Polynucleotide separations on polymeric separation media

Non-polar polymeric separation media, such as beads or monoliths, are suitable for chromatographic separation of mixtures of polynucleotides when the surfaces of the media are unsubstituted or substituted with a hydrocarbon group having from one to 1,000,000 carbons and when the surfaces are substantially free from mutivalent cation contamination. The polymeric media provide efficient separation of polynucleotides using Matched Ion Polynucleotide Chromatography. Methods for maintaining and storing the polymeric media include treatment with multivalent cation binding agents.

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. Pat. 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 for 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. 
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 p resent 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 uses a mobile phase containing a counterion agent and a 
water-soluble organic solvent. Examples of a suitable organic solvent 
include alcohol, nitrile, 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, etramethylammonium acetate, 
tetrapropylammonium acetate, etrabutylammonium acetate, 
dimethydiethylammon ium 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 polyethylene; 
polyesters; polyurethanes; polyamides; olycarbonates; and substituted 
polymers including fluorosubstituted thylenes commonly known under the 
trademark TEFLON. The base polymer can also be mixtures of polymers, 
non-limiting examples of which include poly(styrene-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. In 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 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, 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 multivalent cation capture resin before the solutions 
enter the column to protect the separation medium from multivalent cation 
contamination. The multivalent capture resin is preferably cation exchange 
resin and/or 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.degree. C. to 90.degree. C. The flow 
rate of mobile phase preferably is adjusted to yield a back-pressure not 
greater than 5000 psi. The method referably employs an organic solvent 
that is water soluble. The method also referably 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 cation 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.sub.1-6 alkyl vinylbenzene and divinylbenzene. The bead can 
contain functional groups such as polyvinyl alcohol, hydroxy, nitro, 
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, he bead is octadecyl modified 
poly(ethylvinylbenzene-divinylbenzene) or poly(styrene-divinylbenzene). 
The bead can also contain crosslinking divinylmonomer such as divinyl 
benzene or butadiene. 
In 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, nitrile, 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, propyidiethylammonium 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 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 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 
polynucleotides, 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 surface with a solution containing 
a multivalent cation binding agent. In a preferred embodiment, the 
solution has a temperature of about 50.degree. C. to 90.degree. 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.degree. C. to 90.degree. C. The 
preferred temperature is about 70.degree. C. to 80.degree. 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, .alpha.-furildioxime, nioxime, salicylaldoxime, 
dimethylglyoxime, .alpha.-furildioxime, cupferron, 
.alpha.-nitroso-.beta.-naphthol, nitroso-R-salt, diphenylthiocarbazone, 
diphenylcarbazone, eriochrome black T, PAN, SPADNS, 
glyoxal-bis(2-hydroxyanil), murexide, .alpha.-benzoinoxime, mandelic acid, 
anthranilic acid, ethylenediamine, glycine, triaminotriethylamine, 
thionalide, triethylenetetramine, ethylenediaminetetraacetic acid (EDTA), 
metalphthalein, arsonic acids, .alpha.,.alpha.'-bipyridine, 
4-hydroxybenzothiazole, 8-hydroxyquinaldine, 8-hydroxyquinoline, 
1,10-phenanthroline, picolinic acid, quinaldic acid, 
.alpha.,.alpha.',.alpha."-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, nitrile, 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 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, 
methyihexylammonium 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 riethylammonium 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, .alpha.-furildioxime, nioxime, salicylaldoxime, 
dimethylglyoxime, .alpha.-furildioxime, cupferron, 
.alpha.-nitroso-.beta.-naphthol, nitroso-R-salt, diphenylthiocarbazone, 
diphenylcarbazone, eriochrome black T, PAN, SPADNS, 
glyoxal-bis(2-hydroxyanil), murexide, .alpha.-benzoinoxime, mandelic acid, 
anthranilic acid, ethylenediamine, glycine, triaminotriethylamine, 
thionalide, triethylenetetramine, EDTA, metalphthalein, arsonic acids, 
.alpha.,.alpha.'-bipyridine, 4-hydroxybenzothiazole, 8-hydroxyquinaldine, 
8-hydroxyquinoline, 1,10-phenanthroline, picolinic acid, quinaldic acid, 
.alpha.,.alpha.',.alpha."-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 alcohols, nitriles, 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, propyidiethylammonium 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.

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 
surfaces 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 resent 
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. Pat. 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 
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 
he resolution of 257- and 267-base pair double stranded DNA fragments of a 
pUC18 DNA-HaeIII 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. Operational 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 interfere 
with the separation process. A pretreatment of a porous bead to render it 
nonporous can be effected with any 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 the 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. 
