Modified microporous membrane for non-specific and sequence-specific nucleic acid capture and methods of use

BACKGROUND OF THE DISCLOSURE

[0002] The present disclosure relates to articles of manufacture comprising a multi-layer composite microporous membrane, wherein at least one layer of the membrane has associated therewith highly electropositive solid phase hydrophilic materials useful for highly efficient and irreversible binding of nucleic acids, optionally modified with sequence specific peptide nucleic acids (PNA's); methods of fabricating such articles of manufacture; and methods of using such articles of manufacture to identify, separate and/or amplify target nucleic acid and to optionally store the membrane and bound nucleic acid for archival purposes.

[0003] Detection of Nucleic Acid Through Use of Probe Complementarity

[0004] The molecular structure of nucleic acids provides for specific detection by means of complementary base pairing of oligonucleotide probes or primers to sequences that are unique to specific target organisms or tissues. Since all biological organisms or specimens containing nucleic acid of specific and defined sequences, a universal strategy for nucleic acid detection has extremely broad applications in a number of diverse research and development areas as well as commercial industries. The potential for practical uses of nucleic acid detection has been greatly enhanced by the description of methods to amplify or copy, with fidelity, precise sequences of nucleic acid found at low concentration to much higher copy numbers, so that they are more readily observed by available detection methods.

[0005] Amplification of Nucleic Acid

[0006] The original amplification method is the polymerase chain reaction (PCR) described by Mullis et al. (U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188, all of which are specifically incorporated herein by reference). Subsequent to the introduction of PCR, a wide array of strategies for amplification have been described. See, for example, U.S. Pat. No. 5,130,238 to Malek, nucleic acid sequence based amplification (NASBA); U.S. Pat. No. 5,354,668 to Auerbach, isothermal methodology; U.S. Pat. No. 5,427,930 to Buirkenmeyer, ligase chain reaction; and U.S. Pat. No. 5,455,166 to Walker, strand displacement amplification (SDA); all of which are specifically incorporated herein by reference. Some of these amplification strategies, such as SDA or NASBA, require a single stranded nucleic acid target. The target is commonly rendered single stranded via a melting procedure using high temperature prior to amplification.

[0007] Extraction of Nucleic Acid From Sample

[0008] Prior to nucleic acid amplification and detection, the target nucleic acid must be extracted and purified from the biological specimen such that inhibitors of amplification reaction enzymes are removed. Further, a nucleic acid target that is freely and consistently available for primer annealing must be provided. A wide variety of strategies for nucleic acid purification are known. These include, for example, phenol-chloroform extraction and/or ethanol precipitation (Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; high salt precipitation (Dykes (1988) Electrophoresis 9:359-368); proteinase K digestion (Grimberg et al. (1989) Nucleic Acids Res. 22:8390); chelex and other boiling methods (Walsh et al. (1991) Bio/techniques 10:506-513); and solid phase binding and elution (Vogelstein and Gillespie (1979) Proc. Nat. Acad. Sci. USA 76:615-619), all of which teachings are specifically incorporated herein by reference and representative of knowledge attributable to one of ordinary skill in the relevant area of art.

[0009] Analysis of Nucleic Acid in Complex Samples

[0010] The analysis of nucleic acid targets, therefore, generally consists of three steps: nucleic acid extraction/purification from biological specimens, direct probe hybridization and/or amplification of the specific target sequence, and specific detection thereof. In currently employed conventional protocols each of these three steps is performed separately, making nucleic acid analysis labor intensive. Further, numerous manipulations, instruments and reagents are necessary to perform each step of the analysis.

[0011] For analysis purposes, nucleic acid must frequently be extracted from extremely small specimens in which it is difficult, if not impossible, to obtain a second confirmatory specimen. Examples include analysis of crime scene evidence or fine needle biopsies for clinical testing. In such examples, the extent of the genetic testing and confirmation through replica testing is, thus, limited by the nucleic acid specimen size. Using conventional extraction protocols for these small specimens, the nucleic acid is often lost or yields are such that only a single or few amplification analyses are possible.

[0012] Specimens that contain high levels of endogenous or background nucleic acid such as blood are extremely difficult to analyze for the presence of low level specific targets. Solid phases with high nucleic acid avidity can be utilized to irreversibly capture oligonucleotide or probe sequences. By changing buffer conditions these materials can then selectively capture target sequences even in the presence of high levels of background nucleic acid.

[0013] Nucleic Acid Binding to Solid Phase Supports

[0014] The requirements for binding of DNA and other nucleic acid to solid phases and subsequently being able to elute them therefrom have been described by Boom (U.S. Pat. No. 5,234,809, specifically incorporated herein by reference) and Woodard (U.S. Pat. Nos. 5,405,951, 5,438,129, 5,438,127, all of which are specifically incorporated herein by reference). Specifically, DNA binds to solid phases that are electropositive and hydrophilic.

