Method for rapid purifiction of nucleic acids using layered ion-exchange membranes

The present invention relates to a device method and kit for the convenient and rapid isolation and purification of nucleic acids, proteins, peptides, carbohydrates and oligosaccharides from heterogeneous biological samples. The device comprises a membrane assembly comprised of layers of microporous, polymeric membranes functionalized with ion-exchange groups. The device is reusable for like samples, relatively inexpensive compared to currently available separation techniques and is disposable, thereby avoiding cross-contamination of biological samples.

BACKGROUND OF THE INVENTION 
Processing of nucleic acids for further studies, including sequencing, 
hybridization or PCR (polymerase chain reaction), will require isolation 
and purification of the plasmid DNA from chromosomal DNA, RNA, proteins 
and other contaminants present in bacterial cells. Most of these 
manipulations will require highly purified plasmid DNA. 
A number of plasmid purification methods have been developed over the 
years. These methods include organic solvent extraction, separation 
according to buoyant density, size exclusion chromatography, and 
ion-exchange chromatography. 
Alkaline lysis/organic solvent extraction has been the classic method to 
obtain purified plasmid DNA. The procedure involves numerous extraction 
and precipitation steps using organic solvents such as chloroform or 
phenol. Although an excellent method for obtaining significant quantities 
of highly purified plasmid DNA, it has serious drawbacks. The procedure is 
tedious and lengthy. There is often organic solvent contamination in the 
final product that must be removed before further studies. The procedure 
also has significant safety concerns due to the use of noxious chemicals 
in the process. 
Separation according to buoyant density is another popular method of 
plasmid purification and provides high purity DNA. This method involves 
mixing a crude preparation containing the plasmid DNA with ethidium 
bromide dye and then over-layering the sample on top of a cesium chloride 
(CsCl) solution of higher buoyant density. This mixture is centrifuged at 
high speed to form a gradient of CsCl of increasing buoyant density. 
Macromolecules in the sample separate according to their buoyant density 
in the CsCl gradient, and a discreet band of plasmid DNA can be isolated 
from other contaminants, such as bacterial DNA. RNA is collected on the 
bottom of the tube as a pellet. However, this procedure is extremely time 
consuming, involving often 24 to 48 hours of centrifugation for a single 
sample, in order to form the CsCl gradient. Furthermore, the resulting 
band of isolated plasmid DNA is contaminated with ethidium bromide and 
residual CsCl and must be subjected to multiple organic washes prior to 
use. 
Chromatographic procedures are also typical methods for purifying plasmid 
DNA. These methods include size exclusion chromatography, where separation 
is based on the size and solution conformation differences between linear 
and circular DNA, and ion-exchange chromatography, where separation is 
based on charge density differences between nucleic acids and other 
contaminating macromolecules. 
Ion-exchange protocols have gained widespread acceptance as the method of 
choice for rapid isolation and purification of plasmid DNA. Using an 
appropriate buffer system, the anionic plasmid DNA is adsorbed to an 
ion-exchange support matrix which has been functionalized with anion 
exchange groups that bear a net positive charge under the buffer 
conditions used. The bound DNA can then be released from the ion-exchange 
surface by increasing the concentration of a suitable counter-ion, e.g., 
chloride (Cl.sup.-). Variations on anionic exchange separations are 
described, for example, in U.S. Pat. No. 4,997,932 (porous beads with an 
anion-exchange surface for purifying nucleic acid); U.S. Pat. No. 
4,935,342 (anion-exchange column); Warren and Merion, BioChrom. 3:118-126 
(1988) (purification of synthetic oligonucleotides by anion-exchange high 
performance liquid chromatography using DEAE resin columns). 
SUMMARY OF THE INVENTION 
The present invention relates to a device for the convenient and rapid 
isolation and purification of nucleic acids, proteins, peptides, 
carbohydrates and oligosaccharides and other biological molecules 
available to ion-exchange from heterogeneous biological samples. The 
device comprises a membrane assembly comprised of layers of microporous, 
polymeric membranes functionalized with ion-exchange groups. In preferred 
embodiments, the device is adapted for use with a syringe or centrifuge. 
