U.S. patent application number 11/555975 was filed with the patent office on 2007-05-24 for materials and methods for the purification of polyelectrolytes, particularly nucleic acids.
This patent application is currently assigned to INVITROGEN CORPORATION. Invention is credited to Folim G. Halaka.
Application Number | 20070117972 11/555975 |
Document ID | / |
Family ID | 22897475 |
Filed Date | 2007-05-24 |
United States Patent
Application |
20070117972 |
Kind Code |
A1 |
Halaka; Folim G. |
May 24, 2007 |
Materials and Methods For the Purification of Polyelectrolytes,
Particularly Nucleic Acids
Abstract
This invention describes materials and methods usable for the
purification of polyelectrolytes, such as nucleic acids and
proteins. The materials of the invention are separation media that
possess pH-dependent groups with pKa value in the range of about 5
to about 7. Separation of the nucleic acids or proteins from a
separation medium is effected at a neutral or higher pH.
Inventors: |
Halaka; Folim G.; (Lake
Forest, IL) |
Correspondence
Address: |
INVITROGEN CORPORATION;C/O INTELLEVATE
P.O. BOX 52050
MINNEAPOLIS
MN
55402
US
|
Assignee: |
INVITROGEN CORPORATION
Carlsbad
CA
|
Family ID: |
22897475 |
Appl. No.: |
11/555975 |
Filed: |
November 2, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10436813 |
May 13, 2003 |
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11555975 |
Nov 2, 2006 |
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09840570 |
Apr 23, 2001 |
6562573 |
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10436813 |
May 13, 2003 |
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09238343 |
Jan 27, 1999 |
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09840570 |
Apr 23, 2001 |
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07772346 |
Oct 7, 1991 |
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09238343 |
Jan 27, 1999 |
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Current U.S.
Class: |
536/124 ;
210/799 |
Current CPC
Class: |
B01J 39/19 20170101;
C08L 5/12 20130101; C08B 37/0039 20130101; B01J 20/262 20130101;
B01J 20/28097 20130101; B01J 20/26 20130101; C12N 15/1006 20130101;
B01J 20/28009 20130101; B01J 20/265 20130101; B01J 41/13 20170101;
C07K 1/18 20130101 |
Class at
Publication: |
536/124 ;
210/799 |
International
Class: |
C08B 37/00 20060101
C08B037/00; B01D 17/02 20060101 B01D017/02 |
Claims
1. A polymeric separation medium for recovery of a polyelectrolyte
from a liquid, which separation medium contains pendant,
pH-dependent groups exhibiting a pKa value in the range of about 5
to about 7.
2. The separation medium of claim 1 wherein the ph-dependant groups
are basic groups.
3. The separation medium of claim 2 wherein the pendant basic
groups are derived from a base which is a member of the group
consisting of pyridine, quinoline, imidazole, and pyrimidine.
4. The separation medium of claim 1 wherein the pendant groups are
acid groups.
5. The separation medium of claim 4 wherein the pendant acidic
groups are a member of the group consisting of a carboxylic group
and a phenolic group.
6. The separation medium of claim 1 wherein the polymer is a
polysaccharide.
7. The separation medium of claim 6 wherein the polysaccharide is a
member of the group consisting of agarose, dextran, and
cyclodextrin.
8. The separation medium of claim 1 wherein the separation medium
is a water-insoluble substrate.
9. The separation medium of claim 8 wherein the substrate includes
magnetizable particles.
10. A composition suitable for recovery of a polyelectrolyte from a
liquid by convective flow, which composition includes: (a) a liquid
permeable porous support; (b) a porous separation medium contained
within the pores of the porous support, said medium having pendant,
pH-dependent groups exhibiting a pKa value in the range of about 5
to about 7.
11. The composition of claim 10 wherein an inert casing encloses
the composition, which casing includes an inlet and outlet
ports.
12. The composition of claim 10 wherein the pH-dependent groups are
organic bases.
13. The composition of claim 12 wherein the bases are selected from
the group consisting of pyridine quinoline, imidazole, and
pyrimidine.
14. The separation medium of claim 10 wherein the pendant groups
are organic acid groups.
15. The separation medium of claim 14 wherein the acids are members
of the group consisting of a carboxylic group and a phenolic
group.
16. The composition of claim 10 wherein the polymeric separation
medium is a polysaccharide.
17. The composition of claim 13 wherein the polysaccharide is a
member of the group consisting of agarose, dextran, and a
cyclodextrin.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. patent application
Ser. No. 09/840,570 filed on 23 Apr. 2001, now U.S. Pat. No.
