U.S. patent application number 09/309599 was filed with the patent office on 2001-07-05 for method of separating nucleic acids by means of liquid chromatography.
Invention is credited to KITAMURA, TAKASHI, NAKATANI, SHIGERU.
Application Number | 20010007026 09/309599 |
Document ID | / |
Family ID | 14965701 |
Filed Date | 2001-07-05 |
United States Patent
Application |
20010007026 |
Kind Code |
A1 |
KITAMURA, TAKASHI ; et
al. |
July 5, 2001 |
METHOD OF SEPARATING NUCLEIC ACIDS BY MEANS OF LIQUID
CHROMATOGRAPHY
Abstract
Disclosed is a method for separating nucleic acids by
hydrophobic interaction chromatography. The purpose is to provide a
method for separating and purifying nucleic acids by hydrophobic
interaction chromatography, which enables to separate nucleic acids
such as plasmids and DNA fragments in a shorter time.
Inventors: |
KITAMURA, TAKASHI;
(KUMAGE-MACHI, JP) ; NAKATANI, SHIGERU;
(SHINNANYO-SHI, JP) |
Correspondence
Address: |
JACOBSON PRICE HOLMAN & STERN PLLC
400 SEVENTH STREET N W
WASHINGTON
DC
20004
|
Family ID: |
14965701 |
Appl. No.: |
09/309599 |
Filed: |
May 11, 1999 |
Current U.S.
Class: |
536/25.4 |
Current CPC
Class: |
C12N 15/101 20130101;
B01J 20/287 20130101 |
Class at
Publication: |
536/25.4 |
International
Class: |
C07H 021/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 1998 |
JP |
10-127665 |
Claims
What is claimed is:
1. A method for separating nucleic acids by hydrophobic interaction
chromatography.
2. The separating method of claim 1, wherein a packing material
with a functional group introduced to adsorb nucleic acids that
exhibit certain hydrophobicity is used for the packing material to
be used for the hydrophobic interaction chromatography.
3. The separating method of claim 1 or 2, wherein packing materials
with functional groups introduced to adsorb nucleic acids different
in hydrophobicity, respectively, are combined for the packing
materials to be used for hydrophobic interaction chromatography to
separate nucleic acids.
4. The separating method of claim 3, wherein a packing material
introduced a functional group with weaker hydrophobicity and a
packing material introduced a functional group with stronger
hydrophobicity are combined in order of this in the hydrophobic
interaction chromatography to use the packing materials different
in hydrophobicity in combination.
5. The separating method of any of claims 1 through 4, wherein the
average particle diameter of packing material to be used for
hydrophobic interaction chromatography is within a range from 2 to
500 .mu.m.
6. The separating method of any of claims 1 through 5, wherein the
average pore diameter of packing material to be used for
hydrophobic interaction chromatography is within a range from 500
to 4000 angstroms.
7. A method of separating nucleic acids using hydrophobic
interaction chromatography and ion exchange chromatography in
combination.
8. The separating method of claim 7, wherein a packing material
with a functional group introduced to adsorb nucleic acids that
exhibits certain hydrophobicity is used for the packing material to
be used for hydrophobic interaction chromatography to separate, and
then ion exchange chromatography is used to separate nucleic
acids.
9. The separating method of claim 7 or 8, wherein packing materials
for the hydrophobic interaction chromatography are combined in a
way that the hydrophobicity is increased in sequence from a packing
material introduced a functional group with the weakest
hydrophobicity, successively to a packing material with the second
weakest hydrophobicity to separate, and then ion exchange
chromatography is used to separate nucleic acids.
10. The separating method of any of claims 7 through 9, wherein the
average particle diameter of packing materials to be used for
hydrophobic interaction chromatography and ion exchange
chromatography is within a range from 2 to 500 .mu.m.
11. The separating method of any of claims 7 through 10, wherein
the pore diameter of packing material to be used for ion exchange
chromatography is within a range from 1500 to 4000 angstroms.
12. The separating method of claim 1 or 7, wherein the nucleic
acids to be separated is long chain nucleic acid
13. The separating and purifying method of any of claims 1 through
9, wherein the functional groups to be added to adsorb nucleic
acids have one or more kinds of compounds selected from a group
consisting of following compounds (a) through (c) as major
components in the packing materials to be used for the hydrophobic
interaction chromatography. Compounds (a): These may be of long
chain or branched. Saturated hydrocarbon groups or unsaturated
hydrocarbon groups with carbon atoms of 2 to 20 (however, aromatic
ring may be contained in the hydrocarbon group). Compounds (b):
Compounds represented by a following structural formula 1 (however,
n=0-20 and methylene group may be of straight chain or branched,
m=0-3 and hydrocarbon may be of straight chain or branched, and A
is c= O group or ether group, but methylene group may be bonded
directly to base material without A). Compounds (c): Ether group of
alkylene glycol with carbon atoms of 2 to 20, which consists of
repeating units of 0 to 10 (however, the opposite end of functional
group reacted with base material may be OH group left as it is or
may be capped with alkyl group with carbon atoms of 1 to 4). 2
Description
BACKGROUND OF THE INVENTION
[0001] The present invention relates to a method for separating and
purifying nucleic acids to be used for the genome analysis or the
gene manipulation. In more detail, it relates to a method for
separating long chain nucleic acids represented by particular DNA
contained in the cells of animals, human, etc. that are effective
for the gene therapy to be utilized for the therapy of genetic
disease etc. due to DNA abnormality, by utilizing liquid
chromatography, for example, plasmids, in the bacteria, organelle
DNA, phage DNA, etc.
[0002] In recent years, the gene therapy is attracting an
attention, and a method for separating and purifying a large
quantity of long chain nucleic acids such as plasmids and DNA
fragments to be used for gena therapy in simpler way and in shorter
time is desired eagerly.