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. Pat. Nos. 5,334,310; 5,453,185; 5,522,994 (to Frechet). 
Monolith or rod columns are commercially available form Merck & Co 
(Darmstadt, 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-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. Pat. 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.degree. C.-80.degree. C., 
preferably 30.degree. C.-60.degree. 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 ratio 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 
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. Pat. 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, 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 polymer is preferably unsubstituted or substituted with methyl, ethyl, 
or hydrocarbon having from 23 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 
poly(styrene-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. Pat. No. 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, 
Applicants 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. Pat. 
No. 5,772,889 to Gjerde (1998), and in co-pending U.S. patent applications 
Ser. No. 09/081,040 (filed May 18, 1998) and No. 09/080,547 (filed May 18, 
1998) 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, morin, 
rhodizonic acid, thionalide, thiourea, .alpha.-furildioxime, nioxime, 
salicylaldoxime, dimethylglyoxime, .alpha.-furildioxime, cupferron, 
.alpha.-nitroso-.beta.-naphthol, nitroso-R-salt, diphenylthiocarbazone, 
diphenylcarbazone, eriochrome black T, PAN, SPADNS, 
glyoxal-bis(2-hydroxyanil), murexide, .alpha.-benzoinoxime, mandelic acid, 
anthranilic acid, ethylenediamine, glycine, triaminotriethylamine, 
thionalide, triethylenetetramine, EDTA, metalphthalein, arsonic acids, 
.alpha.,.alpha.'-bipyridine, 4-hydroxybenzothiazole, 8-hydroxyquinaldine, 
8-hydroxyquinoline, 1,10-phenanthroline, picolinic acid, quinaldic acid, 
.alpha.,.alpha.',.alpha."-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 Complexing 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.times.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. 
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. 
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 quatemary 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 acetate, 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 (TEAA) and triethylammonium 
hexafluoroisopropyl alcohol being most preferred. 
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.degree. C. to 90.degree. C.; more 
preferably, 30.degree. C. to 80.degree. C.; most preferably, 50.degree. C. 
to 75.degree. 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, nitriles, 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.degree. 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 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 Ser. No. 09/129,105 filed 
Aug. 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.degree. C. and 70.degree. C. The 
optimum temperature for performing DMIPC is 54.degree. C. to 59.degree. 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 HaeIII 
digest on a MIPC column at 50.degree. C. provided a good separation of all 
the DNA fragments in the digest. However, a standard 209 bp 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.degree. 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 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 cation binding solution at an elevated 
temperature in the range of 50.degree. C. to 80.degree. C. In a preferred 
embodiment the column is washed with a solution containing EDTA, TEAA, and 
acetonitrile, in the 70.degree. C. to 80.degree. C. temperature range. In 
a specific embodiment, the solution contains 0.032 M EDTA, 0.1 M TEAA, 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. In 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.degree. 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 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 0.1M TEAA 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 HaeIII digest (Sigma/Aldrich Chemical Co.) was 
applied at 50.degree. C. (not shown). In order to further test the column 
performance, a mixture of homoduplexes and heteroduplexes in a 209 bp DNA 
standard was applied to the column under DMIPC conditions of 56.degree. 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 MIPC system. The chromatography shown in FIG. 19 
was performed at 56.degree. C. The column used in FIG. 19 was the same 
column used in the separation shown in FIG. 18 and for separating the 
standard pUC18 HaeIII digest. 
FIG. 20 shows the separation of homoduplexes and heteroduplexes of the 
standard mutation detection mixture at 56.degree. C. on the same column 
used to generate the chromatograms in FIGS. 18 and 19. However, in FIG. 20 
the column was washed for 45 minutes with a solution comprising 32 mM EDTA 
and 0.1M TEAA in 25% acetonitrile at 75.degree. C. priorto sample 
application. FIG. 20 shows four cleanly resolved peaks representing the 
two homoduplexes and the two heteroduplexes of the standard 209 bp 
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. DMIPC 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 1.0. 
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 
correction 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 aspects are discussed 
by Snyder, et al., in Introduction to Modern Liquid Chromatography, 
2.sup.nd 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: 
EQU 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 bp Lambda DNA fragment and the corresponding 
100 bp 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. Pat. No. 5,585,236 to Bonn and in U.S. Pat. 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" (CEC) 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. Pat. Nos. to Dadoo 
5,378,334 (1995), U.S. Pat. No. 5,342,492 (1994), and U.S. Pat. No. 
