U.S. patent application number 10/282855 was filed with the patent office on 2003-03-27 for process for performing polynucleotide separations.
This patent application is currently assigned to Transgenomic, Inc.. Invention is credited to Gjerde, Douglas T., Haefele, Robert M., Togami, David.
Application Number | 20030057154 10/282855 |
Document ID | / |
Family ID | 27373893 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030057154 |
Kind Code |
A1 |
Gjerde, Douglas T. ; et
al. |
March 27, 2003 |
Process for performing polynucleotide separations
Abstract
The invention recognizes the deleterious effects of trace, and
even undetectable amounts of multivalent cations on the separation
of mixtures of polynucleotides, especially double stranded
polynucleotides, and provides an improved method for separating
such mixtures on wide pore, non-polar separation media by
eliminating multivalent cations from the all aspects of the
separation process. This is accomplished by using components in the
separation process which are materials which do not release metal
cations. In addition, the use of cation capture resins and other
methods to remove residual traces of multivalent cations from
eluting solvents, sample solutions, separation media, and system
components is described. It is also important to remove any traces
or organic contaminants from solvents solutions and system parts.
Taking similar steps to remove residual traces of multivalent
cations and organic impurities from the separation process, the
invention may also be used in a batch process to separate mixtures
of polynucleotide fragments.
Inventors: |
Gjerde, Douglas T.;
(Saratoga, CA) ; Haefele, Robert M.; (Campbell,
CA) ; Togami, David; (San Jose, CA) |
Correspondence
Address: |
KEITH JOHNSON, ESQ.
TRANSGENOMIC, INC.
12325 EMMETT STREET
OMAHA
NE
68164
US
|
Assignee: |
Transgenomic, Inc.
San Jose
CA
|
Family ID: |
27373893 |
Appl. No.: |
10/282855 |
Filed: |
October 28, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10282855 |
Oct 28, 2002 |
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09705084 |
Nov 2, 2000 |
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6471866 |
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09705084 |
Nov 2, 2000 |
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09324350 |
Jun 2, 1999 |
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6156206 |
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09324350 |
Jun 2, 1999 |
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09081039 |
May 18, 1998 |
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5972222 |
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09324350 |
Jun 2, 1999 |
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08748376 |
Nov 13, 1996 |
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5772889 |
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Current U.S.
Class: |
210/635 ;
210/656; 210/659; 435/6.12; 536/25.4 |
Current CPC
Class: |
B01D 15/12 20130101;
B01D 15/38 20130101; B01J 45/00 20130101; B01D 15/366 20130101;
C12N 15/101 20130101; G01N 30/14 20130101; G01N 2030/528
20130101 |
Class at
Publication: |
210/635 ;
210/656; 210/659; 435/6; 536/25.4 |
International
Class: |
B01D 015/08 |
Claims
What is claimed is:
1. A method for separating a mixture of polynucleotide fragments
comprising a) applying a solution of said fragments and counterion
reagent to a column containing, separation media having a non-polar
surface, wherein said separation media have a pore size greater
than 30 Angstroms and an average diameter of 1-100 microns; b)
eluting said fragments with a gradient eluting solvent of
increasing organic component concentration containing a counterion
agent; wherein surfaces which are contacted by the solution of the
fragments and the eluting solvent are materials which do not trap
or release multivalent metal cations therefrom.
2. A method of claim 1 wherein the solution of said fragments and
the eluting solvent are contacted with a multivalent cation capture
resin to remove any multivalent cations therein before entering the
column.
3. A method of claim 2 wherein said separation media have been
treated to remove residual traces of multivalent cations from the
surfaces thereof.
4. A method of claim 3 wherein the solution or said fragments and
eluting solvent have been contacted with a multivalent cation
capture resin before entering the column.
5. A method of claim 4 wherein the polynucleotide fragments are
double stranded or single stranded.
6. A method of claim 5 wherein the fragments having more than 5
base pairs are separated on the basis of size or polarity.
7. A method of claim 4 wherein said separation media are organic
polymer.
8. A method of claim 4 wherein said separation media have inorganic
substrates selected from the group consisting of inorganic
substrates, silica, zirconia, and alumina.
9. A method of claim 8 wherein the non-polar surface is an organic
polymer supported on the inorganic substrate.
10. A method of claim 8 wherein the non-polar surface includes long
chain hydrocarbon groups having from 8 to 24 carbons bound to the
inorganic substrate.
11. A method of claim 10 wherein residual polar groups of the
inorganic substrate have been end capped with trimethylsilyl
chloride or hexamethyldisilazane.
12. A method of claim 4, wherein the surfaces contacted by the
solution of polynucleotide fragments and eluting solvent are
titanium, coated stainless steel, organic polymer or combinations
thereof.
13. A method of claim 12 wherein traces of residual multivalent
metal cations have been removed from said surfaces by treating said
surfaces with a solution comprising aqueous acid and chelating
agent.
14. A method of claim 12 wherein organic contaminants have been
removed from said surfaces.
15. A method of claim 1 wherein said solution of polynucleotide
mixture and eluting solvent contain a chelating agent, whereby any
trace of multivalent metal cations in the solutions are
captured.
16. A method of claim 4 wherein said solution of polynucleotide
mixture and eluting solvent contain a chelating agent, whereby any
trace of multivalent metal cations in the solutions are
captured.
17. A method of claim 4 wherein the eluting solvent has been
treated to remove oxygen therefrom.
18. A method of claim 4 wherein said method for separating said
mixture of polynucleotides comprises Matched Ion Polynucleotide
Chromatography.
19. A batch process for separating polynucleotide fragments having
a selected size from a mixture of polynucleotide fragments
including fragments of said selected size comprising a) applying a
solution of said polynucleotide fragments and a counterion agent to
non-polar separation media having a non-polar surface, wherein said
separation media have a pore size greater than 30 Angstroms and an
average diameter of 1-100 microns; b) contacting the separation
media with a first eluting solvent and counterion agent, the first
eluting solvent having a concentration of organic component
sufficient to release from the separation media all polynucleotide
fragments having a size smaller than the selected size and removing
the first eluting solvent from the separation media; and c)
contacting the separation media with a second eluting solvent
having a concentration of organic component sufficient to release
from the separation media the polynucleotide fragments having the
selected size and removing the second eluting solvent from the
separation media; wherein surfaces which are contacted by the
solution of polynucleotide fragments and the eluting solvent are
material which does not trap or release multivalent metal cations
therefrom.
20. A batch process of claim 19 wherein the separation media are
rinsed with fresh first eluting solvent following step b) to remove
residual released polynucleotide fragments therefrom.
21. A batch process of claim 19 wherein the separation media are
rinsed with fresh second eluting solvent following step c) to
remove residual released polynucleotide fragments of selected size
therefrom.
22. A batch process of claim 19 wherein the solution of
polynucleotide mixture and eluting solvent have been contacted with
a multivalent cation capture resin before contacting the separation
media.
23. A batch process of claim 19 wherein the polynucleotide mixture
is double stranded or single stranded.
24. A batch process of claim 22 wherein said separation media have
been treated to remove residual traces of multivalent cations
therefrom.
25. A batch process of claim 24 wherein the solution of
polynucleotide mixture and eluting solvent have been contacted with
a multivalent cation capture resin before contacting the separation
media.
26. A batch process of claim 25 wherein the separation media are
contained in a column, a web, a membrane, or container.
27. A batch process of claim 26 wherein said separation media are
organic polymer or inorganic substrates selected from the group
consisting of inorganic substrates, silica, zirconium, and
alumina.
28. A batch process of claim 27 wherein the non-polar surface is an
organic polymer supported on the inorganic substrate.
29. A batch process of claim 27 wherein the non-polar surface
includes long chain hydrocarbons having from 8-24 carbons bonded
the inorganic substrate.
30. A batch process of claim 29 wherein any residual polar groups
of the inorganic substrate have been end capped with trimethylsilyl
chloride or hexamethyldisilazane.
