, and sodium chloride with a pH of about 7.0 to about 8.5. 1. A method for separating and purifying plasmid DNA, which comprises subjecting it to gel filtration chromatography on a hydrophilic semi-rigid crosslinked vinyl polymer gel that has hydroxyl groups and ether bonds and swells in an aqueous solution. (2) The size of the plasmid DNA to be purified and separated is approximately 3
12 kbp to about 12 kbp. (3) The hydrophilic semi-rigid vinyl polymer gel used is approximately 1,
Claim 1 which is a spherical gel having a giant average pore diameter of 000 Å or more and an average particle diameter of about 100 μm or less
) or (2). (4) The separation and purification method according to any one of claims (1) to (3), wherein at least a portion of the RNA is previously digested with ribonuclease and then subjected to gel filtration chromatography.