[0003] Here, for using nucleic acids for the therapy of human, it
is desirable that the nucleic acids can be separated and purified
keeping the same structure (higher-order structure) as that when
they exist in organisms. Here, since the enzyme reaction is
utilized for the recombination of nucleic acids, the nucleic acids
are required to have been separated and purified to the extent that
they can become the substrate for reaction. Also, in order to avoid
the adverse effect of impurities on human body, the nucleic acids
are required to have been separated and purified up to high
purity.
[0004] For separating and purifying long chain nucleic acids such
as DNAs and plasmids contained in the cells and bacteria, chemical
treatment methods have been used most frequently, so far.
[0005] Among various long chain nucleic acids used for the gene
therapy, in particular, plasmid is currently utilized in many
cases, because of limited cleavage sites by particular restriction
enzyme and relatively easy recombination manipulation. In the
following, a general example of purifying a plasmid from
Escherichia coli will be shown.
[0006] First, the cell wall is digested by treating with lysozyme
for a short time, and RNase to degrade RNAs of Escherichia coli is
added. Next, a mixed solution of NaOH and sodium dodecylsulfate
(SDS) is added for the purpose of dissolving the cytoplasmic
membrane. NaOH partially denatures DNAs and partially degrades RNAs
and SDS acts to dissolve the membrane and denature proteins.
Successively, SDS-protein complex and cell debris are precipitated
by adding 5N potassium acetate (pH 4.8). At this time, pH is
important for both to neutralize NaOH used in said manipulation and
to renature plasmid. Thereafter, centrifugation is applied to
remove the precipitates, thus obtaining aiming plasmids in
supernatant.
[0007] In a series of these manipulations (hereinafter referred to
as pretreatment process), it is important to mix slowly and firmly.
If adding violent vibration during this manipulation, then the
bacterial chromosomal DNA is cut off to small fragments so that
they cannot aggregate, causing them to contaminate the plasmid.
[0008] Successively, isopropanol is added to the supernatant, and
the mixture is centrifuged to precipitate and concentrate plasmids.
Finally, protein is removed from plasmid fraction by precipitating
with phenol and chloroform, and plasmid is precipitated with
alcohol.
[0009] Through a series of manipulations as described above, it is
possible to obtain plasmid with relatively high purity
(hereinafter, said method of separating and purifying nucleic acid
is referred to as chemical separating method). However, with the
chemical separating method, separating and purifying process is
complicated and a large quantity of organic solvent must be used,
hence it poses many problems of treatment of waste solvents and
others.
[0010] Besides the chemical separating and purifying method, there
is a method of separating plasmids by electrophoresis. This method
is a technique having the highest resolution at the moment. The
electrophoretic method includes paper electrophoresis and gel
electrophoresis, and gel electrophoresis is common currently. The
electrophoretic method has an advantage of obtaining plasmid with
very high purity, while it has many problems of long separation
time, difficult collection, low sample loading, etc. Consequently,
it is a present situation that the electrophoretic separation is
used only when the purity of plasmid fraction purified by said
chemical separating and purifying method is desired to improve
further.
[0011] For solving the problems in chemical separating and
purifying method and electrophoretic separation as explained above,
a method of separating and purifying nucleic acids that utilizes
liquid chromatography has been used recently. So far, there are
examples, wherein long chain nucleic acids such as plasmids were
separated and purified by using ion exchange chromatography and
reversed phase chromatography.
[0012] With the method of separating and purifying nucleic acids
utilizing liquid chromatography, there are good points of simple
manipulation compared with chemical separating method, easy
collection of nucleic acids and no necessity of using organic
solvent etc. With said conventional method using ion exchange
chromatographic method or reversed phase chromatographic method
alone, however, there is a problem that the nucleic acids with
sufficiently high purity, in particular, long chain nucleic acids
such as plasmids cannot be obtained in large quantity.
[0013] Therefore, the invention aims at providing a separating
method that utilizes liquid chromatography, which enables to
separate a large quantity of long chain nucleic acids such as
plasmids and DNAs in a shorter time.
SUMMARY OF THE INVENTION
[0014] The invention of claim 1 of the present application having
been made in view of the purpose aforementioned provides a method
of separating nucleic acids characterized by using hydrophobic
interaction chromatography. And, the invention of claim 7 of the
present application provides a method for separating and purifying
nucleic acids characterized by using hydrophobic interaction
chromatography and ion exchange chromatography in combination.
BRIEF DESCRIPTION OF THE DRAWING
[0015] FIG. 1 is a chromatogram showing the result of separating a
cleared lysate of Escherichia coli by means of hydrophobic
interaction chromatography in Example 1.
[0016] FIG. 2 is a chromatogram showing the result of separating an
eluate of hydrophobic interaction chromatography by means of ion
exchange chromatography in Example 1.
[0017] FIG. 3 is a schematic diagram of the images of gel
electrophoresis showing the results of purity assay of plasmid
fractions obtained in Example 1 and Comparative Example 1.
[0018] FIG. 4 is a chromatogram showing the result of separating a
cleared lysate of Escherichia coli separated by means of ion
exchange chromatography in Comparative Example 1.
[0019] FIG. 5 is a chromatogram showing the result of separating a
cleared lysate of Escherichia coli by means of hydrophobic
interaction chromatography in Example 2.
[0020] FIG. 6 is a chromatogram showing the result of separating an
eluate of hydrophobic interaction chromatography by means of ion
exchange chromatography in Example 2.
[0021] FIG. 7 is a chromatogram showing the result of separating a
cleared lysate of Escherichia coli by means of ion exchange
chromatography in Comparative Example 2.
EXAMPLE 2
[0022] In Figures,
1 numeral 1 peak of impurities numeral 2 peak of plasmid-fraction
numeral 3 DNA size marker numeral 4 plasmid-fraction obtained by
comparative Example 1 numeral 5 plasmid-fraction obtained by
Example 1 numeral 6 commercial purified plasmid
DETAILED DESCRIPTION OF THE INVENTION
[0023] In following, the invention will be illustrated in detail on
the case of separating and purifying a plasmid being a long chain
nucleic acid from Escherichia coli cultured in large quantity as an
example. In such case, the inventive methods are useful for
separating and purifying nucleic acids such as plasmids and DNAs
being aiming products.
