Charge-modified hydrophobic membrane materials and method for making the same

A hydrophobic material having a crosslinked, cationic charge-modifying coating such that the majority of the ion exchange capacity of the material is provided by fixed formal positive charge groups is disclosed. The material is produced by contacting a hydrophobic substrate with a mildly alkaline, aqueous organic solvent solution into which has been dissolved a cationic charge-modifying agent. The charge-modifying agent comprises a water soluble, organic polymer having a molecular weight of greater than about 1000, wherein the polymer chain contains both fixed formal positive charge groups and halohydrin groups. Materials of the type described herein can be used in a variety of applications including macromolecular blotting and filtration.

BACKGROUND OF THE INVENTION 
Microporous membranes have been demonstrated to have utility in a wide 
variety of applications. As such, numerous processes have been developed 
to produce such membranes. For example, U.S. Pat. No. 3,876,738 described 
a process for preparing a microporous membrane by quenching a solution of 
a film forming polymer in a non-solvent system for the polymer. European 
Patent Application No. 0 005 536 describes a similar process. 
Commercially available microporous membranes, comprising for example nylon, 
are available from Pall Corporation, Glen Cove, N.Y. under the trademark 
ULTIPOR N.sub.66. Additionally, microporous membranes made of cellulose 
acetate, cellulose nitrate or mixtures thereof are widely available from a 
variety of sources. Other membranes, comprising yvinylidene fluoride, 
(PVDF), are available under the trademark Durapore.RTM. (Millipore 
Corporation, Bedford, Mass.). The nylon and nitrocellulose membranes 
exhibit hydrophilic properties, while the PVDF membranes are hydrophobic. 
It is possible, however, to coat the PVDF membranes with materials which 
render them hydrophilic. These hydrophilic Durapore.RTM. membranes are 
also available from the Millipore Corporation. 
For certain applications, notably filtration and macromolecular transfer, 
it has been suggested that the performance of the material could be 
increased by providing an ionic functional group attached to the membrane 
surface which would serve to provide a fixed formal positive charge to the 
membrane. Such charge-modified membranes have been suggested for 
macromolecular transfer applications (e.g., DNA and blotting) in U.S. Pat. 
Nos. 4,512,896 and 4,601,828. Additionally, charge-modified membranes have 
been suggested for use as filtration materials in U.S. Pat. Nos. 4,473,474 
and 4,673,504. In each of these, however, the invention is limited to 
methods for charge-modifying hydrophilic membranes and the use of the 
same. In fact, the latter two patents each provide an example describing 
unsuccessful attempts to charge-modify hydrophobic membranes. These 
attempts led to the conclusion in each of the patents that hydrophobic 
polymer membranes were not amenable to charge-modification by the methods 
attempted and described. 
As such, the charge-modified microporous membranes used for macromolecular 
blotting and filtration applications have utilized hydrophilic membranes 
as starting materials. 
The term "macromolecular blotting" as used herein refers to processes for 
transferring biological macromolecules such as nucleic acids and proteins 
from electrophoresis gels to some type of immobilizing matrix. 
Historically, nitrocellulose was used as a suitable blotting matrix. Of 
particular importance is nucleic acid blotting such as DNA blotting. A 
variety of DNA blotting techniques have been developed. The most common is 
referred to as "Southern Blotting". In this technique, DNA fragments are 
separated by chromatographic techniques and then denatured while still in 
the gel. The gel is neutralized and placed between wicking paper which is 
in contact with a buffer reservoir. Nitrocellulose is then placed on top 
of the gel and dry blotting papers are placed on top of the 
nitrocellulose. As the buffer flows into the gel, DNA is eluted and binds 
to the nitrocellulose, thereby transferring the DNA fragment pattern onto 
the nitrocellulose. The fragment pattern can then be detected using 
hybridization techniques employing labelled nucleic acids which are 
complementary to the specific bound fragments. 
Since the development of the Southern blotting technique, a number of 
variations and improvements on the technique have been developed. For 
example, if the blotting paper is derivatized with diazobenzyloxymethyl 
groups, thereby forming a material commonly referred to as DBM-paper, RNA 
and proteins can be covalently attached to the material. 
Aminophenylthioether coated papers activated to the diazo form, 
(DPT-paper), can also be used to bind DNA, RNA and proteins. Other 
immobilization methods have used high salt or alkaline conditions in 
efforts to improve binding of DNA, RNA and proteins. 