6,562,573, which is a continuation-in-part of U.S. patent
application Ser. No. 09/238,343 filed on 27 Jan. 1999, now
abandoned, which, in turn, is a continuation-in-part of U.S. patent
application Ser. No. 07/772,346 filed on 7 Oct. 1991, now
abandoned.
FIELD OF THE INVENTION
[0002] This invention relates to separation and purification of
polyelectrolytes. In particular, the present invention relates to
the purification of biochemical materials such as proteins and more
particularly nucleic acids.
BACKGROUND OF THE INVENTION
[0003] Purification of molecular species constitutes a crucial part
to their production and utility. This is particularly important in
the biotechnology and diagnostics fields. The present invention
describes polymeric separation media and methods useful for the
purification of molecular species that are polyelectrolytes, such
as proteins and nucleic acids . The terms nucleic acid and
polynucleotide are used interchangeably, and are used here to
signify either a deoxyribonucleic acid (DNA) or a ribonucleic acid
(RNA). Unless otherwise specified, the terms polynucleotide and
oligonucleotide are used interchangeably.
[0004] There is a large body of publications dealing with
synthesis, functionalization, and use of ion-exchangers for
chromatography and biomolecule purification. (The following patents
are herein incorporated by reference in their entirety.) BACKUS et
al (U.S. Pat. Nos. 5,582,988 and 5,622,822, and CA 2157968)
describes a method for isolation of nucleic acids from a lysate by
contacting with polymers containing basic groups, such as
polyethyleneimine. However, an acidic medium was required for
binding, and a strong alkali and heat were found to be the agents
most successful in releasing , or eluting, the nucleic acids from
the polymer.
[0005] COLPAN et al (DE patent number 4139664) describe a method
for isolating nucleic acid from cells--by lysis of the cells, then
elution of nucleic acid fixed to separation medium surface.
However, the method recovers the nucleic acids by eluting with a
buffer of high ionic strength.
[0006] HENCO et al (DE 3639949) describes a method for the
Separation of long-chain nucleic acids--using a porous separation
medium to fix the nucleic acids. The invention uses selective salt
elution to first wash off the short chain nucleic acids whereas the
long-chain nucleic acids are subsequently removed from the anion
exchanger using a washing solution of high ionic strength.
[0007] SELIGSON and SHRAWDER (U.S. Pat. No. 4,935,342) describe the
classical method of isolation of nucleic acids by chromatography on
anion exchanger using salt gradients. After the nucleic acids
become bound to the ion exchange material and washed, the bound
nucleic acids are eluted by passing through the column a salt
solution of high molarity.
[0008] GANNON (EP 366438) describes the separation of nucleic acid
from protein by contacting with cation exchanger at pH below
isoelectric point of a protein, i.e., it binds the proteins not the
DNA.
[0009] Similar principles are offered in U.S. Pat. No. 3,433,782,
where selective elution steps effected with varied molarities of
LiCLO4 or NaClO4.
[0010] Similar principles are offered in U.S. Pat. No. 434,324,
where purification of deoxyribonucleic acid is accomplished by
using anion exchange material, washing with weak ionic salt
solution and elution with strong ionic salt solution.
[0011] Similar principles are offered by Bourque and Cohen (WO
9514087), where detection of charged oligonucleotides is
accomplished by adsorbing on an ion exchange resin, eluting with a
high salt buffer and detection. The oligonucleotides bind to the
anion exchange resin at 40-65.degree. C., while the desorbing from
the resin with a high salt buffer is performed at 40-65.degree.
C.
[0012] BRUCE et al (Patent Number WO 9411103; GB 9223334) describes
magnetizable polymer-based particles derived with ligand having
direct binding affinity for nucleic acids etc., for the separation
of nucleic acids. The polymer is agarose. The ligand is one capable
of assuming a positive charge at pH 7 or below, and is capable of
reversibly binding directly to a negatively charged group or moiety
in the target molecules. The selected ligands are amines, such as
dimethylaminoethyl, and triethanolamine; all have a pKa higher than
in our invention.
[0013] ADRIAANSE et al (EP 389063 A) describe a method that is
widely adapted as diagnostics kits in the art, namely the isolation
of nucleic acid using chaotropic agents for nucleic acid binding to
solid phase. The use of high concentration of chaotropes, e.g.,
guanidinium thiocyanate, forces the DNA to precipitate and interact
with many surfaces. The present invention avoids the use of highly
concentrated chaotropes during purification.