[0015] Since conventional purification methods require elution of the bound nucleic acid, these solid phase materials are widely considered to be of little use for DNA purification. In fact, considerable effort has been expended to derive solid phase materials sufficiently electropositive and hydrophilic to adequately bind nucleic acid and yet allow for its elution therefrom (See, for example, U.S. Pat. Nos. 5,523,392, 5,525,319 and 5,503,816 all to Woodard, and all of which are specifically incorporated herein by reference).

[0016] Solid-phase reversible immobilization (SPRI) is a widely used technique for purifying nucleic acid of interest. SPRI uses carboxyl-coated magnetic particles (that form the base material for most magnetic particle manufacture) to bind nucleic acid. Under conditions of high polyethylene glycol and salt concentration, SPRI magnetic particles have been found to bind both single- and double-stranded DNA, including PCR products. The nucleic acid typically may be eluted with water, 10 mM Tris or formamide.

[0017] Other types of functionalized particles may be used for binding template nucleic acid molecules, such as hydroxylated beads and reverse phase resins. These particles are available from a wide variety of commercial sources (e.g., Ansys, Waters, and Varian).

[0018] U.S. Pat. No. 4,921,805 discloses a capture reagent bound to a solid support useful for the separation and isolation of nucleic acids from complex unpurified biological solutions. The nucleic acid capture reagent comprises a molecule capable of intercalation into a DNA helix, and is attached to the solid support via a molecular linker. The capture reagent-nucleic acid complexes are isolated from the sample by centrifugation, filtration or by magnetic separation. Nucleic acids are separated from the isolated complexes by, for example, treating the capture reagent-nucleic acid complexes with dilute alkali.

[0019] Solid-phase amplification systems are also known. The so-called DIAPOPS (Detection of Immobilized Amplified Product in One Phase System) combines solid phase PCR and detection by hybridization. DIAPOPS is used to covalently bind a PCR primer to a well. Nucleic acid is covalently bound to the solid phase by a carbodiimide condensation reaction. Manipulation is simplified and contamination diminished since the transfer of the amplicon from the amplification system to the detection system is eliminated.

[0020] “Standard” solid phase-anchored amplification techniques use specific oligonucleotides coupled to a solid phase as primers for cDNA synthesis (prepared from a mRNA molecule). This amplification results in the production of a cDNA that is covalently linked to a solid phase such as agarose, acrylamide, magnetic, or latex beads. A solid phase with cDNA attached, generated using oligo (dT) as a primer, contains sequence information similar to a cDNA library; thus, it represents a “solid phase library.” The cDNA that is attached to the solid phase can be used directly as a template for PCR or can be modified enzymatically prior to the PCR or other amplification procedure. Oligonucleotides that are attached to a solid phase can also function in affinity purification of RNA. RNA isolated this way can be directly reverse transcribed, using the primer that is coupled to the solid phase. Subsequent amplification can employ this primer with or without additional internal primers. Since the cDNA is coupled to a solid phase, changing buffer conditions or primer composition is conveniently achieved by washing the solid phase and re-suspending it in a different PCR mixture.

[0021] Multiple-sample nucleic acid hybridization analysis has been conducted on a variety of filter and solid support formats (see G. A. Beltz et al., in Methods in Enzymology, Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press, New York, Chapter 19, pp. 266-308 (1985)). One format, the so-called “dot blot” hybridization, involves the non-covalent attachment of target nucleic acid to a filter, which is subsequently hybridized with a radioisotope labeled probe(s). “Dot blot” hybridization gained widespread use, and many versions were developed (see M. L. M. Anderson and B. D. Young, in Nucleic Acid Hybridization—A Practical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press, Washington D.C., Chapter 4, pp. 73-111, (1985)). The “dot blot” hybridization has been further developed for multiple analysis of genomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for the detection of overlapping clones and the construction of genomic maps (U.S. Pat. No. 5,219,726).

[0022] Carboxylated latex beads having a plurality of first and second nucleic acids are used in the so-called “Bridge Amplification” technique to similarly allow amplification, separation and detection in the same system. Such system is described in detail in U.S. Pat. No. 5,641,658, the disclosure of which is hereby incorporated specifically by reference.

[0023] Polyamides

[0024] Presently, extensive use is made of polyamide matrices, in particular nylon matrices, as solid support for immobilization and hybridization of nucleic acid. Various types of polyamide matrices are known to bind nucleic acid irreversibly and are far more durable than nitrocellulose. As nucleic acid can be immobilized on polyamide matrices in buffers of low ionic strength, transfer of nucleic acid from gels to such matrices can be carried out electrophoretically, which may be performed if transfer of DNA by capillary action or vacuum is inefficient.