Other attempts to improve the binding process have concentrated on the 
blotting substrate by replacing nitrocellulose, for example, with other 
hydrophilic materials such as Nylon 66. Additionally, U.S. Pat. Nos. 
4,512,896 and 4,673,504, previously described, suggest other materials 
such as hydrophilic PVDF for use as blotting substrates. These substrates, 
however, are again limited to producing hydrophilic materials which have 
been charge-modified. While these materials are an improvement over 
nitrocellulose, DBM-paper and DPT-paper, their charge retention, during 
hybridization and recycling, and performance under alkaline conditions 
could still be improved upon. 
SUMMARY OF THE INVENTION 
This invention pertains to charge-modified, hydrophobic substrates and 
methods for making and using the same. More specifically, this invention 
pertains to charge-modified, hydrophobic, microporous membranes 
characterized by having the majority of the ion exchange capacity of the 
material provided by fixed formal positive charge groups. These materials 
exhibit a combination of ionic and hydrophobic properties rendering them 
highly effective for macromolecular adsorption applications under a 
variety of conditions. 
The fixed formal positive charge results, in one embodiment, largely from 
quaternary ammonium functional groups which can be provided on the 
hydrophobic surface by contacting the hydrophobic material with a solution 
containing a polyamine or polyamido-polyamine epichlorohydrin cationic 
resin to provide a surface coating. There is no need to provide a 
secondary charge-modifying agent to stabilize the coating or enhance the 
cationic charge on the microporous surface. 
The materials of the instant invention are particularly well suited for 
applications such as macromolecular blotting and filtration. In the case 
of macromolecular blotting, the combination of hydrophobic and ionic 
effects provides a material them during hybridization, and maintain signal 
strength throughout a number of repeated recycling stages.

DETAILED DESCRIPTION OF THE INVENTION 
The cationic charge-modified substrates of this invention comprise 
hydrophobic, microporous membranes which have been charge-modified with 
fixed formal charge groups having a net positive charge. The performance 
of these charge-modified membranes results from a combination of the 
hydrophobic and ionic surface effects. 
The term "microporous membrane" as used herein defines a substantially 
isotropic porous membrane having an average pore size of at least 0.05 
microns or an initial bubble point (IBP) in water of less than 120 psi. A 
maximum pore size useful for the invention is about 10 microns. The term 
"isotropic" or "symmetrical" as used herein means that the pore structure 
is substantially uniform throughout the membrane. In addition, 
"anisotropic" or "asymmetrical" membranes are also available formed as a 
composite between a thin layer of a material having a small average pore 
size supported by a more porous structure. 
While membranes useful for this invention include those comprising 
hydrophobic polypropylene, polyethylene, polysulfone and 
polytetrafluoroethylene (PTFE), membranes comprising polyvinylidene 
fluoride (PVDF) are preferred. PVDF membranes are known in the art, being 
described, for example, in U.S. Pat. Nos. 4,203,848 and 4,203,847, both of 
Grandine, 2nd, the teachings of which are incorporated herein by 
reference. Furthermore, these hydrophobic membranes are commercially 
available as, for example, Immobilon P.RTM. microporous membranes 
(Trademark of Millipore Corporation, Bedford, Mass.). 
The charge-modifying agent used in this invention is a water soluble 
organic polymer having a molecular weight greater than about 1000, wherein 
the polymer comprises monomers, the majority of which have been reacted 
with epichlorohydrin. The epichlorohydrin serves to convert tertiary amine 
structures previously resident on the polymer to structures having a 
quaternary functionality. This . provides a stable coating which comprises 
a quaternary ammonium fixed formal charge capable of conferring a net 
positive charge to the hydrophobic substrate. 
The charge-modifying agent is coated onto the contacted surfaces of the 
hydrophobic microporous membrane. The term "coated" as used herein refers 
to charge-modifying agents which are sufficiently attached to the membrane 
so that they will not significantly extract under the intended conditions 
of use. 
The charge-modifying resins are preferably polyamine epichlorohydrin 
cationic resins, such as those described in U.S. Pat. Nos. 2,926,116; 
2,926,154; 3,224,986; 3,311,594; 3,332,901; 3,382,096; and 3,761,350, of 
which the teachings of each are incorporated herein by reference. 