[0024] Compound to be used for the synthesis of base materials that
are used for the packing material for hydrophobic interaction
chromatography and ion exchange chromatography to be used in the
invention may be any compounds, if various functional groups that
exhibit hydrophobicity or various ion exchange groups can be
introduced by a post-reaction after the base materials were
synthesized in both cases. For example, as monofunctional monomers,
styrene, o-halomethylstyrene, m-halomethylstyrene,
p-halomethylstyrene, o-haloalkylstyrene, m-haloalkylstyrene,
p-haloalkylstyrene, .alpha.-methylstyrene,
.alpha.-methyl-o-halomethylstyrene,
.alpha.-methyl-m-halomethylstyrene,
.alpha.-methyl-p-halomethylstyrene,
.alpha.-methyl-o-haloalkylstyrene,
.alpha.-methyl-m-haloalkylstyrene,
.alpha.-methyl-p-haloalkylstyrene, o-hydroxymethylstyrene,
m-hydroxymethylstyrene, p-hydroxymethylstyrene,
o-hydroxyalkylstyrene, m-hydroxyalkylstyrene,
p-hydroxylalkylstyrene, .alpha.-methyl-o-hydroxymethylstyrene,
.alpha.-methyl-m-hydroxymethylstyr- ene, .alpha.
-methyl-p-hydroxymethylstyrene, .alpha.-methyl-o-hydroxyalkyl-
styrene, .alpha.-methyl-m-hydroxyalkylstyrene,
.alpha.-methyl-p-hydroxyalk- ylstyrene, glycidyl methacrylate,
glycidyl acrylate, hydroxyethyl acrylate, hydroxymethacrylate,
vinyl acetate, etc. can be exemplified.
[0025] Here, as examples of haloalkyl groups substituted on
aromatic ring, halogens such as Cl, Br, I and F and straight chain
and/or branched saturated hydrocarbons with carbon atoms of 2 to 15
are mentioned.
[0026] As polyfunctional monomers, divinylbenzene, trivinylbenzene,
divinyltoluene, trivinyltoluene, divinylnaphthalene,
trivinylnaphthalene, ethylene glycol dimethacrylate, ethylene
glycol diacrylate, diethylene glycol dimethacrylate, diethylene
glycol diacrylate, methylenebismethacrylamide,
methylenebisacrylamide, etc. can be exemplified.
[0027] As described above, for the compounds to be used in the
invention, there is no special restriction, provided it is possible
to introduce various functional groups that exhibit hydrophobicity
or various ion exchange groups by the post-reaction, but, in order
to minimize the influence on aiming products desired to separate
due to the hydrophobicity exhibited by the base material itself, or
the swelling or shirinking of the base material itself due to the
change in salt concentration and the change in pH value, it is
particularly preferable to prepare the base material using
relatively hydrophilic monomers, for example, glycidyl
methacrylate, glycidyl acrylate, hydroxyethyl acrylate,
hydroxymethacrylate, vinyl acetate, etc.
[0028] How to commonly make the base material using said monomers
is as follows (how to make the base material is not confined to a
method shown here):
[0029] First, monofunctional monomer and polyfunctional monomer are
weighed out at an appropriate ratio and precisely weighed-out
diluent (solvent used for the purpose of adjusting the pores in
particles formed) and similarly precisely weighed-out
polymerization initiator are added, followed by well stirring. And,
the mixture is submitted to so-called oil-in-water type suspension
polymerization wherein the mixture is added into an aqueous
solution dissolved suspension stabilizer weighed out precisely
beforehand, and oil droplets with aiming size are formed by mixing
with stirrer, and polymerization is conducted by gradually warming
mixed solution.
[0030] The ratio of monofunctional monomer to polyfunctional
monomer is not particularly restricted, and, to 1 mol of
monofunctional monomer, around 0.01 to 0.2 mol of polyfunctional
monomer are used in the case of making relatively soft particles
(base material) and around 0.2 to 0.5 mol of that in the case of
making hard particles; in the case of making harder particles,
polyfunctional monomer alone may be used. The polymerization
initiator is also not particularly restricted, and azobis type
and/or peroxide type being used commonly are used. The suspension
stabilizer is also not particularly restricted, and, if possible to
prevent the aggregation among oil droplets themselves, any of ionic
surfactants, nonionic surfactants and polymers with amphipathic
property or mixtures of these can be used.
[0031] The diameter of formed particles is also not particularly
restricted and particles with appropriate diameter of 2 to 500
.mu.m may be selected in line with the use purpose. For example,
when aiming at analysis, the particle diameter is better to be 2 to
30 .mu.m, more preferably around 2 to 10 .mu.m. When aiming at
large scale purification of nucleic acids with high purity, it is
around 10 to 100 .mu.m and, when separating the aiming product from
crude stock solution, it may be 100 to 500 .mu.m, more preferably
around 200 to 400 .mu.m. For adjusting the particle diameter, the
rotational speed of stirrer may be adjusted during polymerization;
when particles with small diameter are needed, the number of
revolutions may be increased and, when large particles are desired,
the number of revolutions may be decreased. Here, since the diluent
to be used is used for adjusting pores in formed particles, the
selection of diluent is particularly important. As a fundamental
concept, for the solvent to be used for polymerization, adjustment
is made by variously combining a solvent that is poor solvent for
monomer with a solvent that is good solvent for monomer. The size
of pore diameter may be selected appropriately depending on the
molecular size of nucleic acids designed to separate, but it is
preferable to be within a range of 500 to 4000 angstroms for the
packing material for hydrophobic interaction chromatography and
within a range from 1500 to 4000 angstroms for the packing material
for ion exchange chromatography. In the hydrophobic interaction
chromatography, for separating nucleic acids with different
hydrophobicity preferable by utilizing packing materials with
different hydrophobicity, respectively, the surface modification of
the base material is important. The applicable hydrophobic group is
not particularly restricted as long as it does not deviate from the
purpose to separate nucleic acids with different hydrophobicity
with packing materials with different hydrophobicity, respectively,
but hydrophobic groups having one or more kinds of compounds
selected from a group consisting of following compounds (a) through
(c) as major components are particularly preferable.
