Note: Descriptions are shown in the official language in which they were submitted.
CA 02286350 1999-09-30
WO 98/48913 PCTIUS98108388
TITLE OF THE INVENTION
POLYNUCLEOTIDE SEPARATIONS ON NONPOROUS POLYMER BEADS
FIELD OF THE INVENTION
The present invention is directed to the separation of polynucleotides
using nonporous polymeric beads.
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 poiynucleotides 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) ace becoming more
important.
High quality materials for double stranded DNA separations previously
have been based on polymeric substrates disclosed in U.S. Patent No.
5,585,236, to Bonn et al., 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.
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
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 poiynucleotides with improved
separation and efficiency.
Another object of the present invention is to provide a method for
separating polynucleotides using nonporous polymer beads having a non-
reactive, non-polar surface.
It is another object of this invention to provide the chromatographic
separation of polynucleotides using nonporous polymeric beads 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 beads which can be
unsubstituted, methyl-substituted, ethyl-substituted, hydrocarbon-substituted,
or hydrocarbon polymer-substituted.
Yet another object of the present invention is to provide improved
polymer beads 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 summary, the method of this invention for separating a mixture of
polynucleotides comprises flowing a mixture of polynucleotides having up to
_2_
CA 02286350 1999-09-30
WO 98/48913 PCTIUS98/08388
1500 base pairs through a separation column containing beads having an
average diameter of 0.5 to 100 microns, and separating the mixture of
polynucleotides using Matched Ion Polynucleotide Chromatography (as
defined hereinbelow). 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 methacryfates; polyolefins such as polypropylene and
polyethylene; polyesters; polyurethanes; polyamides; poiycarbonates; 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). The polymer
can be unsubstituted, or substituted with a moiety selected from the group
consisting of methyl, ethyl, hydrocarbon, or hydrocarbon polymer. The
hydrocarbon polymer optionally has from 23 to 1,000,000 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 (as defined hereinbelow) 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 addition to the beads themselves being substantially metal-free, we
have also found that, to achieve optimum peak separation, the separation
-3-
CA 02286350 1999-09-30
WO 98148913 PCTIUS98108388
column and all process solutions held within the column or flowing through
the column should be 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 eluent 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 resin bed from multivalent cation contamination. The
multivalent capture resin is preferably cation exchange resin andlor chelating
resin.
The method of the present invention can be used to separate double
stranded poiynucleotides having up to about 1500 to 2000 base pairs. In
many cases, the method is used to separate polynucieotides 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 is performed at a temperature within the range of 20°C
to
90°C to yield a back-pressure not greater than 5000 psi. The method
also
preferably employs an organic solvent that is water soluble. The solvent is
preferably selected from the group consisting of alcohols, nitrites,
-4-
. .~x~ .... ~ . r ...
CA 02286350 2004-O1-16
dimethylformamide, esters, and ethers. The method also preferably employs
a counter ion agent selected from trialkylamine acetate, trialkylamine
carbonate, and trialkylamine phosphate. The most preferred counter ion
agent is triethylammonium acetate or triethylammonium hexafluoroisopropyl
alcohol.
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. 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,_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 substituted with a moiety such as
methyl, ethyl, hydrocarbon, or hydrocarbon polymer. In preferred
embodiments, the bead is octadecyl modified poly(ethylvinylbenzene-
Attorney Docket No. TRAM-078 -5-
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
divinylbenzene) or polystyrene-divinylbenzene). The bead can also contain
crosslinking divinylmonomer such as divinyl benzene or butadiene.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic representation of how the DNA Separation
Factor is measured.
FIG. 2 is a MIPC separation of pUCl8 DNA-Haelll digestion fragments
on a column containing alkylated polystyrene-divinylbenzene) beads. Peaks
are labeled with the number of base pairs of the eluted fragment.
FIG. 3 is a MIPC separation of pUCl8 DNA-Haelll digestion fragments
on a column containing nonporous 2.1 micron beads of underivatized
polystyrene-divinylbenzene).
FIG. 4 is a Van't Hoff plot of In k vs. 1/T j°K-'Jwith alkylated
polystyrene-divinylbenzene) beads showing positive enthalpy using
acetonitriie as the solvent.
FIG. 5 is a Van't Hoff plot of In k vs. 1/T ~°K -'J with
underivatized
poiy(styrene-divinylbenzene) beads showing positive enthalpy using
acetonitriie as the solvent.
