Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
POLYNUCLEOTIDE SEPARATIONS ON POLYMERIC SEPARATION MEDIA
FIELD OF THE INVENTION
The present invention is directed to the separation of polynucleotides using
non-
polar separation surfaces, such as the surfaces of polymeric beads and
surfaces within
molded monoliths, which are substantially free from contamination with
multivalent cations.
BACKGROUND OF THE INVENTION
Separations of polynucleotides such as DNA have been traditionally performed
using slab gel electrophoresis or capillary electrophoresis. However, liquid
chromatographic separations of polynucleotides are becoming more important
because of
the ability to automate the analysis and to collect fractions after they have
been separated.
Therefore, columns for polynucleotide separation by liquid chromatography (LC)
are
becoming more important.
High quality materials for double stranded DNA separations previously have
been
based on polymeric substrates disclosed in U.S. Patent No. 5,585,236, to Bonn,
et al.
(1996), which showed that double-stranded DNA can be separated on the basis of
size
with selectivity and performance similar to gel electrophoresis using a
process
characterized as reverse phase ion pairing chromatography (RPIPC). However,
the
chromatographic material described was limited to nonporous beads substituted
with alkyl
groups having at least 3 carbons because Bonn, et al. were unsuccessful in
obtaining
separations using polymer beads lacking this substitution. Additionally, the
polymer beads
were limited to a small group of vinyl aromatic monomers, and Bonn et al. were
unable to
effect double stranded DNA separations with other materials.
A need continues to exist far chromatographic methods for separating
polynucleotides with improved separation efficiency and resolution.
SUMMARY OF THE INVENTION
Accordingly, one object of the present invention is to provide a
chromatographic
method for separating polynucleotides with. improved separation and
efficiency.
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Another object of the present invention is to provide a method for separating
polynucleotides using nonporous polymer separation media, such as beads or
monoliths
(e.g., rods), having non-reactive, non-polar surfaces.
It is another object of this invention to provide the chromatographic
separation of
polynucleotides using nonporous polymeric separation media made from a variety
of
different polymerizable monomers.
It is a further object of this invention to provide the chromatographic
separation of
polynucleotides using polymeric separation media which can be unsubstituted,
methyl-
substituted, ethyl-substituted, hydrocarbon-substituted, or hydrocarbon
polymer-
substituted.
Yet another object of the present invention is to provide improved polymer
separation media by including steps to remove contamination occurring during
the
manufacturing process.
Still another object of the invention is to provide a method for separating
polynucleotides using a variety of different solvent systems.
These and other objects which will become apparent from the following
specification have been achieved by the present invention.
In one aspect, the invention is a method for separating a mixture of
polynucleotides
by applying a mixture of polynucleotides having up to 1500 base pairs to a
polymeric
separation medium having non-polar surfaces which are substantially free from
contamination with multivalent cations, and eluting the mixture of
polynucleotides. The
preferred surfaces are nonporous. The non-polar surfaces can be enclosed in a
column. In
the preferred embodiment, precautions are taken during the production of the
medium so
that it is substantially free of multivalent cation contaminants and the
medium is treated, for
example by an acid wash treatment and/or treatment with multivalent cation
binding agent,
to remove any residual surface metal contaminants. The preferred separation
medium is
characterized by having a DNA Separation Factor (defined hereinbelow) of at
least 0.05.
The preferred separation medium is also characterized by having a Mutation
Separation
Factor (as defined hereinbelow) of at least 0.1. In the preferred embodiment,
the
separation is made by Matched Ion Polynucleotide Chromatography (MIPC, as
defined
hereinbelow). Examples of non-polar surfaces include the surfaces of polymer
beads and
the surfaces of interstitial spaces within a polymeric monolith. The elution
step preferably
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uses a mobile phase containing a counterion agent and a water-soluble organic
solvent.
Examples of a suitable organic solvent include alcohol, nitrite,
dimethylformamide,
tetrahydrofuran, ester, ether, and mixtures of one or more thereof, e.g.,
methanol, ethanol,
2-propanol, 1-propanol, tetrahydrofuran, ethyl acetate, acetonitrile. The most
preferred
organic solvent is acetonitrile. The counterion agent is preferably selected
from the group
consisting of lower alkyl primary amine, lower alkyl secondary amine, lower
alkyl tertiary
amine, lower trialkyammonium salt, quaternary ammonium salt, and mixtures of
one or
more thereof. Non-limiting examples of counterion agents include octylammonium
acetate, octadimethylammonium acetate, decylammonium acetate,
octadecylammonium
acetate, pyridiniumammonium acetate, cyclohexylammonium acetate,
diethylammonium
acetate, propylethylammonium acetate, propyldiethylammonium acetate,
butylethylammonium acetate, methylhexylammonium acetate, tetramethylammonium
acetate, tetrapropylammonium acetate, tetrabutylammonium acetate,
dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium
acetate,
tributylammonium acetate, tetraethylammonium acetate, tetrapropylammonium
acetate,
tetrabutylammonium acetate, and mixtures of any one or more of the above. The
counterion agent includes an anion, e.g., acetate, carbonate, bicarbonate,
phosphate,
sulfate, nitrate, propionate, formate, chloride, perchlorate, or bromide. The
most preferred
counterion agent is triethylammonium acetate or triethylammonium
hexafluoroisopropyl
alcohol.
One embodiment of the invention provides a method for separating a mixture of
polynucleotides, comprising applying a mixture of polynucleotides having up to
1500 base
pairs to polymeric separation beads having non-polar surfaces which are
substantially free
from contamination with multivalent cations, and eluting said mixture of
polynucleotides. In
a particular embodiment of the separation medium, the invention provides a
method for
separating a mixture of polynucleotides comprising flowing a mixture of
polynucleotides
having up to 1500 base pairs through a separation column containing polymer
beads
which are substantially free from contamination with multivalent cations and
having an
average diameter of 0.5 to 100 microns, and separating the mixture of
polynucleotides.
The beads are preferably made from polymers, including mono- and di-vinyl
substituted
aromatic compounds such as styrene, substituted styrenes, alpha-substituted
styrenes
and divinylbenzene; acrylates and methacrylates; polyolefins such as
polypropylene and
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polyethylene; polyesters; polyurethanes; polyamides; polycarbonates; and
substituted
polymers including fluorosubstituted ethylenes commonly known under the
trademark
TEFLON. The base polymer can also be mixtures of polymers, non-limiting
examples of
which include polystyrene-divinylbenzene) and poly(ethylvinylbenzene-
divinylbenzene).
The polymer can be unsubstituted, or substituted with a hydrocarbon such as an
alkyl
group having from 1 to 1,000,000 carbons. In a preferred embodiment, the
hydrocarbon is
an alkyl group having from 1 to 24 carbons. tn more preferred embodiment, the
alkyl group
has 1-8 carbons. The beads preferably have an average diameter of about 1 - 5
microns.
In the preferred embodiment, precautions are taken during the production of
the beads so
that they are substantially free of multivalent cation contaminants and the
beads are
treated, for example by an acid wash treatment, to remove any residual surface
metal
contaminants. The beads of the invention are characterized by having a DNA
Separation
Factor of at least 0.05. In a preferred embodiment, the beads are
characterized by having
a DNA Separation Factor of at least 0.5. Also in a preferred embodiment, the
beads are
characterized by having a Mutation Separation Factor of at least 0.1. The
preferred
method used in the separation is made by MIPC. In one embodiment, the beads
are used
in a capillary column to separate a mixture of polynucleotides by capillary
electrochromatography. In other embodiments, the beads are used to separate
the
mixture by thin-layer chromatography or by high-speed thin-layer
chromatography.
' In addition to the beads (or other media) themselves being substantially
metal-free,
Applicants have also found that to achieve optimum peak separation the inner
surfaces of
the separation column (or other container) and all process solutions held
within the column
or flowing through the column are preferably substantially free of multivalent
cation
contaminants. This can be achieved by supplying and feeding solutions entering
the
separation column with components which have process solution-contacting
surfaces
made of material which does not release multivalent cations info the process
solutions
held within or flowing through the column, in order to protect the column from
multivalent
cation contamination. The process solution-contacting surfaces of the system
components
are preferably material selected from the group consisting of titanium, coated
stainless
steel, and organic polymer.
For additional protection, multivalent cations in mobile phase solutions and
sample
solutions entering the column can be removed by contacting these solutions
with
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multivalent ration capture resin before the solutions enter the column to
protect the
separation medium from multivalent ration contamination. The multivalent
capture resin is
preferably ration exchange resin andlor chelating resin. The method of the
present
invention can be used to separate double stranded polynucleotides having up to
about
1500 to 2000 base pairs. In many cases, the method is used to separate
polynucleotides
having up to 600 bases or base pairs, or which have up to 5 to 80 bases or
base pairs.
The mixture of polynucleotides can be a polymerase chain reaction product. The
method
preferably is performed at a temperature within the range of 20°C to
90°C. The flow rate of
mobile phase preferably is. adjusted to yield a back-pressure not greater than
5000 psi.
The method preferably employs an organic solvent that is water soluble. The
method also
preferably employs a counterion agent.
In another aspect, the present invention provides a polymeric bead having an
average bead diameter of 0.5-100 micron. Precautions are taken during the
production of
the beads so that they are substantially free of multivalent ration
contaminants and the
beads are treated, for example by an acid wash treatment, to remove any
residual surface
metal contaminants. In one embodiment, the beads are characterized by having a
DNA
Separation Factor of at least 0.05. In a preferred embodiment, the beads are
characterized
by having a DNA Separation Factor of at least 0.5. In a preferred embodiment,
the beads
are characterized by having a Mutation Separation Factor of at least 0.1. The
bead
preferably has an average diameter of about 1-10 microns, and most preferably
has an
average diameter of about 1-5 microns. The bead can be comprised of a
copolymer of
vinyl aromatic monomers. The vinyl aromatic monomers can be styrene, alkyl
substituted
styrene, alpha-methylstyrene or alkyl substituted alpha-methylstyrene. The
bead can be a
copolymer such as a copolymer of styrene, C~_g alkyl vinylbenzene and
divinylbenzene.
The bead can contain functional groups such as polyvinyl alcohol, hydroxy,
vitro, halogen
(e.g. bromo), cyano, aldehyde, or other groups that do not bind the sample.
The bead can
be unsubstituted or having bound thereto a hydrocarbon group having from 1 to
1,000,000
carbons. In one embodiment, the hydrocarbon group is an alkyl group having
from 1 to 24
carbons. In another embodiment, the hydrocarbon group has from 1 to 8 carbons.
In
preferred embodiments, the bead is octadecyl modified poly(ethylvinylbenzene-
divinylbenzene) or polystyrene-divinylbenzene). The bead can also contain
crosslinking
divinylmonomer such as divinyl benzene or butadiene.
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!n yet another embodiment, the invention is a method for separating a mixture
of
polynucleotides comprising flowing a mixture of polynucleotides having up to
1500 base
pairs through a polymeric monolith, and separating the mixture of
polynucleotides using
MIPC. In this embodiment, the non-polar separation surfaces are the surfaces
of interstitial
spaces of a polymeric monolith. An example of such a monolith is a polymeric
rod
prepared within the confines of a chromatographic column. The monolith of the
invention
is characterized by having a DNA Separation Factor of at least 0.05. In a
preferred
embodiment, the monolith is characterized by having a DNA Separation Factor of
at least
0.5. The monolith is preferably characterized by having a Mutation Separation
Factor of at
least 0.1. The mobile phase used in the separation preferably includes an
organic solvent
as exemplified by alcohol, nitrite, dimethylformamide, tetrahydrofuran, ester,
ether, and
mixtures. thereof. Examples of suitable solvents include methanol, ethanol, 2-
propanol, 1-
propanol, tetrahydrofuran, ethyl acetate, acetonitrile, and mixtures thereof.
The most
preferred organic solvent is acetonitrile. The mobile phase preferably
includes a counterion
agent such as lower primary, secondary and tertiary amines, and lower
trialkyammonium
salts, or quaternary ammonium salts. More specifically, the counterion agent
can be
octylammonium acetate, octadimethylammonium acetate, decylammonium acetate,
octadecylammonium acetate, pyridiniumammonium acetate, cyclohexylammonium
acetate, diethylammonium acetate, propylethylammonium acetate,
propyldiethylammonium acetate, butylethylammonium acetate, methylhexylammonium
acetate, tetramethylammonium acetate, tetraethylammonium acetate,
tetrapropylammonium acetate, tetrabutylammonium acetate,
dimethydiethylammonium
acetate, triethylammonium acetate, tripropylammonium acetate, tributylammonium
acetate, tetrapropylammonium acetate, tetrabutylammonium acetate, and mixtures
of any
one or more of the above. The counterion agent includes an anion, e.g.,
acetate,
carbonate, bicarbonate, phosphate, sulfate, nitrate, propionate, formats,
chloride,
perchlorate, and bromide. However, the most preferred counterion agent is
triethylammonium acetate.