The device is suitably designed for purification of microgram quantities 
in good yield and high level of purity. The device is reusable for like 
samples, is relatively inexpensive compared to currently available 
separation techniques and is disposable, thereby avoiding 
cross-contamination of biological samples. 
The present invention also relates to a method for isolation and 
purification of nucleic acids such as DNA and RNA from heterogeneous 
biological samples. It further relates to the rapid isolation and 
purification of plasmids from crude cell lysates resulting in high yields 
of bioreactive DNA suitable for further manipulation with a minimum of 
preparation. Proteins, peptides, carbohydrates and oligosaccharides can 
also be purified from heterogeneous biological samples according to the 
methods of the present invention. 
The present invention further relates, but is not limited to, a kit 
comprised of a device housing a multi-layer, microporous ion exchange 
membrane assembly and premeasured volumes of liquid or powdered reagents 
suitable for use with biological samples containing nucleic acids, 
proteins, peptides, or other bioactive molecules.

DETAILED DESCRIPTION OF THE INVENTION 
The device and method of the present invention are intended for use with a 
wide range of biological specimens, including, but not limited to, human 
bodily fluids and tissues, blood, urine, saliva, sperm, cell suspensions, 
bacterial cultures and virus particles, cells infected with viruses, 
tissue cultures and cell lines. The test sample may be prepared by known 
procedures, such as those used for lysing cells and/or viruses to obtain 
the nucleic acids. A standard methodology for lysis of cells is by 
alkaline extraction. Birnboim and Doly, Nucleic Acids Research, 1523-1523 
(1979) and Molecular Cloning--A Laboratory Manual, 2nd Edition, Sanbrook 
et al. (Eds.), Cold Spring Harbor Laboratory Press (1989). 
In order that the invention may be better understood, specific embodiments 
of devices for rapid isolation and purification of biomolecules will now 
be described in more detail with reference to the accompanying drawings. 
The foregoing and other objects, features and advantages of the invention 
will be apparent from the following more particular description of the 
preferred embodiments of the drawings in which like reference characters 
refer to the same parts throughout the different views. The drawings are 
not necessarily to scale, emphasis instead being placed upon illustrating 
the principles of the invention. 
FIG. 1A shows a device comprised of a circular housing 2 having an inlet 4 
for sample delivery and an outlet 6 for sample collection. The device can 
be attached to a syringe 8 containing the sample to be purified by a 
connector 12, such as a female Luer-Lok.TM.. Located within the housing 2 
is the membrane assembly 10 comprised of a plurality of multiporous 
membranes functionalized with ion-exchange groups. The internal surface of 
the housing is shown in FIG. 1B. Radial 3 and circumferential 5 channels 
contact the stacked membrane assembly 10 and provide support thereto and 
permit uniform flow of sample fluid over the membrane assembly. 
Membranes used in the present invention should have a pore size (e.g., 0.1 
to 12 microns) which facilitates high flow rate of sample under minimal 
pressure. The number of membranes stacked within the device should be 
restricted such that significant external mechanical force is not required 
to draw the sample through the device. The preferred number of membranes 
is from about one to about 20 layers, and preferably from one to about 10. 
The term "significant external mechanical force" is intended to mean a 
force which would require high pressure (e.g., high pressure liquid 
chromatography (HPLC)) to draw the sample through the membranes. One 
advantage of the present devices is that minimal backpressure is produced, 
thus, sample can freely flow through the membrane(s) with minimal positive 
or negative pressure. Although significant external pressure is not 
required, it should be understood that external pressure (i.e., 
centrifugal force, peristaltic pumps, HPLC or FPLC systems) may be used as 
a matter of experimental design or investigator choice. 
The membrane (described in further detail below) is functionalized with 
ion-exchange groups capable of binding positively or negatively charged 
substances. By choosing an appropriate ion-exchange group and compatible 
buffering solutions, selected substances contained in a heterogeneous 
specimen can be isolated and purified by being adsorbed by the support, 
followed by desorption with a suitable counter-ion. 