31. A batch process of claim 26, wherein the surfaces contacted by
the solution of polynucleotide fragments and eluting solvent are
comprised of material selected from the group consisting of
titanium, coated stainless steel, and organic polymer, or
combinations thereof.
32. A batch process of claim 31 wherein traces of residual
multivalent metal cations have been removed from said surfaces by
treating said surfaces with a solution comprising aqueous acid and
chelating agent.
33. A batch process of claim 31 wherein organic contaminants have
been removed from said surfaces.
34. A batch process of claim 25 wherein said solution of
polynucleotide mixture and eluting solvent contain a chelating
agent whereby any trace of multivalent metal cations in the
solution are captured.
35. A batch process of claim 19 wherein the eluting solvent has
been treated to remove oxygen therefrom.
Description
[0001] This is a continuation-in-part application of Ser. No.
08/748,376 filed Nov. 13, 1996. This application is filed under
35-U.S.C. .sctn.111(a) and claims priority from copending, commonly
assigned provisional applications Serial No. 60/049,123 filed Jun.
10, 1997; and Serial No. 60/063,835 filed Oct. 30, 1997 under 35
U.S.C. .sctn.111(b).
FIELD OF THE INVENTION
[0002] This invention is directed to the separation of
polynucleotide fragments by liquid chromatography. More
specifically, the invention is directed to a system and method,
which enhances the chromatographic separation of polynucleotides on
non-polar, wide pore separation media.
BACKGROUND OF THE INVENTION
[0003] Separation of polynucleotide mixtures is a focus of
scientific interest, and numerous researchers have been attempting
to achieve technical improvements in various aspects of
polynucleotide separation. Anion exchange separation and reverse
phase ion pair chromatography are among the most frequently used
methods for separating polynucleotide mixtures.
[0004] Samples containing mixtures of polynucleotides can result
from total synthesis of polynucleotides, cleavage of DNA with
restriction endonucleases or RNA, as well as polynucleotide samples
which have been multiplied or amplified using polymerase chain
reaction (PCR) techniques or other amplifying techniques.
[0005] Previous work has focused on developing rapid, high
resolution separations, developing separations based on the size of
the polynucleotide fragment rather than the base sequence of the
fragment, and on developing the ability to collect separated pure
fractions of polynucleotides.
[0006] W. Bloch (European patent publication No. EP 0 507 591 A2)
demonstrated that, to a certain extent, length-relevant separation
of polynucleotide fragments was possible on nonporous anion
exchanger separation media using eluting solvents containing
tetramethylammonium chloride (TMAC). Y. Ohimya et al. (Anal.
Biochem., 189:126-130 (1990)) disclosed a method for separating
polynucleotide fragments on anion exchange material carrying
trimethylammonium groups. Anion exchangers with diethylaminoethyl
groups were used by Y. Kato et al. to separate polynucleotide
fragments (J. Chromatogr., 478:264 (1989)).
[0007] U.S. Pat. No. 5,585,236 (1996) to Bonn et al. describes a
method for separating polynucleotides using what was characterized
as reverse phase ion pair chromatography (RPIPC) utilizing columns
filled with non-polar, nonporous polymeric beads. High resolution,
rapid separations were achieved using an ion pairing agent
(triethylammonium acetate), and acetonitrile/water eluting solvent
gradient. This work is important because it is the first example of
a size dependent, sequence independent chromatographic separation
of double-stranded polynucleotides by Matched Ion Polynucleotide
Chromatography (MIPC). Such separations are comparable to those
effected by gel electrophoresis, which is currently the technology
most widely used for polynucleotide separations. Bonn's work makes
it possible to automate separations of polynucleotides based on
their size alone. This method differs from traditional reverse
phase processes. Therefore, the term Matched Ion Polynucleotide
Chromatography (MIPC) has been applied to the Bonn process to
distinguish it from previously known reverse phase processes.
[0008] The invention of parent application Ser. No. 08/748,376 is
based on the discovery that trace levels of multivalent metal ions,
even when present below the limits of detection, interfere with the
MIPC separation process. Special steps to prevent, remove or
complex any trace multivalent ions result in enhanced separation of
polynucleotides and lower the detection threshold. The inventions
of provisional applications Serial No. 60/049,123 filed Jun. 10,
1997; and Serial No. 60/063,835 filed Oct. 30, 1997 under 35 U.S.C.
.sctn.111(b) are based on the discovery that nitric acid passivated
stainless steel, titanium, and PEEK (polyetherether ketone)
surfaces were, contrary to popular belief, sources of multivalent
metal ion contamination in the MIPC process. The deleterious effect
of multivalent metal cations on polynucleotide separations as
observed herein has not been previously reported. We believe that
all chromatographic processes which are capable of separating
polynucleotides on non-polar, wide pore separation media are
impaired by the interference of multivalent metal ions.
SUMMARY OF THE INVENTION
[0009] Therefore, the invention provides an improved method for
separating a mixture of polynucleotide fragments wherein
multivalent cations are eliminated from the all aspects of the
separation process. The method comprises applying a solution of
said fragments and counterion agent to a column containing
separation media having a non-polar surface, wherein said
separation media have a pore size greater than 30 Angstroms and an
average diameter of 1-100 microns. Separation of said fragments is
accomplished by eluting said fragments with an eluting solvent
gradient of increasing organic component concentration containing a
counterion agent. Surfaces which are contacted by the solution of
the fragments and the eluting solvent are materials which do not
release multivalent metal cations therefrom, said materials having
been washed to remove traces of organic contaminants therefrom. The
method further comprises contacting the solution of said fragments
and the eluting solvent with a multivalent cation capture resin to
remove any multivalent cations therein before entering the
column.
[0010] In a preferred embodiment of the invention, the separation
media have been treated to remove residual traces of multivalent
cations from the surfaces therefrom.
[0011] An optimum embodiment of the invention comprises contacting
the solution of said fragments and eluting solvent with a
multivalent cation capture resin before entering the column,
treating the separation media to remove residual traces of
multivalent cations from the surfaces therefrom, and ensuring that
surfaces which are contacted by the solution of the fragments and
the eluting solvent are materials which do not release multivalent
metal cations therefrom and cleaning said surfaces to remove any
traces of organic contaminants therefrom.
[0012] In one embodiment, the polynucleotide fragments are double
stranded, having more than 5 base pairs. Such fragments are
separated by size or by polarity.
[0013] In another embodiment of the invention, the polynucleotide
fragments are single stranded having 2 or more nucleotides. Such
fragments are separated by size and by polarity.
[0014] The separation media are organic polymer, or an inorganic
substrate selected from the group consisting of inorganic
substrates, silica, zirconia, and alumina. The inorganic substrates
support a non-polar material on their surface. Said non-polar
material may be organic polymer or long chain, C1 to C24
hydrocarbon groups bound to the inorganic substrate, wherein
residual polar groups of the substrate are end capped with
trimethylsilyl chloride or hexamethyldisilazane.
[0015] In a preferred embodiment, surfaces which are contacted by
the solution of polynucleotide fragments and eluting solvent are
titanium, coated stainless steel, organic polymer or combinations
thereof. Removal of traces of residual multivalent metal cations
from the separation process is further ensured by treating said
surfaces with a solution comprising aqueous acid and chelating
agent, by adding a chelating agent to the solution of
polynucleotide mixture and eluting solvent, and by treating the
eluting solvent to remove oxygen therefrom.
[0016] In one embodiment, the improved method for separating said
mixture of polynucleotides comprises Matched Ion Polynucleotide
Chromatography.