[0025] Two basic types of polyamide membranes are commercially available: unmodified nylon and charge-modified nylon. Charge-modified nylon is preferred for transfer and hybridization as its increased positively charged surface has a greater capacity for binding nucleic acids. See, e.g., U.S. Pat. No. 4,473,474, the disclosure of which is herein incorporated specifically by reference. Generally, nylon membranes must be treated, however, to immobilize the DNA after it has been transferred, as by way of thorough drying, or exposure to low amounts of ultraviolet irradiation (at 254 nm), and such immobilization is not irreversible.

[0026] Nylon Filter Membranes

[0027] Polyamide membranes, and in particular nylon membranes, offer many advantages in the filtration of materials in general. Nylon, as other polyamides, has a natural hydrophilicity, but a narrow wicking rate. It is also particularly strong. In addition, nylon can be cast as a liquid film and then converted to a solid film that presents a microporous structure when dried (See, e.g., U.S. Pat. No. 2,783,894). Such microporous structures permit micron and submicron size solid particles to be separated from fluids and provide an exceedingly high effective surface area for filtration. Microporous polyamide structures may be manufactured so as to be multi-layered or multi-layered so as to provide for different filter characteristics in each layer. See, e.g., U.S. Pat. No. 6,090,441 (the “'441 patent”), the disclosure of which is hereby incorporated specifically by reference.

[0028] Labeled Probes

[0029] The detection of amplified nucleic acid for clinical use relies largely on hybridization of the amplified product and detection with a probe labeled with a variety of enzymes and luminescent reagents. U.S. Pat. No. 5,374,524 to Miller, specifically incorporated herein, describes a nucleic acid probe assay that combines nucleic acid amplification and solution hybridization using capture and reporter probes. These techniques require multiple reagents, several washing steps, and specialized equipment for detection of the target nucleic acid. Moreover, these techniques are labor intensive and require technicians with considerable expertise in molecular biology techniques.

[0030] Nucleic acids modified with biotin (U.S. Pat. No. 4,687,732 to Ward et al.; European Patent No. 063879; both specifically incorporated herein), digoxin (European Patent No. 173251, specifically incorporated herein) and other haptens have also been used. For example, U.S. Pat. No. 5,344,757 to Graf, specifically incorporated herein, uses a nucleic acid probe containing at least one hapten as a label for hybridization with a complementary target nucleic acid bound to a solid membrane. The sensitivity and specificity of these assays is based on the incorporation of a single label in the amplification reaction which can be detected using an antibody specific to the label. The usual case involves an antibody conjugated to an enzyme. Furthermore, the addition of substrate generates a calorimetric or fluorescent change which can be detected with an instrument.

[0031] Attachment of Oligonucleotides to Solid Supports

[0032] Mechanisms for attachment of oligonucleotides to microparticles in hybridization assays and for the purification of nucleic acids is well known in the art. European Patent No. 200133, specifically incorporated herein, describes the attachment of oligonucleotides to water-insoluble particles less than 50 micrometers in diameter used in hybridization assays for the capture of target nucleotides; U.S. Pat. No. 5,387,512 to Wu, specifically incorporated herein, describes the use of oligonucleotide sequences covalently bound to microparticles as probes for capturing PCR amplified nucleic acids. U.S. Pat. No. 5,328,825, to Findlay, specifically incorporated herein, also describes an oligonucleotide linked by way of a protein or carbohydrate to a water-insoluble particle. The oligonucleotide probe is covalently coupled to the microparticle or other solid support. The sensitivity and specificity of all of the above-reference patents is based on hybridization of the oligonucleotide probe to the target nucleic acid.

[0033] Detection of Nucleic Acid at Low Copy Number in Complex Samples

[0034] Using the current nucleic acid hybridization formats and stringency control methods, it remains difficult to detect low copy number (i.e., 1-100,000) nucleic acid targets even with the most sensitive reporter groups (enzymes, fluorophores, radioisotopes, etc.) and associated detection systems (fluorometers, luminometers, photon counters, scintillation counters, etc.)

[0035] This difficulty is caused by several underlying problems associated with direct probe hybridization. One problem relates to the stringency control of hybridization reactions. Hybridization reactions are usually carried out under stringent conditions in order to maximize hybridization specificity. Methods of stringency control involve primarily the optimization of temperature, ionic strength, and denaturants in hybridization and subsequent washing procedures. Unfortunately, the application of these stringency conditions results in a concomitant decrease in the number of hybridized probe/target complexes remaining for detection.

