JPH03108661A - Separating method of substance relevant to nucleic acid - Google Patents

Abstract

PURPOSE:To enable efficient separation and refining of substances relevant to a nucleic acid by an ion exchange treatment by a method wherein a liquid containing the substances relevant to the nucleic acid which have specific molecular weights are passed through a column filled up with a cross-linking glucomannan ion-exchanger. CONSTITUTION:First, a cross-linking glucomannan ion-exchanger which is a spherical particle of a particle size 10 to 1,000mum and of which an ion exchange capacity is 0.01 to 5meq/g-gel and an exclusion limit molecular weight 1,000,000 or above is filled up in an appropriate column, and a sample is injected in an ion-exchange column thus obtained. For the injection of the sample, the sample containing substances relevant to a nucleic acid which are to be separated is diluted to be 0.5 to 1,000mug/ml by an appropriate eluant, and the injected sample is absorbed on the ion-exchanger. Subsequently, the eluant is made to flow at a flow rate of LV 10 to 1,000cm/H. On the occasion, the salt concentration, pH or the like of the eluant is varied to give a concentration gradient to components. Thereby the substances relevant to the nucleic acid in the sample adsorbed on the ion-exchanger are eluted sequentially from the one of a smaller molecular weight, and by taking out an eluate for each fraction, therefore, the substances relevant to the nucleic acid in the sample can be separated and refined.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a method for separating nucleic acid-related substances, particularly
The present invention relates to a method for easily and efficiently adsorbing, separating, and purifying nucleic acid-related substances by ion exchange treatment from a solution containing nucleic acid-related substances such as giant DNA with a molecular weight of 1 million or more, such as A.

[Prior Art] Nucleic acid-related substances with small molecular weights, such as nucleic acid bases, nucleosides, and nucleotides, have conventionally been separated and purified using styrene-based ion exchangers. Furthermore, oligonucleotides having a larger molecular weight than nucleotides are separated and purified using cross-linked dextran ion exchangers, etc., which have a looser network structure than styrene-based ion exchangers. All of these separation and purification mechanisms are based on the ion exchange mode based on the electric charge of the phosphate groups of nucleic acid bases and nucleotides.

On the other hand, nucleic acid-related substances having a larger molecular weight than oligonucleotides are separated by gel filtration mode or ion exchange mode using agarose-based or synthetic polymer-based gel filtration agents or ion exchangers. Among these, the ion exchange mode achieves a degree of separation and throughput that cannot be achieved with the gel filtration mode by appropriately adjusting the adsorption and elution conditions for the target substances nucleotides, DNA, and RNA. Because it is possible to
It is seen as promising as an indispensable separation method in this field.

[Problems to be Solved by the Invention] However, in the above-mentioned ion exchange mode, when the target substance to be treated is a huge NA with a molecular weight of 1 million or more, sufficient treatment cannot be achieved for practical purposes. In the ion exchangers currently available on the market, the pore diameter is too small for the target substance, and the ion exchange groups inside the pores are not used effectively, resulting in extremely low adsorption capacity. Ion exchange groups outside the pores can effectively act on large DNAs, etc., but the effective ion exchange capacity of only the ion exchange groups outside the pores, that is, the adsorption capacity of giant DNAs, etc., is practically insufficient for separation and purification. It is difficult. Moreover, non-specific adsorption to giant DNA etc. tends to occur, resulting in problems such as a poor recovery rate.

One way to solve these problems is to eliminate the pores of the ion exchanger and increase the external surface area, thereby increasing the size of the giant D.
Methods have been devised to separate and purify biopolymers with large molecular weights such as NA.

According to this method, the adsorption capacity for large DNA etc. is improved compared to conventional ion exchangers, and separation and purification is possible, but on the other hand, it has the following drawbacks. That is, since this method increases only the specific surface area without using pores, the ion exchanger needs to have a very small particle size of 3 μm or less. In order to obtain a practical flow rate using such a particulate ion exchanger, it is essential that the particle size of the ion exchanger be extremely uniform and that high pressure be applied during liquid passage. Become. However, among the ion exchangers currently on the market, those in the form of fine particles with a uniform particle size are extremely expensive. Therefore, it is economically disadvantageous to use such an ion exchanger. Furthermore, passing a liquid containing unstable biopolymers through a column packed with such an ion exchanger under high pressure may lead to deterioration or destruction of the biopolymers. Another problem is that it is difficult to recover the waste while preserving its original condition. In addition, when using particles with a small particle size, it becomes necessary to use a filter with even finer openings than is normally used, and in reality, liquids containing nucleic acid-related substances such as giant DNA are treated with fine particles. It becomes difficult to pass liquid through a column packed with a ion exchanger. For this reason, techniques using particulate ion exchangers are currently applied exclusively to protein analysis.