In the preferred embodiment, precautions are taken during the production of
the
polymeric monolith so that it is substantially free of multivalent cation
contaminants and the
monolith is treated, for example, by an acid wash treatment, to remove any
residual
surface metal contaminants. In one embodiment, the monolith is characterized
by having
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a DNA Separation Factor of at least 0.05. In a preferred embodiment, the
monolith is
characterized by having a DNA Separation Factor of at least 0.5. Also in a
preferred
embodiment, the monolith is characterized by having a Mutation Separation
Factor of at
least 0.1.
In another aspect, the present invention is a method for treating the non-
polar
surface of a polymeric medium used for separating polynculeotides, such as the
surface of
beads in a MIPC column or the interstitial spaces in a polymeric monolith, in
order to
improve the resolution of polynucleotides, such as dsDNA, separated on said
surface. This
treatment includes contacting the surtace with a solution containing a
multivalent cation
binding agent. In a preferred embodiment, the solution has a temperature of
about 50°C
to 90°C. An example of this treatment includes flowing a solution
containing a multivalent
cation binding agent through a MIPC column, wherein the solution has a
temperature of
about 50°C to 90°C. The preferred temperature is about
70°C to 80°C. In a preferred
embodiment, the multivalent cation binding agent is a coordination compound,
examples
of which include water-soluble chelating agents and crown ethers. Specific
examples
include acetylacetone, alizarin, aluminon, chloranilic acid, kojic acid,
morin, rhodizonic
acid, thionalide, thiourea, a-furildioxime, nioxime, salicylaldoxime,
dimethylglyoxime, a-
furildioxime, cupferron, a-nitroso-~3-naphthol, nitroso-R-salt,
diphenylthiocarbazone,
diphenylcarbazone, eriochrome black T, PAN, SPADNS, glyoxal-bis(2-
hydroxyanil),
murexide, a-benzoinoxime, mandelic acid, anthranilic acid, ethytenediamine,
glycine,
triaminotriethylamine, thionalide, triethylenetetramine,
ethylenediaminetetraacetic acid
(EDTA), metalphthalein, arsonic acids, a,a'-bipyridine, 4-
hydroxybenzothiazole, 8-
hydroxyquinaldine, 8-hydroxyquinoline, 1,10-phenanthroline, picolinic acid,
quinaldic acid,
a,a',a"-terpyridyl, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol,
salicylic acid, tiron,
4-chloro-1,2-dimercaptobenzene, dithiol, mercaptobenzothiazole, rubeanic acid,
oxalic
acid, sodium diethyldithiocarbarbamate, and zinc dibenzyldithiocarbamate.
However, the
most preferred chelating agent is EDTA. In this aspect of the invention, the
solution
preferably includes an organic solvent as exemplified by alcohol, nitrite,
dimethylformamide, tetrahydrofuran, ester, ether, and mixtures thereof.
Examples of
suitable solvents include methanol, ethanol, 2-propanol, 1-propanol,
tetrahydrofuran, ethyl
acetate, acetonitrile, and mixtures thereof. The most preferred organic
solvent is
acetonitrile. In one embodiment, the solution can include a counterion agent
such as lower
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primary, secondary and tertiary amines, and lower trialkyammonium salts, or
quaternary
ammonium salts. More specifically, the counterion agent can be octylammonium
acetate,
octadimethylammonium acetate, decylammonium acetate, octadecylammonium
acetate,
pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium
acetate,
propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium
acetate, methylhexylammonium acetate, tetramethylammonium acetate,
tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium
acetate,
dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium
acetate,
tributylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium
acetate,
and mixtures of any one or more of the above. The counterion agent includes an
anion,
e.g., acetate, carbonate, bicarbonate, phosphate, sulfate, nitrate,
propionate, formate,
chloride, perchlorate, and bromide. However, the most preferred counterion
agent is
triethylammonium acetate.
In yet a further aspect, the invention provides a method for storing a medium
used
for separating polynucleotides, e.g., the beads of a MIPC column or a
polymeric monolith,
in order to improve the resolution of double stranded DNA fragments separated
using the
medium. In the case of a MIPC column, the preferred method includes flowing a
solution
containing a multivalent cation binding agent through the column prior to
storing the
column. In a preferred embodiment, the multivalent cation binding agent is a
coordination
compound, examples of which include water-soluble chelating agents and crown
ethers.
Specific examples include acetylacetone, alizarin, aluminon, chloranilic acid,
kojic acid,
morin, rhodizonic acid, thionalide, thiourea, a-furildioxime, nioxime,
salicylaldoxime,
dimethylglyoxime, a-furildioxime, cupferron, a-nitroso-~i-naphthol, nitroso-R-
salt,
diphenylthiocarbazone, diphenylcarbazone, eriochrome black T, PAN, SPADNS,
glyoxal-
bis(2-hydroxyanil), murexide, a-benzoinoxime, mandelic acid, anthranilic acid,
ethylenediamine, glycine, triaminotriethylamine, thionalide,
triethylenetetramine, EDTA,
metalphthalein, arsonic acids, a,a'-bipyridine, 4-hydroxybenzothiazole, 8-
hydroxyquinaldine, 8-hydroxyquinoline, 1,10-phenanthroline, picolinic acid,
quinaldic acid,
a,a',a"-terpyridyl, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol,
salicylic acid, tiron,
4-chloro-1,2-dimercaptobenzene, dithiol, mercaptobenzothiazole, rubeanic acid,
oxalic
acid, sodium diethyldithiocarbarbamate, and zinc dibenzyldithiocarbamate.
However, the
most preferred chelating agent is EDTA. In this aspect of the invention, the
solution
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preferably includes an organic solvent as exemplified by alcohols, nitrites,
dimethylformamide, tetrahydrofuran, esters, and ethers. The most preferred
organic
solvent is acetonitrile. The solution can also include a counterion agent such
as lower
primary, secondary and tertiary amines, and lower trialkyammonium salts, or
quaternary
ammonium salts. More specifically, the counterion agent can be octylammonium
acetate,
octadimethylammonium acetate, decylammonium acetate, octadecylammonium
acetate,
pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium
acetate,
propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium
acetate, methylhexylammonium acetate, tetramethylammonium acetate,
tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium
acetate,
dimethydiethylammonium.acetate, triethylammonium acetate, tripropylammonium
acetate,
tributylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium
acetate,
and mixtures of any one or more of the above. The counterion agent includes an
anion,
e.g., acetate, carbonate, bicarbonate, phosphate, sulfate, nitrate,
propionate, formate,
chloride, perchlorate, and bromide. However, the most preferred counterion
agent is
triethylammonium acetate.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of how the DNA Separation Factor is
measured.
FIG. 2 is a MIPC separation of pUC18 DNA-Haelll digestion fragments on a
column
containing alkylated polystyrene-divinylbenzene) beads. Peaks are labeled with
the
number of base pairs of the eluted fragment.
FIG. 3 is a MIPC separation of pUC18 DNA-Haelll digestion fragments on a
column
containing nonporous 2.1 micron beads of underivatized polystyrene-
divinylbenzene).
FIG. 4 is a Van't HofF plot of In k vs. 1/T(°K'~jwith alkylated
poly(styrene-
divinylbenzene) beads showing positive enthalpy using acetonitrile as the
solvent.
FIG. 5 is a Van't Hoff plot of In k vs. 1/T [°K-~j with underivatized
poly(styrene-
divinylbenzene) beads showing positive enthalpy using acetonitrile as the
solvent.
FIG. 6 is a Van't Hoff plot of In k vs. 1/T (°K -~j with alkylated
poly(styrene-
divinylbenzene) beads showing negative enthalpy using methanol as the solvent.
FIG. 7 is a separation using alkylated beads and acetonitrile as solvent.
FIG. 8 is a separation using alkylated beads and 50.0% methanol as the
solvent.
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FIG. 9 is a separation using alkylated beads and 25.0% ethanol as the solvent.
FIG. 10 is a separation using alkylated beads and 25.0% vodka (100 proofi~ as
the
solvent.
FIG. 11 is a separation using alkylated beads and 25.0% 1-propanol as the
solvent.
FIG. 12 is a separation using alkylated beads and 25.0% 1-propanol as the
solvent.
FIG. 13 is a, separation using alkylated beads and 10.0% 2-propanol as the
solvent.
FIG. 14 is a separation using alkylated beads and 10.0% 2-propanol as the
solvent.
FIG. 15 is a separation using alkylated beads and 25.0% THF as the solvent.
FIG. 16 is a combination isocratic/gradient separation on a non-alkylated
polystyrene-divinylbenzene) beads.
FIG. 17 shows a schematic representation of a hybridization to form homoduplex
and heteroduplex.
FIG. 18 is an elution profile showing separation of a 209 base pair
homoduplex/heteroduplex mutation detection mixture performed by DMIPC at
56°C.
FIG. 19 is an elution profile of another injection of the same 209 by mixture
and
using the same column as in FIG. 18, but after changing the guard cartridge
and replacing
the pump-valve filter.
FIG. 20 is an elution profile of another injection of the same 209 by mixture
and
using the same column as in FIG. 19, but after flushing the column with 0.1 M
TEAR, 25%
acetonitrile, and 0.32 M EDTA for 45 minutes at 75°C.
FIG. 21 is a DMIPC elution profile of a 100 by PCR product from a wild-type
strand
of Lambda DNA.
FIG. 22 is a DMIPC elution profile of a hybridized mixture containing a Lambda
DNA strand containing a mutation and wild type strand.
FIG. 23 illustrates an elution profile obtained using a monolithic capillary
column.
FIG. 24 illustrates an elution profile of a 20 nucleotide fragments from the
monolithic
capillary column used for FIG. 23 after the column was treated with EDTA.
FIG. 25 illustrates an elution profile of a mixture containing a 20 mer
oligonucleotide
and a double stranded DNA standard.
FIG. 26 illustrates an elution profile using a monolithic column after
injection of a
209 base pair double stranded DNA fragment.
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DETAILED DESCRIPTION OF THE INVENTION
In its most general form, the subject matter of the present invention concerns
the
separation of polynucleotides. e.g. DNA, utilizing a stationary separation
medium having
non-polar surfaces. The preferred surfaces are essentially free from
multivalent cation
contamination which can trap polynucleotides. The separation is performed on
the
stationary surface. The surface can be porous, but preferably any surface
pores are of a
size which excludes the smallest polynucleotide being analyzed.
The medium can be enclosed in a column. In one embodiment, the non-polar
surtaces comprise the surfaces of polymeric beads. In an alternative
embodiment, the
surfaces comprise the surfaces of interstitial spaces in a molded polymeric
monolith. For
purposes of simplifying the description of the invention and not by way of
limitation, the
separation of polynucleotides using nonporous beads, and the preparation of
such beads,
will be primarily described herein, it being understood that other separation
surfaces, such
as the interstitial surfaces of polymeric monoliths, are intended to be
included within the
scope of this invention. Monoliths such as rods contain polymer separation
media which
have been formed inside a column as a unitary structure having through pores
or
interstitial spaces which allow eluting solvent and analyte to pass through
and which
provide the non-polar separation surface.
In general, the only requirement for the separation media of the present
invention is
that they must have a surface that is either intrinsically non-polar or be
bonded with a
material that forms a surface having sufficient non-polarity to interact with
a counterion
agent.
In one aspect, the subject matter of the present invention is the separation
of
polynucleotides utilizing columns filled with nonporous polymeric beads having
an average
diameter of about 0.5 -100 microns; preferably, 1 - 10 microns; more
preferably, 1 - 5
microns. Beads having an average diameter of 1.0 - 3.0 microns are most
preferred.
In U.S. Patent No. 5,585,236, Bonn et al. had characterized the nucleic acid
separation process as reverse phase ion pairing chromatography (RPIPC).
However,
since RPIPC does not incorporate certain essential characteristics described
in the
present invention, another term, Matched Ion Polynucleotide Chromatography
(MIPC), has
been selected. MIPC as used herein, is defined as a process for separating
single and
double stranded polynucleotides using non-polar beads, wherein the process
uses a
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counterion agent, and an organic solvent to elute the nucleic acid from the
beads, and
wherein the beads are characterized as having a DNA Separation Factor of at
least 0.05.
In a preferred embodiment, the beads have a DNA Separation Factor of at least
0.5. In an
optimal embodiment, the beads have a DNA Separation Factor of at least 0.95.
The performance of the beads of the present invention is demonstrated by high
efficiency separation by MIPC of double stranded and single stranded DNA.
Applicants
have found that a useful criterion for measuring performance of the beads is a
DNA
Separation Factor. This is measured as the resolution of 257- and 267-base
pair double
stranded DNA fragments of a pUC18 DNA-Haelll restriction digest and is defined
as the
ratio of the distance from the valley between the peaks to the top of the
peaks, over the
distance from the baseline to the top of the peaks. Referring to the schematic
representation of FIG. 1, the DNA Separation Factor is determined by measuring
the
distance "a" from the baseline to the valley "e" between the peaks "b" and "c"
and the
distance "d" from the valley "e" to the top of one of the peaks "b" or "c". If
the peak heights
are unequal, the highest peak is used to obtain "d." The DNA Separation Factor
is the
ratio of d/(a+d). The peaks of 257- and 267-base pairs in this schematic
representation
are similar in height. In one embodiment, beads of the present invention have
a DNA
Separation Factor of at least 0.05. Preferred beads have a DNA Separation
Factor of at
least 0.5.