5,310,463 (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-.mu.m 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., 2800V/cm, 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 of Pharmacy, 
19.sup.th 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 V/cm) is applied. Solvent accumulating at the top of the plate is 
removed by suction. Applicants have surprisingly discovered that dDNA 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 
(.DELTA.H.sub.sorp). Two compounds (in this case, DNA fragments of 
different size) can only be separated if they have different 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: 
##EQU1## 
The partition coefficient (K) and the retention factor (k) are related 
through the following equations: 
##EQU2## 
the quotient V.sub.m /V.sub.s is also called phase volume ratio (.PHI.). 
Therefore: 
EQU k=K.PHI. 
To calculate the sorption enthalpies, the following fundamental 
thermodynamic equations are necessary: 
##EQU3## 
By transforming the last two equations, one obtains the Van't Hoff 
equation: 
##EQU4## 
From a plot In k versus 1/T, the sorption enthalpy .DELTA.H.sub.sorp can 
be obtained from the slope of the graph (if a straight line is obtained). 
.DELTA.S.sub.sorp can be calculated if the phase volume ratio (.PHI.) is 
known. 
The Sorption Enthalpy .DELTA.H.sub.sorp is positive (.DELTA.H.sub.sorp &gt;0) 
showing the separation is endothermic using acetonitrile as the solvent 
(FIGS. 3 and 4), and using methanol as the solvent, the Sorption Enthalpy 
.DELTA.H.sub.sorp is negative (.DELTA.H.sub.sorp &lt;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. Most liquid chromatographic separations show exothermic 
plots. 
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 poly(styrene-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.degree. C.). Freshly distilled styrene (33.7 g) and 0.2184 g of 
potassium peroxodisulfate (K.sub.2 S.sub.2 O.sub.8) 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.degree. 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 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.degree. C. for 4 hours and mixed by sonication during the entire time 
period until a fine emulsion of &lt;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.degree. C. 
Following the removal of acetone, the swollen beads were further grown by 
the addition of 310 g of a ethyidivinylbenzene 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.degree. C. for about 7 hours, 
73.degree. C. for about 2 hours, and 83.degree. C. for about 12 hours), 
leading to further increases in the degree of polymerization (&gt;500). The 
pore size of beads prepared in this manner was below the detection limit 
of mercury porosimetry (&lt;30 .ANG.). 
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 Wash 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.degree. C. for 12 hours. 
EXAMPLE 3 
Standard Procedure for Testing the Performance of Separation Media 
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-HaeIII 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 .mu.L, 
containing a total mass of DNA of 0.25 .mu.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 TEAA, pH 7.0; Eluent B: 
0.1 M TEAA, 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.degree. C. The pH was 7.0. 
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 
poly(styrene-divinylbenzene). 
EXAMPLE 4 
Sorption Enthalpy Measurements 
Four fragments (174 bp, 257 bp, 267 bp, and 298 bp, found in 5 .mu.L pUC18 
DNA-HaeIII digest, 0.04 .mu.g DNA/.mu.L) were separated under isocratic 
conditions at different temperatures using octadecyl modified, nonporous 
poly(styrene-divinylbenzene) polymer beads. The separation was carried out 
using a Transgenomic WAVE.TM. DNA Fragment Analysis System equipped with a 
DNASep.TM. column (Transgenomic, Inc., San Jose, Calif.) under the 
following conditions: Mobile phase: 0.1 M triethylammonium acetate, 14.25% 
(v/v) acetonitrile at 0.75 mL/min, detection at 250 nm UV, temperatures at 
35, 40, 45, 50, 55, and 60.degree. 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 (.DELTA.H.sub.sorp &gt;0). 
The same experiments on non-alkylated poly(styrene-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 1/T showing the retention 
factor k is decreasing with increasing temperature (FIG. 6). This 
indicates the retention mechanism is based on an exothermic process 
(.DELTA.H.sub.sorp &lt;0). 
EXAMPLE 5 
Separations with alkylatedpoly(styrene-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.times.4.6 mm I.D.; 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.degree. C. The sample was 5 .mu.L (=0.2 .mu.g 
pUC18 DNA-HaeIII digest). 
Repeating the procedure of FIG. 7 replacing the acetonitrile with 50.0% 
methanol in 0.1 M (TEAA) gave the separation shown in FIG. 8. 
Repeating the procedure of FIG. 7 replacing the acetonitrile with 25.0% 
ethanol in 0.1 M (TEM) 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 (TEAA) 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.times.4.6 mm I.D.; mobile phase 0.1 M tetraethylacetic acid (TEAA), pH 
7.3; gradient: 12-18% 0.1 M TEAA 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.degree. C. The sample was 5 .mu.L (=0.2 .mu.g pUC18 DNA-HaeIII 
digest). 