In addition, methods other than liquid chromatography (liquid chromatography) include electrophoresis using agarose gel, etc.
It is used as a means of separating nucleic acid-related substances based on their molecular weight. Although this method has a good degree of separation, it has disadvantages such as being more complicated to operate than liquid chromatography, and having difficulty increasing the throughput and being unsuitable for preparative separation.

For these reasons, at present, the separation of giant DNA and the like is carried out using the conventional ultracentrifugation method over a long period of time.

The present invention solves the above-mentioned conventional problems, and easily and efficiently separates and purifies nucleic acid-related substances such as giant DNA from a solution containing nucleic acid-related substances with a molecular weight of 1 million or more by ion exchange Ig1 phantom immersion. The purpose of the present invention is to provide a method for separating nucleic acid-related substances.

Means for Solving Problem cii] The method for dividing nucleic acid-related substances according to claim (1) is characterized in that the molecular weight is 1
The nucleic acid-related substance according to claim (2), characterized in that the nucleic acid-related substance is separated by passing a solution containing 1,000,000 or more nucleic acid rJA fast substances through a column packed with a cross-linked glucomane ion exchanger. The separation method is defined in claim (1)
The method is characterized in that the liquid containing the nucleic acid-related substance further contains protein.

That is, the present inventors discovered that a giant DNA with a molecular weight of 1 million or more
A method for easily adsorbing and separating high-molecular-weight nucleic acid-related substances, such as substances, using a simple liquid chromatography device and method, without the need for high-pressure liquid passage, with high processing efficiency, and at low cost with high separation efficiency. As a result of intensive study, Unexamined Japanese Patent Publication No. 1-94
It was discovered that the cross-linked glucomane ion exchanger disclosed in No. 949 is particularly effective in separating large nucleic acid-related substances with a molecular weight of 1 million or more, and in particular in separating and purifying multiple DNAs, and completed the present invention. Ta.

The present invention will be explained in detail below.

The crosslinked glucomannan ion exchanger according to the present invention is produced by adding diethylaminoethyl groups (hereinafter referred to as rDEAE groups), carboxymethyl groups (hereinafter referred to as rCM groups), sulfonate groups to crosslinked glucomannan spherical particles according to a conventional method. It can be prepared by introducing an ion exchange group such as a propyl group (hereinafter abbreviated as "r-sp group") or a quarternary polyaminoethyl group (hereinafter abbreviated as "rQAE group"). In addition to the above-mentioned ion exchange groups,
Any known ion exchange group such as a sulfomethyl group, a primary to quaternary aminoethyl group, a phosphoric acid group, etc. can be applied.

To introduce an ion exchange group, for example, crosslinked glucomannan spherical particles are immersed in an alkaline solution in advance, and then reacted with a reagent that has a halogen end group and provides the ion exchange group to be introduced. These reagents include, for example, 2-
Chlorotriethylamine hydrochloride, chloroacetic acid 1, chloromethane sulfonate, phosphoryl chloride, etc. can be used, and as the alkali, sodium hydroxide, potassium hydroxide, etc. can be used.

Note that the crosslinked glucomannan spherical particles are disclosed in, for example, Japanese Patent Application Laid-Open No.
It can be manufactured as follows according to the method disclosed in Japanese Patent Application No.-94949. That is, the prepared glucomannan is acetylated, and then the glucomannan ester obtained by the acetylation is dissolved in an organic solvent, and a diluent (porosity forming agent) is added to prepare a glucomannan ester solution. This glucomannan ester solution is added to an aqueous medium, stirred and suspended to form spheroids, and then the organic solvent is removed by evaporation to solidify (particles). The mixture is saponified in a homogeneous system and further crosslinked with a crosslinking agent to obtain crosslinked glucomannan particles.

In the present invention, in preparing such crosslinked glucomannan particles, by appropriately adjusting the amount of diluent added before particleization, the particleization stirring speed, etc., the particle size is 10 to 500.
Cross-linked glucomannan particles are obtained by obtaining cross-linked glucomannan spherical particles with a swelling degree of 1.5 μm or more, an exclusion limit molecular weight of 100,000 or more in terms of polyethylene glycol (PEG), and introducing an ion exchange group into these particles. Preferably, a mananium ion exchanger is used. That is, such cross-linked glucomannan spherical particles have high pressure resistance, low swelling/shrinkage rate, and excellent acid/alkali resistance and heat resistance. The ion exchanger has almost the same excellent properties and is very suitable as an ion exchanger for use in the present invention.