[0014] NELSON et al (EP 281390) offered a method for the separation
of small nucleotides from larger ones by binding to polycationic
support--which does not retain smaller sequences for hybridization
assays. The bound nucleic acids were apparently eluted, if needed,
by 50% formamide, or other salts.
[0015] JP 06335380 A describes a carrier for bonding nucleic acid,
which has hybrid-forming base sequence fixed over surface of
insoluble solid fine particles. The base of binding is the
interaction between complimentary sequences of polynucleotides.
[0016] U.S. Pat. No. 4,672,040 also describes a silanized magnetic
particles, for use in nucleic acid hybridization, where the
principle of binding is the interaction between the hybrid-forming
base sequence fixed over surface of insoluble solid fine
particles.
[0017] MACFARLANE (U.S. Pat. No. 5,300,635), uses quaternary amine
surfactants--for isolating nucleic acids from a biological sample
by forming complexes which can be dissociated.
[0018] HILL (WO 8605815) also describes the use of magnetized
nucleic acid sequence comprising single or double-stranded nucleic
acid linked to magnetic or magnetizable substance.
[0019] HORNES and KORSNES (U.S. Pat. No. 5,512,439) also describe
the detection and quantitative determination of target RNA or
DNA--by contacting sample with magnetic particles carrying
5'-attached DNA probe.
[0020] REEVE (U.S. Pat. No. 5,523,231) describes a method of making
a product solution containing a nucleic acid by treating a starting
solution containing the nucleic acid by the use of suspended
magnetically attractable beads which do not specifically bind the
nucleic acid, by precipitating the nucleic acid out of the starting
solution in the presence of the suspended magnetic beads whereby a
nucleic acid precipitate becomes aggregated with and entraps the
beads, followed by separating the precipitate and the entrapped
beads and adding a liquid to the precipitate and the entrapped
beads to re-dissolve the nucleic acid and re-suspend the beads.
SUMMARY OF THE INVENTION
[0021] The present invention is directed to polymeric separation
media and to methods useful for the purification of
polyelectrolytes, particularly polynucleotides.
[0022] In contrast to the prior art methods, the present invention
provides mild conditions that avoid the unfavorable, and sometimes
harsh conditions otherwise required to bind and elute biomolecules
in related art. As mentioned above, prior nucleic acid isolation or
purification methods include steps such as heating, and reagents
such as strong alkalis, or highly concentrated salts and
chaotropes. In addition to being automation and operator
unfriendly, these steps/reagents require additional efforts to
implement, neutralize, or remove.
[0023] The polymeric separation media possess multiple pendant
groups, i.e., functional groups, whose protonation state is
pH-dependent. Consequently, the amount of charge created on the
separation medium can be controlled by adjustment to the pH of the
buffer solution where the polymeric separation medium is suspended.
As an example, if the pendant groups are basic (B) and possess a
pKa of 7, then, at neutral pH (i.e., pH=7), 50% of the groups will
acquire positive charges (BH.sup.+). Since the pH scale is
logarithmic, then, at pH=8, the percent of the positively charged
groups will drop to 10%, and to 1% at pH=9. Similarly, if the basic
groups possess a pKa of 6, then at pH=9, only 0.1% of the groups
will be positively charged, and so forth.
[0024] It is an aspect of this invention that at neutral pH, basic
groups with pKa as stated above can bind, preferentially strongly,
to polyelectrolytes with high negative charge density, such as
polynucleotides. At low amount of the positive charges, the
separation medium will bind preferably to polyelectrolytes with the
most negative charges, such as DNA. Other polyelectrolytes such as
proteins, even though they may possess negative charges, will not
bind as strongly. The difference in binding strength is attributed
to the nature of the charge distribution on polynucleotides and
proteins, with polynucleotides possessing multiple, repeated
negative charges. As the polymeric separation medium possesses
multiplicity of positive charges, even when not totally protonated,
multiple electrical interaction (attraction) can occur with the
multiple negative charges on the polynucleotides. Bound proteins
can be washed off the separation medium by the choice of the pH of
the wash buffer. Elution may also be carried in multiple steps,
each with an increasing pH, where proteins would elute first, while
DNA elutes last. In a preferred embodiment, a negatively charged
polyelectrolyte binds to the separation medium at pH 7, as
discussed above, and can be eluted in steps with change of the pH
to 8 to 10, thus washing off the proteins at pH 8, while eluting
DNA at the higher pH. In the above scheme, DNA and protein
molecules can be selectively separated form a mixture thereof.