[0036] Another problem relates to the high complexity of DNA in most samples, particularly in human genomic DNA samples. When a sample is composed of an enormous number of sequences that are closely related to the specific target sequence, even the most unique probe sequence has a large number of partial hybridizations with non-target sequences.

[0037] A third problem relates to the unfavorable hybridization dynamics between a probe and its specific target. Even under the best conditions, most hybridization reactions are conducted with relatively low concentrations of probes and target molecules. In addition, a probe often has to compete with the complementary strand for the target nucleic acid.

[0038] A fourth problem for most present hybridization formats is the high level of non-specific background signal. This is caused by the affinity of DNA probes to almost any material.

[0039] These problems, either individually or in combination, lead to a loss of sensitivity and/or specificity for nucleic acid hybridization in the above described formats. This is unfortunate because the detection of low copy number nucleic acid targets is necessary for most nucleic acid-based clinical diagnostic assays.

[0040] Available Combined Purification/Amplification Systems

[0041] Qiagen, one market leader in nucleic acid sample preparation, produces and markets a variety of DNA and RNA sample preparation devices. Typically such devices are based upon glass fiber sheets where the biological sample must be clarified prior to its being applied to the binding matrix. The nucleic acid is typically captured in the presence of high salt buffer (anion exchange); the nucleic acid extensively washed; and the nucleic acid recovered by exposing the bound nucleic acid to a low ionic strength solution (e.g., Tris-EDTA (10 mM Tris-HCl, pH 7.5-8.0; 1 mM EDTA) or deionized water). The nucleic acid is then transferred to another vessel for amplification or further analysis. Other companies selling nucleic acid sample preparation devices include: Millipore (a membrane-based size exclusion ultra-filtration system), Promega, Bio-Rad, Invitrogen, and MWG (anion exchange-based systems).

[0042] A simplified, combined purification and amplification system is available from CpG-Biotech of Lincoln Park, N.J. (http://www.cpg-biotech.com/). This system utilizes a proprietary cell lysis solution (Release-IT™), which permits cell lysis and amplification to occur in the same reaction tube. Release-IT sequesters cell lysis products that might inhibit polymerases and the supplier claims that this improves the specificity and amplification yield. The CpG-Biotech Release-IT system eliminates the need for a separate genomic DNA purification step prior to amplification.

[0043] Combined Purification, Amplification and Detection Systems

[0044] Combined purification, amplification, and detection systems are also known in the art. Such systems permit the processes of isolation and purification of nucleic acids from complex samples, amplification of target nucleic acid, and detection of the amplified products to occur in a self-contained environment.

[0045] U.S. Pat. No. 5,955,351 discloses a self-contained device integrating nucleic acid extraction, amplification, and detection. The system integrates the extraction and amplification of the nucleic acid allowing both procedures to be performed in one chamber, detection in another chamber and collection of waste in yet another chamber. The reaction chambers are functionally distinct, sequential and compact. Xtrana, Inc. (Denver, Colo.) manufactures an embodiment of such a device, referred to as the SCIP cartridge. U.S. Pat. No. 6,153,425 similarly discloses a self-contained device integrating nucleic acid extraction, amplification and detection. Such device comprises a first hollow elongated cylinder with a single closed end and a plurality of chambers therein, and a second hollow elongated cylinder positioned contiguously inside the first cylinder capable of relative rotation. Sample is introduced into the second cylinder for extraction. The extracted nucleic acid is bound to a solid phase, and therefore not eluted from the solid phase by the addition of wash buffer. Amplification and labeling takes place in the second cylinder. Finally, the labeled, amplified product is reacted with microparticles conjugated with receptor specific ligands for detection of the target sequence.

[0046] A commercial product known as Xtra Amp™ (Xtrana, Inc., Denver, Colo.) permits nucleic acid extraction, amplification and detection to be performed in a single microcentrifuge tube. Xtra Amp employs a proprietary material, known commercially as Xtra Bind™, to extract and irreversibly bind nucleic acid in a sample. As is disclosed in U.S. Pat. No. 6,291,166 (the “'166 patent”), the disclosure of which is hereby incorporated specifically by reference, Xtra Bind is capable of binding both DNA and RNA in single-stranded form. Captured nucleic acid can be amplified directly on the solid phase material by a variety of amplification strategies including those requiring single-strand initiation. Specific selection of low copy nucleic acid targets present in complex specimens can be performed by binding specific hybridization probes to the solid phase beads.