FIG. 6 is a Van't Hoff plot of In k vs. 1/T [°K ~'] with alkylated
polystyrene-divinylbenzene) beads showing negative enthalpy using
methanol as the solvent.
FIG. 7 is a separation using alkylated beads and acetonitrile as
solvent.
FIG. 8 is a separation using alkylated beads and 50.0% methanol as
the solvent.
-6-
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
FIG. 9 is a separation using alkylated beads and 25.0% ethanol as the
solvent.
FIG. 10 is a separation using alkylated beads and 25.0% vodka (100
proof) as the solvent.
FiG. 11 is a separation using alkylated beads and 25.0% 1-propano! as
the solvent.
FIG. 12 is a separation using alkylated beads and 25.0% 1-propanol as
the solvent.
FIG. 13 is a separation using alkylated beads and 10.0% 2-propanol as
the solvent.
FIG. 14 is a separation using alkylated beads and 10.0% 2-propanol as
the solvent.
FIG. 15 is a separation using alkylated beads and 25.0% THF as the
solvent.
FIG. 16 is a combination isocratic/gradient separation on a non-
alkylated polystyrene-divinylbenzene) beads.
DETAILED DESCRIPTION OF THE INVENTION
In its most general form, the subject matter of the present invention is
the separation of poiynucleotides utilizing columns filled with nonporous
polymeric beads having an average diameter of about 0.5 -100 microns;
preferably, 1 - 10 microns; more preferably, 1 - 5 microns. Beads having an
average diameter of 1.0 - 3.0 microns are most preferred.
In U.S. Patent No. 5,585,236, Bonn et al. had characterized the nucleic
acid separation process as reverse phase ion pairing chromatography
(RPIPC). However, since RPIPC does not incorporate certain essential
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
characteristics described in the present invention, another term, Matched Ion
Polynucieotide 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 counter
ion agent, and an organic solvent to desorb the nucleic acid from the beads,
and wherein the beads are characterized as having a DNA Separation Factor
of at least 0.05. fn 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. We have found that the best criterion for measuring performance of the
beads is a DNA Separation Factor. This is measured as the resolution of
257- and 2fi7-base pair double stranded DNA fragments of a pUCl8 DNA-
Haelll restriction digest wherein the distance from the valley between the
peaks to the top of one of the peaks, over the distance from the baseline to
the valley. 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.
_g_
... ...._..~_~.....,.._ _ , .
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
Without wishing to be bound by theory, we 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 0.05 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 eluent and other materials
can enter the bead structure. Pores are often interconnected so that fluid
entering one pore can exit from another pore. We 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
separations that have very long retention times. In MIPC, however, the beads
are "nonporous" and the polynucleotides do not enter the bead structure.
-9-
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
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.
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. (J. W. Goodwin, J. Hearn, C. C. Ho and R. H. Ottweill, Colloid
& Polymer Sci., (1974), 252:464-471 ). 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
-10-
..w_~.._ . r. ~. »_..
CA 02286350 1999-09-30
WO 98/48913 PCT/US98108388
by substitution with various groups to produce the nonporous polymeric
beads of the present invention.
The seed beads produced by emulsion polymerization can be enlarged
by any known process for increasing the size of the polymer beads. For
example, polymer beads can be enlarged by the activated swelling process
disclosed in U.S. Patent No. 4,563,510. The enlarged or swollen polymer
beads are further swollen with a crosslinking polymerizable monomer and a
polymerization initiator. Polymerization increases the crosslinking density of
the enlarged polymeric bead and reduces the surface porosity of the bead.
Suitable crosslinking monomers contain at least two carbon-carbon double
bonds capable of polymerization in the presence of an initiator. Preferred
crosslinking monomers are divinyi 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
CA 02286350 1999-09-30
WO 98148913 PCT/US98/08388
initiator, generally, about 10°C - 80°C, preferably 30°C -
60°C. Optionally, the
temperature of the mixture can be increased by 10 - 20% and the mixture
heated for an additional 1 to 4 hours. The 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. Patent No. 4,563,510 can be modified through substitution of the
polymeric beads with methyl or ethyl groups or with a hydrocarbon polymer
group or can be used in its unmodified state. The polymer beads can be
aikylated with 1 or 2 carbon atoms by contacting the beads with an alkylating
agent, such as methyl iodide or ethyl iodide. Alkyfation is achieved by mixing
-12-
,.