[0025] It is an important aspect of this invention, and one of its
most preferred embodiments to purify nucleic acids, that the
binding step of the positively charged polymeric separation medium
occur at a neutral pH where the separation medium is not maximally
charged. If the separation medium carries a high positive charge
density, then the binding to the highly negatively charged DNA
would be too strong to be separated without recourse to harsh
conditions, as observed in the prior art listed above.
[0026] A purification method using such a separation medium is
capable of discriminating between the binding ability of the
pH-dependent groups to the different solutes present in a mixture.
In addition to selectively binding polyelectrolytes, the separation
medium can also selectively elute polyelectrolytes.
[0027] Toward operating under mild conditions, an aspect of the
present invention is that the pendant groups carry a certain amount
of positive charges at neutral pH. It is clear from the discussion
above that pendant groups with pKa of about 6 to 7 can bind
polynucleotides, and quite strongly at neutral pH. As such, the
binding step can thus be accomplished at neutral pH.
[0028] In a preferred embodiment, the polymeric separation medium
is water-insoluble. In another preferred embodiment, the polymeric
separation medium encapsulates a magnetic core, so that it can be
separated by the application of a magnetic field.
[0029] In another preferred embodiment the polymeric separation
medium contains pendant groups that exhibit pKa values of less that
about 7, and the medium (buffer) containing the polymeric
separation medium is buffered so that the pH of the buffer has a
neutral value, thus causing the pendant groups of the polymeric
separation medium to be not completely protonated (positively
charged) at pH of at least 7.
[0030] Preferred pendant basic groups are derived from a base which
is a member of the group consisting of pyridine, quinoline,
imidazole, and pyrimidine.
[0031] Preferred pendant acidic groups are members of the group
consisting of a carboxylic group and a phenolic group.
BRIEF DESCRIPTION OF THE DRAWINGS
[0032] FIGS. 1(A) through 1(D) illustrate the formation of a
polymeric medium over a period of time and embodying this
invention. The medium is made by slowly adding water, 11, on top of
a 2.5% (W/V) agarose solution in 8M urea, 12. A thin layer of gel,
13, which forms at the interface of the agarose and water layers,
grows in thickness until all the agarose gels as time progresses
from FIG. 1(A) to FIG. 1(D), and as described in Example 1;
[0033] FIG. 2 is a schematic illustration of a cylindrical
configuration of a porous support, 21, embodied within an inert
casing, 22, after the pores of the support have been impregnated
with the gel material, 23, which can be modified to carry
pH-dependent groups as in FIG. 4(A). The path of the liquid flow
through an inlet port 24, the gel material, 23, and the exit port,
25 is indicated by arrows;
[0034] FIG. 3 is a schematic illustration of a flat membrane
configuration of a porous support, 31, embodied within a casing,
32, after the pores of the support have been impregnated with the
polymeric medium, 33. This medium can be modified to carry
pH-dependent groups as in FIG. 4(A), and showing the path of the
liquid through an inlet port 34, the gel material, 33, and the exit
port, 35;
[0035] FIG. 4(A) is a schematic of a polymeric separation medium,
41, provided with pendant groups, RH that are pH dependent. The
polymeric separation medium may be one of polysaccharide, silica,
or other polymers that can be functionalized to attach the groups.
These are acid-base groups, and depending on the ambient pH value,
the groups can be protonated, as shown (RH), or deprotonated (R).
The groups can be a basic nitrogen, which can be protonated
(--NH.sup.+) for polynucleotide purification, or the groups can be
a deprotonated carboxyl (--COO.sup.-) or a deprotonated phenolic
(--C.sub.6H.sub.4--O.sup.-) for the purification of positively
charged proteins. FIG. 4(B) shows a schematic where the polymeric
separation medium, 41, surrounds a magnetic core, 42, such as
paramagnetic iron oxide.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0036] The polymeric separation media possess functional groups
that are pH dependent, such as organic acids and organic bases. The
pKa of the pH-dependent groups is chosen to be near or slightly
below the neutral pH range, e.g., 5 to 7. For organic bases,
usually containing nitrogen atoms, the pKa is that of the
conjugated acid (base+proton). In the case of organic bases, if the
pH is lowered to at or below the pKa, the bases acquire protons to
form the conjugate acids. The separation medium thus acquires
positive electrical charges, and bind negatively charged
polyelectrolytes, such as nucleic acids and negatively charged
proteins. The unbound solutes can be separated from the separation
medium, and the separation medium can be washed with a low pH
buffer. Raising the pH to a value above the pKa of the organic base
will force the separation medium to loose its positive charges
(protons), and thus release the bound nucleic acids in a pure
form.