[0047] Peptide Nucleic Acids

[0048] The structure of DNA identified by Watson and Crick in 1953 has had a great impact on life sciences such as molecular biology, biochemistry, etc. DNA is a biopolymer with four different bases of adenine (A), cytosine (C), guanine (G), thymine (T), sugar (deoxyribose) and phosphate, to build a very stable double helix structure: The phosphate-sugar forms the backbone, and nucleotide bases attached to the sugars are paired with complementary bases, such as A to T, and G to C, in the opposing paired strand, which pairing is stabilized by hydrogen bond formation between the complementary bases in the double helix. The specific/complementary hydrogen bonds between bases plays a very important role in nucleotide drug treatment strategies such as antisense and gene therapy, in particular, for genetic disease, cancer and cardiac diseases.

[0049] Peptide nucleic acid monomers have a N-(2-aminoethyl) glycine backbone to which adenine, cytosine, guanine, or thymine bases are linked by amide bonds. See FIG. 7. Peptide nucleic acids are synthesized by creating an amide bond between an amino group of the backbone and a carboxyl group of another peptide nucleic acid monomer. Currently, peptide nucleic acid monomers protected by an acid-labile t-butyloxycarbonyl protecting group or alkali-labile fluoromethyloxycarbonyl protecting group are commercially available, where exocyclic amino groups of adenine, cytosine and guanine are protected by acid-stable dipenylmethyloxycarbonyl or benzyloxycarbonyl protecting groups.

[0050] Peptide nucleic acid synthesis is generally carried out in a similar manner as the oligonucleotide synthesis method conventionally known in the art. See Acc. Chem. Res. 24:278 (1991); see also U.S. Pat. No. 6,357,163, the disclosure of which is hereby incorporated specifically by reference. Nielson et al. synthesized oligopeptide nucleic acid by using a solid-phase matrix as follows: First, the amino group on the solid support is reacted with the carboxyl group of the specified base (A, C, G or T), whose amino group in the backbone is protected by acid- or base-labile functional groups, in order to link to each other. Next, the resultant structure is treated with acid or base to eliminate the amino protecting groups to reveal the amino group, which is subsequently reacted with the carboxyl group of the peptide nucleic acid of specified base, whose amino group in the backbone is protected by acid- or base-labile functional groups, in order to link to each other in the form of an amide bond. The steps are repeated to obtain an oligonucleotide of desired base sequence and number, and finally treated with strong acid to separate the exocyclic amino protecting group from the solid support by chemical reaction. This method is desirable in a sense that it assures complete reaction of excessive peptide nucleic acids (5 equivalents) as much as possible and provides easy purification of peptide nucleic acid on an organic-solvent-resistant solid support by filtering the residual monomers and reactants and washing with organic solvent.

[0051] The DNA mimetic, peptide nucleic acid (PNA), has the potential to detect single-base substitution in sample DNA. Peptide nucleic acid is a fully synthetic DNA-recognizing ligand with a neutral peptide-like backbone that is structurally homomorphous to the deoxyribose phosphate backbone of DNA, and purine- and pyrimidine-based nucleobases (i.e., adenine, cytosine, thymine and guanine). Sequence specific hybridization of PNA to complementary DNA occurs through Watson-Crick H-bonding between the nucleobases.

[0052] The neutrality of the PNA backbone results in stronger binding of PNA to DNA as compared to DNA-DNA binding. Using the mutations associated with cystic fibrosis (CF) as a model system, it has been demonstrated, for example, that PNA can distinguish normal and mutant sequences in the CF gene.

SUMMARY OF THE INVENTION

[0053] In one aspect, the present invention provides a multi-layer, composite microporous membrane, wherein at least one layer of the membrane comprises a highly electropositive material operatively positioned on or within the microporous membrane, and wherein the material has associated therewith a sequence-specific peptide nucleic acid (PNA), wherein the PNA is capable of associating with target nucleic acid to form a PNA-nucleic acid complex. Preferably, each layer of the microporous membrane comprises a polymeric material. Preferably, the polymeric material is a polyamide. More preferably, the polyamide is nylon. More preferably still, the nylon is nylon 6,6. A preferred method for preparation of the nylon membrane of the invention is a phase inversion process. Preferably, the polymeric material of the membranes will have a high surface area. More preferably, the surface area of the microporous membrane is at least 60 m2/g. In an alternative embodiment, the multi-layer membrane of the invention comprises a reinforcing material. Preferably, the reinforcing material is a polyolefin.

[0054] In another aspect of the invention, the highly electropositive hydrophilic material of which one layer of the multi-layer membrane is comprised, comprises an element selected from the group consisting of: silicon (Si), boron (B), titanium (Ti) and aluminum (Al). Preferably, the highly electropositive material has been rendered hydrophilic by treatment with functionalizing groups capable of imparting sufficient hydrophilicity to the electropositive material. Preferably, the electropositive material is functionalized with hydroxyl (—OH) groups. In one embodiment, the electropositive material is in a crystalline form. In an alternative embodiment, the electropositive material is in an amorphous form. Preferably, the electropositive material is an oxide. More preferably, the electropositive material is aluminum oxide. In yet another alternative embodiment, the electropositive material is in an elemental state.