[0032] Compounds (a): These may be of long chain or branched.
Saturated hydrocarbon groups or unsaturated hydrocarbon groups with
carbon atoms of 2 to 20 (however, aromatic ring may be contained in
the hydrocarbon group).
[0033] Compounds (b): Compounds represented by a following
structural formula 1 (however, n=0-20 and methylene group may be of
straight chain or branched, m=0-3 and hydrocarbon group may be of
straight chain or branched, and A is C=O group or ether group, but
methylene group may be bonded directly to base material without
A).
[0034] Compounds (c): Ether group of alkylene glycol with carbon
atoms of 2 to 20, which consists of repeating units of 0 to 10
(however, the opposite end of functional group reacted with base
material may be OH group left as it is or may be capped with alkyl
group with carbon atoms of 1 to 4). 1
[0035] When modifying the surface using compounds dividable into
three categories as above, they may be used solely or in mixture.
In following, one example of different ways to use them will be
explained, taking alkyl groups that belong to Compounds (a) as an
example. For example, for separating compounds with high
hydrophobicity such as RNA originating from Escherichia coli and
RNA in the cells of human and animals, alkyl groups with carbon
atoms of 2 to 15 are particularly suitable. Moreover, in the case
of compounds with relatively low hydrophobicity such as DNAs
originating from Escherichia coli and DNAs in the cells of human
and animals, alkyl groups with carbon atoms of 4 to 18 are
particularly suitable. Furthermore, in the case of compounds with
low hydrophobicity like plasmids, alkyl groups with carbon atoms of
6 to 20 are suitable. Upon separating these compounds, compounds
may be selected appropriately to modify the surface without being
confined to said exemplification. The reason for this is that the
degree of hydrophobicity of packing material varies depending on
the concentration of salt in medium or the concentration of salt in
eluent for adsorption. Moreover, this is because of that, even with
the same functional group, the degree of hydrophobicity of packing
material differs depending on the amount of the group introduced
into the base material.
[0036] The pore diameter of the base material for hydrophobic
interaction chromatography is particularly preferable to be 500 to
4000 angstroms, but it can be selected appropriately from said
range depending on the molecular size of nucleic acids desired to
separate. In general, since the retention of nucleic acids on the
packing material and the adsorption capacity (sample leading)
differ depending on the pore diameter, it is preferable to use a
base material with large pore diameter for nucleic acids with large
molecular size and a base material with small pore diameter for
nucleic acids with small molecular size.
[0037] Next, one example of the methods for reacting these
hydrophohic groups with base material will be described. In the
case of the base material being styrene base and reacting with
compounds in the first and second category, using
halogen-containing compound B and/or carbonyl halide C and catalyst
such as FeCl.sub.3, SnCl.sub.2 or AlCl.sub.3, and utilizing
Friedel-Craft reaction, it is possible to add directly to aromatic
ring in base material as dehalogenated compound B and/or acylated
compound C. In the case of the base material being particle
containing halogen group, for example, using compounds with OH
contained in functional group to be added, like butanol, and
utilizing Williamson reaction with alkali catalyst such as NaOH or
KOH, it is possible to introduce the functional group through ether
bond. In the case of the functional group desired to add being
amino group-containing compound, like hexylamine, it is possible to
add using alkali catalyst such as NaOH or KOH and utilizing
dehalogenic acid reaction. In the case of the base material
containing OH group, inversely, if introducing epoxy group, halogen
group or carbonyl halide group beforehand into the functional group
desired to add, it is possible to introduce the functional group
through ether or ester bond. In the case of the base material
containing epoxy group, if reacting with compound with OH group or
amino group contained in the functional group desired to add, it is
possible to introduce the functional group through ether or amino
bond. Moreover, in the case of the functional group desired to add
containing halogen group, it is possible to add the functional
group through ether bond using acid catalyst. Since the proportion
of functional group to be introduced into base material is
influenced by the hydrophobicity of subject product desired to
separate, it cannot be restricted, but, in general, packing
material with around 0.05 to 4.0 mmol of functional group added per
1 g of dried base material is suitable.
[0038] With respect to the surface modification, a method of adding
the functional group through post-reaction after formation of base
material (particles) has been exemplified above, but no difference
exists, even if a method may be adopted, wherein the base material
is formed after polymerization using monomers with said functional
groups added before polymerization, thus posing no particular
problem. In addition, the base material to be used may also be
porous silica gel. As an example of the method of manufacturing
silica gel, silane coupling may also be conducted, using a compound
such as alkyltrimethoxysilane, directly onto particles manufactured
according to the method described in "Latest High-Speed Liquid
Chromatography", page 289 ff. (written by Toshio Nambara and Nobuo
Ikegawa, published by Tokyo Hirokawa Bookstore in 1988). Or, after
conducted the silane coupling using epoxy group-containing silane
coupling agent, the functional group may be added according to the
method aforementioned. As for the proportion of functional group to
be introduced, packing material with around 0.05 to 4.0 mmol of
functional group added per 1 g of dried base material is
suitable.
[0039] Next, one example of the methods of separating and purifying
a nucleic acid using these packing materials will be described.