[0037] On the other hand, if the functional group is an organic
acid, and the pH is raised to at, or above, its pKa, the separation
medium is made to acquire negative electrical charges, and thus
binds to positively charged polyelectrolytes, such as positively
charged proteins. The unbound solutes can be separated from the
separation medium, and the separation medium can be washed with a
high pH buffer. Lowering the pH to a value below the pKa of the
acidic groups will force the groups to loose their negative charges
(by acquiring protons), and thus release the bound
polyelectrolytes. Although it is clear from the above discussion
that the separation medium can be modified to acquire positive or
negative charges by the choice of the functional groups,
henceforth, the focus will be on the cases where the separation
medium is functionalized with basic groups, to act as nucleic acid
purifiers.
[0038] In addition to choosing the condition of binding, in this
invention, the pKa of the basic groups is chosen to be near or
slightly below neutral pH to avoid the harsh conditions that will
be required otherwise to release polyelectrolytes, especially
biomolecules. When the pKa is around 5 to 7, then polyelectrolytes
can bind at pH 7, as discussed above, and can be eluted by a change
of the pH to 8 to 10 (weakly basic). This distinguishes this
invention from the prevailing methods of purifying proteins and
nucleic acids, which employ harsh unfavorable conditions of high
salt concentrations and/or highly alkaline solutions to release
bound molecules, particularly in the case of nucleic acids
purification.
[0039] The basic group in a particularly preferred embodiment is
p(2-chloroethyl) pyridine (4-picolyl chloride), and similar
pyridine, quinoline, imidazole, and pyrimidine derivatives, with
near neutral pKa, such as 2-ethyl benzimidazole (6.18), 2-methyl
benzimidazole (6.19), 2-phenyl benzimidazole (5.23), isoquinoline
(5.42), papaverine (6.4), pyrimidine (6.35), phenanthridine (5.58),
p-phenitidine (5.20), 2-picoline (5.97), 3-picoline (5.68),
4-picoline (6.02), pilocarpine (6.87), 2-amino pyridine (6.82),
2-benzyl pyridine (5.13), 2,5-diamino pyridine (6.48), 2,3-dimethyl
pyridine (6.57), 2,4-dimethyl pyridine (6.99), 3,5-dimethyl
pyridine (6.15), 2-ethyl pyridine (5.89), benzimidazole (5.532),
quinoline (4.9), 8-hydoroxy quinoline (5.0), 6-methoxy quinoline
(5.03), 2-methyl quinoline (5.83), 4-methyl quinoline (5.67),
2-aminothiazole (5.36), p-toluidine (5.08).
[0040] For acidic groups, in a preferred embodiment, the groups are
carboxylic or phenolic, having a pKa of about 7. Therefore, at
pH=8, ninety percent of the groups will acquire negative charges,
and thus bind to positively charged polyelectrolytes. In a
preferred embodiment, for example, a positively charged
polyelectrolyte binds to the separation medium at pH 7, and is
eluted by a buffer solution of pH value of about 5 (weakly
acidic).
[0041] The present invention also describes polymeric separation
media suitable for implementing purification, by chemically
attaching the above mentioned pH-dependent functional groups to
such polymeric separation media, as in FIG. 4(A). In preferred
embodiments, the polymeric separation medium is one of
polysaccharide, silica, or polyacrylamide polymers. The
polysaccharide is preferably an agarose, a dextran, or a
cyclodextrin, and derivatives thereof. Typical cyclodextrins are
the .alpha.-, .beta.- and .gamma.-cyclodextrins. In a preferred
method of purification, the sample containing the nucleic acids to
be purified is equilibrated with the polymeric separation medium in
particulate form (gel particles) carrying pH-dependent groups at pH
near the pKa of the groups, where the nucleic acids can
preferentially bind to the gel particles. The particles are allowed
to separate from the sample solution by gravitational settling or
centrifugation, and the unbound solution is aspirated. The
particles are washed with a buffer also having a pH near the pKa of
the basic groups, and the wash buffer is again aspirated after
separation from the particles. The bound nucleic acids may be
eluted by a buffer having pH approximately 3 units higher than the
pKa of the basic groups, thus releasing the nucleic acids in pure
form.