The separation shown in FIG. 12 was obtained using octadecyl modified, 
nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 
50.times.4.6 mm I.D.; mobile phase 0.1 M TEAA, pH 7.3; gradient: 15-18% 
0.1 M TEAA 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 mL/min, detection UV at 260 nm, and column temp. 51.degree. 
C. The sample was 5 .mu.L (=0.2 .mu.g pUC18 DNA-HaeIII digest). 
The separation shown in FIG. 13 was obtained using octadecyl modified, 
nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 
50.times.4.6 mm I.D.; mobile phase 0.1 M TEAA, pH 7.3; gradient: 35-55% 
0.1 M TEAA 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 mL/min, detection UV at 260 nm, and column temp. 51.degree. 
C. The sample was 5 .mu.L (=0.2 .mu.g pUC18 DNA-HaeIII digest). 
The separation shown in FIG. 14 was obtained using octadecyl modified, 
nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 
50.times.4.6 mm I.D.; mobile phase 0.1 M TEA.sub.2 HPO.sub.4, pH 7.3; 
gradient: 35-55% 0.1 M TEA.sub.2 HPO.sub.4 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 mL/min, detection UV at 260 
nm, and column temp. 51.degree. C. The sample was 5 .mu.L (=0.2 .mu.g 
pUC18 DNA-HaeIII digest). 
The separation shown in FIG. 15 was obtained using octadecyl modified, 
nonporous poly(ethylvinylbenzene-divinylbenzene) beads as follows: Column: 
50.times.4.6 mm I.D.; mobile phase 0.1 M TEAA, pH 7.3; gradient: 6-9% 0.1 
M TEAA 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 .degree. C. The sample 
was 5 .mu.L (=0.2 .mu.g pUC18 DNA-HaeIII digest). 
EXAMPLE 6 
Isocratic/gradient separation of ds DNA 
The following is an isocratic/gradient separation of ds DNA using nonporous 
poly(styrene-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 TEAA, and 14.25% acetonitrile at 0.75 mL/min at 40.degree. 
C. on 50.times.4.6 mm cross-linked poly(styrene-divinylbenzene) column, 
2.1 micron. 5 .mu.L of pUC18 DNA-HaeIII digest (0.2 .mu.g) was injected 
under isocratic conditions and 257, 267 and 298 base pairs DNA eluted 
completely resolved as shown in FIG. 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 poly(styrene-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.degree. 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.degree. C. to complete the reaction (2 hours). 
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.degree. C. 
The polymer beads are packed into a 50.times.4.6 mm i.d 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.degree. C. When the temperature had dropped to &lt;4.degree. 
C., 50 g of poly(styrene-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.degree. 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.degree. C. 
The polymer beads are packed into a 50.times.4.6 mm i.d 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.times.8 mm I.D.) sealed with a rubber nut plug at 
the bottom end. The tube is then sealed at the top end with a rubber nut 
plug and the contents are allowed to polymerize at 70.degree. 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 Wash Treatment 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 
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: 
EQU 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 bp Lambda DNA fragment and the corresponding 
100 bp 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: 
______________________________________ 
COMPONENT VOLUME 
______________________________________ 
Pfu 10X Buffer (Cat. No. 
5 .mu.L 
600153-82, Stratagene, Inc., 
La Jolla, CA) 
100 .mu.M dNTP Mix 4 .mu.L 
Primer 1 7.5 .mu.L 
(forward) 
Primer 2 8.5 .mu.L 
(reverse) 
H.sub.2 O 19.5 .mu.L 
Lambda DNA Template 5 .mu.L 
PFUTurbo .TM. 0.5 .mu.L 
(600250, Stratagene) 
______________________________________ 
The PCR tubes were placed into a thermocycler (PTC-100 Prograrmmable 
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 94.degree. C. 2 minutes 
2 94.degree. C. 1 minute 
3 58.degree. C. 1 minute 
4 72.degree. C. 1 minute 
5 Go to Step 2, 34X 
6 72.degree. C. 10 minutes 
7 End 
______________________________________ 
The DIMPC conditions used for the mutation detection separations are shown 
below: 
Eluent A: 0.1 M TEAA; Eluent B: 0.1 M TEAA, 25% Acetonitrile; Flow rate: 
0.90 mL/min; Gradient: 
______________________________________ 
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-Jun. 2, 
1997, Brno, Czech Republic, Poster no. 29. The 100 bp Lambda fragment 
sequence (base positions 32011-32110) was used as a standard (available 
from FMC Corp. available from FMC Corp. BioProducts, Rockland, Me.). The 
mutation was at position 32061. The chart below lists the primers used: 
______________________________________ 
Primers 
______________________________________ 
Forward Primer: 
5'-GGATAATGTCCGGTGTCATG-3' 
Reverse Primer: 
3'-GGACACAGTCAAGACTGCTA-5' 
______________________________________ 
FIG. 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. 