First, as the eluents to be used for the hydrophobic interaction
chromatography of the invention, at least two types of eluents
consisting of eluent A containing high-concentration of salt and
eluent B containing low-concentration of salt are used. The eluting
method switching stepwise from eluent A to eluent B and the
gradient eluting method continuously changing the composition from
eluent A to eluent B can be used. For the buffers and salts to be
used in these eluents, those used usually for the hydrophobic
interaction chromatography can be used. For the eluent A containing
high-concentration of salt, aqueous solution with salt
concentration of 1.0 to 4.5M and pH value of 6 to 8 is particularly
preferable. For the eluent B containing low-concentration of salt,
aqueous solution with salt concentration of 0.01 to 0.5M and pH
value of 6 to 8 is particularly preferable. For the salts, ammonium
sulfate and sodium sulfate can be exemplified.
[0040] In the invention, it is particularly preferable to conduct
the hydrophobic interaction chromatography by combining a packing
material introduced the functional group with weak hydrophobicity
with a packing material introduced the functional group with strong
hydrophobicity in sequence. This method is suitable particularly
for the separation of plasmid. For example, in the medium cultured
Escherichia coli in large quantity, various components different in
hydrophobicity such as polysaccharides, Escherichia coli genome
DNA, RNAs plasmids and proteins are contained, and, according to
the inventors' knowledge, there are differences in the
hydrophobicity even among nucleic acids themselves; proteins that
become impurities have higher hydrophobicity compared with plasmids
being aiming product. Hence, by connecting columns packed with
various packing materials different in hydrophobicity in order from
lower hydrophobicity, plasmid can be separated and purified
efficiently. Concretely, after adsorbing sequentially onto the
packing materials with increasingly higher hydrophobicity in order
from higher hydrophobicity of components in medium, column with
aiming component adsorbed alone is detached and eluted.
[0041] In the invention, it is preferable to separate and purify
nucleic acids by hydrophobic interaction chromatography and ion
exchange chromatography in combination for efficiently obtaining
nucleic acids with high purity in large quantity. Here, for the
hydrophobic interaction chromatography, packing material etc. as
described above can be used. Moreover, here, as the hydrophobic
interaction chromatography, it is particularly preferable to
connect columns packed with various packing materials different in
hydrophobicity in order from lower hydrophobicity.
[0042] The packing material to be used for ion exchange
chromatography for purifying the aiming plasmic having been
separated beforehand by means of hydrophobic interaction
chromatography further to higher purity is preferable to have
relatively large pore diameter, particularly within a range from
1500 to 4000 angstroms. How to commonly make the base material used
for ion exchange chromatography is as described above, and the
surface modification to introduce ion exchange groups to these base
materials can be performed by publicly known method.
[0043] As the eluents to be used for the ion exchange
chromatography, at least two types of eluents consisting of eluent
C containing low-concentration of salt and eluent D containing
high-concentration of salt are used. The eluting method switching
stepwise from eluent C to eluent D and the gradient eluting method
continuously changing the composition from eluent C to eluent D can
be used. For the buffers and salts to be used in these eluents,
those used usually for the ion exchange chromatography can be used.
For the eluent C containing low-concentration of salt, aqueous
solution with concentration of buffer of 10 to 50 mM and pH value
of 6 to 9 is particularly preferable. For the eluent D containing
high-concentration of salt, aqueous solution with 0.1 to 2M sodium
salt added to eluent C is particularly preferable. For the sodium
salts, sodium chloride and sodium sulfate can be mentioned.
[0044] Moreover, a component other than buffer can be contained in
both eluents, and, in particular, chelating agent for bivalent
metal ion, for example, ethylenediamine-tetraacetic acid is
particularly in the case of separating plasmids preferable, since
it can inhibit the degradation of plasmids due to DNA-degrading
enzymes in the lysate of Escherichia coli. The concentration of
chelating agent for bivalent metal ion is preferably 0.1 to 100
mM.
[0045] And, in the particularly preferable embodiment of the
invention, the eluent A with high salt concentration prepared
according to the method aforementioned is passed through columns of
hydrophobic interaction chromatography, which are connected in
order from lower hydrophobicity. After reached stationary state,
medium of Escherichia coli etc. omitted the degrading manipulation
of RNA with degrading enzyme etc. is injected into column.
Successively, the eluent A is passed through to flow out the
compounds that were not adsorbed in any column outside the system.
Thereafter, column with aiming compound adsorbed alone is detached
and aiming product is eluted by the stepwise method or gradient
method. Following this, the eluent C is passed through said ion
exchange column, and, after reached stationary state, the elute
containing aiming product is injected as it is. Thereafter, using
the eluent D, aiming product is eluted by stepwise method or
gradient method to obtain purified product.
[0046] The invention provides a method for separating and purifying
nucleic acids by simple manipulation. Concretely, in the preferable
embodiment of the invention using columns wherein columns packed
respectively with packing materials different in hydrophobicity are
connected in order from lower hydrophobicity, aiming nucleic acids,
in particular, long chain nucleic acids such as plasmids can be
separated and purified simply in large quantity only by passing the
solution from pretreatment process in the conventional
manipulation. Besides, in the invention, it is also possible to
separate and purify nucleic acids by passing the solution from
pretreatment process that was omitted the degrading manipulation of
Escherichia coli-originated RNA with degrading enzyme in the
conventional pretreatment process directly through the columns of
hydrophobic chromatography.
[0047] In the invention, if using the hydrophobic interaction
chromatography and the ion exchange chromatography in combination,
which is preferable in particular, it is possible to separate and
purify aiming nucleic acids with high purity, in particular, long
chain nucleic acids such as plasmids in large quantity by simple
manipulation.
[0048] As described, according to the inventive method for
separating nucleic acids, aiming products with high purity can be
obtained in large quantity by simpler manipulation over
conventional method.
[0049] In following, the invention will be illustrated in more
detail based on the examples, but the invention is not confined to
these examples.