[0042] In another preferred embodiment, the above polymeric
separation medium surrounds a magnetic core, such as paramagnetic
iron oxide particles, as described schematically in FIG. 4(B). For
example, magnetic particles may be coated by polymers, using
techniques known in the art, to form magnetized separation media in
particulate form. The polymer may be subsequently functionalized
with pH-dependent groups as discussed herein. In another preferred
method of purification, the sample containing the nucleic acids to
be purified is equilibrated with the magnetized particles carrying
basic groups at a pH near or slightly above the pKa of the groups
(e.g., pKa=6, and pH.about.7), where they preferentially bind
nucleic acids. Using a magnet, the particles are separated from the
solution. The particles are washed with a buffer, also having a pH
near the pKa of the basic groups and the particles are again
separated using a magnet, and the wash buffer is aspirated. The
bound nucleic acids may be eluted from the particles by a buffer
having pH approximately 3 units higher than the pKa of the basic
groups, thus releasing the nucleic acids in pure form.
[0043] In another embodiment, the separation medium is a gel that
pervades the pores of a porous support to form a continuous phase.
The porous support is fabricated into a device for the separation
using convective flow. The device is formed by imbibing a
dissolved, gellable polymer into the pores of the porous support.
In a preferred embodiment, the polymer is dissolved by the use of
chaotropes. Chaotropes are small solutes that influence the
solution behavior of a solvent and its dissolving power. Chaotropes
can give the solution the capacity to dissolve polymers that are
normally sparingly soluble in the pure solvent. For example, the
polymer is a polysaccharide and the chaotrope is urea, guanidinium
salts, or KI. In a preferred embodiment, agarose was dissolved in
water solution containing 8M urea. This process yields solutions of
agarose, which do not gel at room temperature. If water is
carefully added to such a solution, a thin gel layer forms at the
interface between the agarose solution and the water. The gel layer
prevents the migration of the polysaccharide, but still allows
further migration of the much smaller urea molecules. As the urea
molecules leave the agarose solution into the mainly water medium,
the agarose further gels until all the agarose solution turns into
gel. The resulting gel can be further chemically modified to attach
the pH-dependent groups described above to be used as a separation
medium. This process is schematically depicted in FIG. 1(A) through
FIG. 1(D), and further explained in Examples 1 through 3.
[0044] The porous support can take several forms, preferably a
cylindrical shape or a flat shape, as shown in FIGS. 2 and 3,
respectively. The polysaccharide solution just described is pushed
or drawn by vacuum to impregnate the pores of a porous support, and
the excess solution is drained off. The chaotrope (urea) is allowed
to migrate from the polysaccharide solution by diffusion through
the pores of the support when soaking in water. As the
concentration of urea in the pores is lowered, the agarose gels in
the pores of the support. The agarose thus becomes entrapped in the
pores of the support. This gelling process appears to be unique and
gives a stable and reproducible pore-size distribution that is
useful for the purpose of this invention. The resulting gel in the
above process can be further locked inside the pores by
cross-linking of the agarose with a cross-linking agent such as
epichlorohydrin and 1,2-dibromo propane.
[0045] The porous support material may be made of a number of
polymeric, glass, metallic and ceramic materials. In a preferred
embodiment, the porous support may be fabricated from ultra high
density polyethylene, or polypropylene composition, that is formed
into the shape and configuration as shown in FIGS. 2 and 3, which
can be obtained from Porex Company, located at Fairburn, Ga.
[0046] In another preferred method of purification, the sample
containing the nucleic acids to be purified is passed through
polymeric separation medium containing basic groups, formed in the
pores of the porous support. The process is carried out where the
sample and the polymeric separation medium are equilibrated at a pH
near or slightly above the pKa of the groups (e.g., pKa=6, and
pH.about.7), whereby the nucleic acids bind to the polymeric
separation medium. Wash buffer is then flown to wash off unbound
solutes. Wash buffers may have the same pH as the binding buffer
(.about.7). The bound nucleic acids may be eluted by a buffer
having pH approximately 3 units higher than the pKa of the basic
groups, thus releasing the nucleic acids in substantially pure
form.