Without wishing to be bound by theory, Applicants believe that the beads which
conform to the DNA Separation Factor as specified herein have a pore size
which
essentially excludes the polynucleotides being separated from entering the
bead. As used
herein, the term "nonporous" is defined to denote a bead which has surface
pores having a
diameter that is less than the size and shape of the smallest DNA fragment in
the
separation in the solvent medium used therein. Included in this definition are
polymer
beads having these specified maximum size restrictions in their natural state
or which
have been treated to reduce their pore size to meet the maximum effective pore
size
required. Preferably, all beads which provide a DNA Separation Factor of at
least 0.5 are
intended to be included within the definition of "nonporous" beads.
The surface conformations of nonporous beads of the present invention can
include
depressions and shallow pit-like structures which do not intertere with the
separation
process. A pretreatment of a porous bead to render it nonporous can be
effected with any
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material which will fill the pores in the bead structure and which does not
significantly
interfere with the MIPC process.
Pores are open structures through which mobile phase and other materials can
enter the bead structure. Pores are often interconnected so that fluid
entering one pore
can exit from another pore. Applicants believe that pores having dimensions
that allow
movement of the polynucleotide into the interconnected pore structure and into
the bead
impair the resolution of separations or result in separations that have very
long retention
times. In MIPC, however, the beads are "nonporous" and the polynucleotides do
not enter
the bead structure.
The term polynucleotide is defined as a linear polymer containing an
indefinite
number of nucleotides, linked from one ribose (or deoxyribose) to another via
phosphoric
residues. The present invention can be used in the separation of RNA or of
double- or
single-stranded DNA. For purposes of simplifying the description of the
invention, and not
by way of limitation, the separation of double-stranded DNA will be described
in the
examples herein, it being understood that all polynucleotides are intended to
be included
within the scope of this invention.
Chromatographic efficiency of the column beads is predominantly influenced by
the
properties of surface and near-surface areas. For this reason, the following
descriptions
are related specifically to the close-to-the-surface region of the polymeric
beads. The
main body and/or the center of such beads can exhibit entirely different
chemistries and
sets of physical properties from those observed at or near the surface of the
polymeric
beads of the present invention.
In another embodiment of the present invention, the separation medium can be
in
fihe form of a polymeric monolith such as a rod-like monolithic column. The
monolithic
column is polymerized or formed as a single unit inside of a tube as described
in the
Examples hereinbelow. The through pore or interstitial spaces provide for the
passage of
eluting solvent and analyte materials. The separation is performed on the
stationary
surface. The surface can be porous, but is preferably nonporous. The form and
function
of the separations are identical to columns packed with beads. As with beads,
the pores
contained in the rod must be compatible with DNA and not trap the material.
Also, the rod
must not contain contamination that will trap DNA.
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The molded polymeric rod of the present invention is prepared by bulk free
radical
polymerization within the confines of a chromatographic column. The base
polymer of the
rod can be produced from a variety of polymerizable monomers. For example, the
monolithic rod can be made from polymers, including mono- and di-vinyl
substituted
aromatic compounds such as styrene, substituted styrenes, alpha-substituted
styrenes
and divinylbenzene; acrylates and methacrylates; polyolefins such as
polypropylene and
polyethylene; polyesters; polyurethanes; polyamides; polycarbonates; and
substituted
polymers including fluorosubstituted ethylenes commonly known under the
trademark
TEFLON. The base polymer can also be mixtures of polymers, non-limiting
examples of
which include poly(glycidyl methacrylate-co-ethylene dimethacrylate),
poly(styrene-
divinylbenzene) and poly(ethylvinylbenzene-divinylbenzene. The rod can be
unsubsituted
or substituted with a substituent such as a hydrocarbon alkyl or an aryl
group. The alkyl
group optionally has 1 to 1,000,000 carbons inclusive in a straight or
branched chain, and
includes straight chained, branch chained, cyclic, saturated, unsaturated
nonionic
functional groups of various types including aldehyde, ketone, ester, ether,
alkyl groups,
and the like, and the aryl groups includes as monocyclic, bicyclic, and
tricyclic aromatic
hydrocarbon groups including phenyl, naphthyl, and the like. In a preferred
embodiment,
the alkyl group has 1-24 carbons. In a more preferred embodiment, the alkyl
group has 1-
8 carbons. The substitution can also contain hydroxy, cyano, nitro groups, or
the like which
are considered to be non-polar, reverse phase functional groups. Methods for
hydrocarbon
substitution are conventional and well-known in the art and are not an aspect
of this
invention. The preparation of polymeric monoliths is by conventional methods
well known
in the art as described in the following references: Wang et al. (J.
Chromatog. A 699:230
(1994)), Petro et al. (Ana. Chem. 68:315 (1996)), and the following U.S.
Patent Nos.
5,334,310; 5,453,185; 5,522,994 (to Frechet). Monolith or rod columns are
commercially
available form Merck & Co (Darmstadfi, Germany).
The nonporous polymeric beads of the present invention are prepared by a two-
step process in which small seed beads are initially produced by emulsion
polymerization
of suitable polymerizable monomers. The emulsion polymerization procedure of
the
invention is a modification of the procedure of Goodwin, et al. (Colloid &
Polymer Sci.,
252:464-471 (1974)). Monomers which can be used in the emulsion polymerization
process to produce the seed beads include styrene, alkyl substituted styrenes,
alpha-
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methyl styrene, and alkyl substituted alpha-methyl styrene. The seed beads are
then
enlarged and, optionally, modified by substitution with various groups to
produce the
nonporous polymeric beads of the present invention.
The seed beads produced by emulsion polymerization can be enlarged by any
known process for increasing the size of the polymer beads. For example,
polymer beads
can be enlarged by the activated swelling process disclosed in U.S. Patent No.
4,563,510.
The enlarged or swollen polymer beads are further swollen with a crosslinking
polymerizable monomer and a polymerization initiator. Polymerization increases
the
crosslinking density of the enlarged polymeric bead and reduces the surface
porosity of
the bead. Suitable crosslinking monomers contain at least two carbon-carbon
double
bonds capable of polymerization in the presence of an initiator. Preferred
crosslinking
monomers are divinyl monomers, preferably alkyl and aryl (phenyl, naphthyl,
etc.) divinyl
monomers and include divinyl benzene, butadiene, etc. Activated swelling of
the
polymeric seed beads is useful to produce polymer beads having an average
diameter
ranging from 1 up to about 100 microns.
Alternatively, the polymer seed beads can be enlarged simply by heating the
seed
latex resulting from emulsion polymerization. This alternative eliminates the
need for
activated swelling of the seed beads with an activating solvent. Instead, the
seed latex is
mixed with the crosslinking monomer and polymerization initiator described
above,
together with or without a water-miscible solvent for the crosslinking
monomer. Suitable
solvents include acetone, tetrahydrofuran (THF), methanol, and dioxane. The
resulting
mixture is heated for about 1 - 12 hours, preferably about 4 - 8 hours, at a
temperature
below the initiation temperature of the polymerization initiator, generally,
about 10°C
80°C, preferably 30°C - 60°C. Optionally, the temperature
of the mixture can be increased
by 10 - 20% and the mixture heated for an additional 1 to 4 hours. The rafiio
of monomer
to polymerization initiator is at least 100:1, preferably about 100:1 to about
500:1, more
preferably about 200:1 in order to ensure a degree of polymerization of at
least 200.
Beads having this degree of polymerization are sufficiently pressure-stable to
be used in
high pressure liquid chromatography (HPLC) applications. This thermal swelling
process
allows one to increase the size of the bead by about 110 - 160% to obtain
polymer beads
having an average diameter up to about 5 microns, preferably about 2 - 3
microns. The
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thermal swelling procedure can, therefore, be used to produce smaller particle
sizes
previously accessible only by the activated swelling procedure.
Following thermal enlargement, excess crosslinking monomer is removed and the
particles are polymerized by exposure to ultraviolet light or heat.
Polymerization can be
conducted, for example, by heating of the enlarged particles to the activation
temperature
of the polymerization initiator and continuing polymerization until the
desired degree of
polymerization has been achieved. Continued heating and polymerization allows
one to
obtain beads having a degree of polymerization greater than 500.
In the present invention, the packing material disclosed by Bonn et al. or
U.S.
Patent No. 4,563,510 can be modified through substitution of the polymeric
beads with
alkyl groups or can be used in its unmodified state. For example, the polymer
beads can
be alkylated with 1 or 2 carbon atoms by contacting the beads with an
alkylating agent,
such as methyl iodide or ethyl iodide. Alkylation is achieved by mixing the
polymer beads
with the alkyl halide in the presence of a Friedel-Crafts catalyst to effect
electrophilic
aromatic substitution on the aromatic rings at the surface of the polymer
blend. Suitable
Friedel-Crafts catalysts are well-known in the art and include Lewis acids
such as
aluminum chloride, boron trifluoride, tin tetrachloride, etc. The beads can be
hydrocarbon
substituted by substituting the corresponding hydrocarbon halide for methyl
iodide in the
above procedure, for example.
The term alkyl as used herein in reference to the beads of the present
invention is
defined to include alkyl and alkyl substituted aryl groups, having from 1 to
1,000,000
carbons, the alkyl groups including straight chained, branch chained, cyclic,
saturated,
unsaturated nonionic functional groups of various types including aldehyde,
ketone, ester,
ether, alkyl groups, and the like, and the aryl groups including as
monocyclic, bicyclic, and
tricyclic aromatic hydrocarbon groups including phenyl, naphthyl, and the
like. Methods for
alkyl substitution are conventional and well-known in the art and are not an
aspect of this
invention. The substitution can also contain hydroxy, cyano, nitro groups, or
the like which
are considered to be non-polar, reverse phase functional groups.
The chromatographic material reported in the Bonn patent was limited to
nonporous
beads substituted with alkyl groups having at least 3 carbons because Bonn et
al. were
unsuccessful in obtaining separations using polymer beads lacking this
substitution.
Additionally, the polymer beads were limited to a small group of vinyl
aromatic monomers,
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and Bonn et al. were unable to effect double stranded DNA separations with
other
materials.
In the present invention, it has now been surprisingly discovered that
successful
separation of double stranded DNA can be achieved using underivatized
nonporous beads
as well as using beads derivatized with alkyl groups having 1 to 1,000,000
carbons.
The base polymer of the invention can also be other polymers, non-limiting
examples of which include mono- and di-vinyl substituted aromatics such as
styrene,
substituted styrenes, alpha-substituted styrenes and divinylbenzene; acrylates
and
methacrylates; polyolefins such as polypropylene and polyethylene; polyesters;
polyurethanes; polyamides; polycarbonates; and substituted polymers including
fluorosubstituted ethylenes commonly known under the trademark TEFLON. The
base
polymer can also be mixtures of polymers, non-limiting examples of which
include
polystyrene-divinylbenzene) and poly(ethylvinylbenzene-divinylbenzene).
Methods for
making beads from these polymers are conventional and well known in the art
(for
example, see U.S. Patent 4,906,378). The physical properties of the surface
and near-
surface areas of the beads are the predominant influence on chromatographic
efficiency.
The polymer, whether derivatized or not, must provide a nonporous, non-
reactive, and
non-polar surface for the MIPC separation.
In an important aspect of the present invention, the beads and other media of
the
invention are characterized by having low amounts of metal contaminants or
other
contaminants that can bind DNA. The preferred beads of the present invention
are
characterized by having been subjected to precautions during production,
including a
decontamination treatment, such as an acid wash treatment, designed to
substantially
eliminate any multivalent cation contaminants (e.g. Fe(III), Cr(III), or
colloidal metal
contaminants). Only very pure, non-metal containing materials should be used
in the
production of the beads in order that the resulting beads will have minimum
metal content.
In addition to the beads themselves being substantially metal-free,
Applicanfis have
also found that, to achieve optimum peak separation during MIPC, the
separation column
and all process solutions held within the column or flowing through the column
are
preferably substantially free of multivalent cation contaminants. As described
in commonly
owned U.S. Patent No. 5,772,889 to Gjerde (1998), and in co-pending U.S.
Patent
Applications No. 09/081,040 (filed May 78, 1998) and No. 09/080,547 (filed May
18, 1998)
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this can be achieved by supplying and feeding solutions that enter the
separation column
with components which have process solution-contacting surfaces made of
material which
does not release multivalent cations into the process solutions held within or
flowing
through the column, in order to protect the column from multivalent cation
contamination.
The process solution=contacting surfaces of the system components are
preferably
material selected from the group consisting of titanium, coated stainless
steel, passivated
stainless steel, and organic polymer.