In the present invention, the crosslinked glucomane ion exchanger used in particular is a spherical particle with a particle size of 10 to 1000 μm,
Ion exchange capacity is 0.01-5 meq/g-gel
It is preferable that the % exclusion limit molecular weight is 1 million or more (in terms of PEG).

The crosslinked glucomannan ion exchanger according to the present invention, which is obtained by introducing ion exchange groups into the crosslinked glucomannan particles, is insoluble in water and acidic or alkaline aqueous solutions, and has high physical strength and physical properties. Excellent in Furthermore, compared to other hydrophilic gels, the exclusion limit molecular weight range is significantly wider.
Furthermore, even spherical particles with a high exclusion limit molecular weight of 1 million or more exhibit significantly higher pressure resistance than other types of gels.

The exclusion limit molecular weight referred to here refers to the limit molecular weight that cannot enter the micropores of the filler particles.
) was injected, extruded with an eluent, and measured using a differential refractometer.
The amount of eluate is measured using a spectrophotometer or the like, and the amount of each eluate is plotted against the molecular weight of the eluate sample, and the molecular weight is indicated by the molecular weight at the bending point of the obtained spelling line.

In carrying out the method of the present invention, a cross-linked glucomanone ion exchanger is first packed into a suitable column, and a sample is injected into the resulting ion exchange column. For sample injection, the sample containing the nucleic acid-related substance to be separated is diluted with an appropriate eluent at 0.5~
The eluent was diluted to 1000 μg/ml, injected, and adsorbed onto an ion exchanger.
Flow at a flow rate of m/H. At this time, the salt concentration, pH, etc. of the eluent are changed to create a concentration gradient of the components in the solution. As a result, the nucleic acid-related substances in the sample that have been adsorbed to the ion exchanger are eluted in order of decreasing molecular weight, so by separating the effluent into each fraction, the nucleic acid-related substances in the sample are separated and purified. Suko Aoto is said to be possible.

As the eluent, for example, a Tris-HCl buffer with a pH of 6 to 9, a phosphoric acid solution, etc. can be used, and as a component to create a concentration gradient in such an eluent,
NaCIA%KCl1 etc. can be used.

In the present invention, a liquid containing nucleic acid-related substances with a molecular weight of 1 million or more to be separated may contain only two or more types of nucleic acid-related substances with a molecular weight of 1 million or more; In particular, in the present invention, a solution containing protein together with a nucleic acid-related substance having a molecular weight of 100,000 or more is subjected to pre-treatment for protein removal. It is very advantageous to be able to separate and purify it by continuous chromatography without having to worry about it.

[Function] In ion exchange treatment, in order to have a sufficient throughput without requiring high pressure as in columns packed with non-porous fine particles, it is necessary to use particles with a particle size of about 10 μm or more and large DNA.
It is necessary to use an ion exchanger that has large pores that can sufficiently accommodate large DNA and the like in order to retain ion exchange groups that can effectively act on DNA and the like. In addition to having such huge pores, it also has a pressure-resistant strength that allows liquid to pass through at a flow rate similar to that of conventional liquid black, and it has almost no non-specific adsorption properties that are disadvantageous to biopolymers. In addition, it is required that the ion exchanger be a truly spherical ion exchanger that satisfies all of the requirements of a practically practical low swelling/shrinkage ratio, acid/alkali resistance, and heat resistance. However, JP-A-1-949
The crosslinked glucomane ion exchanger disclosed in No. 49 satisfies all of these required properties and is a very effective ion exchanger for treating high molecular weight nucleic acid-related substances.

That is, the crosslinked glucomannan ion exchanger according to the present invention can be prepared by introducing an ion exchange group into crosslinked glucomannan spherical particles according to a conventional method. is 10-1
000 μm or more, swelling degree 1.5 or more, exclusion limit molecular weight 200 to 100,00 in terms of PEG.
0,000 crosslinked glucomannan spherical particles have giant pores with an exclusion limit molecular weight of 1 million or more,
It has a pressure resistance that far exceeds that of conventional natural polymer packing materials such as dextran and agarose, which have an exclusion limit molecular weight of less than 1 million, and therefore columns packed with this material can easily handle high flow rates. It has the excellent characteristics of having a small expansion/shrinkage ratio of 11 ftl, which is sufficiently practical as a filler for liquid chromatography, and high acid/alkali resistance and heat resistance. Therefore, cross-linked glucomannan ion exchangers, which are made by introducing ion-exchange groups into cross-linked glucomannan spherical particles using an appropriate method, have excellent physical and chemical properties that are almost equivalent to those without ion-exchange groups. It has properties.