[0017] The improved method of the invention may also be practiced
as a batch process for separating polynucleotide fragments having a
selected size from a mixture of polynucleotide fragments including
fragments of said selected size. The batch process method of the
invention comprises applying a solution of said polynucleotide
fragments and a counterion agent to non-polar separation media
having a non-polar surface, wherein said separation media have a
pore size greater than 30 Angstroms and an average diameter of
1-100 microns. The method further comprises contacting the
separation media with a first eluting solvent and counterion agent,
the first eluting solvent having a concentration of organic
component sufficient to release from the separation media all
polynucleotide fragments having a size smaller than the selected
size and removing the first eluting solvent from the separation
media. The selected size fragments are obtained by contacting the
separation media with a second eluting solvent having a
concentration of organic component sufficient to release from the
separation media the polynucleotide fragments having the selected
size and removing the second eluting solvent from the separation
media. Preferably, surfaces which are contacted by the solution of
polynucleotide fragments and the eluting solvent are material which
does not release multivalent metal cations therefrom.
[0018] Following removal of the first eluting solvent, the
separation media are rinsed with fresh first eluting solvent to
remove residual released polynucleotide fragments therefrom. In a
similar manner, following removal of the second eluting solvent the
separation media are rinsed with fresh second eluting solvent to
remove residual released polynucleotide fragments of selected size
therefrom.
[0019] A preferred embodiment of the invention comprises contacting
the solution of polynucleotide mixture and eluting solvent with a
multivalent cation capture resin before contacting the separation
media. In another preferred embodiment the method comprises
treating the separation media to remove residual traces of
multivalent cations therefrom. Optimally the separation media have
been treated to remove residual traces of multivalent cations
therefrom and the solution of polynucleotide mixture and eluting
solvent have been contacted with a multivalent cation capture resin
before contacting the separation media. Said separation media are
contained in a column, a web, a membrane, or container.
[0020] The batch process can be used to separate mixtures of double
stranded polynucleotides or single stranded polynucleotides.
[0021] The separation media are organic polymer, or an inorganic
substrate selected from the group consisting of inorganic
substrates, silica, zirconia, and alumina. The inorganic substrates
support a non-polar material on their surface. Said non-polar
material may be organic polymer or long chain, C1 to C24
hydrocarbon groups bound to the inorganic substrate, wherein
residual polar groups of the substrate are end capped with
trimethylsilyl chloride or hexamethyldisilazane.
[0022] The surfaces contacted by the solution of polynucleotide
fragments and eluting solvent are, preferably, comprised of
material selected from the group consisting of titanium, coated
stainless steel, and organic polymer, or combinations thereof.
Removal of traces of residual multivalent metal cations from the
separation process is further ensured by treating said surfaces
with a solution comprising aqueous acid and chelating agent, by
adding a chelating agent to the solution of polynucleotide mixture
and eluting solvent, and by treating the eluting solvent to remove
oxygen therefrom.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows a guard disk having a one-piece annular
ring.
[0024] FIG. 2 is an exploded view of a guard disk having a
two-piece annular ring and containing three pads of guard disk
material (i.e., a layer or pad of multivalent cation capture resin
which has been incorporated into a fabric or membrane).
[0025] FIG. 3 shows an assembled view of the guard disk of FIG.
2.
[0026] FIG. 4 shows placement of a chelating guard column and
chelating guard disk in a liquid chromatographic system for
polynucleotide separation.
[0027] FIG. 5 shows placement of a chelating guard disk positioned
between a chromatographic separation column and a column top, where
the guard disk is in direct contact with a titanium frit at the top
portion of the separation column.
[0028] FIG. 6. shows the chromatography of a 500 base pair DNA
fragment on PRX-1 (Sarasep, San Jose, Calif.) non-polar, wide pore
polymer separation media which have been washed (see Example 1) to
remove multivalent metal cations thereform.
[0029] FIG. 7 shows the effect on the chromatography result shown
in FIG. 6 when Cr(III) cations were added to the column before
sample injection.
[0030] FIG. 8 shows the effect on the chromatography result of FIG.
7 when a Cr(III) contaminated column was treated with EDTA prior to
sample injection.
[0031] FIG. 9. shows the chromatography of a 500 base pair DNA
fragment on INERTSIL (MetaChem, Torrance, Calif.) C-18 non-polar,
wide pore silica separation media which have been washed with EDTA
to remove multivalent metal cations thereform.
[0032] FIG. 10 shows the effect on the chromatography result shown
in FIG. 9 when Cr(III) cations were added to the column before
sample injection.
[0033] FIG. 11 shows the effect on the chromatography result of
FIG. 10 when a Cr(III) contaminated column was treated with EDTA
prior to sample injection.
[0034] FIG. 12 shows the chromatography of a 20 mer single stranded
DNA fragment standard (Seq 2A, CTGen, Milpitas, Calif.) on INERTSIL
(MetaChem, Torrance, Calif.) C-18 non-polar, wide pore silica
separation media which have been washed with EDTA to remove
multivalent metal cations thereform.
[0035] FIG. 13 shows the effect on the chromatography result shown
in FIG. 12 when Cr(III) cations were added to the column before
sample injection.
[0036] FIG. 14 shows the chromatographic separation at 56.degree.
C. of a 209 base pair DNA standard 4 component heteroduplex and
homoduplex mixture on a freshly packed, untreated DNASep column
(Transgenomic, Inc., San Jose, Calif.).
[0037] FIG. 15 shows the effect on the chromatography result shown
in FIG. 14 when the column was treated with EDTA prior sample
injection.
DETAILED DESCRIPTION OF THE INVENTION
[0038] The term polynucleotide, as used herein, is defined as a
linear polymer containing an indefinite number of nucleotides,
linked from one ribose (or deoxyribose) to another via phosphate
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
hereinafter, it being understood that all polynucleotides are
intended to be included within the scope of this invention.
[0039] The present invention is an improved chromatographic and
batch process method for separating mixtures of polynucleotide
fragments on wide pore non-polar separation media. The improvement
comprises ensuring the removal of all traces of residual
multivalent metal cations from the sample mixture, the eluting
solvent as well as components of the chromatographic or batch
process equipment which contact said sample mixture and eluting
solvent. The inventor's observation and demonstration that even
traces of multivalent metal cations below the level of detection,
degrade polynucleotide separations on wide pore, non-polar
separation media and that removal of said cations results in
improved separation efficiency and column life is both surprising
and novel.
[0040] The system used to implement the method of the invention
comprises a liquid chromatographic system. The liquid
chromatography system comprises a column containing a separation
bed of non-polar, wide pore separation media held in the column
between porous frits positioned at each end of the column. Other
components of the liquid chromatography system include an injection
valve and one or more eluting solvent supply means. Eluting solvent
supply means is (are) connected to the injection valve, and the
injection valve is connected to the inlet of the chromatographic
separation column, by means of conduit (e.g., tubing), as
illustrated in FIG. 5.
[0041] The chromatography system components mentioned hereinabove
and variations thereof are well known in the chromatography art and
described in detail in references cited hereinbelow.
[0042] In a preferred embodiment of the invention, the mixture of
polynucleotide fragments is separated by Matched Ion Polynucleotide
Chromatography (MIPC). The term "Matched Ion Polynucleotide
Chromatography" as used herein is defined as a process for
separating single and double stranded polynucleotides using
non-polar, wide pore separation media, wherein the process uses a
counterion agent, and an organic solvent to release the
polynucleotides from the separation media.
[0043] The pores of the separation media may be contiguous, i.e.,
extend from one surface of the media to another surface of the
media. The pores of the separation media may also be
non-contiguous, i.e., extend into the media at one point on the
surface but not through to another point on the surface.
[0044] The non-polar, wide pore separation media can be an
inorganic substrate, including silica, zirconia, alumina, or other
material; or can be polymeric, including crosslinked resins of
polystyrene, polyacrylates, polyethylene, or other organic
polymeric material. The non-polar, wide pore separation media can
also be a "rod column" or "monolith column". Such columns contain
silica or polymer separation media which have been formed inside
the column as a continuous structure which has through pores or
interstitial spaces which allow eluting solvent and analyte to pass
through. The only requirement for the non-polar, wide pore
separation media is that they must have a surface that is either
intrinsically hydrophobic or be bonded with a material that forms a
surface having sufficient hydrophobicity to interact with a
counterion agent.