Sample fluid enters the inlet 4 and is directed toward the membrane 
assembly 10. Flow of sample into the membrane assembly 10 may be 
facilitated by positive pressure, such as that generated when pushing on 
the plunger of a syringe 8 attached to the inlet 4. Alternately, sample 
flow may be facilitated by negative pressure such as attaching the outlet 
6 to a vacuum source which draws the sample through the membrane assembly 
10. 
As the sample flows through the device, under appropriate conditions, the 
substance of interest is retained by adsorption to the ion-exchange 
surface. By adjusting the conditions for purification, the majority of 
contaminating substances will not be retained by the membrane, or will be 
adsorbed so loosely that they are easily displaced by additional sample 
buffer or by small increases in the counter-ion concentration insufficient 
to desorb the biomolecules of interest. After the contaminating substances 
are washed through the device, the retained material is released from the 
membrane by increasing the counter-ion concentration. The optimal 
adsorption and desorption conditions for a desired substance can be 
readily determined with a minimum of experimentation by one of ordinary 
skill in the art. 
In another preferred embodiment of the present invention, shown in FIGS. 2A 
and 2B, the device comprises a small, cylindrical cup 14 with an opening 
at the top 16 to receive sample fluid, and the bottom embracing the 
membrane assembly 10 with outlet 6. Sample cup 14 is placed in a 
collecting vessel 18 having an insertable cap 20 hinged to the upper open 
end of the collection vessel 18 and insertable in the top 16 of the cup 
14. As shown in FIG. 2B, the collecting vessel 18 is suitable for use in 
an ultracentrifuge. Under operating conditions, the sample cup 14 is 
filled with sample fluid and the cup 14/collecting vessel 18 combination, 
in closed position, can be placed into a centrifuge and subjected to 
centrifugal force sufficient to draw the sample fluid through the membrane 
assembly 10 into the collecting vessel 18. The initial filtrate containing 
contaminants is discarded, and the membrane is washed with an appropriate 
rinsing solution. This filtrate is also drawn through the membrane 
assembly 10 by centrifugal force, collected and discarded. The desired 
substance is then released from the membrane with appropriate elution 
buffer containing a high concentration of counter-ions. 
In another preferred embodiment, shown in FIG. 3, for use with small 
volumes of test fluid, the sample cups 14, as described above, are 
miniaturized and connected in a multi-well plate-like format 22. Each of 
the sample cups 14 comprise an inlet 16 for receiving sample and a bottom 
embracing the membrane assembly 10. The multi-sample cup plate 22 fits 
within a multi-well collection vessel 24, to which a vacuum source 26 may 
be applied. Sample fluids are added to the sample cups 14, vacuum is 
applied, and the sample is drawn through the membrane assembly 10. 
Contaminating substances are washed through the membrane assembly 10 with 
appropriate wash buffer and discarded. The desired substance is then 
released from the membrane using an appropriate elution buffer as 
described above, and collected in the multi-well collection vessel 24. 
This embodiment is particularly well-suited for selecting clones of 
interest. 
In yet another embodiment of the present invention, shown in FIG. 4, the 
device may be enlarged to accommodate large volumes of sample fluid. The 
device comprises a circular housing 2 having an inlet 4 and an outlet 6, 
each being fitted with a removable protective cap 28 to maintain moist 
state of membrane during storage. Within the circular housing 2 resides a 
membrane assembly 10 comprising a plurality of multiporous membranes that 
have been functionalized with ion-exchange groups layered with a filter 
support 32. The membrane assembly 10 is attached to the circular housing 2 
by gaskets 34. A vacuum source is connected to the outlet 28, the hose 
adapter 30, and sample fluid, wash buffer, and elution buffer is drawn 
through the membrane assembly 10 as previously described in a preferred 
embodiment. 