EXAMPLES
Example 1
[0047] 25 grams of agarose (Sigma Chemical Co., St. Louis, Mo.,
product number A9918) were dissolved in one liter of 8M urea
solution by heating the solution with stirring until the solution
became clear, forming layer 12, in FIG. 1. The medium is made by
slowly adding water, 11, on top of a 2.5% (W/V) agarose solution in
8M urea, 12. A thin layer of gel, 13, which forms at the interface
of the agarose and water layers, grows in thickness until all the
agarose gels as time progresses from FIG. 1(A) to FIG. 1(D), and as
described in Example 1. This solution remained ungelled after
cooling to room temperature for a period of over 150 days. Upon
slow addition of water, layer 11 in FIG. 1, to the top of this
solution and standing unperturbed for a few hours, a gelled layer,
13, forms at the interface. This is indicated by the appearance of
a white color. This layer plays an important role in preventing
further mixing of the water with the agarose in the chaotropic
solution. Water and urea molecules can cross this layer, but the
agarose molecules, being considerably larger, remain in the bottom
layer. At longer times, more urea molecules diffused to the top
layer, until the whole bottom part became gelled, as shown in FIGS.
1(A) to 1(D). This solution behavior of agarose was repeated with
quantities of agarose concentrations varying from 0.5 to 4% (in 8M
urea solution). This solution behavior was also reproduced with 3M
Nal solution and buffered 8M guanidine hydrochloride solution. The
agarose dissolved to form a clear solution that did not gel at room
temperature, however, it became yellowish with time in the case of
NaI, presumably due to the oxidation of iodide ions. In the devices
that were constructed using this approach, a gelled layer is formed
at the surface of the porous support, preventing migration of the
entrapped polymer from the pores. The gel structure of the
entrapped polymer becomes more predictable as a result of this
process.
Example 2
[0048] An ultra high molecular weight polyethylene (UHMWPE) porous
cylinder, 9.5 mm O.D., 4.8 mm I.D., and 35 mm long was used, with
one end sealed and the other open. This cylinder had nominal pore
size of 20 microns and void volume (empty space in the walls of the
cylinder) of approximately 50%. The inner bore of the cylinder was
blocked by a rod of the same diameter as the ID of the cylinder to
prevent the agarose solution from filling the bore. Agarose (2.5%
w/v) was solubilized in 8M Urea as in Example 1. The agarose
solution was vacuum drawn into the pores of the cylinder using a
vacuum pump. The cylinder was soaked in water overnight. Referring
to the schematic in FIG. 2, this cylinder is numbered 21. The
agarose gelled in the pores of the cylinder as the urea diffused
out to form the gel, 23. The rod was removed from the cylinder. A
luer fitting attachment, 24, was sealed to the open end of the
cylinder, and was used to attach the device to syringes or
peristaltic pumps for the purpose of processing fluids. The device
was placed in a casing, 22. The liquid permeability decreased from
approximately 1800 for the gel-free device to about 8 after this
step, indicating filling of the pores with the gel.
Example 3
[0049] The agarose-filled cylinder in Example 2 was equilibrated in
1M NaOH for 4 hours. The NaOH was drained out and the agarose was
cross-linked using 3% epichlorohydrin in 95% (VIV) ethanol/water
mixture for 16 hours at room temperature. The resulting
cross-linked gel can be used a filtration device. The liquid
permeability of the device after cross-linking increased slightly
to about 10.
Example 4
[0050] The agarose in the cylinder of Examples 3 was allowed to
react with 5% (W/V) p-(2-chloroethyl) pyridine (4-picolyl chloride)
in 50% (W/V) reagent alcohol-water for 16 hours. This cylinder
produced a device with a weak base that is suitable for DNA
exchange, since the picolyl group has a pKa of about 6, it can bind
DNA at neutral pH's and release DNA at moderately high pH (9).
[0051] Example 5.
[0052] The agarose was cross-linked and re-equilibrated in 10% NaOH
in a cylinder as in Examples 2-3. The cylinder was then allowed to
react with 10% (W/V) chloroacetic acid in 95% (W/V) reagent
alcohol-water for 16 hours. This cylinder produced a device with
carboxyl group modified agarose as a weak cation exchanger.
[0053] Example 6
[0054] The cylinder in Example 5 was equilibrated with 15 mM Tris
buffer at pH=7. Aliquots of 5 ml of cytochrome c at 1 mg/ml in the
same buffer were loaded through the wall of the cylinder by closing
one end of the cylinder. Total protein bound was 32 mg. The
cylinder acquired the color of the protein. The device was washed
with buffer, and the protein was eluted using aliquots of 500 mM
buffered sodium chloride solution. As in Example 5, FIG. 8 shows
the bind-elution histogram for cytochrome c.