[0055] According to the practice of the present invention, the PNA-nucleic acid complex formed with the target nucleic acid comprises deoxyribonucleic acid (DNA). Preferably, the nucleic acid is complementary deoxyribonucleic acid (cDNA). Alternatively, the nucleic acid is ribonucleic acid (RNA). In this alternative embodiment, the ribonucleic acid is, preferably, messenger ribonucleic acid (mRNA). In yet another aspect of the invention, the PNA can be radiolabeled to facilitate identification or recognition of the PNA-nucleic acid complexes. In an alternative version of this embodiment, the target nucleic acid can be radiolabeled to facilitate identification or recognition of the PNA-nucleic acid complexes.

[0056] In yet another aspect of the present invention, the multi-layer composite membrane comprises at least one layer comprising a highly electropositive material capable of associating with one or more nucleic acids. In such an embodiment, wherein highly electropositive hydrophilic material of the least one layer is free of association with a sequence-specific peptide nucleic acid (PNA). In addition, the present invention contemplates that the multi-layer composite membrane comprise at least one layer void of any highly electropositive material. Consistent with the invention, each of the layers is individually characterized in terms of porosity, average pore size, pore size distribution, three-dimensionality of pore distribution, and loading with heterogeneous materials.

[0057] In another aspect, the present invention provides a method of making the a multi-layer composite membrane, wherein the method comprises the steps of combining a highly electropositive hydrophilic material with a microporous membrane dope during formation of the dope; modifying the hydrophilic material by association with a sequence specific peptide nucleic acid (PNA); and using the combined dope and highly electropositive hydrophilic material to form the membrane. In this embodiment, the invention contemplates that the electropositive material is modified with PNA prior to combining the electropositive material with the membrane dope. In this fashion, the PNA may be synthesized using the electropositive material as a solid-phase medium. Alternatively, the electropositive material is modified with PNA prior to formation of the membrane and after mixing of the electropositive material with the membrane dope. In yet another alternative, the electropositive is modified with PNA after formation of the membrane.

[0058] In another embodiment, the present invention provides a method for confirming the presence of target nucleic acid in a sample known to comprise the target nucleic acid, wherein the sample is derived from animal and vegetable tissues and cells containing nucleic acid and other substances, the method comprising the steps of (a) providing the multi-layer microporous membrane of the invention; (b) contacting the sample with the membrane, wherein two or more layers of the membrane comprise a highly electropositive hydrophilic material, wherein at least one layer is free of the highly electropositive hydrophilic material, wherein at least one layer comprising the hydrophilic material further comprises PNA associated with the electropositive material, and wherein the PNA is capable of hybridizing with the target nucleic acid; (c) subjecting the membrane to conditions sufficient to hybridize the nucleic acid to the PNA; (d) removing all non-hybridized substances from the membrane; (e) treating the membrane so as to dissociate the hybridized nucleic acid from the PNA; and (f) collecting the nucleic acid. Alternatively, the method comprises the further step of storing the membrane containing the nucleic acid hybridized to the PNA before execution of steps (e) and (f). This embodiment of the invention contemplates that the target nucleic acid is hybridized to the PNA under stringent hybridization conditions. According to this embodiment, the sample contacts the at least one layer free of the highly electropositive material subsequent to contacting the at least one layer comprising a highly electropositive material associated with the PNA. Furthermore, this embodiment of the invention contemplates that the sample will contact the at least one layer comprising highly electropositive material having PNA capable of hybridizing the specific nucleic acid associated therewith prior to contacting a layer free of the highly electropositive hydrophilic material, and prior to contacting a layer comprising highly electropositive material free of association with PNA. According to the method of the invention, the target nucleic acid comprises DNA. Preferably, the target nucleic acid comprises complementary DNA (cDNA). In an alternative aspect, the target nucleic acid comprises RNA. Preferably, the target nucleic acid comprises messenger RNA (mRNA). According to the practice of the method of the invention, the nucleic acid-containing sample is derived from a nucleic acid/protein mixture, a biotechnical preparation of bacteria or viruses, a bodily fluid or matter, animal or vegetable tissue, a cell lysate or homogenate, or degradation products thereof.