FIG. 5 illustrates another embodiment of the device which has been adapted 
for attachment to a syringe 8 or chromatographic column. The membrane 
assembly 10 is positioned within a miniaturized sample cup 14 which has an 
inlet 4 that is capable of receiving a syringe 8. The miniaturized size of 
the device permits separation of nanogram to microgram samples. 
The ion-exchange membranes contained within the device of this invention 
are critical to achieving highly pure products. Functionalized groups 
attached to the membrane can be either weak or strong cationic (negatively 
charged) or anionic (positively charged) exchangers. A number of well 
known anionic and cationic surface chemistries can be exploited to 
functionalize porous membrane materials suitable for use with this 
invention. Among the anionic chemistries, diethylaminoethyl (DEAE; weak) 
functionalized cellulose membrane is preferred for nucleic acid 
purification. Other anionic chemistries include, but are not limited to, 
quaternary methyl amine (QMA; strong) and phosphate (strong). Cationic 
groups that can be used included but are not limited to carboxymethyl (CM; 
weak) and sulfylpropyl (SP; strong). The membrane or solid support itself 
can be selected from the following microporous membrane materials such as, 
but not limited to, cellulose, nylon, polyvinylidene fluoride (PVDF), 
polypropylene or other porous material, provided that the porosity of the 
material adequately permits sample to flow through the membrane without 
significant external mechanical force. The membrane material should also, 
when derivatized, have an adequate number of ion-exchange groups on its 
surface to selectively adsorb the biomolecules of interest without 
significant product loss. 
The microporous membranes of the present invention are especially 
advantageous because the high surface area is readily accessible to bind 
the desired biomolecules within the confines of a small, easy-to-use, 
disposable device. As such, use of the device should require less volume 
of expensive matrix than conventional ion-exchange chromatography 
performed in columns, where the column must be of sufficient length to 
ensure adequate absorption of the sample. The membranes further provide 
adequate binding capacity to bind nucleic acids under conditions which 
allow undesired molecules (i.e., proteins, RNA) to flow through. 
In a preferred embodiment, the devices of this invention will house a 
plurality of DEAE-derivatized cellulose membranes, i.e., from about one to 
about 20 membranes, stacked one on top of the other to form a column 
having a short bed depth. The preferred configuration of each membrane is 
circular or disc-shaped which are then placed within the housing in 
stacked or spiral configuration. Large open pores within the membrane 
(e.g., 12,000 .ANG. diameter) allow free passage of large biological 
molecules through the membrane at high flow rates. The number of membranes 
stacked within the device should provide a short bed length to allow the 
device to be used with low pressure systems, such as a simple vacuum 
manifold or hand held syringe. The short bed depth reduces the quantity of 
eluant (e.g., 3-5 mls) required to elute the biomolecules of interest in a 
more concentrated form compared to traditional ion-exchange 
chromatography. The quantity of eluant used, however, will vary depending 
upon the sample used and the configuration of the device selected. 
DEAE-derivatized membranes used in devices of this invention are physically 
and chemically stable to a wide range of solutions (pH 2-12), including 
but not limited to, urea, guanidine hydrochloride, ethylene glycol and 
detergents (e.g., non-ionic, cationic or zwitterionic). Anionic 
detergents, however, should be avoided as they may bind to the 
DEAE-derivatized membrane. Compatible organic solvents that may be used 
include, but are not limited to, 50% methanol, 20% ethanol, 20% propanol, 
20% butanol, 8M urea, 8M guanidine hydrochloride, 50% ethylene glycol, 50% 
glycerol, 10% trifluoroacetic acid, 30% acetonitrile and 50% 
dimethylsulfoxide. These percentages represent known tolerable levels, 
however, higher titer solutions may be acceptable without compromising 
membrane integrity. 
The above-described devices containing the ion-exchange membranes can be 
used to separate and purify nucleic acids, such as single-stranded DNA, 
double-stranded DNA, genomic DNA, phage lambda DNA, plasmid DNA and RNA, 
as well as proteins, peptides, carbohydrates and oligosaccharides from 
heterogeneous biological samples. For effective binding of the desired 
substance to the membrane, the substance itself must be in an optimally 
ionized configuration. The net charge of the substance, determining 
whether it will bind to a cationic or anionic exchanger depends on the 
nature of ionic groups on the molecules surface exposed to solvent and the 
pH and ionic strength of the buffer with which it is in equilibrium. 