[0045] As used herein, the term "non-polar, wide pore separation
medium" is defined to denote any material which has surface pores
having a diameter that is greater than 30 Angstroms or the
approximate size and shape of the smallest polynucleotide fragment
in the separation in the solvent medium used therein or greater,
and is capable of separating polynucleotide fragments. Although the
non-polar, wide pore separation medium may be any shape, those
comprising alkylated wide pore polymer beads bead having an average
diameter of 1-100 microns are preferred. Such particles are
described in the references cited hereinbelow.
[0046] Regardless of the source or composition of the non-polar,
wide pore reverse phase separation media described above, special
precautions are taken to ensure that they are substantially free of
multivalent cation contaminants. For example, the separation media
are washed with acid followed by methanol to ensure removal of
residual multivalent cation contaminants. The separation media can
also be washed with EDTA or other chelating agent.
[0047] The concepts, materials, systems and methods related to
chromatography on non-polar, wide pore separation media are well
known and are described in detail in the following references:
Chromatography Today, by Colin F. Poole and Salwa K. Pool, Elsevier
(1991); Introduction to Modern Liquid Chromatography, L. R. Snyder
and J. J. Kirland, J. Wiley and Sons, Inc. (1979). These references
and references contained therein are incorporated in their entirety
herein.
[0048] Non-polar, wide pore separation media and their use for the
separation of polynucleotide mixtures are well known in the art and
are commercially available, e.g., Hamilton HPLC Application
Handbook, (1993), Hamilton Company, Inc., 4970 Energy Way, Reno,
Nev. 89502. This, and references contained therein, are
incorporated in their entirety herein. Another reference, which is
incorporated in its entirety herein, describing polynucleotide
separations on non-polar, wide pore separation media is
Chromatography, 5.sup.th edition, Part B, edited by E. Heftmann,
Elsevier (1992). Separation of tRNA and DNA fragment mixtures on
non-polar, wide pore silica particles is described by R. Bischoff
and L. W. McLaughlin, Analytical Biochemistry, 155, 526-533 (1985)
and S. Eriksson, et. al., J. Chromatography, 359, 265-274
(1986).
[0049] Monolith or rod columns are commercially avialable form
Merck & Co (Darmstadt, Germany) and described in the following
references: U.S. Pat. No. 5,453,185 to J. M. J. Frechet and F.
Svec; M. Petro, et. al., Analytical Chemistry, 68, 315-321 (1996).
The references cited above and the references contained therein are
incorporated in their entirety herein.
[0050] The components of the liquid chromatography system have
surfaces (i.e., "process solution contacting surfaces") which
contact process solutions held within the components (e.g., the
eluting solvent supply means) or flowing through the components
(e.g., the porous frits, chromatographic column, injection valve,
and conduits). The term "process solution" as used herein refers to
any solution (such as the polynucleotide mixture solution and the
eluting solvent) which is contained within or flows through any
component of the liquid chromatography system during the
chromatographic process. The term "process solution contacting
surface" refers to any surface of a liquid chromatography system
which contacts said process solutions.
[0051] The process solution contacting surfaces of the porous frits
on either end of the separation column must be made of material
which does not release multivalent cations into solutions flowing
through the column, or collect said cations from other sources. The
material is preferably titanium, coated stainless steel, or organic
polymer, or combinations thereof, but is most preferably acid
treated titanium as described hereinbelow. The term "coated
stainless steel" as used herein refers to stainless steel that has
been coated so that it does not release, or is prevented from
releasing, multivalent cations. A non-limiting example of a coating
material is polytetrafluoroethylene (i.e., Teflon.RTM.). "Coated
stainless steel" as used herein also refers to stainless steel that
has been pre-treated with an agent such as EDTA or phosphoric acid
which forms coordination complexes with multivalent metal ions.
[0052] "Passivated stainless steel" as used herein refers to
stainless steel that has been treated with an agent that removes
oxidized metals and also metals that are easily oxidized such as
iron. The most common passivating agent for stainless steel is
nitric acid. Nitric acid will removed any oxidized metals, but will
also remove iron that is located on the surface of the metal,
leaving other metals such as chromium and nickel. Some chelating
agents can coat and passivate. EDTA will first coat oxidized metals
especially colloidal iron oxide particles. As treatment continues,
the EDTA will bind and dissolve the iron oxide. However, as
individual iron molecules leave the particle, other chelating
molecules must coat the newly exposed surfaces for the surface to
remain suitable for polynucleotide separations. A chelating agent
does not passivate in the sense that it will only coat metal ions
for which it is specific and will not dissolve non-oxidized metals.
However, a chelating agent may, eventually, dissolve oxidized
metals.
[0053] The chelating agents used depend upon the type of ion
contamination which is present. For example, Tiron chelating agent
is selective for titanium and iron oxides. EDTA is selective for
most metal oxides at pH 7. Other chelating agents include
cupferron, 8 hydroxyquinoline, oxine, and various iminodiacetic
acid derivatives. If the chelating agents are to be used as
passivating reagents as well as coating reagents, then it is
important that the metal ion chelate complex, for example,
EDTA-metal ion complex, is soluble in the fluid. Chelating agents
that form insoluble complexes, for example 8-hydroxyquinoline,
perform coating functions only.
[0054] Without wishing to be bound by theory, it is believed that
oxidized and positively charged metals, such as oxides of iron on
the surface of stainless steel can trap negatively charged
molecules such as DNA leading to degradation of the chromatographic
separation, and that the pre-treatment masks or shields these
surface charges. EDTA can be added, for example, in an amount
sufficient to shield any surface sites which would interfere with
the chromatographic separation. In one embodiment, a solution of a
metal chelating agent such as EDTA can be applied in a batch
process to coat the surface, for example by a single injection of
EDTA solution into the HPLC system. In another embodiment, EDTA is
included as an additive in the eluting solvent in an amount
sufficient to complex the metal ions present.
[0055] Other components of the liquid chromatography system are
preferably titanium, coated stainless steel, or organic polymer
such as polyetherether ketone (PEEK) or polyethylene. The preferred
system tubing (i.e., conduit) is titanium, PEEK, or other polymeric
material, with an inner diameter of 0.007". The preferred eluting
solvent inlet filters are composed of non-polar, porous,
non-stainless steel material, which can be PEEK, polyethylene, or
other polymeric material. The preferred solvent pump is also made
of a non-stainless steel material; the pump heads, check valves,
and solvent filters are preferably titanium, PEEK, or other
polymeric material. The preferred means for removing oxygen from
the eluting solvent is an inline degasser placed prior to the pump
inlet. The sample injection valve is also preferably titanium,
PEEK, or other polymeric material. A standard detector and eluting
solvent reservoirs can be used, with no modifications
necessary.
[0056] Materials such as titanium, PEEK and other organic polymers
such as polyethylene, have been generally considered to be inert
and preferred for the chromatographic separation of biological
molecules. We have discovered that these materials, while inert for
the prior art processes, can be a source of contaminants which
interfere with the chromatographic separation of polynucleotides on
non-polar, wide pore separation media. We have also observed that
the interference with separation of polynucleotides by these
materials becomes more apparent during separations carried out at
elevated temperatures, e.g. 56.degree. C. as compared to 50.degree.
C.
[0057] In a preferred embodiment of the present invention, all of
the process solution-contacting surfaces are subjected to a
multivalent cation removal treatment to remove any potential source
of multivalent cation contamination. These surfaces include the
column inner surface, porous frits, conduits, eluting solvent
supply system, injector valves, mixers, pump heads, and fittings. A
non-limiting example of a multivalent cation removal treatment is
an acid wash treatment. This wash treatment can include flushing or
soaking and can include sonication. An example of an acid wash
treatment is sonication of a PEEK or titanium frit in the presence
of aqueous nitric acid solution, followed by sonication in water
until a neutral pH is achieved. Other treatments include contacting
the surfaces with chelating agents such as EDTA, pyrophosphoric
acid, or phosphoric acid (e.g. 30% by weight phosphoric acid).