It is required that the charge-modifying agent contain halohydrin 
substituents as well as fixed formal positive charge groups. Agents 
containing epichlorohydrin substitutents and quaternary ammonium ions are 
most preferred. One such charge-modifying agent is the polyamine 
epichlorohydrin resin available commercially as R4308 resin (Trademark of 
Hercules Corp., Wilmington, Del.). In this resin, the charged nitrogen 
atom forms part of a heterocyclic grouping, and is bonded through a 
methylene moiety to a pendent chlorohydrin group. Each monomer group of 
Hercules R4308 is represented by the following general formula: 
##STR1## 
Another preferred charge modifying agent is Polycup 1884 (Trademark of 
Hercules Corp., Willington, Del.), the individual monomers of which are 
represented by the following general formula: 
##STR2## 
It must be pointed out, however, that ions other than ammonium ions, such 
as sulfonium, phosphonium or the like which form fixed formal positive 
charge groups can be used in the practice of this invention as well. 
In the broadest sense, the process of this invention is directed to 
cationically charge-modifying a hydrophobic organic polymeric material, 
such as for example, a microporous membrane in a manner such that the 
majority of the ion exchange capacity of the material is provided by fixed 
formal positive charge groups. The process comprises applying to the 
membrane a charge-modifying amount of the cationic charge-modifying agent 
which coats the membrane structure and is cross-linked through the 
halohydrin substituent and residual tertiary amines in the resin. In its 
broadest embodiment, the process comprises the steps of: 
(a) providing a hydrophobic material substrate; 
(b) contacting the substrate with a charge-modifying solution, said 
solution comprising a charge modifying agent which comprises an organic 
polymer having a molecular weight of greater than about 1000, the polymer 
having a polymer chain having fixed formal positive charge groups as a 
result of halohydrin substitution; said charge-modifying agent being 
dissolved in an alkaline, water-miscible, organic solvent solution; and 
(c) curing the cationic charge-modified, hydrophobic substrate. 
As stated previously, the hydrophobic material substrate can be any of a 
number of hydrophobic membranes, for example, polypropylene, polyethylene, 
polysulphone, PTFE or the like. Hydrophobic PVDF is preferred. The 
charge-modifying agent is preferably a polyamine epichlorohydrin resin and 
is present in the aqueous solution of the water-miscible organic solvent 
as between about 0.5% and 5% solids by weight, however, a solution of 
between about 1% and 3% solids by weight is preferred. The organic solvent 
can be any of the lower alcohols routinely used in industrial processing. 
Methanol, ethanol, isopropanol and mixtures thereof are preferred. This 
alcohol/water mixture is typically a 10-50% mixture by weight depending 
upon the solvent. The mixture is rendered mildly alkaline by the addition 
of a material such as NaOH. Preferably the solution is at a pH of between 
about 9.0 and 12.0. 
Unlike other charge-modified, hydrophobic membranes known in the art, the 
charge-modified, hydrophobic membranes produced by the process of this 
invention are not subjected to a treatment with a secondary 
charge-modifying material such as tetraethylenepentamine. Historically, 
secondary charge-modifying agents have been suggested as a means to 
enhance the cationic charge of the primary charge-modifying agent as well 
as a means to crosslink the primary charge-modifying agent and enhance its 
bonding to the membrane surface. 
The fixed formal positive charge groups as utilized in this invention are 
desirable in that they confer, to the substrate, excellent performance in 
binding macromolecules. When used in conjuction with hydrophobic 
membranes, the ion exchange capacity of the fixed formal positive charge 
groups and the hydrophobic nature of the membrane combine to provide 
excellent performance in applications in which it is desired to cause 
binding of macromolecules. Such applications include macromolecular 
blotting as well as filtration. 
In their blotting applications, the cationically charged-modified 
hydrophobic membranes of this invention are used in the same manner that 
nitrocellulose, DBM-paper, DPT-paper and charge modified hydrophilic 
membranes have been used in the past. However, the combination of 
hydrophobic properties and the fixed formal positive charge provides the 
membranes of this invention with improved performance over the currently 
used blotting matrices. Furthermore, the materials of the present 
invention have been found to be effective when used under conditions in 
which blotting materials such as nylon have proven less than satisfactory, 
i.e., environments having high ionic strength. 
Additionally, the materials of the present invention have a number of 
further advantages over traditional matrix materials such as 
nitrocellulose. For example, when used as a material for DNA blotting, 
nitrocellulose immobilizing matrices require high salt conditions in the 
buffer solution to perform adequately. The high salt conditions, however, 
lead to a high current flow requirement, i.e., up to about 5 amperes. 