Depending upon the identity of the sample and with a minimum of 
experimentation, it is possible to determine the buffer of choice for 
maximum binding of the desired substance to the membrane. For nucleic acid 
samples, the buffer of choice would be from about 20-100 mM (20 mM 
preferred) Tris HCl with from about 0M to about 1M salt (NaCl). For 
separation and purification of protein and peptide samples, it is 
desirable to use from about 20 mM Tris HCl with from 10 mM to about 0.5M 
salt (pH 4-10). 
According to the method of the invention, sample containing the desired 
substance is first diluted, reconstituted or resolubilized in the buffer 
of choice. The sample fluid is then introduced into an equilibrated device 
through the inlet opening and permitted to flow through the membrane 
assembly using minimal positive or negative pressure. The flow rate of the 
test fluid through the membrane may be rapid due to the large surface area 
available to bind the desired substance, but may be increased by 
regulating the pressure exerted on the device. Binding of the desired 
substance takes place almost instantaneously, and remains intact until 
released with the proper elution buffer containing an increased 
concentration of the appropriate counter-ion. 
After the desired substance is retained by the membrane assembly, a second 
buffer is washed through the membrane assembly to selectively remove 
contaminants from the membrane. This wash buffer will typically be of a 
higher ionic strength than the binding buffer, yet not of sufficient ionic 
strength to elute the desired substance. 
The elution buffer is typically of a higher ionic strength than the wash 
buffer. Optimal pH and salt concentration of the eluting buffer can be 
easily determined with a minimum of experimentation. The elution buffer 
selectively releases the retained substance from the membrane. 
Consequently, when the desired substance is released or eluted from the 
membrane, it is contained in a buffer compatible with subsequent 
biological procedures. The desired substance will be essentially free from 
contaminants and sufficiently concentrated for further processing due to 
smaller buffer volumes required for the purification. 
In a particularly preferred method, plasmid DNA can be isolated from a 
crude bacterial cell lysate, in a stepwise manner, by first removing 
biomolecules and cellular contaminant material that do not bind to the 
ion-exchange membrane(s), including proteins and RNA. Any residual RNA is 
then removed from the bound DNA during a subsequent wash step. The 
conditions of loading and elution can be modified such that the eluted RNA 
is essentially pure and free of RNA and protein contamination. In the 
final elution step, the purified plasmid DNA is released from the membrane 
and recovered for further experimental manipulation and/or 
characterization. Thus, the process yields purified DNA or usable RNA, 
which can subsequently be used for restriction enzyme digestion, cloning, 
ligation, sequencing or other applications requiring high purity DNA. 
Depending upon the particular application, the device may be reused for 
multiple purifications of like sample by first flushing the device with an 
appropriate elution buffer having high salt concentration or low pH which 
is suitable for releasing tightly bound molecules on the membrane. 
Thereafter, the device should be re-equilibrated by washing the membrane 
with a buffer of low ionic strength, and preferably one which is 
essentially of neutral pH. If reuse of the device is not imminant, the 
device can be stored in an appropriate preservation solution, such as 
10-20% (v/v) alcohol (e.g., ethanol or isopropanol) by itself, or in 
combination with an appropriate buffer (e.g., 20 mM Tris (pH 6.0-9.0), 
0.1-0.5M NaCl and 1-100 mM EDTA). A particularly preferrred equilibration 
buffer will contain 0.4M NaCl, 20 mM Tris (pH 7.0), 10 mM EDTA (pH 8.0) 
and 15% isopropanol. Other storage solutions which preserve the 
ion-exchange membrane can be determined by routine experimentation. 
Equilibration, as discussed above, is necessary to remove the preservation 
solution prior to use. 