[0059] In an alternative aspect, the present invention contemplates that the nucleic acid not hybridized to the PNA is collected separately. Additionally, the at least one layer free of highly electropositive hydrophilic material may further comprise a bacteriocide. In addition, the at least one layer free of highly electropositive hydrophilic material may further comprise a cell lysing agent. Also contemplated in the practice of the method of the present invention is the further step of determining the quantity of target nucleic acid present in the sample. According to this aspect of the invention, the quantity of target nucleic acid present is determined by a method selected from the group consisting of fluorescence, chemiluminescence, and radioisotopic assay. Still further, the method of the present invention contemplates that determination of the quantity of target nucleic acid present in the sample is performed prior to the steps of treating the hybridized target nucleic acid to dissociate it from the PNA, and collecting the target nucleic acid.

[0060] In another alternative embodiment, the present invention provides a method for the combined separation and amplification of target nucleic acid comprising the steps of (a) providing the multi-layer composite microporous membrane of the invention, wherein a layer comprising the hydrophilic material has been treated to associate a PNA with the material, wherein the PNA so associated is capable of hybridizing with target nucleic acid; (b) contacting a sample known to comprise the target nucleic acid with the membrane under conditions sufficient to hybridize the nucleic acid to the PNA; (c) amplifying the hybridized nucleic acid; and (d) collecting at least a portion of the amplified nucleic acid. According to the practice of the method of the invention, amplification of the target nucleic acid is achieved by a polymerase chain reaction (PCR) technique. Alternatively, amplification of the target nucleic acid is achieved by isothermic methods. Preferably, the isothermic method is selected from the group consisting of NASBA, RCAT and SDA. This embodiment of the invention also contemplates that the method further comprises the step of dissociating the hybridized nucleic acid prior to collection of the amplified nucleic acid. In addition, the method contemplates that at least one of the layers of the microporous membrane is free of association with PNA.

[0061] In still another alternative embodiment, the present invention contemplates a method for separating target nucleic acid from a sample suspected of containing the nucleic acid comprising the steps of (a) providing the multi-layer composite microporous membrane of the invention, wherein PNA associated with highly electropositive material in at least one layer of the membrane is capable of hybridizing to the nucleic acid of interest; (b) contacting the membrane with the sample under conditions sufficient to permit hybridization of the nucleic acid of interest with the PNA; and (c) removing the nucleic acid hybridized to the PNA. In general, the nucleic acid-containing sample is derived from a bodily fluid or matter, animal or vegetable tissue, or a cell lysate or homogenate, or degradation products thereof. Alternatively, the nucleic acid-containing sample comprises a nucleic acid/protein mixture, or a biotechnical preparation of bacteria or viruses.

[0062] According to this aspect of the present invention, the nucleic acid of interest interacts with the PNA under stringent hybridization conditions. Also contemplated by the present invention is a self-contained device for extraction, amplification and detection of nucleic acid of interest comprising the membrane of the invention.

[0063] In still another alternative embodiment, the present invention provides a multi-layer, composite microporous membrane, wherein one or more of the layers of the membrane have been modified so as to confer on the layer a capability to associate therewith target nucleic acid. Preferably, the one or more of the layers has been physically modified so as to confer on the layer the capability to associate therewith target nucleic acid. More preferably, the physical modification comprises addition of one or more heterogeneous substances to the layer. According to this aspect of the invention, the one or more heterogeneous substances are added to the layer prior to fabrication of the multi-layer membrane. Alternatively, the one or more heterogeneous substances are added to the layer after fabrication of the multi-layer membrane.

[0064] In accord with an additional aspect of the invention, the one or more heterogeneous substances comprise a hydrophilic, highly electropositive material. Alternatively, the one or more heterogeneous substances comprise sequence specific peptide nucleic acid. In an alternative aspect of this embodiment of the invention, the one or more of the layers has been chemically modified so as to confer on the layer the capability to associate therewith target nucleic acid. Preferably, chemical modification of the one or more layers comprises charge modification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The above description of the present disclosure will be more fully understood with reference to the following detailed description when taken in conjunction with the accompanying drawings, wherein:

[0066]
FIG. 1 is an illustration of a method for isolating and amplifying nucleic acids from a crude biological sample using a nylon membrane imbued with a highly electropositive solid phase hydrophilic material.

[0067]
FIGS. 2A, 3A and 4A are scanning electron photomicrographs of a microporous membrane of the present disclosure illustrating the membrane imbued with a highly electropositive solid phase hydrophilic material at 500×, 2,500×, and 5,000×;

[0068]
FIGS. 2B, 3B and 4B are scanning electron photomicrographs of a control microporous membrane free of the highly electropositive solid phase hydrophilic material of FIGS. 2A, 3A and 4A at 500×, 2,500×, and 5,000×;

[0069]
FIG. 5 is a photograph of an agarose gel stained with ethidium bromide;

[0070]
FIG. 6 is a photograph of the lower portion of the gel of FIG. 5;

[0071]
FIG. 7 is a comparison of the structures of peptide nucleic acids (PNA's) and deoxyribonucleic acid (DNA);

[0072]
FIG. 8 is a schematic representation of an embodiment of the present invention illustrating a three-layer composite membrane material where one layer comprises PNA, a second layer is free of both PNA and a highly electropositive hydrophilic material, and a third layer comprises a heterogeneous, highly electropositive, hydrophilic material.