[0058] PEEK and titanium can be treated with dilute acids including
nitric and hydrochloric acids. PEEK is not compatible with
concentrated sulfuric or concentrated nitric acids. Titanium is not
compatible with concentrated hot hydrochloric acid. Treatment with
a chelating agent can be performed before, but preferably after
treatment with an acid. 20 mM tetrasodium EDTA is a preferred
chelating agent treatment.
[0059] The preferred treatment for titanium frits is sonication for
10 minutes with cold hydrochloric acid, sonication with water until
neutral pH, 2 hour sonication with 0.5 M tetrasodium EDTA, storage
several days in 0.5 M tetrasodium EDTA, sonication with water until
neutral pH, and then washing with methanol, followed by drying.
Preferred treatment for PEEK frits is sonication for 15-30 minutes
each with THF, concentrated hydrochloric acid, 20% nitric acid,
sonication with water until neutral pH, and then washing with
methanol, followed by drying. Although this is a preferred
treatment method, the effectiveness of this treatment of PEEK frits
can depend on the vendor and lot of material treated. The success
of the treatment also depends on the temperature of the
polynucleotide separation with higher column temperatures requiring
the most complete removal of contamination. If the ionic
contaminant is organic, then organic solvents or a combination of
organic solvents and acids can be used. Also, organic ionic
contaminants can require detergents, soaps or surfactants for
removal from the surface. Nonionic contaminants such as greases and
oils will also contaminate the separation column, generally leading
to poor peak shape, but depending upon the size of the fragment.
Nonionic organic contaminants such as oils will require detergents,
soaps or surfactants to remove. Column tubing can be treated under
sonication with Decalin (D5039, Sigma) to remove silicon greases
and oils. Removal of colloidal metal oxides such as colloidal iron
oxide can require repeated or continuous treatment as the surface
of the particle is dissolved and new metal oxides are exposed.
[0060] The preferred embodiment of the liquid chromatography system
of the present invention utilizes methods to minimize the exposure
of all process solution contacting surfaces to oxygen. Dissolved
oxygen within the eluting solvent, for example, can react with
exposed metals on these surfaces to form oxides which will
interfere with the chromatographic separation.
[0061] The liquid chromatography system preferably employs a
degassing method for essentially removing dissolved oxygen from the
eluting solvent prior to contact with the rest of the
chromatography system. Examples of degassing methods include
sparging of the eluting solvent with an inert gas such as argon or
helium, or filtering the eluting solvent under vacuum. A preferred
method uses a vacuum type degasser which employs inline passage of
the eluting solvent over one side of an oxygen permeable membrane
system where the other side is subjected to a vacuum. An example of
a suitable four channel vacuum type degasser is Degaset.TM., Model
6324 (MetaChem Technologies, Torrance, Calif.).
[0062] In another embodiment of the invention, a stainless steel
HPLC system can be used if a component for removing multivalent
cations, herein referred to as a "multivalent cation capture
resin," is also used. A multivalent cation capture resin is
preferably a cation exchange resin or chelating resin. Any suitable
cation exchange resin or chelating resin can be used. Preferred
cation exchange and chelating resins are described hereinbelow.
[0063] Cation exchange resins having an ion exchange moiety
selected from the group consisting of iminodiacetate,
nitriloacetate, acetylacetone, arsenazo, hydroxypyridinone, and
8-hydroxyquinoline groups are particularly preferred. Cation
exchange resins having hydroxypyridinone groups are especially
useful for removing iron from the system. Cation exchange resins
having iminodiacetate groups are particularly preferred for use in
the present invention because of their wide availability in resin
format.
[0064] A chelating (i.e., coordination binding) resin is an organic
compound which is capable of forming more than one non-covalent
bond with a metal. Chelating resins include iminodiacetate and
crown ethers. Crown ethers are cyclic oligomers of ethylene oxide
which are able to interact strongly with alkali or alkaline earth
cations and certain transition metal cations. A cavity in the
center of the molecule is lined with oxygen atoms which hold
cations by electrostatic attraction. Each ether has a strong
preference for cations whose ionic radius best fits the cavity.
[0065] The multivalent cation capture resin is preferably contained
in a guard column, guard cartridge, or guard disk. Guard columns
and cartridges are frequently used to protect liquid chromatography
columns from contamination and are widely available. In their
normal use, guard columns and cartridges typically contain packing
material which is similar to the stationary phase of the separation
column. However, for use in the present invention, the guard column
or cartridge must contain a multivalent cation capture resin. The
guard disc or guard column must contain particles which trap the
metal ions.
[0066] For use in the system of the present invention, the guard
cartridge or column should be sufficiently large to provide
adequate cation capture capacity, but must be small enough to allow
effective gradient elution to be used. A preferred guard cartridge
has a void volume of less than 5 mL, more preferably, less than 1
mL, so that the eluting solvent gradient is not delayed by more
than 5 minutes and, preferably, less than 1 minute. The preferred
cartridge has a 10.times.3.2 mm bed volume.
[0067] Guard disks are described in detail in U.S. Pat. No.
5,338,448, which is incorporated herein by reference in its
entirety. For use in the present invention, a guard disk comprises
a layer or pad of a multivalent cation capture resin which has been
incorporated into a fabric or membrane so that the resin is not
separable from the guard disk under liquid flow conditions present
during the performance of chromatographic separations.
[0068] In its preferred form, the guard disk is circular, having a
rigid annular outer ring or collar for easy handling. The annular
ring can be constructed of any suitable material which is inert to
the chromatographic separation, such as inert conventional
engineering plastic. The only requirement for the material is that
it must be inert to the eluting solvent and sample and have
sufficient dimensional stability. The rigid annular outer ring of
the guard disk can comprise a single rigid annular outer ring
encircling a disk-shaped pad of guard disk material. As used
herein, the term "guard disk material" refers to a layer or pad of
multivalent cation capture resin which has been incorporated into a
fabric or membrane.
[0069] As shown in FIG. 1, one or more pads of guard disk material
2 are placed in the rigid annular ring 4. For example, the fabric
can be cut to a circular diameter which securely contacts the inner
diameter surface of the annular ring. As the disk holder is
tightened against the disk, the top and bottom surfaces of the
holder seal against the collar of the guard disk. Sealing pressure
from the guard disk holder is, therefore, applied against the
collar of the disk which prevents the material of the guard disk
pad from being crushed.
[0070] Alternatively, the rigid annular outer ring can comprise two
flanged rings, as shown in FIGS. 2 and 3, an outer flanged ring 6
and an inner flanged ring 8, where the inner flanged ring is
insertable within the flange of the outer ring, forming a press-fit
two-piece collar around one or more pads of guard disk material 10.
Preferably, the inner diameter (a) of the inner flanged ring will
have the same diameter as the separation column bed.
[0071] In the two-piece annular ring embodiment shown in FIG. 3,
one or more pads of guard disk material 10 having a diameter
greater than the inner diameter (b) of the outer flanged ring 6 are
positioned within the flanges of the outer ring. The inner flanged
ring 8 is then inserted into the outer ring to form a press-fit
two-piece annular ring in which the guard disk pad(s) is (are)
frictionally held within the press-fit ring or collar. Preferably,
the inner diameter (b) of the outer flanged ring and the inner
diameter (a) of the inner flanged ring are substantially the
same.