EXAMPLE 1
[0050] (1) Preparation of packing material for hydrophobic
interaction chromatography
[0051] Employing a packing material for gel filtration
chromatography (G600OPW (from Tosoh Corp.)) with average particle
diameter of 20 .mu.m and average pore diameter of 2000 angstroms as
the base material, the packing material for the hydrophobic
interaction chromatography was prepared. A mixture of 20 g of
G600OPW washed and substituted with 1,4-dioxane, 20 g of
1,4-dioxane and 1 g of 1,2-epoxybutane was stirred and mixed for 6
hours at 45.degree. C. to obtain a packing material (hereinafter
referred to as Butyl-6PW) having butyl group as a functional group
of weak hydrophobicity. Similarly, a mixture of 20 g of G6000PW, 20
g of 1,4-dioxane and 1 g of 1,2-epoxyoctane was stirred and mixed
for 6 hours at 45.degree. C. to obtain a packing material
(hereinafter referred to as octyl-6PW) having octyl group as a
functional group of strong hydrophobicity. Each packing material
was packed into a stainless steel column with inner diameter of 7.5
mm and length of 7.5 cm.
[0052] (2) Separation of a plasmid by hydrophobic interaction
chromatography
[0053] After Escherichia coli having pBR322 as a plasmid was
cultured for 16 hours at 37.degree. C., the medium was subjected to
centrifugal separation for 20 minutes at 4.degree. C. and 8000 rpm.
The precipitated Escherichia coli was suspended into 10 ml of 25 mM
Tris hydrochloric acid buffer (pH 7.5) containing 100 mg of
lysozyme, 50 mM glucose and 10 mM ethylenediamine-tetraacetic acid
(hereinafter referred to as EDTA), which was stirred and then
allowed to stand for 5 minutes at room temperature to dissolve cell
wall. Then, 20 ml of 0.2N sodium hydroxide solution containing 1%
sodium dodecylsulfate were added thereto, and, after mixed gently,
the mixture was allowed to stand for 10 minutes under cooling with
ice to dissolve cytoplasmic membrane.
[0054] Next, 15 ml of 3M sodium acetate buffer (pH 5.4) were added
thereto, and the mixture was stirred slowly and allowed to stand
for 30 minutes under cooling with ice. Then, after subjected to
centrifugal separation for 20 minutes at 4.degree. C. and 10000
rpm, the supernatant was collected to obtain a cleared lysate of
Escherichia coli. After equal volume of 0.1M sodium phosphate
buffer (pH 7.0) containing 4M ammonium sulfate was added to the
crushed liquor of Escherichia coli, this mixture was subjected to a
hydrophobic interaction chromatography.
[0055] Into tandem columns of Butyl-6PW column and Octyl-6PW column
linked in series, which were equilibrated with 0.1M sodium
phosphate buffer (pH 7.0) containing 2M ammonium sulfate and 1 mM
EDTA, 3 ml of the crushed liquor of Escherichia coli containing
ammonium sulfate were injected, and then 0.1M sodium phosphate
buffer (pH 7.0) containing 2M ammonium sulfate and 1 mM EDTA was
fed into the tandem columns for 20 minutes at flow rate of 1 ml/min
to elute the impure substances outside the columns. Following this,
after detached Butyl-6PW column outside the flow path system, 0.1M
sodium phosphate buffer (pH 7.0) containing 1 mM EDTA was fed for
15 minutes at flow rate of 1 ml/min into only Octyl-6PW column. As
a result, a chromatogram as shown in FIG. 1 was obtained. In FIG.
1, numeral 1 shows a peak of impurities and numeral 2 shows a peak
of plasmid-containing fraction. The column eluate corresponding to
the peak 2 was collected and purified further by means of ion
exchange interaction chromatography as shown below.
[0056] (3) Separation of a plasmid by a combined use of hydrophobic
interaction chromatography and ion exchange chromatography
[0057] As a packed column for the ion exchange chromatography,
DEAE-5PW (trade name, from Tosoh Corp., inner diameter of 7.5 mm,
length of 7.5 cm) was used. Into DEAE-5PW column equilibrated with
20 mM Tris hydrochloric acid buffer (pH 7.5) containing 0.6M sodium
chloride and 1 mM EDTA, 3 ml of plasmid fraction was injected, and
then 20 mM Tris hydrochloric acid buffer (pH 7.5) containing 0.6M
sodium chloride and 1 mM EDTA was fed into the column for 35
minutes at flow rate of 1 ml/min to elute the impure substances
outside the column. Then, the elution was conducted by a gradient
method wherein the concentration of sodium chloride in 20 mM Tris
hydrochloric acid buffer (pH 7.5) containing 1 mM EDTA was changed
continuously from 0.6M to 0.8M over 30 minutes at flow rate of 1
ml/min. As a result, a chromatogram as shown in FIG. 2 was
obtained. In FIG. 2, numeral 1 shows peaks of impurities and
numeral 2 shows a peak of plasmid-containing fraction. The column
eluate corresponding to the peak 2 was collected and the purity was
examined by agarose gel electrophoresis. When dyeing the gel after
the electrophoresis with ethidium bromide, electrophoretic images
as shown in FIG. 3 were obtained. In FIG. 3, numeral 3 shows a DNA
size marker, numeral 5 shows a plasmid fraction obtained by the
present purifying method, and numeral 6 shows an electrophoretic
image of commercial purified pBR322. As evident from the diagram,
high-purity supercoil type plasmid could be obtained by simple
manipulation according to the present purifying method.
Comparative Example 1
[0058] For comparison, purification of a plasmid pBR322 was
conducted from the cleared lysate of Escherichia coli by means of
ion exchange interaction chromatography alone.