These high current flows can generate heat which adversely affects the DNA 
being studied. Since high salt conditions are not required when using the 
materials of this invention, the current can be maintained in the 
milliampere range and thermal damage is therefore minimized or avoided 
entirely. 
Overlaying of blots or transferred electrophoretograms can be carried out 
in the same manner with the materials of the present invention as they 
have been carried out in the past with other immobilizing matrix 
materials. Furthermore, because the binding ability of the materials of 
the instant invention is superior to that of the prior art materials, the 
instant materials exhibit enhanced ability to retain bound macromolecules 
during hybridization as well as the ability to maintain a strong signal 
throughout repeated recycling stages. While the material of the invention 
has been found to be particularly useful in connection with the transfer 
of macromolecules from chromatographic gel substrates, it can be used in 
connection with substantially all other macromolecular blotting 
techniques, including specifically those techniques in which transfer 
occurs via convection or diffusion. 
Charge-modified, hydrophobic membrane materials produced by the process 
previously described herein do not wet instantly when immersed into 
aqueous solutions. Rather, in applications in which the materials must be 
wet prior to use, such as in macromolecular blotting applications, it is 
necessary to prewet the materials with a water-miscible organic solvent 
either neat or in an aqueous solution. As before, the water miscible 
organic solvent can be any of the lower alcohols routinely used 
industrially (i.e., methanol, ethanol, isopropanol). These alcohols are 
preferably used in aqueous solutions with the ratio of alcohol to water 
varying from about 10-50% by weight depending upon the alcohol chosen. 
As an alternative to an aqueous alcohol prewet step, membrane materials 
which will be used in applications requiring prewetting can be produced 
with an added surfactant or wetting agent incorporated into the matrix. In 
this case, the hydrophobic membrane is contacted with an aqueous solution 
of surfactant or wetting agent during the step of applying the 
charge-modifying coating or subsequent to the curing step. Suitable 
surfactant solutions include aqueous solutions of either ionic or 
non-ionic detergents such as Tween-20 (Trademark of ICI United States, 
Inc.) or Pluronic F-68 (Trademark of BASF Corp.). Suitable wetting agents 
include tetraglyme (tetraethylene glycol dimethyl ether), glycerin and the 
like. Such treatments render the material of the invention wettable as 
soon as it is contacted with aqueous media. 
The invention will now be more specifically described in the following 
examples. 
EXAMPLES 
The examples described below illustrate the process of making and using 
cationically charge modified hydrophobic membranes. These membranes have 
utility for many applications including macromolecular blotting and 
filtration. 
Broadly, the cationically charge modifying agents are coated onto a 
hydrophobic organic polymeric membrane by the following procedures: The 
hydrophobic membrane is first pre-wet in a water miscible organic solvent 
(i.e., alcohol) followed by exchange with water. This membrane is then 
coated by placing it in an aqueous solution of the charge modifying agent. 
Alternately, the process involves contacting the hydrophobic membrane 
substrate with an aqueous solution of a water miscible organic solvent 
(i.e., alcohol) which contains the charge modifying agent. The coating can 
be applied by dipping, slot coating, transfer rolling, spraying and the 
like. The coated membrane is then dried and cured under restraint. 
Suitable methods for drying and curing include the use of a heat transfer 
drum, hot air impingement, radiational heating or a combination of these 
methods. 
EXAMPLE 1A 
Preparation of Charge Modified Microporous Membrane 
Sheets of microporous membranes (Immobilon P.TM., a hydrophobic 
polyvinylidene fluoride, 0.45 micron pore size and Durapore.TM., a 
hydrophilic polyvinylidene fluoride, 0.65 micron pore size, both available 
from Millipore Corporation, Bedford, Mass.) 15.0.times.25.0 cm were coated 
with a 3% (V/V) solution of Hercules R4308 adjusted to pH 10.56 with NaOH 
for 1 h at room temperature. In the case of the hydrophobic substrate the 
membrane was first pre-wet in methanol and exchanged with water before 
placing in the coating solution. The hydrophilic Durapore was placed 
directly into the coating solution. After coating, the membranes were air 
dried overnight and then placed between sheets of polyester film and 
heated under restraint at 121.degree. C. for 3 min. 