[0072] Alternatively, the rigid annular outer ring can be
incorporated into the guard disk holder or chromatographic column
cap. The annular ring is a flange that is part of one or both sides
of the disk holder or the column cap. In this case, the guard disk
does not have an outer ring. A circle of the guard disk sheet
material is placed into the holder or column cap. The flange in the
holder column cap is annular so that, when the holder or column cap
is tightened, the flange pinches or seals the outer annular portion
of the guard disk. The center portion of the guard disk not pinched
is in a chamber or depression in the holder or cap. Fluid flows
through the center portion, allowing the guard disk to retain
particulate or strongly adsorbed material, but fluid cannot flow
around the disk or past the edges. The function of the guard disk
is exactly the same as when the collar is part of the guard disk
itself. However, in this case, the collar is part of the holder or
column cap.
[0073] In a most preferred embodiment of the invention, a
multivalent cation capture resin contained in a guard column, guard
cartridge, or guard disk is placed upstream of the separation
column. Most preferably, the guard column, cartridge, or disk
containing the resin is placed upstream of the sample injection
valve. Although this is preferably a guard disk, a guard cartridge
or column can be used as long as the dead volume of the cartridge
or column is not excessive and an effective eluting solvent
gradient can be produced.
[0074] Optimally, a guard disk, column, or cartridge can be placed
before the injection valve and a second guard disk, column, or
cartridge also placed between the sample injection valve and the
separation column. In certain cases, the second guard disk (or
cartridge or column) can be avoided if the contaminants are
sufficiently cleaned by a guard column placed upstream of the
injection valve, or if the contaminants are avoided through the use
of non-metal or titanium components throughout the HPLC system.
[0075] Placement of a chelating guard column and chelating guard
disk in a liquid chromatography system for polynucleotide
separation is illustrated in FIG. 4. The eluting solvent reservoirs
12 contain eluting solvent inlet filters 14 which are connected to
the solvent pump 16 by system tubing 18. The solvent pump 16 is
connected to a chelating column 20 by system tubing 18. The
chelating column 20 is connected to the sample injection valve 22
by system tubing 18. The sample injection valve has means for
injecting a sample (not shown). The sample injection valve 22 is
connected to a chelating guard disk 24 by system tubing 18. The
chelating guard disk 24 is connected to the inlet (not shown) of
the separation column 26 by system tubing 18. Detector 28 is
connected to the separation column 26. As discussed above, the
system tubing, eluting solvent inlet filters, solvent pump, sample
injection valve, and separation column are preferably made of
titanium, coated stainless steel, or organic polymer. The material
is preferably treated so that it does not release multivalent
cations. The treatment can include treatment with nitric acid,
phosphoric acid, pyrophosphoric acid, or chelating agents. In
cases, where components of the HPLC do not release metal ion
contaminants and are suitable for polynucleotide separations in
general and MIPC in particular, then use of the chelating cation
exchange guard column or guard disc is not necessary.
[0076] In operation, eluting solvent from the eluting solvent
reservoirs 12 is pumped through eluting solvent inlet filters 14 by
solvent pump 16. By way of system tubing 18, the eluting solvent
stream flows through chelating column 20, through sample injection
valve 22, through chelating guard disk 24, then into separation
column 26. Detector 28 is located downstream from separation column
26.
[0077] FIG. 5 illustrates a specific embodiment of the invention in
which a chelating guard disk is placed in direct contact with a
titanium frit at the top portion of a chromatographic separation
column. Column top 30 has conventional fittings for receiving
eluting solvent and sample through inlet tubing 32. The column top
or cap 30 is fitted and sealably attached to column body 34
containing chromatographic bed 36 using a conventional fitting 38
(e.g., threaded) or any equivalent fitting capable of tightly
sealing the column top to the column body. The column top 30 is
adapted to receive the chelating guard disk 40 in a sealing cavity
42. In this embodiment, the guard disk 40 is in direct contact with
a titanium column frit 44, which is located at the upstream end of
the column body 34 to prevent disturbance of the chromatographic
bed 36 when the column top 30 is removed to observe the guard
disk.
[0078] In operation, solvent pump 46 pumps elution solvent to
sample injection valve 48 into column top 30 through chelating
guard disk 40 and then through titanium frit 44 before entering
chromatographic bed 36. Eluting solvent pressure upstream from the
guard disk is measured by pressure transducer 50 which is
electrically connected to a display device 52.
[0079] As discussed above, a chelating guard column, cartridge, or
disk can be used in conjunction with a conventional, stainless
steel liquid chromatography system, or with a system containing
non-metal or titanium components in order to provide extra
protection against ionic contaminants. For additional column
protection, an eluting solvent containing 0.1 mM tetrasodium EDTA
or other chelating solution can be used during the performance of
polynucleotide separations.
[0080] In another aspect of the invention the non-polar, wide pore
separation media have been washed to remove any traces of residual
multivalent metal cations from the surface thereof. Preferred
washing solvents comprise tetrahydrofuran, hydrochloric acid, and
water. An example of a preferred washing procedure is described in
Example 1.
[0081] The methods of the invention comprise using the improved
systems described above to separate mixtures of polynucleotide
fragments, particularly double-stranded polynucleotide fragments.
The methods of the present invention can be used to separate
polynucleotide fragments having up to about 1500 base pairs using
non-polar, wide pore separation media under the chromatography
conditions described herein.
[0082] The most preferred method of the invention comprises
contacting a solution of a mixture of polynucleotide fragments
containing a counterion agent with a multivalent metal cation
capture resin, followed by application of said solution to a
separation column containing non-polar, wide pore separation media
wherein said particles have been washed to remove any traces of
residual multivalent cation therefrom. Optimally, the process
solution contact surfaces of the system have been passivated, as
described hereinabove, to remove multivalent metal cations
therefrom. In an optimum configuration, a guard cartridge or guard
column containing multivalent cation capture resin is placed at the
front of the column or in line between the eluting solvent
reservoir and the solvent pump(s) to protect said column and the
separation media contained therein from any traces of residual
multivalent metal cations in the eluting solvent. The
polynucleotide fragments are separated by releasing said fragments
from the separation media using an eluting solvent comprising an
organic component, water, and a counterion agent. The separation of
the polynucleotide components is based on the size or polarity of
the fragments. By way of example only, the fragments are released
from the separation media in order of size by increasing the
concentration of organic component in the eluting solvent. The
concentration of the organic component can be increased in stepwise
fashion by means of a step gradient, or continuously, by means of a
continuous gradient.
[0083] The methods used to capture multivalent cations and prevent
their presence in the chromatography system, are essential in order
to achieve high resolution separations of polynucleotides,
especially double stranded DNA, and also to greatly extend the
useful life of the separation media. Evidence demonstrating the
detrimental effect of multivalent metal cation contamination on the
chromatographic separation of both single stranded polynucleotide
fragments and double stranded polynucleotide fragments on
non-polar, wide pore silica and polymer separation media is
presented in Examples 2-7 and FIGS. 6-15.
[0084] Example 2 describes a polynucleotide fragment separation on
non-polar, wide pore organic polymer separation media using the
optimized method of the invention compared to a deliberate
contamination of the system with multivalent metal cations. While a
sharp peak is obtained using the optimized method of the invention,
the peak is completely absent when the chromatography system was
deliberately contaminated with multivalent metal cations.
[0085] Example 3 is identical to Example 2, except that non-polar,
wide pore silica separation media were used in the
chromatography.
[0086] In Example 4, a series of three separations of a 500 base
pair DNA fragment was performed using non-polar, wide pore polymer
separation media are described. In the first separation, the
separation media was washed to remove multivalent cations as
described in Example 1 prior to sample injection, and a sharp peak
was obtained as shown in FIG. 6. Deliberate contamination of this
separation media with Cr(III) cations resulted in a complete loss
of the sample peak as shown in FIG. 7. Treatment of the separation
particles with EDTA solution to remove the Cr(III) and any other
ions which may have been present, partially restored the sample
peak as shown in FIG. 8.