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
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 "hydrocarbon" as used herein is defined to include alkyl and
alkyl substituted aryl groups, having from 23 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 tricyctic aromatic hydrocarbon groups including
phenyl, naphthyl, and the like. Methods for hydrocarbon substitution are
conventional and well-known in the ari 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
term "hydrocarbon polymer" as used herein is defined to be a polymer having
a hydrocarbon composition and from 23 to 1,000,000 carbons.
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 tacking 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.
-13-
CA 02286350 1999-09-30
WO 98148913 PCT/US98/08388
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
methyl, ethyl, hydrocarbon, or hydrocarbon polymer substitution.
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; polyoiefins such as
polypropylene and polyethylene; polyesters; polyurethanes; poiyamides;
polycarbonates; and substituted polymers including fluorosubstituted
ethyienes commonly known under the trademark TEFLON. The base
polymer can also be mixtures of polymers, non-limiting examples of which
include polystyrene-divinylbenzene) and poly(ethylvinylbenzene-
divinylbenzene). Methods for making beads from these polymers are
conventional and well known in the art (for example, see U.S. Patent
4,906,378). The physical properties of the surface and near-surface areas of
the beads are the predominant influence on chromatographic efficiency. The
polymer, whether derivatized or not, must provide a nonporous, non-reactive,
and non-polar surface for the Matched ion Polynucleotide Chromatographic
separation.
The beads of the invention are also 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
-14-
_. _._......~_.~..~.-...-.~..w.~~._._.._.._._ . t t . .__
CA 02286350 2004-O1-16
eliminate any multivalent cation contaminants (e.g. Fe(111), Cr(111), 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, we
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 should be 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
I' components are preferably material selected form the group consisting of
titanium, coated stainless steel, passivated stainless steel, and organic
polymer.
For additional protection, multivalent cations in eluent 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 resin bed from multivalent cation contamination. The
multivalent capture resin is preferably cation exchange resin and/or chelating
resin.
Attorney Docket No. TRAN I-078 -15- '
CA 02286350 1999-09-30
WO 98/48913 PCTILJS98/08388
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 sonification are
typically used to improve packing density.
For example, to pack a 4.6 x 50 mm i.d. column, 2.0 grams of beads
can be suspended in 10 mL of methanol with the aid of sonification. 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 polynucfeotides having up to about-1500 to 2000 base pairs. fn
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.
-16-
~.
CA 02286350 1999-09-30
WO 98/48913 PCT/LJS98/08388
In a preferred embodiment, the separation is by Matched Ion
Pofynucleotide Chromatography (MIPC). The nonporous beads of the
invention are used as a reverse phase material that will function with counter
ion agents and a solvent gradient to produce the DNA separations. fn MIPC,
the polynucleotides are paired with a counter ion and then subjected to
reverse phase chromatography using the nonporous beads of the present
invention. Counter ion agents that are volatile, such as trialkyiamine
acetate,
trialkylamine carbonate, trialkylamine phosphate (TEAAHPO,), etc., 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°C to 90°C; more preferably,
30°C to
80°C; most preferably, 50°C to 75°C. 14. The temperature
is selected to yield
a back pressure not exceeding 5000 psi. In general, separation of single-
stranded fragments should be performed at higher temperatures.
We have found that the temperature at which the separation is
performed affects the choice of 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 transport of the DNA between the mobile phase and the
stationary phase. Acetonitrile and 1-propanol are preferred solvents in these
-1?-
CA 02286350 2004-O1-16
cases. Finally, the toxicity (and cost) of the solvent can be important. In
this
case, methanol is preferred over acetonitrile and 1-propanol is preferred over
methanol.
When the separation is performed at a temperature within the above
range, an organic solvent that is water soluble is preferably used, for
example, alcohols, nitrites, dimethylformamide (DMF), tetrahydrofuran (THF),
esters, and ethers. Water soluble solvents are defined as those which exist
as a single phase with aqueous systems under all conditions of operation of
the present invention. Solvents which are particularly preferred for use in
the
method of this invention include methanol, ethanol, 2-propanol, 1-propanol,
tetrahydrofuran (THF), and acetonitrile, with acetonitrile being most
preferred
overall.