There are two places where multivalent cation binding agents, e.g., chelators,
are
used in MIPC separations. In one embodiment, these binding agents can be
incorporated
into a solid through which the mobile phase passes. Contaminants are trapped
before
they reach places within the system that can harm the separation. In these
cases, the
functional group is attached to a solid matrix or resin (e.g., a flow-through
cartridge, usually
an organic polymer, but sometimes silica or other material). The capacity of
the matrix is
preferably about 2 mequiv./g. An example of a suitable chelating resin is
available under
the trademark CHELEX 100 (Dow Chemical Co.) containing an iminodiacetate
functional
group.
In another embodiment, the multivalent cation binding agent can be added to
the
mobile phase. The binding functional group is incorporated into an organic
chemical
structure. The preferred multivalent cation binding agent fulfills three
requirements. First,
it is soluble in the mobile phase. Second, the complex with the metal is
soluble in the
mobile phase. Multivalent cation binding agents such as EDTA fulfill this
requirement
because both the chelator and the multivalent cation binding agent-metal
complex contain
charges which make them both water-soluble. Also, neither precipitate when
acetonitrile,
for example, is added. The solubility in aqueous mobile phase can be enhanced
by
attaching covalently bound ionic functionality, such as, sulfate, carboxylate,
or hydroxy. A
preferred multivalent cation binding agent can be easily removed from the
column by
washing with water, organic solvent or mobile phase. Third, the binding agent
must not
interfere with the chromatographic process.
The multivalent cation binding agent can be a coordination compound. Examples
of
preferred coordination compounds include water soluble chelating agents and
crown
ethers. Non-limiting examples of multivalent cation binding agents which can
be used in
the present invention include acetylacetone, alizarin, aluminon, chloranilic
acid, kojic acid,
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morin, rhodizonic acid, thionalide, thiourea, a-furildioxime, nioxime,
salicylaldoxime,
dimethylglyoxime, a-furildioxime, cupferron, a-nitroso-~3-naphthol, nitroso-R-
salt,
diphenylthiocarbazone, diphenylcarbazone, eriochrome black T, PAN, SPADNS,
glyoxal-
bis(2-hydroxyanil), murexide, a-benzoinoxime, mandelic acid, anthranilic acid,
ethylenediamine, glycine, triaminotriethylamine, thionalide,
triethylenetetramine, EDTA,
metalphthalein, arsonic acids, a,a'-bipyridine, 4-hydroxybenzothiazole, 8-
hydroxyquinaldine, 8-hydroxyquinoline, 1,10-phenanthroline, picolinic acid,
quinaldic acid,
a,a',a"-terpyridyl, 9-methyl-2,3,7-trihydroxy-6-fluorone, pyrocatechol,
salicylic acid, tiron,
4-chloro-1,2-dimercaptobenzene, dithiol, mercaptobenzothiazole, rubeanic acid,
oxalic
acid, sodium diethyldithiocarbarbamate, and zinc dibenzyldithiocarbamate.
These and
other examples are described by Perrin in Organic Gomplexing Reagents:
Structure,
Behavior, and Application to Inorganic Analysis, Robert E. Krieger Publishing
Co. (1964).
In the present invention, a preferred multivalent cation binding agent is
EDTA.
To achieve high resolution chromatographic separations of polynucleotides, it
is
generally necessary to tightly pack the chromatographic column with the solid
phase
polymer beads. Any known method of packing the column with a column packing
material
can be used in the present invention to obtain adequate high resolution
separations.
Typically, a slurry of the polymer beads is prepared using a solvent having a
density equal
to or less than the density of the polymer beads. The column is then filled
with the
polymer bead slurry and vibrated or agitated to improve the packing density of
the polymer
beads in the column. Mechanical vibration or sonication are typically used to
improve
packing density.
For example, to pack a 50 x 4.6 mm I.D. column, 2.0 grams of beads can be
suspended in 10 mL of methanol with the aid of sonication. The suspension is
then
packed into the column using 50 mL of methanol at 8,000 psi of pressure. This
improves
the density of the packed bed.
The separation method of the invention is generally applicable to the
chromatographic separation of single stranded and double stranded
polynucleotides of
DNA and RNA. Samples containing mixtures of polynucleotides can result from
total
synthesis of polynucleotides, cleavage of DNA or RNA with restriction
endonucleases or
with other enzymes or chemicals, as well as nucleic acid samples which have
been
multiplied and amplified using polymerase chain reaction techniques.
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The method of the present invention can be used to separate double stranded
polynucleotides having up to about 1500 to 2000 base pairs. In many cases, the
method
is used to separate polynucleotides having up to 600 bases or base pairs, or
which have
up to 5 to 80 bases or base pairs. .
In a preferred embodiment, the separation is by Matched Ion Polynucleotide
Chromatography (MIPC). The nonporous beads of the invention are used as a
reverse
phase material that will function with counterion agents and a solvent
gradient to effect the
DNA separations. In MIPC, the polynucleotides are paired with a counterion and
then
subjected to reverse phase chromatography using the nonporous beads of the
present
invention.
There are several types of counterions suitable for use with MIPC. These
include a
mono-, di-, or trialkylamine that can be protonated to form a positive counter
charge or a
quaternary alkyl substituted amine that already contains a positive counter
charge. The
alkyl substitutions may be uniform (for example, triethylammonium acetate or
tetrapropylammonium acetate) or mixed (for example, propyldiethylammonium
acetate).
The size of the alkyl group may be small (methyl) or large (up to 30 carbons)
especially if
only one of the substituted alkyl groups is large and the others are small.
For example
octyldimethylammonium acetate is a suitable counterion agent. Preferred
counterion
agents are those containing alkyl groups from the ethyl, propyl or butyl size
range.
The purpose of the alkyl group is to impart a nonpolar character to the
polynucleic
acid through a matched ion process so that the polynucleic acid can interact
with the
nonpolar surface of the separation media. The requirements for the extent of
nonpolarity
of the counterion-DNA pair depends on the polarity of the separation media,
the solvent
conditions required for separation, the particular size and type of fragment
being
separated. For example, if the polarity of the separation media is increased,
then the
polarity of the counterion agent may have to change to match the polarity of
the surface
and increase interaction of the counterion-DNA pair. Triethylammonium acetate
is
preferred although quaternary ammonium reagents such as tetrapropyl or
tetrabutyl
ammonium salts can be used when extra nonpolar character is needed or desired.
In
general, as the polarity of the alkyl group is increased, size specific
separations, sequence
independent separations become more possible. Quaternary counterion reagents
are not
volatile, making collection of fragments more difficult.
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In some cases, it may be desired to increase the range of concentration of
organic
solvent used to perform the separation. For example, increasing the alkyl
length on the
counterion agent will increase the nonpolarity of the counterion-DNA pair
resulting in the
need to either increase the concentration of the mobile phase organic solvent,
or increase
the strength of the organic solvent type, e.g. acetonitrile is about two times
more effective
than methanol for eluting polynucleic acids. There is a positive correlation
between
concentration of the organic solvent required to elute a fragment from the
column and the
length of the fragment. However, at high organic solvent concentrations, the
polynucleotide could precipitate. To avoid precipitation, a strong organic
solvent or a
smaller counterion alkyl group can be used. The alkyl group on the counterion
reagent
can also be substituted with halides, nitro groups, or the like to moderate
polarity.
The mobile phase preferably contains a counterion agent. Typical counterion
agents include trialkylammonium salts of organic or inorganic acids, such as
lower alkyl
primary, secondary, and lower tertiary amines, lower trialkyammonium salts and
lower
quaternary alkyalmmonium salts. Lower alkyl refers to an alkyl radical of one
to six carbon
atoms, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl, isoamyl, n-
pentyl, and
isopentyl. Examples of counterion agents include octylammonium acetate,
octadimethylammonium acetate, decylammonium acetate, octadecylammonium
acetate,
pyridiniumammonium acetate, cyclohexylammonium acetate, diethylammonium
acetate,
propylethylammonium acetate, propyldiethylammonium acetate, butylethylammonium
acetate, methylhexylammonium acetate, tetramethylammonium acetate,
tetraethylammonium acetate, tetrapropylammonium acetate, tetrabutylammonium
acetate,
dimethydiethylammonium acetate, triethylammonium acetate, tripropylammonium
acetate,
tributylammonium acetate, tetrapropylammonium acetafie, and tetrabutylammonium
acetate. Although the anion in the above examples is acetate, other anions may
also be
used, including carbonate, phosphate, sulfate, nitrate, propionate, formate,
chloride, and
bromide, or any combination of cation and anion. These and other agents are
described
by Gjerde, et al. in Ion Chromatography, 2nd Ed., Dr. Alfred Huthig Verlag
Heidelberg
(1987). Counterion agents that are volatile are preferred for use in the
method of the
invention, with triethylammonium acetate (TEAR) and triethylammonium
hexafluoroisopropy) alcohol being most preferred.
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To achieve optimum peak resolution during the separation of DNA by MIPC using
the beads of the invention, the method is preferably performed at a
temperature within the
range of 20°C to 90°C; more preferably, 30°C to
80°C; most preferably, 50°C to 75°C.
The flow rate is selected to yield a back pressure not exceeding 5000 psi. In
general,
separation of single-stranded fragments should be performed at higher
temperatures.
Applicants have found that the temperature at which the separation is
performed
affects the choice of organic solvents used in the separation. One reason is
that the
solvents affect the temperature at which a double stranded DNA will melt to
form two
single strands or a partially melted complex of single and double stranded
DNA. Some
solvents can stabilize the melted structure better than other solvents. The
other reason a
solvent is important is because it affects the distribution of the DNA between
the mobile
phase and the stationary phase. Acetonitrile and 1-propanol are preferred
solvents in
these cases. Finally, the toxicity (and cost) of the solvent can be important.
In this case,
methanol is preferred over acetonitrile and 1-propanol is preferred over
methanol.
When the separation is performed at a temperature within the above range, an
organic solvent that is water soluble is preferably used, for example,
alcohols, nitrites,
dimethylformamide (DMF), tetrahydrofuran (THF), esters, and ethers. Water
soluble
solvents are defined as those which exist as a single phase with aqueous
systems under
all conditions of operation of the present invention. Solvents which are
particularly
preferred for use in the method of this invention include methanol, ethanol, 2-
propanol, 1-
propanol, tetrahydrofuran (THF), and acetonitrile, with acetonitrile being
most preferred
overall.
Mixtures of polynucleotides in general, and double stranded DNA in particular,
'are
effectively separated using Matched Ion Polynucleotide Chromatography (MIPC).
MIPC
separations of polynucleotides at non-denaturing temperature, typically less
than about
50°C, are based on base pair length. However, even traces of
multivalent cations
anywhere in the solvent flow path can cause a significant deterioration in the
resolution of
the separation after multiple uses of an MIPC column. This can result in
increased cost
caused by the need to purchase replacement columns and increased downtime.
Therefore, effective measures are preferably taken to prevent multivalent
metal
cation contamination of the separation system components, including separation
media
and mobile phase contacting. These measures include, but are not limited to,
washing
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protocols to remove traces of multivalent cations from the separation media
and
installation of guard cartridges containing cation capture resins, in line
between the mobile
phase reservoir and the MIPC column. These, and similar measures, taken to
prevent
system contamination with multivalent cations have resulted in extended column
life and
reduced analysis downtime.
Recently, MIPC has been successfully applied to the detection of mutations in
double stranded DNA by separating heteroduplexes from homoduplexes as
described in
co-pending U.S. Patent Application No. 09/129,105 filed August 4, 1998 which
is herein
incorporated by reference. Such separations depend on the lower temperature
required to
denature a heteroduplex at the site of base pair mismatch compared to a fully
complimentary homoduplex DNA fragment. MIPC, when performed at a temperature
which is sufficient to partially denature a heteroduplex is referred to herein
as Denaturing
Matched Ion Polynucleotide Chromatography (DMIPC). DMIPC is typically
performed at a
temperature between 52°C and 70°C. The optimum temperature for
performing DMIPC is
54°C to 59°C.
The previously described precautions taken to remove multivalent metal cations
were adequate for maintaining column life, as demonstrated by good separation
efficiency,
under non-denaturing conditions. However, Applicants have surprisingly found
that when
performed at partially denaturing temperature, conditions for effective DMIPC
separations
become more stringent. For example, a separation of a standard pUC18 Haelll
digest on
a MIPC column at 50° C provided a good separation of all the DNA
fragments in the
digest. However, a standard 209 by DYS271 mutation detection mixture of
homoduplexes
and heteroduplexes, prepared as described in Example 15, applied to the same
MIPC
column and eluted under DMIPC conditions, i.e., 56°C, afforded a poor
separation the
mixture components. In order to optimize column life and maintain effective
separation
performance of homoduplexes from heteroduplexes at partially denaturing
temperatures,
as is required for mutation detection, special column washing and storage
procedures are
used in the embodiments of the invention as described hereinbelow.