FIG. 22 is the Lambda mutation analyzed in identical conditions as FIG. 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 WAVE.TM. DNA Fragment 
Analysis System (Transgenomic, Inc., San Jose, Calif.) under the following 
conditions: Column: 50.times.4.6 mm i.d. containing alkylated 
poly(styrene-divinylbenzene) beads (DNASep.RTM., Transgenomic, Inc.); 
mobile phase 0.1 M TEAA (1 M concentrate available from Transgenomic, 
Inc.) (Eluent A), pH 7.3; gradient: 50-53% 0.1 M TEAA 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.degree. C. The 
sample was 2 .mu.L (=0.2 .mu.g DNA, DYS271 209 bp 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.times.4.0 mm, chelating cartridge, part no. 530012 
from Transgenomic, Inc.) and replacing the pump-valve filter (Part no. 
638-1423, Transgenomic, Inc.). The guard cartridge had dimensions of 
10.times.3.2 mm, containing iminodiacetate chelating resin of 2.5 mequiv/g 
capacity and 10 .mu.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 TEAA, 25% acetonitrile, and 32 mM 
EDTA, at 75.degree. 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 bp mutation standard 
containing a mixture of the homozygous mutant DNA fragment (with an A to G 
mutation at position 168) 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, Calif.; the mutation 
is described by Seielstad et al., Human Mol. Genet. 3:2159 (1994)) was 
heated at 95.degree. C. for 3-5 minutes then cooled to 25.degree. 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 mL/min. 25 g 
of the beads prepared in Example 1 were suspended in 150-160 g of 
1-chlorooctadecane (product no. 0235, TCI America, Portland, Oreg.) using 
a bow shaped mixer (use a 250 mL wide neck Erlenmeyer flask). The 
temperature was set to 50-60.degree. 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.degree. 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 AlCl.sub.3 powder 
(product no. 06218, Fluka, Milwaukee, Wis.) and continued for 16-18 h. 
After the reaction, the polymer was separated from excess 1 
-chlorooctadecane by centrifugation followed by consecutive washing steps: 
______________________________________ 
Addition Comment 
______________________________________ 
50 mL conc. HCl, 50-60 mL n-heptane 
4 repetitions, with recycled 
heptane 
100 mL H.sub.2 O, 50-60 mL n-heptane 
1 repetition, with fresh heptane 
50 mL conc. HCl, 50-60 mL n-heptane 
1 repetition, with fresh heptane 
100 mL H.sub.2 O, 50-60 mL n-heptane 
1 repetition, fresh heptane 
150 mL H.sub.2 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. 
100 mL THF 3 repetitions 
100 mL THF/n-heptane 
1 repetition 
100 mL n-heptane 1 repetition 
100 mL THF 1 repetition 
100 mL CH.sub.3 OH 4 repetitions 
______________________________________ 
In the steps where aqueous solvents (HCl or H.sub.2 O) were used, the 
polymer was shaken for 30 seconds with the aqueous phase before adding 
n-heptane. n-Heptane was then added and the mixture was shaken vigorously 
for 2 min. 
After the final polymeric beads were dried at 40-50.degree. C. for 2-3 hr 
it was ready for packing. 
EXAMPLE 17 
Column packing procedure 
After weighing out 1.2 grams of oven dried polymeric beads, form a slurry 
with 10 mL tetrahydrofuran (THF) and place in a sonicator under a fume 
hood for 15 min. The add 5 mL of THF and 5 mL of methanol (MeOH) and 
sonicate an additional 10 min. Pre-fill a packing assembly with 20 mL 
MeOH. Pour the slurry slowly into the packing assembly. Turn on a Haskel 
pump (Haskel International, Inc., Burbank, Calif.) and slowly increase 
packing pressure to 5000 psi for the initial packing phase. After 10 min, 
slowly increase packing pressure to 9000 psi and set the secondary packing 
phase for 20 min. After 20 min, change the packing eluent from MeOH to 
0.05 M Na.sub.4 EDTA. The set the final packing phase for 40 min. 
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. 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.