We have determined that the chromatographic separations of double.
stranded DNA fragments exhibit unique Sorption Enthalpies (OHso,P). Two
compounds (in this case, DNA fragments of different size) can only be
separated if they have different partition coefficients (f~. The Nernst
partition
coefficient is defined as the concentration of an analyte (A) in the
stationary
phase divided by its concentration in the mobile phase: -
K = Lls
tAlm
The partition coefficient (f~ and the retention factor (k) are related through
the
following equations:
K - nA Vm and k - nAs
n(A)m VS n (A)m
the quotient Vm/VS is also called phase volume ratio (~). Therefore:
K=k~
Attorney Docket No. TRAM-078 -18-
CA 02286350 2004-O1-16
To calculate the sorption enthalpies, the following fundamental
thermodynamic equations are necessary:
In K= - ~G$o,~, In k= - oGSa,~ + In ~ and ~Gso,p = OHSa,p - TOSso,p
RT RT
By transforming the last two equations, we obtain the Van't Hoff equation:
fn k = - ~Hso,P + OSso,~ + In ~
RT R
From a plot In k versus 1/T, the sorption enthalpy ~Hso,P can be obtained from
the slope of the graph (if a straight line is obtained). ASso,~ can be
calculated if
the phase volume ratio (~) is known.
The Sorption Enthalpy ~Hso~ is positive (OH~~P > 0) showing the
separation is endothermic using acetonitrife as the solvent (Figs. 3 and 4),
and using methanol as the solvent, the Sorption Enthalpy ~Hso,P is negative
(~H~,p< 0), showing the separation is exothermic (FIG. 5).
The thermodynamic data (as shown in the Examples hereinbelow)
reflect the relative affinity of the DNA-counter ion 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.
Attorney Docket No. TRAN I-078 -19- .
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
Procedures described in the past tense in the examples below have
been carried out in the laboratory. Procedures described in the present tense
have not yet been carried out in the laboratory, and are constructively
reduced to practice with the filing of this application.
EXAMPLE 1
Preparation of nonporous polystyrene-divinylbenzene) particles
Sodium chloride {0.236 g) was added to 354 mL of deionized water in
a reactor having a volume of 1.0 liter. The reactor was equipped with a
mechanical stirrer, reffux condenser, and a gas introduction tube. The
dissolution of the sodium chloride was carried out under inert atmosphere
(argon), assisted by stirring (350 rpm), and at an elevated temperature
(87°C).
Freshly distilled styrene (33.7 g) and 0.2184 g of potassium peroxodisulfate
(KZSZOB) dissolved in 50 mL of deionized water were then added.
Immediately after these additions, the gas introduction tube was pulled out of
the solution and positioned above the liquid surface. The reaction mixture
was subsequently stirred for 6.5 hours at 87°C. After this, the
contents of the
reactor were cooled down to ambient temperature and diluted to a volume
yielding a concentration of 54.6 g of polymerized styrene in 1000 mL volume
of suspension resulting from the first step. The amount of polymerized
styrene in 1000 mL was calculated to include the quantity of the polymer still
sticking to the mechanical stirrer (approximately 5 - 10 g). The diameter of
the spherical beads in the suspension was determined by light microscopy to
be about 1.0 micron.
-20-
_ _ _._~._.._. _ ~ ~ . __
CA 02286350 1999-09-30
WO 98/48913 PCTIUS98I08388
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 crossfinking.
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°C for 4 hours and
mixed by
sonication during the entire time period until a fine emulsion of < 0.3
microns
was obtained. The mixture of polystyrene seeds, acetone, and 1-
chlorododecane emulsion was stirred for about 12 hours at room
temperature, during which time the swelling of the beads occurred.
Subsequently, the acetone was removed by a 30 minute distillation at
80°C.
Following the removal of acetone, the swollen beads were further
grown by the addition of 310 g of a ethyldivinylbenzene and divinylbenzene
(DVB) (1:1.71 ) mixture also containing 2.5 g of dibenzoylperoxide as an
initiator. The growing occurred with stirring and with occasional particle
size
measurements by means of light microscopy.