The ion-exchange device, after multiple use, can be regenerated by 
successive washings with water, acid (e.g., 0.5N HCl) and base (e.g., 0.5N 
NaOH). As discussed above, the regenerated device can be stored in 
preservative solution for later use. 
The invention is further directed to kits for purification and isolation of 
biomolecules. The kit will comprise a device having a plurality of 
ion-exhange membranes and a plurality of buffer solutions of varying ionic 
strength to adsorb and then release sample of interest from the membrane. 
A particularly preferred kit will comprise a DEAE derivatized membrane 
(e.g., cellulose) and reagents for nucleic acid purification. More 
particularly, the kit will comprise an equilibration buffer (as described 
in detail above), a wash buffer and an elution buffer. For plasmid 
purification, a preferred wash buffer will comprise: 400 mM NaCl, 20 mM 
Tris (pH 7.0), 10 mM EDTA (pH 8.0) and 15% isopropanol; and the elution 
buffer will comprise: 0.6M-4M NaCl (2.0M preferred), 20 mM Tris (pH 7.0), 
20 mM EDTA (pH 8.0) and 15% isopropanol. It should be recognized, however, 
that modification and optimization of these buffers can be ascertained by 
the investigator without undue experimentation and will depend upon sample 
type, membrane choice, number of ion-exchange membranes in the device and 
identity of the ion-exchange used. The kit may optionally contain reagents 
for cell lysis. 
There are a number of advantages using ion-exchange membranes for 
biomolecule separation that offer improvements to traditional ion-exchange 
column methodologies. The various configurations of the device of this 
invention permit the purification and recovery of small quantities of 
sample. For example, microliter quantities of sample can be conveniently 
recovered using the ultracentrifuge cup embodiment. This is particularly 
useful for further manipulation of the sample, such as by PCR. Further, 
the devices can be regenerated and reused multiple times for purification 
of like samples, but are designed to be disposable when different samples 
of interest are being investigated. The disposability aspect of the 
devices eliminate the possibility of cross contamination during the 
purification process. 
The method of this invention is further illustrated by the following 
non-limiting examples: 
EXAMPLE 1 
PLASMID PURIFICATION 
For preparation/growth of transformed cells, general methods outlined by 
Maniatis, "A Molecular Guide to Cloning", were followed. In general, 4 
pellets from 500 ml cultures transformed with pBR322 were grown overnight 
in Luria-briani media with ampicillin (35-50 .mu.g/ml) (LB amp) media. 
Bacterial pellets were obtained by subjecting the liquid cultures to slow 
speed centrifugation (i.e., 10 min. at 3000 rpm in Sorvall Gs 3 Rotor or 
equivalent). 
Ten ml of 50 mM Tris, 10 mM EDTA (pH 8.0), containing 100 .mu.g/ml RNAse A 
was added to each bacterial pellet from the 500 ml overnight culture. The 
buffer was swirled in the centrifuge tube to resuspend the pellets. To 
each tube was added 10 ml 0.2M NaOH, 1.0% SDS. This was mixed by swirling 
and incubated at room temperature for 5 minutes. After the 5 minute 
incubation was complete, 10 ml 2.55M potassium acetate (pH 5.2) was added 
to each pellet, again swirling to mix the components. This was centrifuged 
for 30 minutes in Sorvall SS34 rotor (12,000.times.g) to obtain a clear 
supernatant free from cellular particulate and repeated to remove 
additional debris. 
The supernatant generated in the above procedure was removed from the 
pellet promptly after centrifugation. After pooling all fractions, 20 ml 
was loaded directly on to the pre-equilibrated ion-exchange membrane 
device of the invention (FIG. 4) and a traditional ion-exhange column 
(Qiagen Q500, Qiagen, Inc., Studio City, Calif.) (after equilibration with 
Qiagen equilibration buffer as per protocol). Samples separated using 
Qiagen Q500 followed Qiagen protocol and used Qiagen buffers. The 
ion-exchange membrane device was loaded using a 30 ml syringe to push the 
crude prep through the device. Unbound and weakly binding impurities were 
washed from the device by pushing 20 ml of 20 mM Tris, 10 mM EDTA, 0.4 m 
NaCl, 15% isopropanol through the device. The plasmid DNA was eluted by 
pushing 15 ml of 20 mM Tris, 20 mM EDTA, 2.0M NaCl, 15% isopropanol 
through the device and collecting this eluant in a clean tube. Fifty 
microliters was removed from each fraction, mixed with loading dye and run 
on an agarose gel. 