[0055] Example 7
[0056] Sephadex G-50 beads (polysaccharide consisting of dextran
and cross-linked agarose, Sigma Chemical Co., St, St. Louis, Mo.)
were suspended in dioxane and allowed to react with 5% (W/V)
p(2-chloroethyl) pyridine (4-picolyl chloride) hydrochloride in 95%
(V/V) dioxane-water for 16 hours, with the addition of potassium
carbonate as a base. The liquid was aspirated and the beads were
washed repeatedly with water until the color disappeared. The beads
were functionalized to produce a DNA purifier, since p-ethyl
pyridine (gamma picoline) has a pKa of about 6, it can bind DNA at
neutral pH's and release DNA at moderately high pH (9). As shown in
FIG. 4, the polymeric separation medium may be one of
polysaccharide, silica, or other polymers that can be
functionalized to attach the groups. These are acid-base groups,
and depending on the ambient pH value, the groups can be
protonated, as shown (RH), or deprotonated (R). The groups can be a
basic nitrogen, which can be protonated (--NH.sup.+) for
polynucleotide purification, or the groups can be a deprotonated
carboxyl (--COO.sup.-) or a deprotonated phenolic
(--C.sub.6H.sub.4--O.sup.-) for the purification of positively
charged proteins. FIG. 4 (B) shows a schematic where the polymeric
separation medium, 41, surrounds a magnetic core, 42, such as
paramagnetic iron oxide.
[0057] Example 8
[0058] Approximately 50 mg of the beads in Example 7 were allowed
to equilibrate with 0.3 ml of 30 mM acetate buffer of pH=5.5 in an
eppendorf tube. 2 microgram solution of DNA (PBKSRV plasmid, from
Strategene, .about.8 kb in length) in 0.05 ml of water were added
to the beads and allowed to incubate at room temperature for
approximately 10 minutes with mixing. The beads were centrifuged
and the supernatant aspirated. The beads were washed three times
with 0.5 ml each of the same buffer, centrifuged, and the buffer
was aspirated after each wash. An aliquots of 0.2 ml of alkaline
(pH=9) solution of HEPES buffer was added to the beads to elute the
bound DNA, and the solution was aspirated. Another 0.5 ml elution
step was applied, and again the supernatant was aspirated after
centrifugation. After each aspiration step, the aspirated solution
was saved for later DNA analysis by gel electrophoresis. Gel
electrophoresis results indicate binding of the DNA under the
acidic conditions and DNA release under the slightly alkaline
elution.
[0059] Example 9
[0060] Paramagnetic iron oxide beads coated with silica were
treated to produce a DNA purifier in a method similar to that in
Example 7, by attaching 4-picoline to the silica separation
medium.
[0061] Example 10
[0062] Approximately 50 mg of the magnetic beads in Example 9 were
allowed to equilibrate with 0.3 ml of 30 mM acetate buffer of
pH=5.5 in an eppendorf tube. 2 microgram solution of the DNA used
in Example 8 in 0.05 ml of water were added to the beads and
allowed to incubate at room temperature for approximately 10
minutes with mixing. A magnet was brought in contact with the tube,
where the magnetic beads were attracted to the side of the tube
contacting the magnet, and the supernatant aspirated. The beads
were washed three times with 0.5 ml each of the same buffer, and
after contacting the tube with the magnet, the buffer was aspirated
after each wash. An aliquots of 0.2 ml of alkaline (pH=9) solution
of HEPES buffer was added to the beads to elute the bound DNA,
mixed and the supernatant aspirated after contacting the tube with
the magnet. Another 0.5 ml elution step was applied, and again the
supernatant was aspirated after magnetic separation. After each
aspiration step, the aspirated solution was saved for analysis. Gel
electrophoresis results indicated binding of the DNA under the
acidic conditions and complete release of the DNA under the
slightly alkaline elution.
[0063] Example 11
[0064] Approximately 50 mg of the magnetic beads in Example 9 were
allowed to equilibrate with 0.5 ml of 30 mM acetate buffer of
pH=5.5 in an eppendorf tube. 2 microgram solution of DNA in 0.2 ml
of calf serum was added to the beads and the procedure in Example
10 was followed. Gel electrophoresis results indicate binding of
the DNA under the acidic conditions and complete release of the DNA
under the slightly alkaline elution. The released DNA band in this
example was slightly diffuse, presumably due to DNA degradation by
the action of nucleases present in the serum, a condition that can
be avoided by the addition of a protease or other
nuclease-deactivating substances.
* * * * *