EXAMPLE 1B 
Preparation of Charge Modified Microporous Membrane 
Membrane samples similar to those of Example 1A were coated with a 3% 
solids by wt. solution of Hercules R4308 resin in 25% (V/V) 
isopropanol/water, adjusted to pH 10 using 50% (wt./wt.) NaOH. The 
membrane samples were immersed in this solution for 0.5 to 1.0 min. and 
then removed from the coating solution. Excess resin solution was removed 
by "squeegee" action using wiper bars. The membrane was then dried under 
restraint sandwiched between sheets of polyester film by contact with a 
heat transfer drum at a temperature of 95.degree. C. for a period of 3 
min. Samples of the membrane were characterized for thickness, initial 
methanol bubble point, flow time, BET surface area and ion exchange 
capacity (IEC). The results are summarized in Table 1. 
TABLE 1 
______________________________________ 
Chemical and Physical Characteristics of Charge 
Modified Hydrophobic PVDF Microporous Membrane 
Control 
Charge Modified 
______________________________________ 
Membrane 
Thickness (mils) 4.65 4.68 
Initial Bubble Point (psi).sup.1 
11.2 11.36 
Flow time (cc/min/cm.sup.2).sup.2 
51.2 30.0 
BET Surface Area (m.sup.2 /g) 
6.47 5.5 
IEC Capacity.sup.3 
0 0.137 
______________________________________ 
.sup.1 47 mm diameter disks of the membrane samples were placed in a test 
holder which seals the edge of the disk. Above the membrane and in direct 
contact with its upper face, was a perforated stainless steel support 
screen which prevented the membrane from deforming or rupturing when air 
pressure was applied to it from below. Methanol was then placed above the 
membrane. Air pressure from a regulated supply was then applied beneath 
the membrane and increased until the first stream of air bubbles was 
emitted by the membrane. This pressure is termed the initial bubble point 
in psi. 
.sup.2 Flow time was measured in a similar device to that described above 
following ASTM method #F317/72. 
.sup.3 To measure total IEC capacity, 47 mm disks of membrane samples wer 
placed in 100 ml of 0.1 M HCl in 50% (V/V) methanol for 5 mins followed b 
air drying at room temperature for 1 h. The membrane disks were then wet 
in 100 mL of an 80% (V/V) methanol/water mixture, to which 2 ml of 5 M 
NaNO.sub.3 solution was then added. The chloride ion concentration was 
then estimated by derivative titration with silver nitrate (0.0282 M Ag 
NO.sub.3) using a Fisher computer aided titrimeter. The IEC capacity is 
then expressed as milliequivalents/g wt. of disk. 
EXAMPLE 2 
Binding of Nucleic Acid to Charge Modified Microporous Membranes 
In this example, the binding of DNA to samples of microporous membranes 
coated with the charge modifying agent as described in Example 1A was 
investigated in a dot-blot manifold assay format. In this assay format, a 
sample of membrane forms the base of a well into which liquid can be 
placed for incubation with the membrane. The liquid can then be drawn 
through the membrane under vacuum. In this study a sample of double 
stranded DNA derived from the replicative form (RF) of the bacteriophage 
Ml3, which had been digested with the restriction enzyme Hind III (NE 
Biolabs) was used to compare DNA binding efficiencies of nylon to the 
charge modified PVDF surface. The RF-DNA was isolated as described by 
Messing, J., Methods in Enzymology, 101, Recombinant DNA [part C]:20-78, 
R. Wu, L. Grossman, K. Moldave, eds., Academic Press, New York (1983). The 
DNA was 3'end labelled using a commercial kit (NEN/Dupont, NEK 009) 
utilizing the enzyme terminal deoxynucleotidyl transferase and 
[alpha-.sup.32 P]3'-dATP as a label. Labeled DNA was combined with 
unlabeled material to give a specific activity of 10,000 cpm/ug DNA. The 
DNA was then added to the well of the dot-blot assay manifold in a volume 
of 0.050 mL in one of the following reagent systems: 0.125 M NaOH, 
0.125.times.SSC (standard saline citrate, 18.75 mM NaCL, 2.1 mM Na 
Citrate), 10.times.SSC(1.5 M NaCl, 0.17 M Na Citrate, pH 7.4) and 25 mM Na 
Phosphate buffer (pH 7.4). In the latter two buffers, the DNA was rendered 
single stranded by heating to 100.degree. C. for 5 minutes followed by 
rapid cooling in ice for 10 min. before application to the test membrane 
samples. After 30 min. any residual liquid was drawn through the membrane 
under vacuum. The membranes were then washed in the manifold under low 
vacuum with 0.100 mL of the same sample application buffer, and the 
membrane sheet was then removed and air dried. The individual membrane 
discs were then placed in scintillation counting fluid and the 
incorporation of .sup.32 P was measured. 