[0059] After prepared the cleared lysate of Escherichia coli
similarly to the Example, equal volume of 20 mM Tris hydrochloric
acid buffer (pH 7.5) was added thereto to make a sample for the ion
exchange chromatography. Into the previous DEAE-5PW column
equilibrated with 20 mM Tris hydrochloric acid buffer (pH 7.5)
containing 0.6M sodium chloride and 1 mM EDTA, 3 ml of sample were
injected, and then 20 mM Tris hydrochloric acid buffer (pH 7.5)
containing 0.6M sodium chloride and 1 mM EDTA was fed into the
column for 60 minutes at flow rate of 1 ml/min to elute the impure
substances outside the column. Next, elution was conducted by the
gradient method similarly to the Example. As a result, a
chromatogram as shown in FIG. 4 was obtained. In FIG. 4, numeral 1
shows peaks of impurities and numeral 2 shows a peak of
plasmid-containing fraction. The column effluent corresponding to
the peak 2 was collected and the purity was examined by agarose gel
electrophoresis. As a result, electrophoretic images as shown in
FIG. 3 were obtained. In FIG. 3, numeral 3 shows a DNA size marker,
numeral 4 shows a plasmid fraction obtained in the Comparative
Example, numeral 5 shows a plasmid fraction obtained in the Example
1, and numeral 6 shows an electrophoretic image of commercial
purified pBR322. Many impurities were contained in the plasmid
fraction obtained by means of ion exchange chromatography
alone.
EXAMPLE 2
[0060] (1) Preparation of packing materials for hydrophobic
interaction chromatography
[0061] Employing a packing material for gel filtration
chromatography (G6000 PW (from Tosoh Corp.)) with average particle
diameter of 20 .mu.m and average pore diameter of 2000 angstroms as
the base material, the packing material for the hydrophobic
interaction chromatography was prepared. A mixture of 20 g of G6000
PW washed and substituted with 1,4-dioxane, 20 g of 1,4-dioxane, 1
g of 1,2-epoxyoctane and 0.5 ml of boron trifluoride as catalyst
was stirred and mixed for 6 hours at 45.degree. C. to obtain a
packing material (hereinafter referred to as Octyl-6 PW) having
octyl group for adsorbing plasmid and for hydrophobic interaction
chromatography.
[0062] Next, employing a packing material for gel filtration
chromatography (G5000 PW (from Tosoh Corp.)) with average particle
diameter of 20 .mu.m and average pore diameter of 950 angstroms as
the base material, a packing material with weak hydrohobicity was
prepared. A mixture of 20 g of G5000 PW, 20 g of 1,4-dioxane, 1 g
of 1,2-epoxybutane and 0.5 ml of boron trifluoride as catalyst was
stirred and mixed for 6 hours and at 45.degree. C. to obtain a
packing material (hereinafter referred to as Butyl-5 PW) having
butyl group for adsorbing RNAs and proteins and for hydrophobic
interaction chromatography. Each packing material was packed into a
stainless steel column with inner diameter of 7.5 mm and length of
7.5 cm.
[0063] (2) Separation of a plasmid by a combined use of hydrophobic
interaction chromatography and ion exchange
chromatography--separation by hydrophobic interaction
chromatography
[0064] After Escherichia coli having pBR 322 as a plasmid was
cultured for 16 hours at 37.degree. C., the medium was subjected to
centrifugal separation for 20 minutes at 4.degree. C. and 8000 rpm.
The precipitated Escherichia coli was suspended into 10 ml of 25 mM
Tris hydrochloric acid buffer (pH 7.5) containing 100 mg of
lysozyme, 50 mM glucose and 10 mM ethylenediamine tetraacetate
(hereinafter referred to as EDTA), which was stirred and then
allowed to stand for 5 minutes at room temperature to dissolve cell
wall. Then, 20 ml of 0.2N sodium hydroxide solution containing 1%
sodium dodecylsulfate were added thereto, after mixed gently, the
mixture was allowed to stand for 10 minutes under cooling with ice
to dissolve cytoplasmic membrane.
[0065] Next, 15 ml of 3M sodium acetate buffer (pH 5.4) were added
thereto, and the mixture was stirred slowly and allowed to stand
for 30 minutes under cooling with ice. Then, after subjected to
centrifugal separation for 20 minutes at 4.degree. C. and 10000
rpm, the supernatant was collected to obtain a cleared lysate of
Escherichia coli. After equal volume of 0.1M sodium phosphate
buffer (pH 7.0) containing 4M ammonium sulfate was added to the
cleared lysate of Escherichia coli, this mixture was subjected to a
hydrophobic interaction chromatography.
[0066] Into tandem columns of Butyl-5 PW column and Octyl-6 PW
column linked in series, which were equilibrated with 0.1M sodium
phosphate buffer (pH 7.0) containing 2M ammonium sulfate and 1 mM
EDTA, 3 ml of the cleared lysate of Escherichia coli containing
ammonium sulfate were injected, and then 0.1M sodium phosphate
buffer (pH 7.0) containing 2M ammonium sulfate and 1 mM EDTA was
applied into the tandem columns for 20 minutes at flow rate of 1
ml/min to elute the impure substances outside the columns.
Following this, after detached Butyl-5t PW column outside the flow
path system, 0.1M sodium phosphate buffer (pH 7.0) containing 1 mM
EDTA was fed into only Octyl-6 PW column for 15 minutes at flow
rate 1 ml/min. As the result, chromatogram as shown in FIG. 5 was
obtained. In FIG. 5, numeral 1 shows a peak of impurities and
numeral 2 shows a peak of plasmid-containing fraction. The column
effluent corresponding to the peak 2 was collected and purified
further by means of ion exchange chromatography as shown below.
[0067] (3) Preparation of a packing material for ion
chromatography
[0068] This was prepared by the way described below, employing a
packing material (G6000 PW (from Tosoh Corp.)) for gel filtration
chromatography with average particle diameter of 20 .mu.m and
average pore diameter of 2000 angstroms as base material. A mixture
of 20 g of G6000 PW washed thoroughly with pure water, 40 g of pure
water, 10 g of epichlorohydrin, 10 g of diethylaminoethanol and 5 g
of NaOH was stirred and mixed for twenty-four hours at 40.degree.
C. to obtain anion exchanger (hereinafter referred to as DEAE-6 PW)
having total ion-exchange capacity of 0.05 meq/ml-gel for purifying
plasmid.