In one aspect of this invention, therefore, an aqueous solution of multivalent
cation
binding agent is flowed through the column to maintain separation efficiency.
In order to
maintain the separation efficiency of a MIPC column, the column is preferably
washed with
multivalent cation binding agent solution after about 500 uses or when the
performance
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starts to degrade. Examples of suitable cation binding agents are as described
hereinabove.
The concentration of a solution of the cation binding agent can be between
0.01 M
and 1 M. In a preferred embodiment, the column washing solution contains EDTA
at a
concentration of about 0.03 to 0.1 M.
In another embodiment, the solution contains an organic solvent selected from
the
group consisting of acetonitrile, ethanol, methanol, 2-propanol, and ethyl
acetate. A
preferred solution contains at least 2% organic solvent to prevent microbial
growth. In a
most preferred embodiment a solution containing 25% acetonitrile is used to
wash a MIPC
column. The multivalent cation binding solution can contain a counterion agent
as
described hereinabove.
In one embodiment of a column washing procedure, the MIPC separation column is
washed with the multivalent canon binding solution at an elevated temperature
in the
range of 50°C to 80°C. In a preferred embodiment the column is
washed with a solution
containing EDTA, TEAR, and acetonitrile, in the 70°C to 80°C
temperature range. In a
specific embodiment, the solution contains 0.032 M EDTA, 0.1 M TEAR, and 25%
acetonitrile.
Column washing can range from 30 seconds to one hour. For example, in a high
throughput DMIPC assay, the column can be washed for 30 seconds after each
sample,
followed by equilibration with mobile phase. Since DMIPC can be automated by
computer,
the column washing procedure can be incorporated into the mobile phase
selection
program without additional operator involvement. tn a preferred procedure, the
column is
washed with multivalent cation binding agent for 30 to 60 minutes at a flow
rate preferably
in the range of about 0.05 to 1.0 mL/min.
In one embodiment, a DMIPC column is tested with a standard mutation detection
mixture of homoduplexes and heteroduplexes after about 1000 sample analyses.
If the
separation of the standard mixture has deteriorated compared to a freshly
washed column,
then the column can be washed for 30 to 60 minutes with the multivalent cation
binding
solution at a temperature above about 50°C to restore separation
performance.
Applicants have found that other treatments for washing a column can also be
used
alone or in combination with those indicated hereinabove. These include: use
of high pH
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washing solutions (e.g., pH 10-12), use of denaturants such as urea or
formamide, and
reverse flushing the column with washing solution.
In another aspect, Applicants have discovered that column separation
efficiency
can be preserved by storing the column separation media in the column
containing a
solution of multivalent cation binding agent therein. The solution of binding
agent may also
contain a counterion agent. Any of the multivalent cation binding agents,
counterion
agents, and solvents described hereinabove are suitable for the purpose of
storing a MIPC
column. In a preferred embodiment, a column packed with MIPC separation media
is
stored in an organic solvent containing a multivalent cation binding agent and
a counterion
agent. An example of this preferred embodiment is 0.032 M EDTA and 0.1 M TEAR
in
25% aqueous acetonitrile. In preparation for storage, a solution of
multivalent cation
binding agent, as described above, is passed through the column for about 30
minutes.
The column is then disconnected from the HPLC apparatus and the column ends
are
capped with commercially available threaded end caps made of material which
does not
release multivalent cations. Such end caps can be made of coated stainless
steel,
titanium, organic polymer or any combination thereof.
The effectiveness of the surprising discovery made by Applicants, that washing
a
MIPC column with a multivalent cation binding agent restores the ability of
the column to
separate heteroduplexes and homoduplexes in mutation detection protocols under
DMIPC
conditions, is described in Example 14 and demonstrated in FIGs. 18, 19, and
20. As
described in Example 14, Applicants noticed a decrease in resolution of
homoduplexes
and heteroduplexes during the use of a MIPC column in mutation detection.
However, no
apparent degradation in resolution was observed when a DNA standard containing
pUC18
Haelll digest (Sigma/Aldrich Chemical Co.) was applied at 50°C (not
shown). In order to
further test the column performance, a mixture of homoduplexes and
heteroduplexes in a
209 by DNA standard was applied to the column under DMIPC conditions of
56°C (Kuklin
et al., Genetic Testing 1:201 (1998). It was surprisingly observed the peaks
representing
the homoduplexes and heteroduplexes of the mutation detection standard were
poorly
resolved (FIG. 18).
FIG. 19 shows some improvement in the separation of homoduplexes and
heteroduplexes of the standard mutation detection mixture when a guard
cartridge
containing cation capture resin was deployed in line between the solvent
reservoir and the
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MIPC system. The chromatography shown in FIG. 19 was performed at
56°C. The
column used in FIG. 19 was the same column used in the separation shown in
FIG. 18
and for separating the standard pUClB Haelll digest.
FIG. 20 shows the separation of homoduplexes and heteroduplexes of the
standard .
mutation detection mixture at 56°C on the same column used to generate
the
chromafiograms in FIGs. 18 and 19. However, in FIG. 20 the column was washed
for 45
minutes with a solution comprising 32 mM EDTA and 0.1 M TEAA in 25%
acetonitrile at
75°C prior to sample application. FIG. 20 shows four cleanly resolved
peaks representing
the two homoduplexes and the two heteroduplexes of the standard 209 by
mutation
detection mixture. This restoration of the separation ability, after washing
with a solution
containing a cation binding agent, of the MIPC column under DMIPC conditions
compared
to the chromatograms of FIGs. 18 and 19 clearly shows the effectiveness and
the utility of
the present invention.
In an important aspect of the present invention, Applicants have developed a
standardized criteria to evaluate the performance of a DMIPC separation media.
DM1PC
as used herein, is defined as a process for separating heteroduplexes and
homoduplexes
using a non-polar separation medium (e.g., beads or rod) in the column,
wherein the
process uses a counterion agent, and an organic solvent to desorb the nucleic
acid from
the medium, and wherein the medium is characterized as having a Mutation
Separation
Factor (MSF) of at least 0.1. In one embodiment, the medium has a Mutation
Separation
Factor of at least 0.2. In a preferred embodiment, the medium has a Mutation
Separation
Factor of at least 0.5. In an optimal embodiment, the medium has a Mutation
Separation
Factor of at least 1Ø
The performance of the column is demonstrated by high efficiency separation by
DMIPC of heteroduplexes and homoduplexes. Applicants have found that the best
criterion for measuring performance is a Mutation Separation Factor as
described in
Example 13. This is measured as the difference between the areas of the
resolved
heteroduplex and homoduplex peaks. A correcfiion factor may be applied to the
generated
areas underneath the peaks. The following aspects may affect the calculated
areas of the
peaks and reproducibility of the same: baseline drawn, peak normalization,
inconsistent
temperature control, inconsistent elution conditions, detector instability,
flow rate instability,
inconsistent PCR conditions, and standard and sample degradation. Some of
these
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aspects are discussed by Snyder, et al., in Introduction to Modern Liquid
Chromatography,
2"d Ed, John Wiley and Sons, pp. 542-574 (1979) which is incorporated by
reference
herein.
The Mutation Separation Factor (MSF) is determined by the following equation:
MSF = (area peak 2 - area peak 1)/area peak 1
where area peak 1 is the area of the peak measured after DMIPC analysis of
wild
type and area peak 2 is the total area of the peak or peaks measured after
DMIPC
analysis of a hybridized mixture containing a putative mutation, with the
hereinabove
correction factors taken into consideration, and where the peak heights have
been
normalized to the wild type peak height. Separation particles are packed in an
HPLC
column and tested for their ability to separate a standard hybridized mixture
containing a
wild type 100 by Lambda DNA fragment and the corresponding 100 by fragment
containing an A to C mutation at position 51.
High pressure pumps are used for pumping mobile phase in the systems described
in U.S. Patent No. 5,585,236 to Bonn and in U.S. Patent No. 5,772,889 to
Gjerde. It will
be appreciated that other methods are known for driving mobile phase through
separation
media and can be used in carrying out the separations of polynucleotides as
described in
the present invention. A non-limiting example of such an alternative method
includes
"capillary electrochromatography" (GEC) in which an electric field is applied
across
capillary columns packed with microparticles and the resulting electroosmotic
flow acts as
a pump for chromatography. Electroosmosis is the flow of liquid, in contact
with a solid
surface, under the influence of a tangentially applied electric field. The
technique
combines the advantages of the high efficiency obtained with capillary
electrophoretic
separations, such as capillary zone electrophoresis, and the general
applicability of HPLC.
CEC has the capability to drive the mobile phase through columns packed with
chromatographic particles, especially small particles, when using
electroosmotic flow.
High efficiencies can be obtained as a result of the plug-like flow profile.
In the use of CEC
in the present invention, solvent gradients are used and rapid separations can
be obtained
using high electric fields. The following references describing CEC are each
incorporated
in their entirety herein: Dadoo, et al, LC-GC 15:630 (1997); Jorgenson, et
al., J.
Chromatog. 218:209 (1981); Pretorius, et al., J. Chromatog. 99:23 (1974); and
the
following U.S. Patent Nos. to Dadoo 5,378,334 (1995), 5,342,492 (1994), and
5,310,463
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(1994). In the operation of this aspect of the present invention, the
capillaries are packed,
either electrokinetically or using a pump, with the separation beads described
in the
present specification. In another embodiment, a polymeric rod is prepared by
bulk free
radical polymerization within the confines of a capillary column. Capillaries
are preferably
formed from fused silica tubing or etched into a block. The packed capillary
(e.g., a 150-
pm i.d. with a 20-cm packed length and a window located immediately before the
outlet
frit) is fitted with frits at the inlet and outlet ends. An electric field,
e.g., 2800Vicm, is
applied. Detection can be by uv absorbance or by fluorescence. A gradient of
organic
solvent, e.g., acetonitrile, is applied in a mobile phase containing
counterion agent (e.g.
0.1 M TEAA). to elute the polynucleotides. The column temperature is
maintained by
conventional temperature control means. In the preferred embodiment, all of
the
precautions for minimizing trace metal contaminants as described hereinabove
are
employed in using CEC.
In a related method, mixtures of polynucleotides are separated on thin layer
chromatography (TLC) plates. In this method, the beads of the present
invention are
mixed with a binder and bound to a TLC plate by conventional methods
(Remington: The
Science and Practice ofPharmacy, 19th Edition, Gennaro ed., Mack Publishing
Co. (1995)
pp. 552-554). A fluorophore is optionally included in the mixture to
facilitate detection. The
sample is spotted on the plate and the sample is run isocratically under
capillary flow. In a
preferred embodiment, the sample is run under electroosmotic flow in a process
called
High-Speed TLC (HSTLC). In the case of HSTLC, the plate is first wetted with
solvent
(e.g., acetonitrile solution in the presence of counterion agent) and an
electric field (e.g.,
2000 Vlcm) is applied. Solvent accumulating at the top of the plate is removed
by suction.
Applicants have surprisingly discovered that ds DNA of selected ranges of base
pair length
are separable under isocratic conditions by MIPC using the beads of the
present invention
as described in Example 6. The isocratic solvent conditions for separating a
selected
range of DNA base pair length, as determined using MIPC, are used in the TLC
and
HSTLC methods.
Applicants have determined that the chromatographic separations of double
stranded DNA fragments exhibit unique Sorption Enthalpies (~HSOrp). Two
compounds (in
this case, DNA fragments of different size) can only be separated if they have
different
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partition coefficients (K). The Nernst partition coefficient is defined as the
concentration of
an analyte (A) in the stationary phase divided by its concentration in the
mobile phase:
K = .Lls
~A~m
The partition coefficient (K) and the retention factor (k) are related through
the following
equations:
K - nASV~, and k - nAs
n~A~mUs n~A~m
the quotient Vm/Vs is also called phase volume ratio (~). Therefore:
k = K c~
To calculate the sorption enthalpies, the following fundamental thermodynamic
equations are necessary:
In K = - ~Gsorp , In k = - ~Gso~ + In ~ and ~Gsorp = 01"Isorp - TOSsorp
RT RT
By transforming the last two equations, one obtains the Van't Hoff equation:
In k = - ~HSOrp '~' ~SSO~ + In ~
RT R
From a plot In k versus 9/T, the sorption enthalpy ~Hsorp can be obtained from
the slope of
the graph (if a straight line is obtained). dSsorp can be calculated if the
phase volume ratio
(~) is known.
The Sorption Enthalpy ~Hsorp is positive (~Hsorp > 0) showing the separation
is
endothermic using acetonitrile as the solvent (Figs. 3 and 4), and using
methanol as the
solvent, the Sorption Enthalpy dHsorp is negative (~Hsorp < 0), showing the
separation is
exothermic (FIG. 5).
The thermodynamic data (as shown in the Examples hereinbelow) reflect the
relative affinity of the DNA-counterion agent complex for the beads of the
invention and the
elution solvent. An endothermic plot indicates a preference of the DNA complex
for the
bead. An exothermic plot indicates a preference, of the DNA complex for the
solvent over
the bead. The plots shown herein are for alkylated and non-alkylated surfaces
as
described in the Examples. Mosfi liquid chromatographic separations show
exothermic
plots.