[0087] Example 5 describes an sequence similar to Example 4, except
that non-polar, wide pore silica separation media was used and
washed with EDTA solution prior to sample injection. Once again, a
sharp sample peak was obtained, as shown in FIG. 9, when the sample
was injected onto a column containing cleaned separation media.
Deliberate contamination of the separation media with Cr(III)
resulted in complete loss of the sample peak, as shown in FIG. 10.
Injection of EDTA solution to remove Cr(III), or other multivalent
cations, followed by injection of the 500 base pair DNA sample,
partially restored the sample peak as shown in FIG. 11.
[0088] The deleterious effect of multivalent metal cations on the
chromatographic separation of a 20 mer single stranded DNA standard
is described in Example 6 and shown by the complete loss of
resolution as seen in FIG. 13 (after deliberate Cr(III)
contamination) compared to FIG. 12 (column cleaned with EDTA).
[0089] The deleterious effect of even trace levels of multivalent
cations on demanding chromatographic separation is described in
Example 7 and shown in FIGS. 14 and 15. A standard 4 component
mixture of double stranded DNA consisting of two 209 base pair
homoduplex fragments and two 209 base pair heteroduplex fragments
were chromatographed at 56.degree. C. as described in Example 6 on
a freshly packed column DNASep column (Transgenomic, Inc., San
Jose, Calif.). FIG. 14 shows only partial resolution of the 4
component mixture. However, when the column was treated with EDTA
solution followed by re-injection and elution of the 4 component
homoduplex/heteroduplex DNA mixture as described in Example 6, a
clean separation of all 4 components was achieved, as seen in FIG.
15. This result clearly indicates that trace levels of multivalent
cations were present in a freshly packed column, and that said
cations interfered with the separation of double stranded DNA
fragments.
[0090] The method of the invention can also be used to separate
polynucleotide mixtures in a batch process useful for production
and isolation of pure polynucleotide fragments of a plurality of
selected sizes, on a small or large scale. The method of the
invention comprises contacting a solution of a mixture of
polynucleotide fragments containing a counterion agent with a
multivalent metal cation capture resin, followed by applying said
solution to non-polar, wide pore separation media. The separation
media are held in a container. The container may be a column, a
membrane, a container, or a web. The polynucleotide mixture is held
on the separation media since the concentration of the organic
component of the solvent in which the mixture is dissolved is not
sufficient to release the polynucleotide fragments therefrom. The
separation media are then contacted with a first eluting solvent
and a counterion agent, said first eluting solvent having a
concentration of the organic component sufficient to remove all
polynucleotide fragments from the separation media which are
smaller than the selected size. The eluting solvent is then
separated from the separation media. The separation media are
rinsed with the first eluting solvent to remove any remaining
released polynucleotides. The separation media are then contacted
with a second eluting solvent and counterion agent, said second
eluting solvent having a concentration of the organic component
sufficient to release the polynucleotide fragment having the
selected size from the separation media. The second eluting solvent
is separated from the separation media and the particles are rinsed
with the second eluting solvent. This process can be repeated to
release polynucleotide of any selected size which are present in
the mixture.
[0091] Specific eluting solvent compositions required to elute
polynucleotide fragments of any specific base pair length can be
determined experimentally. For example, isolation of a 102 base
pair fragment from the polynucleotide mixture may be desired, and
said fragment may be eluted with 15.9% acetonitrile-water-0.1M
triethylammonium acetate. In this example, the separation media
holding the polynucleotide mixture may be contacted with 14.6%
acetonitrile-water-0.1M triethylammonium acetate to remove reaction
mixture reagents and additives, as well as all fragments having
less than 102 base pairs. Increasing the acetonitrile concentration
to 15.9% followed by contact of this eluting solvent with the
non-polar, wide pore separation media will release the desired 102
base pair fragment, leaving larger fragments still attached to the
separation media. The desired polynucleotide fragment dissolved in
the eluting solvent is isolated by separating the eluting solvent
from the separation media by filtering, decanting, centrifuging, or
any other compatible liquid/solid separation technique. By using a
step gradient of increasing acetonitrile concentration, larger
particles may be removed in discreet base pair lengths from the
separation media and isolated by repeating the procedure described
hereinabove.
[0092] In a most preferred embodiment of the batch process of the
invention, all the methods and procedures used to remove traces of
multivalent cations from solvents and surfaces which contact
process solution are identical to the methods and procedures
described in separation method of the invention described
hereinabove. All of process solution contract surfaces are of
materials which do not release multivalent cations. Said materials
are identical to those described in the separation method of the
invention hereinabove.
[0093] The methods used to capture multivalent cations and prevent
their presence in the batch process described hereinabove, are
essential in order to achieve high resolution separations of
polynucleotides, especially double stranded DNA, and also to
greatly extend the useful life of the separation media.
[0094] The concepts, materials, systems and methods related to
chromatographic separations on non-polar, wide pore separation
media are well known and are described in detail in the following
references: Chromatography Today, by Colin F. Poole and Salwa K.
Pool, Elsevier (1991); Introduction to Modern Liquid
Chromatography, L. R. Snyder and J. J. Kirland, J. Wiley and Sons,
Inc. (1979). These references and references contained therein are
incorporated in their entirety herein.
[0095] Non-polar, wide pore separation media and their use for the
separation of polynucleotide mixtures are well known in the art and
are commercially available, e.g., Hamilton HPLC Application
Handbook, (1993), Hamilton Company, Inc., 4970 Energy Way, Reno,
Nev. 89502. This, and references contained therein, are
incorporated in their entirety herein. Another reference, which is
incorporated in its entirety herein, describing polynucleotide
separations on non-polar, wide pore reverse phase particles is
Chromatography, 5.sup.th edition, Part B, edited by E. Heftmann,
Elsevier (1992). Separation of tRNA and DNA fragment mixtures on
non-polar, wide pore silica particles is described by R. Bischoff
and L. W. McLaughlin, Analytical Biochemistry, 155, 526-533 (1985)
and S. Eriksson, et. al., J. Chromatography, 359, 265-274 (1986).
Regardless of the source or composition of the non-polar, wide pore
separation media, precautions are taken to ensure that they are
free of multivalent cation contaminants. For example, the
separation media are washed with acid followed by methanol to
ensure removal of residual multivalent cation contaminants.
[0096] 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 been carried out in the laboratory, and
are constructively reduced to practice with the filing of this
application.
EXAMPLE 1
Acid Wash Treatment to Remove Multivalent Metal Cation
Contaminants
[0097] The non-polar, wide pore reverse phase separation media were
washed three times with tetrahydrofuran, then two times with
methanol. The non-polar, wide pore separation media were then
stirred for 12 hours with a mixture containing 100 mL of
tetrahydrofuran and 100 mL of concentrated hydrochloric acid.
Following this acid treatment, the non-polar, wide pore separation
media were washed with tetrahydrofuran/water (1:1) until neutral
(pH 7). The non-polar, wide pore separation media were then dried
at 40.degree. C. for 12 hours.
EXAMPLE 2
Standard Procedure for Demonstrating the Effects of Colloidal Iron
on Non-Polar, Wide Pore Polymer Separation Media
[0098] Non-polar, wide pore PRX-1 separation media (Sarasep, Inc.
San Jose, Calif.) of polystyrene/divinylbenzene polymer having a
pore size of 50-200 Angstroms (average pore size is 80 Angstroms)
and a bead diameter of 5 microns are washed as described in Example
1 and packed in a 4.6.times.50 mm HPLC column. A sample (5 .mu.L,
20 ng) of 80 base pair DNA standard solution from purified pUC18
DNA Hae III restriction enzyme digest (Sigma-Aldrich, D6293) is
injected onto the column. The chromatography is conducted under the
following conditions: Eluting solvent A: 0.1 M Triethylammonium
acetate (TEAA), pH 7.2; Eluting solvent B: 0.1 M TEAA, 25%
acetonitrile; Gradient:
1 Time (min) % A % B 0.0 65 35 3.0 45 55 10.0 35 65 14.0 0 100 16.0
65 35
[0099] The flow rate is 0.75 mL/min, UV detection at 260 nm, column
temp. 51.degree. C. A peak for the 80 base pair DNA fragment is
obtained. Some columns, depending on the packing volume and packing
polarity, may require longer time for elution of some changes in
the driving solvent concentration.