After completion of the swelling and growing stages, the reaction
mixture was transferred into a separation funnel. In an unstirred solution,
the
-21-
CA 02286350 1999-09-30
WO 98/48913 PCTlUS98/08388
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 (fi3°C for about 7 hours,
73°C for about 2
hours, and 83°C for about 12 hours), leading to further increases in
the
degree of polymerization (> 500). The pore size of beads prepared in this
manner was below the detection limit of mercury porosimetry (< 30~).
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°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
pUCl8 DNA-Haelll digest (Sigma-Aldrich, D6293) which contains 11
fragments having 11, 18, 80, 102, 174, 257, 267, 298, 434, 458, and 587
-22-
, i .
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
base pairs, respectively. The standard is diluted with water and five NL,
containing a total mass of DNA of 0.25 p.g, is injected.
Depending on the packing volume and packing polarity, the procedure
requires selection of the driving solvent concentration, pH, and temperature.
The separation conditions are adjusted so that the retention time of the 257,
267 peaks is about 6 to 10 minutes. Any one of the following solvents can be
used: methanol, ethanol, 2-propanol, 1-propanol, tetrahydrofuran (THF), or
acetonitrile. A counter ion agent is selected from trialkylamine acetate,
trialkylamine carbonate, trialkylamine phosphate, or any other type of cation
that can form a matched ion with the polynucieotide 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 %A %B
min
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 mUmin, detection UV at 260 nm, column temp.
50°C. The pH was 7Ø
-23-
CA 02286350 1999-09-30
WO 98/48913 PCT/I1S98/08388
As another example of this procedure using the same separation
conditions as in FIG. 2, FIG. 3 is a high resolution separation of the
standard
DNA mixture on a column containing nonporous 2,1 micron beads of
underivatized polystyrene-divinylbenzene).
EXAMPLE 4
Sorption Enthalpy Measurements
Four fragments (174 bp, 257 bp, 267 bp, and 298 bp, found in 5 NL
pUCl8 DNA-Haelll digest, 0.04 Ng DNA/NL) were separated under isocratic
conditions at different temperatures using octadecyl modified, nonporous
polystyrene-divinylbenzene) polymer beads. The separation was carried out
using a Transgenomic WAVET"" DNA Fragment Analysis System equipped
with a DNASepT"" column (Transgenomic, Inc., San Jose, CA) under the
following conditions: Eluent: 0.1 M triethyiammonium acetate, 14.25% (v/v)
acetonitrile at 0.75 mUmin, detection at 250 nm UV, temperatures at 35, 40,
45, 50, 55, and 60°C, respectively. A plot of In k versus 1/T shows
that the
retention factor k is increasing with increasing temperature (FIG. 4). This
indicates that the retention mechanism is based on an endothermic process
(~Hso,P > 0).
The same experiments on non-alkylated polystyrene-divinylbenzene)
beads gave a negative slope for a plot of In k versus 1/T, although the plot
is
slightly curved (FIG. 5).
The same experiments performed on octadecyl modified, nonporous
polystyrene-divinylbenzene) beads but with methanol replacing the
acetonirile as solvent gave a plot In k versus 1/T showing the retention
factor
-24-
.~
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
k is decreasing with increasing temperature (FIG. 6). This indicates the
retention mechanism is based on an exothermic process (OHso,P < 0).
EXAMPLE 5
Separations with alkylated polystyrene-divinylbenzene) beads
Eluents are chosen to match the desorption ability of the elution
solvent to the attraction properties of the bead to the DNA-counter ion
complex. As the polarity of the bead decreases, a stronger (more organic) or
higher concentration of solvent will be required. Weaker organic solvents
such as methanol are generally required at higher concentrations than
stronger organic solvents such as acetonitrile.
FIG. 7 shows the high resolution separation of DNA restriction
fragments using octadecyl modified, nonporous poly(ethylvinylbenzene-
divinylbenzene) beads. The experiment was conducted under the following
conditions: Column: 50 x 4.6 mm 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% acetonitrife in 1 min; and 100-35%
acetonitrile in 1.5 min. The flow rate was 0.75 mUmin, detection UV at 260
nm, column temp. 51 °C. The sample was 5 pL (= 0.2 pg pUC18 DNA-Haelll
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 (TEAA) gave the separation shown in FIG. 9.
-25-
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
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 octadecyi
modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as
follows: Column: 50 x 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 (Fluent
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 mUmin, detection UV at 260
nm, and column temp. 51°C. The sample was 5 p.L (= 0.2 p.g pUCl8 DNA-
Haelll digest).