Further samples were subjected to analysis for retention of radiolabelled 
DNA under conditions which simulated hybridization analysis as described 
in published literature (Molecular Cloning, pg. 387-389, T. Maniatis, E. 
F. Fritsch and J. Sambrook, eds., Cold Spring Harbor Press, 1982). In 
addition the bound DNA was also subjected to the alkali "stripping" 
conditions (0.4 M NaOH at 42.degree. C. for 30 min.) used to remove bound 
radiolabeled DNA "probe". This simulated the conditions to which the 
membrane would be subjected if the blotted DNA was being reprobed. The 
results of a comparison of a hydrophobic and closely related hydrophilic 
substrate are summarized in Tables 2 and 3. 
TABLE 2 
______________________________________ 
Binding of DNA to Charge Modified Microporous 
Membranes Using a Dot-Blot Manifold Format Assay 
(Initial Retention of DNA) 
% DNA retained of that applied.sup.1 
in the following buffers: 
Alkali.sup.2 
10 .times. SSC.sup.3 
Phosphate.sup.4 
______________________________________ 
Membrane Type: 
R4308 coated hydrophobic 
100 100 70.1 
PVDF 
R4308 coated hydrophilic 
95.2 0 64.7 
PVDF 
Nylon.sup.5 100 95.2 61.1 
______________________________________ 
.sup.1 1 ug of M13 RFDNA in one of the above sample buffer systems was 
applied in a volume of 0.050 mL at a specific activity of 9,000 to 13,000 
cpm/ug DNA. After scintillation counting the ug DNA retained were 
calculated and expressed as % of 1 ug initial applied sample. 
.sup.2 125 mM NaOH, 18,75 mM NaCl, 2.1 mM Na Citrate. 
.sup.3 1.5 M NaCl, 0.17 M Na Citrate pH 7.4. 
.sup.4 25 mm Na Phosphate, pH 7.4. 
.sup.5 Nylon sample used was Genescreen Plus (NEN/DuPont). 
TABLE 3 
______________________________________ 
Retention of Membrane Bound DNA After Exposure to the 
Reagent Conditions of Hybridization Analysis and 
and Alkali Recycling 
% DNA retained on the 
membrane surface.sup.1 
Post- Post- 
Sample Buffer 
Membrane Type hybridization 
stripping 
______________________________________ 
(1) Alkali R4308 coated 81.5 75.6 
hydrophobic PVDF 
R4308 coated 75.0 74.7 
hydrophilic PVDF 
Nylon 99.0 62.2 
(2) 10 .times. SSC 
R4308 coated 68.2 55.2 
hydrophobic PVDF 
R4308 coated 0 0 
hydrophilic PVDF 
Nylon 15.1 3.2 
(3) Phosphate 
R4308 coated 42.9 30.7 
hydrophobic PVDF 
R4308 coated 34.9 30.3 
hydrophilic PVDF 
Nylon 14.4 5.6 
______________________________________ 
.sup.1 Amount of DNA retained after exposure to the reagent conditions of 
hybridization analysis and one cycle of alkali stripping (to simulate 
removal of DNA probe during reprobing) is expressed as % of the initial 
DNA applied (1 ug) to the membrane. 
The results of this study illustrate that charge modification of a 
microporous, hydrophobic PVDF membrane surface can produce a membrane 
exhibiting enhanced DNA binding and retention characteristics under 
conditions which simulate hybridization analysis and reprobing. The 
experimental conditions studied in this example provide a representative 
range of conditions as would likely be experienced by a membrane useful 
for solid phase nucleic acid blotting. Of special note is the complete 
absence of DNA binding to the R4308 coated hydrophilic PVDF surface under 
high ionic strength conditions (i.e. 10.times.SSC), which are used in the 
major application of Southern blotting. In contrast, the hydrophobic 
surface shows excellent binding of DNA. The observed performance advantage 
supports the theory that a hybrid ionic/hydrophobic microenvironment 
promotes more efficient macromolecular blotting. This is an important 
attribute of the charge modified hydrophobic surface. 