[0100] A 0.05M aqueous solution of Fe(Cl).sub.3 is prepared and
allowed to stand at ambient temperature for four hours. A 100 .mu.L
sample of the resulting colloidal iron suspension is injected onto
the column and allowed to stand for five minutes. Subsequent
injection of 5 .mu.L of the above described 80 base pair DNA
standard solution followed by the identical gradient elution
conditions described above, shows a complete absence of any
peak.
EXAMPLE 3
Standard Procedure for Demonstrating the Effects of Colloidal Iron
on Non-Polar, Wide Pore Silica Separation Media
[0101] INERTSIL (MetaChem, Torrance, Calif.), a 51 .mu.m C-18
non-polar, wide pore separation medium having 200 Angstrom pores
was packed in a 4.6.times.50 mm HPLC column and cleaned with 5
injections of 0.1M Na.sub.4EDTA. A 80 base pair DNA standard (5
.mu.L) is injected and eluted as described in Example 2. A peak for
the 80 base pair DNA standard is obtained.
[0102] An injection of the colloidal iron suspension is made as
described in Example 3. Subsequent injection of the 80 base pair
DNA standard and elution as described in Example 3 shows a complete
absence of any peak.
EXAMPLE 4
Standard Procedure for Demonstrating the Effects of Chromium(III)
on the Separation of Double Stranded DNA Using Non-Polar, Wide Pore
Polymer Separation Media
[0103] Non-polar, wide pore PRX-1 separation media (Sarasep, Inc.,
San Jose, Calif.) of polystyrene/divinylbenzene polymer having a 5
micron diameter and a pore size of 50-200 Angstroms (80 Angstrom
average pore size) was washed as described in Example 1 and packed
in 4.6.times.50 mm HPLC column. A sample (5 .mu.L, 20 ng) of 500
base pair DNA standard solution from GeneAmp.sup.R Lambda Control
Reagent, N808-0008, (Perkin Elmer, Foster City, Calif.) was
injected onto the column. The chromatography was conducted under
the following conditions: eluting solvent A; 0.1M triethylammonium
acetate (TEAA), pH 7.2; eluting solvent B; 0.1M TEAA, 25%
acetonitrile gradient:
2 Time (min) % A % B 0.0 60 40 14 0 100 17 60 40
[0104] The flow rate was 0.60 mL/min, UV detection at 260 nm,
column temperature 50.degree. C. A peak for the 500 base pair DNA
fragmetn was obtained, as shown in FIG. 6.
[0105] A 520 ppm aqueous solution of Cr(III) was prepared from
Cr.sub.3(SO.sub.4).sub.2.12H.sub.2O and injected onto the column.
The 17 min. solvent gradient was flowed through the column.
Injection of 5 .mu.L of the 500 base pair DNA standard onto the
column and elution as described above, showed a complete absence of
any peak (FIG. 7).
[0106] A subsequent column cleanup with 3 injections, 10 .mu.L
each, of 0.1M Na.sub.4EDTA was followed by equilibration to a
constant baseline. A re-injection of 5 .mu.L of the 500 base pair
DNA standard showed a reappearance of the a peak (FIG. 8) having an
area of about 50% of the original injection (FIG. 6).
EXAMPLE 5
Standard Procedure for Demonstrating the Effects of Chromium(III))
on the Separation of Double Stranded DNA Using Non-Polar, Wide Pore
Silica Separation Media
[0107] INERTSIL (MetaChem, Torrance, Calif.) 5 micron C-18
non-polar, wide pore (150 Angstrom pores) separation media was
packed into a 4.6.times.50 mm HPLC column and cleaned with 5
injections of 0.1M Na.sub.4EDTA. A 500 base pair DNA standard was
injected onto the column and eluted as described in Example. 4. A
peak fo rthe 500 bas pair standard is shown in FIG. 9.
[0108] Injection of the Cr(III) solution as described in Example 4
followed by elution as described in Example 4, showed a complete
absence of a peak as shown in FIG. 10.
[0109] Three 10 mL injections of Na.sub.4EDTA as described in
Example 4 follwed by reinjection of the 500 base pair DNA standard
and elution as described in Example 4, showed a peak (FIG. 11)
corresponding to the 500 base pair DNA standard
EXAMPLE 6
The Effect of Chromium(III) on the Separation of Single Stranded
DNA Using Non-Polar, Wide Pore Silica Separation Media
[0110] The column described in Example 5 was cleaned with
Na.sub.4EDTA as decribed in Example 5 and equilibrated with 60%A
eluting solvent to a constant bse line. A 20 mer, single stranded
DNA standard (Seq2A purchased from CTGen, Milpitas, Calif.) was
injected onto the column and eluted with the gradient protocol of
Example 4. FIG. 12 shows a major peak corresponding to the 20 mer
standard and some well resolved impurity peaks.
[0111] A single 5 mL injection of the Cr(III) solution described in
Example 4 was followed by re-injection of the 20 mer standard and
elution using the gradient protocol of Example 4. The results seen
in FIG. 13 show a greatly diminished peak corresponding to the 20
mer, and essentially no resolution of the impurities.
[0112] This example clearly show that metal contamination has a
negative effect on the chromatography of single stranded DNA but
not to the same extent as it has on double stranded DNA.
[0113] An additional injection of 5 .mu.L onto the column followed
by another injection of the 20 mer standard as described above, did
result in complete elimination of the 20 mer peak. However, after
10 injections of 0.1M Na.sub.4EDTA to remove metal contamination,
as described above, followed by another injection of the 20 mer
standard, did restore the peak corresponding to the 20 mer
standard. However, the peak shape was distorted and broad.
EXAMPLE 7
The Effect Of Metal Contamination on the Separation of
Heteroduplexes and Homoduplexes
[0114] A freshly packed DNASep column (Transgenomic, Inc., San
Jose, Calif.) was equilibrated with eluting solvent 50%A. A mixture
of DNA 209 base pair standard fragments (Transgenomic, Inc., San
Jose, Calif., Cat. No. 560012) consisting of 2 heteroduplex DNA
fragmetns and 2 homoduplex DNA fragments was injected (5 .mu.L)
onto the column. The mixture was eluted at a flow rate of 0.9
mL/min using the following gradient program:
3 Time (min) % A % B 0.0 50 5 05 47 53 4.0 40 60 5.5 0 100 6.5 50
50 8.5 50 50
[0115] When the chromatography was run at 50.degree. C. a normal
size based separation was observed, i.e., a single peak of
unresolved hetroduplex and homoduplex 209 base pair fragments was
observed.
[0116] FIG. 14 shows the results of a chromatography run at
56.degree. C., on a newly packed column. Three peaks are seen. The
low retention time pair corresponds to the two heteroduplexes. The
large higher retention time peak corresponds to the unresolved
homoduplex peaks. The lack of complete separation of all four
fragments indicated that the column, though freshly packed,
contained multivalent metal cation contaminants.
[0117] Column cleanup with 5, 10 .mu.L injections of 0.1M
Na.sub.4EDTA was followed by washing with 25% actonitrile, 5%
acetic acid, and equilibration to constant base line with eluting
solvent 50%A. Injection of the 209 fragment standard
homoduplex/heteroduplex mixture and elution with the gradient
protocol described above, gave complete separation of the mixture
into four peaks, i.e., two homoduplex and 2 heteroduplex peaks as
depicted in FIG. 15.
[0118] The results described in this example indicate that
multivalent metal cation contamination is more critical at higher
column temperatures, especially as related to the difficult
separation of homoduplex and heteroduplex fragments of identical
base pair length.
* * * * *