The cross-linked glucomane ion exchanger according to the present invention exhibits unparalleled high performance in high-speed separation of biopolymers such as proteins, but its true value is particularly apparent when targeting large DNA etc. Demonstrate. That is, the exclusion limit molecular weight is 100
The cross-linked glucomannan ion exchanger, which is made by introducing ion exchange groups such as DEAE groups into more than 10,000 cross-linked glucomannan spherical particles, has large pores that are unparalleled in other products.
NA and the like can also be easily adsorbed in ion exchange mode. Desorption can then be carried out by varying the buffer conditions of the eluent and the like according to a conventional method. In addition, the cross-linked glucomanion exchanger has rich hydrophilic properties derived from the properties of its parent substance, glucomannan, which is a natural polymer.
Therefore, the substances to be separated have the same function as ordinary ion-exchange chromatographies, and the lower the molecular weight, the lower the molecular weight. They exhibit the property of being easily eluted, and by appropriately varying the buffer conditions, they can be eluted in descending order of molecular weight. Moreover, the recovery rate also shows an extremely high value.

In addition, according to the method of the present invention using a cross-linked glucomane ion exchanger, not only the mutual separation of nucleic acid-related substances including giant DNA etc., but also the separation of protein removal can be easily performed.

That is, conventionally, when there is protein in a solution, direct ion exchange I^
However, the present invention makes it possible to carry out this process using a single column.

By appropriately setting conditions, it is also possible to separate each component of a nucleic acid-related substance and each component of a protein from a liquid containing a nucleic acid-related substance and a protein through a series of liquid chromatography processes.

[Example〕 EXAMPLES The present invention will be explained in more detail with reference to Examples below.

Example 1 [Measurement of adsorption capacity and recovery rate of λ-DNA] Inner diameter 1.2
mmφ, length 50 mmL (inner volume 56.5μi) stainless steel column with exclusion limit of more than 10,000 molecules (P
DEAE-glucomannan spherical particles having an ion exchange capacity of 1.2 ieq/g-ge J2 into which DEAE groups were introduced were packed into cross-linked glucomannan spherical particles (particle size 20 to 44 μm) of EGIM calculation). This column was prepared using a 10mM Tris-HCl buffer (pH 7,8) (hereinafter referred to as rA solution).
) was then stabilized with commercially available λ-DNA (48,5
0.02bp=molecular weight approximately 32 million) was added to the above column.
The flow continued at a flow rate of 1 mf/win (LV-531 am/H). The effluent was directly introduced into a liquid chromatography UV detector, and the absorption at 260 nm was continuously monitored. At the time when the absorbance of the effluent became almost equal to that of the stock solution, the passage of λ-DNA was stopped, the solution was switched to solution A, and the column was washed at the same flow rate.

When the absorbance of the washing effluent returned to almost zero, a solution containing NaCJl dissolved in IM in solution A (hereinafter referred to as rB solution) was added.
), and the λ-DNA adsorbed on DEAE-glucomannan spherical particles was eluted by ion exchange. After the absorbance of the eluent increased once, it decreased, and when the absorbance returned to zero, the flow of the eluent was stopped.

The effluent from the column was collected separately in the adsorption/washing process and the elution process, and the volume and UV absorbance of the liquid were measured. Regarding the stock solution, the amount flowing into the column was actually measured. Then, the adsorption capacity and recovery rate of λ-DNA were determined from the liquid volume of each fraction and the concentration calculated from the UV absorbance at 260 nm.

As a result, the equilibrium adsorption capacity of λ-DNA was 446 μg/r.
Jl-gejl, the recovery rate was 98%.

Experiments were conducted in exactly the same manner using a commercially available ion exchanger rDEAE-Toyopearl 650S (particle size 20 to 40 μm). As a result, the λ-DN of this DEAE-gel
A equilibrium adsorption capacity is 66 p g/1. -geJZ, about one-seventh of the DEAE-glucomannan spherical particles
It was confirmed that this was significantly lower. Furthermore, the recovery rate of λ-DNA was 88%, which was lower than that of DEAE-glucomannan spherical particles.

That is, the DEAE-glucomannan spherical particles according to the present invention hardly cause non-specific adsorption to DNA and can be recovered with a high recovery rate, whereas commercially available products have a large amount of adsorbed DNA that cannot be recovered due to non-specific adsorption. , a large amount of DNA will remain in the column.

Ability ζ Example 2 [The difference between λ-DNA and Co I E 1-DNA
The experiment was conducted under the following conditions.