EXAMPLE 3 
Binding of Nucleic Acid to Charge Modified Microporous Membranes and 
Simulation of Cycles of Reprobing of the Blotted DNA 
In this example, membrane samples produced as described in Example 1A were 
used in a manifold dot-blot assay in a series of five cycles of 
hybridization analysis. The example illustrates the improved performance 
of the charge modified hydrophobic membrane over a currently available 
nylon substrate for this application. 
In this assay, the Ml3-RF DNA isolated as described in Example 2 was 
radiolabeled as follows: (1) after digestion with the Hind III restriction 
enzyme a sample was 3'end labeled and was used to measure DNA binding as 
described in Example 2 using 10.times.SSC high ionic strength conditions; 
and (2) a sample of intact RF-DNA was nick-translated with (alpha-.sup.32 
P) d-ATP using a commercial kit (NEN/DuPont, NEK 004) to achieve a 
specific activity of 10.sup.7 cpm/ug DNA and was used as a probe for 
hybridization analysis. This assay was then carried out as described in 
Example 2 using a dot-blot manifold (BioRad). 
Two sets of samples of DNA digested with Hind III (1 ug in 0.050 mL) were 
absorbed to the test membranes as follows: (1) DNA that had been trace 
labeled with the .sup.32 P 3'end label. (This was used to determine the 
retention of DNA.) (2) DNA that was unlabeled. (This was used to follow 
the hybridization analysis with the nick-translated probe.) After one 
cycle of hybridization as described in Example 2, a set of samples were 
retained for scintillation counting while the rest were then alkali 
"stripped" to remove the radioactive probe DNA. These latter samples were 
then recycled through the hybridization process. This was repeated a total 
of five times. The results are summarized in Tables 4 and 5. 
TABLE 4 
______________________________________ 
DNA Binding/Rentention During Five Cycles of 
Simulated Hybridization Analysis 
Membrane Type 
Cycle # ug DNA Retained.sup.1 
% Tota1.sup.2 
______________________________________ 
R4308 Coated 1 0.65 61.8 
Hydrophobic PVDF 
2 0.26 22.8 
3 0.20 17.5 
4 0.21 18.4 
5 0.17 14.9 
Nylon.sup.3 1 0.22 22.5 
2 0.018 1.8 
3 0.034 3.5 
4 0.027 2.7 
5 0.014 1.9 
______________________________________ 
.sup.1 M13 RFDNA digested with Hind III was .sup.32 P 3' end labeled to a 
specific activity of 15,780 cpm/ug. Samples (1 ug in 0.05 ml) were bound 
to the above membrane samples in a dotblot manifold as described in 
Example 2. The membranes were then removed from the manifold and subjecte 
to cycles of hybridization analysis and "alkali stripping" conditions to 
simulate five cycles of reprobing. The amount of DNA retained was 
estimated by scintillation counting. 
.sup.2 Control values of DNA loaded onto the membrane samples before 
hybridization analysis are as follows: 
R43098 coated hydrophobic PVDF 1.14 ug 
nylon 0.98 ug 
.sup.3 Genescreen Plus nylon was used for comparison. 
TABLE 5 
______________________________________ 
Hybridization Analysis Using a Nick-Translated Probe: 
A Simulation of Five Cycles of Reprobing 
Membrane Type Cycle # CPM Hybridized.sup.1 
______________________________________ 
R4308 coated 1 116,107 
hydrophobic PVDF 
2 113,362 
3 149,768 
4 221,768 
5 113,282 
Nylon.sup.2 1 32,699 
2 40,713 
3 32,580 
4 69,189 
5 22,693 
______________________________________ 
.sup.1 Dotblotted M13 RFDNA was hybridized using "nicktranslated" probe a 
a specific activity of 10 cpm/ug DNA following an established protocol 
(Molecular Cloning, pg. 387-389). After stringency washing, samples of 
membrane (in duplicate) with bound .sup.32 P probe were subjected to 
scintillation counting. The extent of hybridization was expressed as CPM 
bound to the adsorbed unlabeled Hind III digest of Lambda DNA. 
.sup.2 Genescreen Plus nylon used for comparison. 