The separation shown in FIG. 12 was obtained using octadecyl
modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as
follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3;
gradient: 15-18% 0.1 M TEAR and 25.0% 1-propanol (Fluent B) in 2 min, 18-
21 % B in 8 min, 21 % B for 2.5 min; 21-100% B in 1 min; and 100-15% B in
1.5 min. The flow rate was 0.75 mUmin, detection UV at 260 nm, and column
temp. 51 °C. The sample was 5 p.L (= 0.2 ~,g pUC18 DNA-Haelll digest).
The separation shown in FIG. 13 was obtained using octadecyl
modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as
follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAA, pH 7.3;
gradient: 35-55% 0.1 M TEAA and 10.0% 2-propanol (Fluent 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 mUmin, detection UV at 260 nm, and column
temp. 51 °C. The sample was 5 ~L {= 0.2 p,g pUC18 DNA-Haelll digest).
-26-
,.
CA 02286350 1999-09-30
WO 98148913 PCT/US98/08388
The separation shown in FIG. 14 was obtained using octadecyl
modified, nonporous poly(ethylvinylbenzene-divinylbenzene) beads as
follows: Column: 50 x 4.6 mm i.d.; mobile phase 0.1 M TEAZHPO4, pH 7.3;
gradient: 35-55% 0.1 M TEAzHPO4and 10.0% 2-propanoi {Eluent B) in 3 min,
55-65% B in 7 min, 65% B for 2.5 min; 65-100% B in 1 min; and 100-65% B in
1.5 min. The flow rate was 0.75 mUmin, detection UV at 260 nm, and column
temp. 51 °C. The sample was 5 p.L (= 0.2 p,g pUCl8 DNA-Haelll digest).
The separation shown in FIG. 15 was obtained using octadecyl
modified, nonporous poly(ethylvinylbenzene-divinyfbenzene) beads as
follows: Column: 50 x 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 mUrnin, detection UV at 2fi0 nm, and column temp. 51
°C.
The sample was 5 ~.L (= 0.2 p,g pUCl8 DNA-Haelll digest).
EXAMPLE 6
Isocraticlgradient separation of ds DNA
The following is an isocraticlgradient 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 an eluent
of 0.1 M TEAR, and 14.25% acetonitrile at 0.75 mUmin at 40°C on 50 x
4.6
-27-
CA 02286350 1999-09-30
WO 98/48913 PCTIUS98108388
mm cross-linked polystyrene-divinylbenzene) column, 2.1 micron. 5 p.L of
pUCl8 DNA-Haelll digest (0.2 p.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 polystyrene-divinylbenzene) polymer beads were
suspended in 500 g of tetrachloromethane. The suspension was transferred
into a 1000 mL glass reactor (with attached reflux condenser, separation
funnel and overhead stirrer). The mixture was kept at 20°C. Bromine
(100
mL) was added over a period of 20 minutes. After addition was completed,
stirring continued for 60 minutes. The temperature was raised to 50°C
to
complete the reaction {2 hours).
The polymer beads were separated from the tetrachloromethane and
excess bromine by means of centrifugation and cleaned with tetrahydrofuran
(once with 100 mL) and methanol (twice with 100 mL). The polymer beads
were dried at 40°C.
-28-
CA 02286350 1999-09-30
WO 98/48913 PCT/US98/08388
The polymer beads are packed into a 50 x 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°C. When the temperature had dropped to <4°C, 50 g
of
polystyrene-divinylbenzene) polymer beads were added slowly under
continuous stirring. After addition was completed, 50 mL of nitric acid (65%)
was added. The suspension was stirred for three hours, maintaining a
temperature of 5-10°C.
On the next day the reaction was quenched by adding ice to the
suspension. The polymer beads were separated from the acid by means of
centrifugation. The polymer beads were washed to neutrality with water,
followed by washing steps with tetrahydrofurane (four times with 100 mL) and
methanol (four times with 100 mL). The polymer beads were dried at
40°C.
The polymer beads are packed into a 50 x 4.6 mm i.d column and the
DNA Separation Factor is greater than 0.05 as tested by the procedure of
Example 3.
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
-29-
CA 02286350 1999-09-30
WO 98/48913 PCTIUS98/08388
depart from the spirit and scope of the invention as described and claimed
herein.
-30-