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Other features of the invention will become apparent in the course of the
following
descriptions of exemplary embodiments which are given for illustration of the
invention and
are not intended to be limiting thereof.
Procedures described in the past tense in the examples below have been carried
out in the laboratory. Procedures described in the present tense have not yet
been carried
out in the laboratory, and are constructively reduced to practice with the
filing of this
application.
EXAMPLE 1
Preparation of nonporous polystyrene-divinylbenzene) particles
Sodium chloride (0.236 g) was added to 354 mL of deionized water in a reactor
having a volume of 1.0 liter. The reactor was equipped with a mechanical
stirrer, reflux
condenser, and a gas introduction tube. The dissolution of the sodium chloride
was
carried out under inert atmosphere (argon), assisted by stirring (350 rpm),
and at an
elevated temperature (87°C). Freshly distilled styrene (33.7 g) and
0.2184 g of potassium
peroxodisulfate (KZS208) dissolved in 50 mL of deionized water were then
added.
Immediately after these additions, the gas introduction tube was pulled out of
the solution
and positioned above the liquid surface. The reaction mixture was subsequently
stirred for
6.5 hours at 87°C. After this, the contents of the reactor were cooled
down to ambient
temperature and diluted to a volume yielding a concentration of 54.6 g of
polymerized
styrene in 1000 mL volume of suspension resulting from the first step. The
amount of
polymerized styrene in 1000 mL was calculated to include the quantity of the
polymer still
sticking to the mechanical stirrer (approximately 5 - 10 g). The diameter of
the spherical
beads in the suspension was determined by light microscopy to be about 1.0
micron.
Beads resulting from the first step are still generally too small and too soft
(low
pressure stability) for use as chromatographic packings. The softness of these
beads is
caused by an insufficient degree of crosslinking. In a second step, the beads
are enlarged
and the degree of crosslinking is increased.
The protocol for the second step is based on the activated swelling method
described by Ugelstad et al. (Adv. Colloid Interface Sci., 13:101-140 (1980)).
In order to
initiate activated swelling, or the second synthetic step, the aqueous
suspension of
polystyrene seeds (200 ml) from the first step was mixed first with 60 mL of
acetone and
then with 60 mL of a 1-chlorododecane emulsion. To prepare the emulsion, 0.206
g of
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sodium dodecylsulfate, 49.5 mL of deionized water, and 10.5 mL of 1-
chlorododecane
were brought together and the resulting mixture was kept at 0°C for 4
hours and mixed by
sonication during the entire time period until a fine emulsion of < 0.3
microns was
obtained. The mixture of polystyrene seeds, acetone, and 1-chlorododecane
emulsion
was stirred for about 12 hours at room temperature, during which time the
swelling of the
beads occurred. Subsequently, the acetone was removed by a 30 minute
distillation at
80°C.
Following the removal of acetone, the swollen beads were further grown by the
addition of 310 g of a ethyldivinylbenzene and divinylbenzene (DVB) (1:1.71 )
mixture also
containing 2.5 g of dibenzoylperoxide as an initiator. The growing occurred
with stirring
and with occasional particle size measurements by means of light microscopy.
After completion of the swelling and growing stages, the reaction mixture was
transferred into a separation funnel. In an unstirred solution, the excess
amount of the
monomer separated from the layer containing the suspension of the polymeric
beads and
could thus be easily removed. The remaining suspension of beads was returned
to the
reactor and subjected to a stepwise increase in temperature (63°C for
about 7 hours, 73°C
for about 2 hours, and 83°C for about 12 hours), leading to further
increases in the degree
of polymerization (> 500). The pore size of beads prepared in this manner was
below the
detection limit of mercury porosimetry (< 30A).
After drying, the dried beads (10 g) from step two were washed four times with
100
mL of n-heptane, and then two times with each of the following: 100 mL of
diethylether,
100 mL of dioxane, and 100 mL of methanol. Finally, the beads were dried.
EXAMPLE 2
Acid VI/ash Treatment
The beads prepared in Example 1 were washed three times with tetrahydrofuran
and two times with methanol. Finally the beads were stirred in a mixture
containing 100
mL tetrahydrofuran and 100 mL concentrated hydrochloric acid for 12 hours.
After this acid
treatment, the polymer beads were washed with a tetrahydrofuran/water mixture
until
neutral (pH = 7). The beads were then dried at 40°C for 12 hours.
EXAMPLE 3
Standard Procedure for Testing the Performance of Separation Media
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Separation particles are packed in an HPLC column and tested for their ability
to
separate a standard DNA mixture. The standard mixture is a pUC18 DNA-Haelll
digest
(Sigma-Aldrich, D6293) which contains 11 fragments having 11, 18, 80, 102,
174, 257,
267, 298, 434, 458, and 587 base pairs, respectively. The standard is diluted
with water
and five pL, containing a total mass of DNA of 0.25 p,g, is injected.
Depending on the packing volume and packing polarity, the procedure requires
selection of the driving solvent concentration, pH, and temperature. The
separation
conditions are adjusted so that the retention time of the 257, 267 peaks is
about 6 to 10
minutes. Any one of the following solvents can be used: methanol, ethanol, 2-
propanol,
1-propanol, tetrahydrofuran (THF), or acetonitrile. A counterion agent is
selected from
trialkylamine acetate, trialkylamine carbonate, trialkylamine phosphate, or
any other type
of cation that can form a matched ion with the polynucleotide anion.
As an example of this procedure, FIG. 2 shows the high resolution of the
standard
DNA mixture using octadecyl modified, nonporous poly(ethylvinylbenzene-
divinylbenzene)
beads. The separation was conducted under the following conditions: Eluent A:
0.1 M
TEAR, pH 7.0; Eluent B: 0.1 M TEAR, 25% acetonitrile; Gradient:
Time (min)%A %B
0.0 65 35
3.0 45 55
10.0 35 65
13.0 35 65
14.0 0 100
15.5 0 100
16.5 65 35
The flow rate was 0.75 mL/min, detection UV at 260 nm, column temp.
50°C. The
pH was 7Ø
As another example of this procedure using the same separation conditions as
in
FIG. 2, FIG. 3 is a high resolution separation of the standard DNA mixture on
a column
containing nonporous 2.1 micron beads of underivatized polystyrene-
divinylbenzene).
EXAMPLE 4
Sorption Enthalpy Measurements
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Four fragments (174 bp, 257 bp, 267 bp, and 298 bp, found in 5 pL pUC18 DNA-
Haelll digest, 0.04 pg DNA/pL) were separated under isocratic conditions at
different
temperatures using octadecyl modified, nonporous polystyrene-divinylbenzene)
polymer
beads. The separation was carried out using a Transgenomic WAVET"" DNA
Fragment
Analysis System equipped with a DNASepT"" column (Transgenomic, Inc., San
Jose, CA)
under the following conditions: Mobile phase: 0.1 M triethylammonium acetate,
14.25%
lulu) acetonitrile at 0.75 mL/min, detection at 250 nm UV, temperatures at 35,
40, 45, 50,
55, and 60°C, respectively. A plot of In k versus 1/T shows that the
retention factor k is
increasing with increasing temperature (FIG. 4). This indicates that the
retention
mechanism is based on an endothermic process (~HS°rp > 0).
The same experiments on non-alkylated polystyrene-divinylbenzene) beads gave a
negative slope for a plot of In k versus 1/T, although the plot is slightly
curved (FIG. 5).
The same experiments performed on octadecyl modified, nonporous poly(styrene-
divinylbenzene) beads but with methanol replacing the acetonirile as solvent
gave a plot In
k versus 9/T showing the retention factor k is decreasing with increasing
temperature (FIG.
6). This indicates the retention mechanism is based on an exothermic process
(~Hs°rp < 0).
EXAMPLE 5
Separations with alkylated polystyrene-divinylbenzene) beads
Mobile phase components are chosen to match the desorption ability of the
elution
solvent in the mobile phase to the attraction properties of the bead to the
DNA-counterion
complex. As the polarity of the bead decreases, a stronger (more organic) or
higher
concentration of solvent will be required. Weaker organic solvents such as
methanol are
generally required at higher concentrations than stronger organic solvents
such as
acetonitrile.
FIG. 7 shows the high resolution separation of DNA restriction fragments using
octadecyl modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads.
The
experiment was conducted under the following conditions: Column: 50 x 4.6 mm
LD.;
mobile phase 0.1 M TEAA, pH 7.2; gradient: 33-55% acetonitrile in 3 min, 55-
66%
acetonitrile in 7 min, 65% acetonitrile for 2.5 min; 65-100% acetonitrile in 1
min; and 100-
35% acetonitrile in 1.5 min. The flow rate was 0.75 mL/min, detection UV at
260 nm,
column temp. 51°C. The sample was 5 pL (= 0.2 pg pUC18 DNA-Haelll
digest).
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Repeating the procedure of FIG. 7 replacing the acetonitrile with 50.0%
methanol in
0.1 M (TEAR) gave the separation shown in FIG. 8.
Repeating the procedure of FIG. 7 replacing the acetonitrile with 25.0%
ethanol in
0.1 M (TEAA) gave the separation shown in FIG. 9.
Repeating the procedure of FIG. 7 replacing the acetonitrile with 25% vodka
(Stolichnaya, 100 proof) in 0.1 M (TEAR) gave the separation shown in FIG. 10.
The separation shown in FIG. 11 was obtained using octadecyl modified,
nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 50
x 4.6 mm
I.D.; mobile phase 0.1 M tetraethylacetic acid (TEAR), pH 7.3; gradient: 12-
18% 0.1 M
TEAR and 25.0% 1-propanol (Eluent B) in 3 min, 18-22% B in 7 min, 22% B for
2.5 min;
22-100% B in 1 min; and 100-12% B in 1.5 min. The flow rate was 0.75 mL/min,
detection
UV at 260 nm, and column temp. 51°C. The sample was 5 p,L (= 0.2 wg
pUCl8 DNA-Haelll
digest).
The separation shown in FIG. 12 was obtained using octadecyl modified,
nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 50
x 4.6 mm
I.D.; mobile phase 0.1 M TEAR, pH 7.3; gradient: 15-18% 0.1 M TEAR and 25.0% 1-
propanol (Eluent B) in 2 min, 18-21 % B in 8 min, 21 % B for 2.5 min; 21-100%
B in 1 min;
and 100-15% B in 1.5 min. The flow rate was 0.75 mUmin, detection UV at 260
nm, and
column temp. 51°C. The sample was 5 wL (= 0.2 p,g pUCl8 DNA-Haelll
digest).
The separation shown in FIG. 13 was obtained using octadecyl modified,
nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 50
x 4.6 mm
I.D.; mobile phase 0.1 M TEAR, pH 7.3; gradient: 35-55% 0.1 M TEAR and 10.0% 2-
propanol (Eluent B) in 3 min, 55-65 % B in 10 min, 65% B for 2.5 min; 65-100%
B in 1 min;
and 100-35% B in 1.5 min. The flow rate was 0.75 mLlmin, detection UV at 260
nm, and
column temp. 51°C. The sample was 5 p,L (= 0.2 pg pUC18 DNA-Haelll
digest).
The separation shown in FIG. 14 was obtained using octadecyl modified,
nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 50
x 4.6 mm
I.D.; mobile phase 0.1 M TEA2HPOa, pH 7.3; gradient: 35-55% 0.1 M TEA2HP04 and
10.0% 2-propanol (Eluent B) in 3 min, 55-65% B in 7 min, 65% B for 2.5 min; 65-
100% B
in 1 min; and 100-65% B in 1.5 min. The flow rate was 0.75 mUmin, detection UV
at 260
nm, and column temp. 51°C. The sample was 5 ~,L (= 0.2 p,g pUC18 DNA-
Haelll digest).
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The separation shown in FIG. 15 was obtained using octadecyl modified,
nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 50
x 4.6 mm
LD.; mobile phase 0.1 M TEAR, pH 7.3; gradient: 6-9% 0.1 M TEAR and 25.0% THF
(Eluent B) in 3 min, 9-11 % B in 7 min, 11 % B for 2.5 min; 11-100% B in 1
min; and 100-
6% B in 1.5 min. The flow rate was 0.75 mL/min, detection UV at 260 nm, and
column
temp. 51°C. The sample was 5 ~,L (= 0.2 ~g pUCl8 DNA-Haelll digest).