It is clear from the data presented in Table 4 that DNA is better retained 
on the R4308 coated hydrophobic PVDF surface compared to nylon. However, 
in both cases a large amount of the initial DNA adsorbed on these surfaces 
appears to be lost during the first cycle of use and then remains at a 
constant level. The hybridization analysis presented in Table 5 reflects 
the performance advantages of higher DNA retention by the charge modified 
hydrophobic PVDF surface. The improved dot-blot assay performance 
illustrates an important attribute of the hybrid ionic/hydrophobic 
membrane surface. 
EXAMPLE 4 
Dot-Blot Hybridization Analysis Using Charge Modified Microporous Membranes 
In this example, samples of charge modified hydrophobic PVDF membrane, 
prepared by the process outlined in Example IB, were used in a dot-blot 
manifold assay to compare hybridization assay performance of the charge 
modified PVDF surface to nylon. Bacteriophage M13 RF-DNA digested with the 
restriction enzyme Hind III was heat denatured in 10.times.SSC and applied 
to charge modified hydrophobic PVDF and nylon membrane samples in 
10.times.SSC in a dot-blot manifold as described in Example 2. A range of 
concentrations was applied as follows: 0.32, 1.6, 8.0, 40, 200 and 1000 
ng/well. The dot-blotted DNA was then hybridized with a nicktranslated 
.sup.32 P labeled probe prepared as described in Example 3. An example of 
the resulting autoradiogram (Kodak XAR-5 film, 18 h exposure with 
intensifying screens) is shown in FIG. 1. 
It is clear from the autoradiogram reproduced in FIG. 1 that the 
hybridization assay performance on the DNA dot-blotted to the charge 
modified hydrophobic PVDF is superior to that seen on nylon under the 
conditions of this experiment. Using the PVDF surface as little as 8 ng of 
blotted DNA can be detected on the autoradiograph. In contrast, DNA was 
only marginally detectable at the 200 ng level on the nylon membrane 
sample under the same conditions. 
EXAMPLE 5 
Southern Blotting Analysis Using Charge Modified Microporous Membranes 
In the process of Southern blotting DNA fragments are separated by 
electrophoresis in an agarose gel and then transferred to a membrane 
support by capillary action. The following steps can be included in this 
process: (1) acid depurination to improved transfer of large DNA 
molecules, (2) alkali denaturation to render the DNA single stranded for 
transfer and (3) neutralization and equilibration with a high ionic 
strength transfer buffer (10.times.XSSC). For more details see Molecular 
Cloning, pg. 387-389. 
After capillary transfer to the membrane, the blotted DNA is subjected to 
hybridization analysis. In the process of hybridization the following 
steps are carried out: (1) prehybridization is carried out first to reduce 
the non-specific binding of DNA to the membrane surface, (2) the membranes 
are exposed to homologous .sup.32 P labeled DNA probes (single stranded 
DNA which is complementary to the DNA bound to the membrane) under 
conditions which allow hybridization to occur between the probe and its 
target DNA sequences on the membrane, and (3) excess non-specifically 
retained probe is removed by washing under conditions of increasing 
stringency (for details see Molecular Cloning, pg. 387-389). 
In this example two experiments will be described: 
(1) DNA fragments resulting from the Hind III digestion of Lambda DNA were 
labeled at the 5' ends with .sup.32 P dCTP (kit from DuPont/NEN). The 
resulting specific activity of the DNA probe was determined to be 10.sup.6 
cpm/ug DNA, a total of 1 ug was used in this experiment. The retained DNA 
probe was visualized by exposure to X-ray film for 18 hours (FIG. 2). (2) 
In a similar experiment to that shown above, a DNA probe was labeled using 
many small random oligonucleotides as primers for DNA polymerase 
(Pharmacia Kit) to a specific activity of 10.sup.9 cpm/ug DNA and 0.2 ug 
was used for the experiment. The retained DNA probe was visualized by 
exposure to X-ray film for 10 min. (FIG. 3). 
In FIG. 2, the DNA binding capacities of a nylon membrane and the charge 
modified hydrophobic surface are compared. It is clear from the data (with 
the exception of lane 2 for the nylon which is atypical) that the latter 
membrane gave better signal detection under the conditions of this 
experiment. In FIG. 3, essentially the same result is seen using a 
different probe having higher specific activity. The improved Southern 
blotting performance of the charge modified hydrophobic surface is an 
important attribute of this material as compared to nylon using accepted 
Southern blotting techniques. 
Equivalents 
Those skilled in the art will recognize or be able to ascertain, using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described herein. Such equivalents are 
intended to be covered by the following appended claims.