[0069] This packing material was packed into a stainless steel
column with inner diameter of 7.5 mm and length of 7.5 cm for
ion-exchange chromatography.
[0070] (4) Separation of a plasmid by a combined use of hydrophobic
interaction chromatography and ion exchange chromatography
[0071] After 3 ml of a plasmid fraction was injected into a DEAE-6
PW column equilibrated with 20 mM Tris hydrochloric acid buffer (pH
7.5) containing 0.6M sodium chloride and 1 mM EDTA, 20 mM Tris
hydrochloric acid buffer (pH 7.5) containing 0.6M sodium chloride
and 1 mM EDTA was fed into the column for 35 minutes at flow rate
of 1 ml/min to elute impure substances outside the column.
[0072] Then, the elution was conducted by a gradient method wherein
the concentration of sodium chloride in 20 mM Tris hydrochloric
buffer (pH 7.5) containing 1 mM EDTA was changed continuously from
0.6M to 0.8M over 30 minutes at flow rate of 1 ml/min. As a result,
a chromatogram as shown in FIG. 6 was obtained. In FIG. 6, numeral
1 shows peaks of impurities and numeral 2 shows a peak of
plasmid-containing fraction. The column effluent corresponding to
the peak 2 was collected and the purity was examined by agarose gel
electrophoresis. When dying the gel after the electrophoresis with
ethidium bromide, a supercoil type plasmid of high purity could be
obtained in the present Example.
Comparative Example 2
[0073] For comparison, purification of a plasmid pBR 322 from the
cleared lysate of Escherichia coli was conducted by ion-exchange
chromatography alone.
[0074] After the cleared lysate of Escherichia coli was prepared
like as in Example 2, equal volume of 20 mM Tris hydrochloric acid
buffer (pH 7.5) was added thereto to make a sample. Into the
above-mentioned DEAE-6 PW column equilibrated with 20 mM Tris
hydrochloric acid buffer (pH 7.5) containing 0.6M sodium chloride
and 1 mM EDTA, 3 ml of the sample were injected, and thereafter 20
mM Tris hydrochloric acid buffer (pH 7.5) containing 0.6M sodium
chloride and 1 mM EDTA was fed into the column for 60 minutes at
flow rate of 1 ml/min to elute impure substances outside the
column.
[0075] Then, the elution was carried out by the gradient method
likewise as in Example. As the result, a chromatogram shown in FIG.
7 was obtained. In FIG. 7, numeral 1 shows peaks of impurities and
numeral 2 shows a peak of fraction containing plasmid. The column
effluent corresponding to the peak of numeral 2 was collected and
the purity was examined by agarose-gel electrophoresis. As the
result, the plasmid fraction obtained by ion-exchange
chromatography alone was recognized to contain a lot of
impurities.
EXAMPLE 3
[0076] Adsorption capacity of a plasmid
[0077] Adsorption capacity of the packing material for hydrophobic
interaction chromatography employed in Example 2, Octyl-6 PW was
examined. Into a stainless steel column with inner diameter of 6.0
mm and length of 10 mm, the gel was packed and 0.1M sodium
phosphate buffer (pH 7.0) containing 2.0M ammonium sulfate and 0.1
mM EDTA was fed thereinto for 20 minutes at flow rate of 0.64
ml/min to make the column equibrated. Then. about 0.4 mg/ml plasmid
pUC 19 (2686 base pairs) of 4 ml was injected into the column and
non-adsorption fraction which was not adsorbed onto the column was
collected.
[0078] Next, by changing the eluent to 0.1M sodium phosphate buffer
(pH 7.0) containing 0.1 mM EDTA, plasmid adsorbed onto the column
was eluted to collect the adsorption fraction.
[0079] Following this, basing on the calibration curve of plasmid
pUC 19 (an equation which shows the relationship between the
injected quantity of plasmid pUC 19 and area of chromatograph)
which was previously obtained by a gel filtration chromatography on
TSK gel DNA-PW column (from Tosoh Corp.), the quantity of plasmid
pUC 19 contained in every fraction was determined to calculate
adsorption capacity per 1 ml of gel and recovery. With respect to
the packing material for ion exchange chromatography, DEAE-6 PW,
the adsorption capacity was likewise examined, too.
[0080] Except adsorption and disorption eluents all of the others
were conducted under the same condition. As the result, adsorption
quantity of Octyl-6 PW was 1.1 mg/ml and adsorption quantity of
DEAE-6 PW was 2.4 mg/ml. Further, recoveries thereof were 90.3% and
77.3%, respectively.
Comparative Example 3
[0081] For comparison, a packing material of which pore diameter is
smaller than that of Octyl-6 PW was prepared and the comparison of
adsorption quantity was tested. The preparation was made by
employing a packing material for gel filtration chromatography
(G5000 PW (from Tosoh Corp.)) with average particle diameter of 20
.mu.m and average pore diameter of 950 angstroms as a base
material. A mixture of 20 g of G5000 PW, 20 g of 1,4-dioxane, 1 g
of 1,2-epoxyoctane and 0.5 ml of boron trifluoride as catalyst were
stirred and mixed for 6 hours at 45.degree. C. to obtain Octyl-5 PW
having octyl group.
[0082] With respect to packing material for ion-exchange
chromatography, commercially available DEAE-5 PW (average particle
diameter of 20 .mu.m, average pore diameter of 880 angstroms, from
Tosoh Corp.) of which pore diameter is smaller than that of DEAE-6
PW was employed, too. The determination of adsorption capacity of
plasmid was conducted likewise as in the above-mentioned Example.
As a result, adsorption capacity of Octyl-5 PW was 0.6 mg/ml and
adsorption capacity of DEAE-5 PW was 1.2 mg/ml. Recoveries thereof
were 89.9% and 60.6%, respectively.
[0083] As evident from the above results of Example 3 and
Comparative Example, those having larger pore diameter can submit
good results for long chain nucleic acids such as plasmid in both
adsorption capacity and recovery.
* * * * *