EXAMPLE 6
Isocraticlgradient separation of ds DNA
The following is an isocratic/gradient separation of ds DNA using nonporous
polystyrene-divinylbenzene) beads. Isocratic separations have not been
performed in
DNA separations because of the large differences in the selectivity of
DNA/alkylammonium
ion pair for beads. However, by using a combination of gradient and isocratic
elution
conditions, the resolving power of a system can be enhanced for a particular
size range of
DNA. For example, the range of 250-300 base pairs can be targeted by using a
mobile
phase of 0.1 M TEAR, and 14.25% acetonitrile at 0.75 mL/min at 40°C on
50 x 4.6 mm
cross-linked polystyrene-divinylbenzene) column, 2.1 micron. 5 ~,L of pUC18
DNA-Haelll
digest (0.2 wg) was injected under isocratic conditions and 257, 267 and 298
base pairs
DNA eluted completely resolved as shown in FlG. 16. Then the column was
cleaned from
larger fragments with 0.1 M TEAA/25% acetonitrile at 9 minutes. In other
examples, there
might be an initial isocratic step (to condition the column), then a gradient
step (to remove
or target the first group of DNA at a particular size), then an isocratic step
(to separate the
target material of a different size range) and finally a gradient step to
clean the column.
EXAMPLE 7
Bromination of Remaining Double Bonds on the Surface of Poly(Styrene-
Divinylbenzene)
Polymer Beads
50.0 g of a polystyrene-divinylbenzene) polymer beads were suspended in 500 g
of
tetrachloromethane. The suspension was transferred into a 1000 mL glass
reactor (with
attached reflux condenser, separation funnel and overhead stirrer). The
mixture was kept
at 20°C. Bromine (100 mL) was added over a period of 20 minutes. After
addition was
completed, stirring continued for 60 minutes. The temperature was raised to
50°C to
complete the reaction (2 hours).
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The polymer beads were separated from the tetrachloromethane and excess
bromine by means of centrifugation and cleaned with tetrahydrofuran (once with
100 mL)
and methanol (twice with 100 mL). The polymer beads were dried at 40°C.
The polymer beads are packed into a 50 x 4.6 mm ID column and the DNA
Separation Factor is greater than 0.05 as tested by the procedure of Example
3.
EXAMPLE 8
Nitration of a Poly(Styrene-Divinylbenzene~ Polymer Beads
In a 1000 mL glass reactor 150 mL of concentrated nitric acid (65%) were
combined
with 100 mL concentrated sulfuric acid. The acid mixture was cooled to 0-
4°C. When the
temperature had dropped to <4°C, 50 g of polystyrene-divinylbenzene)
polymer beads
were added slowly under continuous stirring. After addition was completed, 50
mL of nitric
acid (65%) was added. The suspension was stirred for three hours, maintaining
a
temperature of 5-10°C.
On the next day the reaction was quenched by adding ice to the suspension. The
polymer beads were separated from the acid by means of centrifugation. The
polymer
beads were washed to neutrality with water, followed by washing steps with
tetrahydrofurane (four times with 100 mL) and methanol (four times with 100
mL). The
polymer beads were dried at 40°C.
The polymer beads are packed into a 50 x 4.6 mm ID column and the DNA
Separation Factor is greater than 0.05 as tested by the procedure of Example
3.
EXAMPLE 9
Preparation of a Non-Polar Organic Polymer Monolith Chromatography Column
A chromatography tube in which the monolith polymeric separation medium is
prepared is made of stainless steel. The monomers, styrene (Sigma - Aldrich
Chemical
Corp.) and divinylbenzene (Dow Chemical Corp.) are dried over magnesium
sulfate and
distilled under vacuum.
To a solution of a 1:1 mixture by volume of the distilled styrene and
divinylbenzene,
containing 1 % by weight (with respect to monomers) of azobisisobutyronitrile
(AIBN), is
added eight volumes of a solution of the porogenic solvent, dodecyl alcohol
and toluene
(70:30). The solution so prepared is bubbled with nitrogen for 15 minutes and
is used to
fill a chromatography tube (50 x 8 mm I.D.) sealed with a rubber nut plug at
the bottom
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end. The tube is then sealed at the top end with a rubber nut plug and the
contents are
allowed to polymerize at 70° C for 24 hours.
Following polymerization, the rubber plugs are replaced by column end fittings
and
the column is connected to an HPLC system. The HPLC instrument has a low-
pressure
mixing quaternary gradient capability. A cartridge or guard column containing
an
iminodiacetate multivalent cation capture resin is placed in line between the
column and
the mobile phase source reservoir. The column is then washed by flowing 100 mL
of
tetrahydrofuran (THF) at 1 mL/min through the column to remove the dodecyl
alcohol and
toluene, thereby creating through-pores in the otherwise solid polymer
monolith.
In this example, all of the flow paths are either titanium, sapphire, ceramic,
or
PEEK, except for the tube body, which is 316 stainless steel. The interior of
the 316
stainless steel tube is passivated with dilute nitric acid prior to use.
EXAMPLE 10
Acid VI/ash Treatmenf To Remove Multivalent Metal Cation Contaminants
The non-polar, organic polymer monolith column is washed by flowing
tetrahydrofuran through the column at a flow rate of 2 mL per minute for 10
minutes
followed by flowing methanol through the column at 2 mL per minute for 10
minutes. The
non-polar, organic polymer monolith column is washed further by flowing a
mixture
containing 100 mL of tetrahydrofuran and 100 mL of concentrated hydrochloric
acid
through the column at 10 mL per minute for 20 minutes. Following this acid
treatment, the
non-polar, organic polymer monolith column is washed by flowing
tetrahydrofuran/water
(1:1) through the column at 2 mL per minute until neutral (pH 7).
EXAMPLE 11
Bromination of Remaining Double Bonds on the Surface of Non-Polar Organic
Polymer
Monolith Column
Any double bonds remaining on the surface of the monolith column prepared in
Example 9 are reacted with bromine as described in Example 7.
EXAMPLE 12
Nitration of a Non-Polar Organic Polymer Monolith Column
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The non-polar organic polymer column prepared in Example 9 is nitrated as
described in Example 8.
EXAMPLE 13
Determination of the Mutation Separation Factor
The Mutation Separation Factor (MSF) is determined by the following equation:
MSF = (area peak 2 - area peak 1 )/area peak 1
where area peak 1 is the area of the peak measured after DMIPC analysis of
wild
type and area peak 2 is the total area of the peak or peaks measured after
DMIPC
analysis of a hybridized mixture containing a putative mutation, with the
hereinabove
correction factors taken into consideration, and where the peak heights have
been
normalized to the wild type peak height. Separation particles are packed in an
HPLC
column and tested for their ability to separate a standard hybridized mixture
containing a
wild type 100 by Lambda DNA fragment and the corresponding 100 by fragment
containing an A to C mutation at position 51.
Depending on the packing volume and packing polarity, the procedure requires
selection of the driving solvent concentration, pH, and temperature. Any one
of the
following solvents can be used: acetonitrile, tetrahydrofuran, methanol,
ethanol, or
propanol. Any one of the following counterion agents can be used:
trialkylamine acetate,
trialkylamine carbonate, and trialkylamine phosphate.
As an example of the determination of the Mutation Separation Factor, FIG. 22
shows the resolution of the separation of the hybridized DNA mixture.
The PCR conditions used with each of the primers are described in the table
below.
All the components were combined and vortexed to ensure good mixing, and
centrifuged.
Aliquots were then distributed into PCR tubes as shown in the following table:
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COMPONENT VOLUME
Pfu 10X Buffer (Cat. 5 ~L
No.
600153-82, Stratagene,
Inc.,
La Jolla, CA)
100 pM dNTP Mix 4 wL
Primer 1 7.5 ~,L
(forward)
Primer 2 8.5 wL
(reverse)
Hz0 19.5 pL
Lambda DNA Template 5 pL
PFUTurbo""'
0.5 p,L
(600250, Stratagene)
The PCR tubes were placed into a thermocycler (PTC-100 Programmable Thermal
Controller from MJ Research, Inc., Watertown, Mass.) and the temperature
cycling
program was initiated. The cycling program parameters are shown in the table
below:
STEP TEMPERATURE TIME
1 94C 2 minutes
2 94C 1 minute
3 58C 1 minute
4 72C 1 minute
5 Go to Step
2, 34X
6 72C 10 minutes
7 End
The DMIPC conditions used for the mutation detection separations are shown
below:
Eluent A: 0.1 M TEAR; Eluent B: 0.1 M TEAR, 25% Acetonitrile; Flow rate: 0.90
mL/min; Gradient:
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Time (min) !A !B
0.0 50.0 50.0
0.1 45.0 55.0
4.6 36.0 64.0
4.7 0.0 100.0
5.2 0.0 100.0
5.3 50.0 50.0
7.8 50.0 50.0
The Lambda sequence has been published by O'Conner et al. in Biophys. J.
74:A285 (1998) and by Garner, et al., at the Mutation Detection 97 4th
International
Workshop, Human Genome Organization, May 29-June 2, 1997, Brno, Czech
Republic,
Poster no. 29. The 100 by Lambda fragment sequence (base positions 32011 -
32110)
was used as a standard (available from FMC Corp. available from FMC Corp.
BioProducts, Rockland, Maine). The mutation was at position 32061. The chart
below lists
the primers used:
Primers
Forward Primer
5'-GGATAATGTCCGGTGTCATG-3'
Reverse Primer:
3'-GGACACAGTCAAGACTGCTA-5'
Figure 21 is a chromatogram of the wild type strand analyzed under the above
conditions. The peak appearing has a retention time of 4.78 minutes and an
area of
98621.
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Figure 22 is the Lambda mutation analyzed in identical conditions as Figure 21
above. Two peaks are apparent in this chromatogram, with retention times of
4.32 and
4.68 minutes and a total area of 151246.
The Mutation Separation Factor is calculated by applying these various peak
areas
to the above MSF equation. Thus, using the definition stated hereinabove, MSF
= (area
peak 2 - area peak 1)/area peak 1, the MSF would be (151246 - 98621)/98621, or
0.533.
EXAMPLE 14
Effect of multivalent cation decontamination measures on sample resolution by
DMIPC
The separation shown in FIG. 18 was obtained using a WAVET"" DNA Fragment
Analysis System (Transgenomic, Inc., San Jose, CA) under the following
conditions:
Column: 50 x 4.6 mm ID containing alkylated polystyrene-divinylbenzene) beads
(DNASep~, Transgenomic, Inc.); mobile phase 0.1 M TEAR (1 M concentrate
available
from Transgenomic, Inc.) (Eluent A), pH 7.3; gradient: 50-53% 0.1 M TEAR and
25.0%
acetonitrile (Eluent B) in 0.5 min; 53-60% B in 7 min; 60-100% B in 1.5 min;
100-50% B in
1 min; 50% B for 2 min. The flow rate was 0.9 mL/min, UV detection was at 254
nm, and
the column temperature was 56°C. The sample was 2 p.L (=0.2 wg DNA,
DYS271 209 by
mutation standard with an A to G mutation at position 168).
FIG. 19 is the same separation as performed in FIG. 18, but after changing the
guard cartridge (20 x 4.0 mm, chelating cartridge, part no. 530012 from
Transgenomic,
)nc.) and replacing the pump-valve filter (Part no. 638-1423, Transgenomic,
Inc.). The
guard cartridge had dimensions of 10 x 3.2 mm, containing iminodiacetate
chelating resin
of 2.5 mequiv/g capacity and 10 ~,m particle size, and was positioned directly
in front of the
injection valve.
FIG. 20 is the same separation as performed in FIG. 19, but after flushing the
column for 45 minutes with 0.1 M TEAR, 25% acetonitrile, and 32 mM EDTA, at
75°C.
EXAMPLE 15
Hybridization of mutant and wild type DNA fragments
A mixture of two homoduplexes and two heteroduplexes was produced by a
hybridization process. In this process, a DYS271 209 by mutation standard
containing a
mixture of the homozygous mutant DNA fragment (with an A to G mutation at
position 168)
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combined with the corresponding wild type fragment in an approximately 1:1
ratio (the
mixture is available as a Mutation Standard from Transgenomic, Inc., San Jose,
CA; the
mutation is described by Seielstad et al., Human MoL Genet. 3:2159 (1994)) was
heated
at 95°C for 3 - 5 minutes then cooled to 25°C over 45 minutes.
The hybridization process
is shown schematically in FIG. 17.
EXAMPLE 16
Alkylation of Poly(Styrene-Divinylbenzene) Polymer Beads
The following procedures were carried out under nitrogen (Air Products, Ultra
Pure
grade, Allentown, PA) at a flow rate of 250-300 mLlmin. 25 g of the beads
prepared in
Example 1 were suspended in 150-160 g of 1-chlorooctadecane (product no. 0235,
TCI
America, Portland, OR) using a bow shaped mixer (use a 250 mL wide neck
Erlenmeyer
flask). The temperature was set to 50-60°C to prevent the 1-
chlorooctadecane from
solidifying. Larger pieces of polymer were broken up to facilitate suspending.
The solution
was mixed using a stirrer (Model RZRI, Caframo, ONT NOH2T0, Canada) with the
speed
set at 2. The polymer suspension was transferred into a three neck bottle
(with reflux
condenser, overhead stirrer and gas inlet). 52-62 g of 1-chlorooctadecane were
used to
rinse the Erlenmeyer flask and were added to the three neck bottle. The bottle
was heated
in an ethylene glycol bath set at 80°C. The solution was mixed using a
stirrer (Caframo)
with the speed set at 0. After 20 minutes, the reaction was started by
addition of 1.1 g
AICI3 powder (product no. 06218, Fluka, Milwaukee, WI) and continued for 16-18
h.