Filling 11: DEAE-glucomannan spherical particles particle size = 1
2 to 20 μm Ion exchange capacity ±1.3 meq/g-geu exclusion limit molecular weight = 10 million or more (PEG equivalent) Column size: 4.6 mmφ x 100 mmL (inner volume 1
．． 66mft) Eluent: Solution A: 10mM Tris-HCl buffer containing 1mM EDTA (pH 8,0) Solution B: Solution containing Na (, Q) dissolved in Solution A to a concentration of 0.5M Load: 100Mg-DNA/mj2-A liquid liquid 18
．． 3μm Flow rate: 0. 2 mIL/min (LV=72.2 cm/H) Sample injection: The following sample was injected into the column stabilized with AfFi at the above load.

Run-1-λ-D N A (48,502 bp - molecular weight approximately 32,000,000) was injected alone Run-2 = Co1E 1- D N A (6,6
46 bp = molecular weight approximately 4.4 million) was injected alone Run-3 = λ-DNA and Co1E 1- DNA
Towo l! Mix at a ratio of 1:1 and inject. Elution conditions: 0 to 60 minutes after sample injection.
The NaCl1 concentration was increased from OM to 100% with a linear gradient from OM to 0.5M.

The results for each Run-1 to Run-3 are shown below.

Run-1: As shown in FIG. 1, a single peak was detected at 44.8 minutes.

Run-2: As shown in FIG. 1, a single peak was detected at 42.9 minutes.

Run-3: Two peaks were detected at 42.9 minutes and 44.8 minutes. From the results of Run-1 and Run-2, it is obvious that these peaks belong to Co1E1-DNA and λ-DNA, respectively, and it is clear that λ-DNA and Co1E1-DNA were well separated. .

Example 3 [DNA separation and protein removal] The experiment was conducted under the following conditions.

Filler: DEAE-glucomannan spherical particles particle size = 12
~20μm Ion exchange capacity = t, 3meq/g-geλ exclusion limit molecular weight = 10 million or more (PEG equivalent) Column size: 6mmφX100mmL (inner volume 2.8
3mλ) Eluent: Solution A 10mM phosphoric acid solution containing 0.1mM EDTA and 0.05M NaCJ2 (PH6,5) Solution B Solution in which NaCf was dissolved in solution A to a concentration of 0.3M Flow rate: 1mJZ /min (LV=212cm/H
) Sample injection: human serum albumin (molecular weight approximately 70,000), ColEl-DNA (molecular weight approximately 4.4 million) λ-DNA into a column stabilized with liquid A.
(molecular weight approximately 32 million) was dissolved in solution A at 100 μC and injected, 10 μg each, a total of 30 μg.

Elution and Results: After sample injection, 2 bed volumes of solution A were passed through the column at the above flow rate, and all human serum albumin flowed out of the column. Next, the content of B solution in the eluent was increased from 0 to 1 over 2 hours.
00% and a linear gradient of NaCj2 concentration from 0.3M to IM was applied.

As a result, two types of DNA were separated and eluted in descending order of molecular weight, and these were able to be completely fractionated.

[Effects of the Invention] As detailed above, according to the method for separating nucleic acid-related substances of the present invention, a crosslinked glucomane ion exchanger is used, and conventional packing materials for ion exchange chromatography do not adsorb,
Nucleic acid-related substances such as giant DNA with a molecular weight of 1 million or more, which are difficult to separate, can be adsorbed, separated, and purified in ion exchange mode without impairing their properties, ie, their activities. Moreover, according to the method of the present invention, the ion exchanger does not need to be in the form of homogeneous or fine particles, so an increase in packing material costs can be prevented, and there is no need for high-pressure liquid passage, leading to column scale-up and Large-scale fractionation can be easily performed.

Therefore, according to the method of the present invention, it is possible to easily and efficiently separate, purify, and recover nucleic acid-related substances of 1 million or more molecules with high processing efficiency and separation efficiency.

In particular, the method of the present invention separates a solution containing nucleic acid-related substances and proteins into each nucleic acid-related substance component and each protein component by a series of chromatographic operations without performing pretreatment such as protein removal in advance. It can be purified and has extremely high industrial utility.

[Brief explanation of drawings]

FIG. 1 is a diagram showing a chromatogram obtained in Example 2.

Claims (2)

[Claims] (1) A method for separating nucleic acid-related substances, characterized in that the nucleic acid-related substances are separated by passing a liquid containing a nucleic acid-related substance with a molecular weight of 1 million or more through a column packed with a cross-linked glucomanone ion exchanger. . (2) The method according to claim 1, wherein the solution containing the nucleic acid-related substance further contains protein.