To remove salt from the purified plasmid DNA, one volume of isopropanol was 
added to the final elution buffer and was centrifuged for 15 min. at 
4.degree. C. The supernatant was poured off, the pellet was rinsed with 
70% ethanol and then dried. The pellet was resuspended in a buffer of 10 
mM Tris and 1 mM EDTA (pH 7.6) and samples were loaded on to an agarose 
gel. 
The quantities of DNA eluted using this method ranged from 343 .mu.g to 483 
.mu.g for the large size devices of this invention (capacity 100-500 
.mu.g), and 178 .mu.g was eluted using the Qiagen Q500 ion-exhange 
column/kit (500 .mu.g capacity) from plasmid preparations. On other 
occasions, separations on Qiagen Q500 yielded 500 .mu.g DNA. Total 
purification time after sample preparation using the device of this 
invention ranged from 30 seconds to 5 minutes, whereas the Qiagen Q500 
column took 15 minutes to 1 hour. 
EXAMPLE 2 
PLASMID PURIFICATION 
For preparation/growth of transformed cells, general methods outlined by 
Maniatis were followed. Several 500 ml cultures transformed with pBR322 
were grown overnight in LB media containing 35-50 .mu.g/ml ampicillin. 
Bacterial pellets were obtained by subjecting the liquid cultures to slow 
speed centrifugation (i.e., 10 min. at 3000 rpm in Sorvall Gs 3 Rotor or 
equivalent). 
Ten ml of 50 mM Tris, 10 mM EDTA (pH 8.0) containing 
Ten ml of 50 mM Tris, 10 mM EDTA (pH 8.0) containing 100 .mu.g/ml RNAse A 
was added to each bacterial pellet from 500 ml overnight culture. The 
buffer was swirled in the centrifuge tubes to resuspend the pellets. To 
each tube was added 10 ml 0.2M NaOH, 1.0% SDS. This was mixed by swirling 
to mix the components. This was centrifuged for 30 minutes in Sorvall SS34 
rotor (12,000.times.g) to obtain a clear supernatant free from cellular 
particulate and repeated to remove additional debris. 
The supernatant generated in the above procedure was removed from the 
pellet promptly after centrifugation. After pooling all fractions, 20 ml 
was loaded directly onto the pre-equilibrated ion-exchange membrane device 
of this invention (FIG. 4). The devices were equilibrated using 10 ml of 
0.4M NaCl, 15% ethanol, 0.15% Triton X 100, and 50 mM Mops (pH 7.0). The 
ion-exchange membrane devices were loaded using a 30 ml syringe to push 
the crude prep through the device. Unbound and weakly bound impurities 
were washed from the device by pushing 10 ml of 0.4M NaCl, 15% ethanol, 
and 50 mM Mops (pH 7.0) through the device. The plasmid DNA was eluted by 
pushing 15 ml of 0.6M NaCl, 15% ethanol and 50 mM Mops (pH 7.0) through 
the device and collecting this eluant in a clean tube. 50 microliters was 
removed from each fraction, mixed with loading dye and run on an agarose 
gel. 
To remove salt from the purified plasmid DNA, one volume of isopropanol was 
added to the final elution buffer and was centrifuged for 15 min. at 
4.degree. C. The supernatant was poured off, the pellet was rinsed with 
70% ethanol and then dried. The pellet was resuspended in a buffer of 10 
mM Tris and 1 mM EDTA (pH 7.6) and samples were loaded onto an agarose 
gel. A230-A300 scans of the final elution fraction show the 260 nm peak 
and the A260/A280 ratio and nucleic acid concentration was 1.76 using the 
device of this invention. Each fraction was run on an 0.8% agarose gel as 
follows: Lane 1=crude prep prior to purification; Lane 2=flow through 
lane; Lane 3=wash fraction lane; Lane 4=eluted pBR322 fraction. 