After the reaction, the polymer was separated from excess 1-chlorooctadecane
by
centrifugation followed by consecutive washing steps:
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Comment
. HCI, 50-60 mL n-heptane4 repetitions, with recycled
heptane
50-60 mL n-heptane 1 repetition, with fresh
heptane
. HCI, 50-60 mL n-heptane1 repetition, with fresh
heptane
50-60 mL n-heptane 1 repetition, fresh heptane
O, no n-heptane 3 repetitions, use plastic
stirrer to break up
chuncks of polymer beads.
Repeat steps 4
and 5 three times. Shake
for two minutes
with no centrifugation.
F 3 repetitions
F/ n-heptane 1 repetition
eptane 1 repetition
F 1 repetition
30H 4 repetitions
ps where aqueous solvents (NCI or H20) were used, the polymer was shaken for
ds with the aqueous phase before adding n-heptane. n-Heptane was then added
Fixture was shaken vigorously for 2 min.
:er the final polymeric beads were dried at 40-50°C for 2-3 hr it was
ready for
EXAMPLE 17
Column packing procedure
r weighing out 1.2 grams of oven dried polymeric beads, form a slurry with 10
lrofuran (THF) and place in a sonicator under a fume hood for 15 min. The add
-IF and 5 mL of methanol (MeOH) and sonicate an additional 10 min. Pre-fill a
~sembly with 20 mL MeOH. Pour the slurry slowly into the packing assembly.
Haskel pump (Haskel International, Inc., Burbank, CA) and slowly increase
ressure to 5000 psi for the initial packing phase. After 10 min, slowly
increase
ressure to 9000 psi and set the secondary packing phase for 20 min. After 20
ge the packing eluent from MeOH to 0.05 M Na4EDTA. The set the final packing
40 min.
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EXAMPLE 18
Preparation of Monolithic Capillary Columns
Fused silica capillary tubing (360~.m OD x 250~m ID, Polymicro Technologies,
Phoenix, AZ) was flushed successively with 0.1 M and 1 M NaOH, water (Milli Q
System,
Millipore, Bedford, MA) and methanol (Anhydrous, EM Science, Gibbstown, NJ) in
order to
deprotonize the surface silanol groups. The capillary tubing was dried by
passing N2
through it.
The capillary tubing was cut in 1 m sections prior to the following surface
treatment:
The capillaries sections were filled with a 50% (v/v) solution of 3-
(trimethoxysilyl)propyl
acrylate (Sigma-Aldrich, St. Louis, MO) and 3-(trimethoxysilyl)propyl
methacrylate (Sigma-
Aldrich), respectively in dimethylformamide (EM Science). This solution
contained also
0.01 % (w/v) 2,2 diphenylpicryl hydrazyl radical (Sigma-Aldrich) in order to
inhibit
polymerization of the acrylate and methacrylate groups, respectively. After
degassing the
solution with He for 20 minutes fihe capillaries were filled with the solution
and put into an
oven at 90°C for about 12 hours. In order to prevent migration of the
solution inside the
tubing during the treatment, one end of each capillary tube was immersed in a
1mL
reservoir of the solution retained in an Eppendorf centrifugation tube. The
open end of the
tube was sealed to the capillary using glue (Super Strength Adhesive, 3M). The
treated
capillary tubing was extensively flushed with dimethylformamide (Omnisolve for
HPLC, EM
Science) and anhydrous methanol (EM Science) and blown dry by passing N2
through the
capillary.
To form a monolithic capillary column, each dry capillary tube (1 m) was
gravity filled
with monomer-porogen-initiator mixture. The following three different recipes
for the
mixture were used in which all chemicals were used without further
purification:
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Capillary monolith C-1 included the following components: 500wL divinylbenzene
(80%) (Sigma-Aldrich); 500FL styrene (98%), (Sigma-Aldrich); 1300pL 1-decanol
(99%),
(Alfa Aesar, Ward Hill. MA); 200~L tetrahydrofuran (99%), (Omnisolve for HPLC,
EM
Science); and 25mg 2,2' azobisisobutyronitrile (Alfa Aesar, Ward Hill. MA).
Capillary monolith C-2 included the following components: 250wL divinylbenzene
(80%); 750wL styrene (98%); 1300wL 1-decanol (98%); 200wL tetrahydrofuran
(99%); and
25mg 2,2' azobisisobutyronitrile.
Capillary monolith C-3 included the following components: 500~L divinylbenzene
(80%); 500p.L styrene (98%); 2600pL 1-decanol (98%); 200p.L tetrahydrofuran
(99%); and
25mg 2,2' azobisisobutyronitrile.
During polymerization, one end of each tube was immersed in a reservoir of
monomer-porogen-initiator mixture retained in a tube as described above. The
mixture
within the tube was polymerized for 24h at 75°C for C-1, 18 h at
80°C for C-2, and 18 h at
90°C for C-3.
In order to remove unreacted monomers, oligomers and the porogen after
polymerization, each monolith was flushed with tetrahydrofuran (Omnisolve, EM
Science)
and methanol (Anhydrous, EM Science). The pump was set to constant pressure at
350
bar. The flow rate was estimated to be 3-6wLlmin. The temperature was
90°C. Flushing
with THF and methanol took approximately 24 hours per 50cm of monolithic
capillary.
EXAMPLE 19
Separation of DNA using a Polystyreneldivinylbenxene Monolithic Capillary
Column
A monolithic capillary column (250 ~,M ID x 145 mm length), prepared as
described
for the C-1 monolith, was used in this example. Chromatography was performed
using an
HPLC system configured with a Dionex GP50 pump (Dionex Corp., Sunnyvale, CA),
a
Hitachi L7200 autosampler (Hitachi Ltd., Tokyo, Japan) fitted with a 100 ~.L
sample loop, a
Hitachi L7300 column oven, a Valco (Valco Instrument Co., Houston, TX)
stainless steel
tee with 10-32 fittings and a Spectra- Physics Model 100 variable wavelength
absorbance
detector fitted with a capillary flow cell adapter. A 100 wm x 70 mm polyimide
coated fused
silica capillary (Polymicro Technologies, Phoenix, AZ) was used for detection
by thermal
removal (burning ofd the polyimide coating to create an optical detection
window.
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Data was acquired using Dionex PeakNet Chromatography workstation with a
Dionex U120 universal interface for digitizing the analog signal from the
absorbance
detector.
The system was configured with an eluent preheat tubing which consisted of 1.5
m
of 0.010 ID x 0.062 OD PEEK tubing (Upchurch Scientific, Oak Habor, WA). The
pre-heat
tubing vuas place in the oven and the oven temperature set to 80°C. In
order to minimize
the distance from the outlet of the monolithic separation capillary to the
detector, the
separation capillary was placed outside the oven. The preheat tubing and
separation
capillary were connected to the stainless steel mixing tee. Connected to the
third port of
the mixing tee was 2 m of 0.010 ID x 0.062 OD PEEK tubing (Upchurch
Scientific).
Connected at the waste end of the tee was a 10-32 PEEK coupler and a 10-32
PEEK plug.
The plug was tightened into the coupler in order to create sufficient
backpressure to cause
flow through the high pressure separation capillary. At approximately 2500
psi, the flow
through the separation capillary and into the detection capillary was about 3
pL/min.
Under these conditions, the majority of the flow (497 p,L/min) passed through
the waste
line port of the mixing tee.
Eluents were prepared using reagent or HPLC grade chemicals and deionized
water. Fluent A consisted of 100 mM triethylammonium acetate (TEAR,
Transgenomic,
Inc., San Jose, CA) and 1 mM tetrasodium ethylenediamine tetraacetic acid
(EDTA).
Fluent B consisted of 100 mM triethylammonium acetate, 1 mM EDTA and 25% (v/v)
acetonitrile.
A sample comprising a 20 mer oligonucleotide (obtained from Operon
Technologies
as described below) was injected onto the system and eluted using the
following gradient:
Time (min) %B
0.0 30
5.0 70
7.0 90
8.1 90
8.2 30
The injection volume was 0.5 p.L (split from 100 p.L). The pressure was 2450
psi.
The temperature of the mixing tee was 47°C. The detection was by UV at
254nm. The
flow rate was 3 ~,L/min (split from 500 wL/min). The 20 mer oligonucleotide,
having a
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sequence of: 5'-CGA CCT CCC TTT ATC CTC CAC AGA TCT CA-3', was obtained from
Operon Technologies (Alameda, CA) as "unpurified" grade and was diluted in TE
buffer
(lOmM Tris-HCI, pH 8.0, 1 mM EDTA) to 100 p,M prior to injection.
Using the system described above, initial injections of the 20 mer single
stranded
synthetic oligonucletotide sample showed no detector response (FIG. 23). Metal
contamination was suspected, possibly from the stainless steel injection
valve, sample
loop or mixing tee. Ten consecutive injections (30 seconds apart) of 100
p,L.of 0.2 M
tetrasodium ethylenediamine tetraacetic acid (EDTA) were performed. Injection
of the 20
mer oligonucleotide still did not reveal a detector response. For additional
cleaning, 1 mM
. EDTA was added to each of the eluents. The system was allowed to run
overnight with
100% eluent B. The 20 mer oligonucleotide was again injected, and eluted as
the peak
labeled "A" as shown in FIG. 24.
In another injection, after the overnight EDTA cleaning, a sample containing a
mixture of single stranded and double stranded DNA was injected onto the
system and
eluted using the following gradient:
Time (min)%B
0.0 30
5.0 70
7.0 90
8.1 90
8.2 30
The injection volume was 0.36 wL (split from 60 p.L). The pressure was 2450
psi.
The temperature of the mixing tee was 47°C. The detection was by UV at
254nm. FIG. 25
was obtained after injection of a mixture containing the 20 mer (12~M) (Operon
Technologies) and a Bio-Rad DNA standard (double stranded DNA ruler, catalogue
no.
170-8203). The concentration of the sdDNA in the injected mixture was 4 nM
based on a
5000 by average length. Peak A corresponds to the 20 mer oligonucleotide and
peak B
corresponds to the dsDNA standard. Under these conditions the 20 mer
oligonucleotide
eluted at 2.3 minutes and the dsDNA standard eluted as a broad
undifferentiated peak at
6.8 minutes.
EXAMPLE 20
Preparation of a Standard Bore Monolithc Separation Column
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A 4.6 mm !D x 50.0 mm length stainless steel column was packed with macro
porous resin beads (27% polystyrene cross-linked with divinylbenzene;
catalogue no.
POL-99-0319, Transgenomic) in methanol (Omnisolve for HPLC, EM Science) at
3000psi
for 20 min. The stainless steel column jacket, end fitting assembly, and
titanium frits were
obtained from Isolation Technologies, Inc., (Hopedale, MA). Ten mL of a
monomer
mixture, as described below, was pumped through the column at a rate of 0.2
mL/min.
The column was sealed with end-plugs and heated at 90°C for 18 h.
The monomer mixture comprised the following components: 2000pL divinylbenzene
(80%) (Sigma-Aldrich, St. Louis, MO); 3000wL styrene (98%), (Sigma-Aldrich);
6500wL 1-
decanol (99%), (Alfa Aesar, Ward Hill. MA); 1000p,L tetrahydrofuran (99%),
(Omnisolve for
HPLC, EM Science); and 120mg 2,2' azobisisobutyronitrile (Alfa Aesar).
In order to remove unreacted monomers, oligomers and the porogen the
capillaries
were flushed for 12 h with tetrahydrofuran (Omnisolve, EM Science) and
methanol
(Anhydrous, EM Science). The pumps were set to constant pressure at 4000 psi.
The flow
rate was 100-250~,L/min at 90°C.
EXAMPLE 21
Separation of DNA using a Polystyreneldivinylbenxene Monolithic Capillary
Column
The column described in Example 20 was used to elute a double stranded DNA
standard. The sample contained a 209 by standard (concentration 0.0025 pg
DNA/~L,
catalogue no. 560077, Transgenomic). The injection volume was 30 p.L. The
mobile
phase (pH 7) included eluent A: 100 mM TEAA in water; and eluent B: 100 mM
TEAA with
25% acetonitrile. The following gradient was used:
Time (min)%B
0 30
3 70
45 90
46 30
The flow rate was 0.2mUmin and the detection was by UV at 254nm. A single peak
at
14.4 min was observed (FIG. 26).
While the foregoing has presented specific embodiments of the present
invention, it
is to be understood that these embodiments have been presented by way of
example only.
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CA 02395101 2002-06-19
WO 01/83072 PCT/US00/11795
It is expected that others will perceive and practice variations which, though
differing from
the foregoing, do not depart from the spirit and scope of the invention as
described and
claimed herein.
49
SUBSTITUTE SHEET (RULE 26)