In order to test the bioactivity of the DNA, the ethanol precipitated DNA 
was restriction enzyme digested and run on a 0.8% agarose gel as follows: 
Lane 0=pBR322 control; Lane 1=Hae ll digest; Lane 2=Hae III digest; Lane 
3=Hind III digest; Lane 4=undigested; Lane 5=undigested and 
unprecipitated. 
In conclusion, the pBR322 DNA eluted using this protocol was RNA free and 
protein free and was restriction enzyme digestible. 
EXAMPLE 3 
SEATION OF HUMAN TRANSFERRIN AND .beta.-LACTOGLOBULIN A 
The performance of a DEAE-derivatized cellulose ion-exchange membrane 
device of this invention (FIG. 4) was compared to the performance of 
MemSep.TM. 1000 DEAE (Millipore Corp., Bedford, Mass.) using a linear 
gradient followed by a step gradient. A 10 mg/ml mix of human transferrin 
and .beta.-lactoglobulin A was used as the starting material for protein 
separations. Separations were performed on a Waters.TM. 650 HPLC system. A 
four buffer system was used and mixing was performed by the HPLC pump 
(Buffer A=100 mM Tris-HCl; Buffer B=100 mM Trizma Base; Buffer C=1.0M 
NaCl; Buffer D=deionized water). Injections of the 10 mg/ml mix ranged 
from 1-100 .mu.l in various runs. 
In both separations, 10 .mu.l (10 .mu.g) of the two protein mix was 
injected in each run. Table 1 shows the gradient setup used for both 
separations. The gradient formed was a curved linear gradient. The greater 
resolution in the resulted from the increased bed depth of the MemSep.TM. 
1000 over the disposable device of the invention. However, the more rapid 
elution of protein peaks using the ion-exchange device of this invention 
resulted from the smaller bed depth of this device. 
A step gradient was also used to separate the peaks of the same two protein 
mixture described above. The gradient parameters are shown in Table 2. 
Both sets of peaks elute in the same steps. However, the peaks from the 
present device, were again eluted sooner than the MemSep peaks generated 
using MemSep.TM. 1000. This is the result of the decreased depth of the 
present device as compared to the MemSep.TM. 1000 bed. 
TABLE 1 
______________________________________ 
Time Flow % A % B % C % D 
______________________________________ 
Initial Conditions 
5.600 12 8 0 80 
0.10 5.600 12 8 0 80 
1.00 5.600 12 8 0 80 
11.00 5.600 12 8 19 61 
15.00 5.600 12 8 80 0 
16.00 5.600 12 8 80 0 
17.00 5.600 12 8 0 80 
18.00 5.600 12 8 0 80 
20.00 0.100 12 8 0 80 
______________________________________ 
TABLE 2 
______________________________________ 
Solution % A % B Volume 
______________________________________ 
1 0 80 4 ml 
2 4 76 4 ml 
3 12 68 4 ml 
4 80 0 4 ml 
______________________________________ 
EXAMPLE 4 
SEATION PERFORMED ON MILLISEP USING A HAND HELD SYRINGE 
The step gradient described in Example 3 was used to separate the two 
components of the protein mix using a hand held syringe rather than an 
expensive HPLC instrumentation. 
The following conditions may be used to perform the syringe operated 
separation. Solutions prepared to contain relative proportion of buffer A 
and B as outlined in Table 1 above (Buffer A: 20 mM Tris pH 8.0; Buffer B: 
20 mM Tris pH 8.0+1.0M NaCl) were used. A four milliliter injection was 
used in order to ensure maximum recovery of the protein peak. 
Equivalents 
Those skilled in the art will recognize, or be able to ascertain using not 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be encompassed by the following claims.