[0073]
FIG. 9, in six panels over six pages, illustrates schematically the practice of the present invention with the membrane of FIG. 8 for the capture and selective release of sequence specific nucleic acid.

[0074]
FIG. 10 illustrates schematically the practice of the present invention with the membrane of FIG. 8, having varying pore sizes between layers, used for the separation of genetic material of interest from a complex biological sample.

DETAILED DESCRIPTION OF THE INVENTION

[0075] Unless indicated otherwise, the terms defined below have the following meanings:

[0076] Xtra Bind™ solid phase matrix available from Xtrana, Inc, Denver, Colo. Xtra Bind is a hydrophilic and electropositive solid phase matrix.

[0077] PCR (Polymerase Chain Reaction). A method for amplifying a DNA base sequence using a heat-stable polymerase and two 20 nucleotide primers, one complementary to the (+)-strand at one end of the sequence to be amplified and the other complementary to the (−)-strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence. PCR also can be used to detect the existence of the defined sequence in a DNA sample.

[0078] Affinity chromatography: A technique of analytical chemistry used to separate and purify a biological molecule from a mixture, based on the attraction of the molecule of interest to a particular ligand which has been previously attached to a solid, inert substance. The mixture is passed through a column containing the ligand attached to the stationary substance, so that the molecule of interest stays within the column while the rest of the mixture continues through to the end. Then, a different chemical is flushed through the column to detach the molecule from the ligand and bring it out separately from the rest of the mixture.

[0079] Hybridization: a single strand of a nucleic acid molecule (DNA or RNA) is joined with a complementary strand of nucleic acid, again DNA or RNA, to form a double-stranded molecule (or one which is partly double-stranded, if one of the original single strands is shorter than the other).

[0080] Probe: A single-stranded nucleic acid molecule with a known nucleotide sequence which is labeled in some way (for example, radioactively, fluorescently, or immunologically) and used to find and mark certain DNA or RNA sequences of interest to a researcher by hybridizing to it.

[0081] Rolling Circle Amplification (RCA): an amplification process driven by DNA polymerase which can replicate circular oligonucleotide probes with either linear or geometric kinetics under isothermal (single temperature) conditions. In the presence of two suitably designed primers, a geometric amplification occurs via DNA strand displacement and hyperbranching to generate 1012 or more copies of each circle in 1 hour. In addition to grossly amplifying a signal, this method—called Exponential-RCA—is adequately sensitive to detect point mutations in genomic DNA. Additional information is available on the Molecular Staging Website at www.molecularstaging.com.

[0082] cDNA: complementary DNA—synthesized from an RNA template using reverse transcriptase.

[0083] Reverse transcriptase: an enzyme found in retroviruses that enables the virus to make DNA from viral RNA.

[0084] mRNA: messenger RNA—RNA that serves as a template for protein synthesis.

[0085] Nucleotide: A subunit of DNA or RNA consisting of a nitrogenous base (adenine, guanine, thymine, or cytosine in DNA; adenine, guanine, uracil, or cytosine in RNA), a phosphate molecule, and a sugar molecule (deoxyribose in DNA and ribose in RNA). Thousands of nucleotides are linked to form a DNA or RNA molecule.

[0086] Oligonucleotide: a compound comprising a nucleotide linked to phosphoric acid. When polymerized, it gives rise to a nucleic acid.

[0087] Primer: a short pre-existing polynucleotide chain to which new deoxyribonucleotides can be added by DNA polymerase.

[0088] Template: a molecular mold or pattern for the synthesis of another molecule. Specifically, the DNA molecule from which a PCR or amplification product is generated.

[0089] Intercalating dye: a planar dye molecule that binds to nucleic acid in a non-covalent fashion by inserting itself between the stacked bases of the nucleic acid helix. Fluorescent dyes, like ethidium bromide, can be used to visualize DNA and RNA molecules in gel matrices.

[0090] The present disclosure enables the practice of the present invention to overcome many of the problems associated with the isolation of nucleic acids from large, complex sample volumes, and the amplification of such isolated nucleic acids.

[0091] The invention of the present disclosure combines the attributes of highly electropositive hydrophilic materials that irreversibly bind with one or more nucleic acids with those of microporous membranes having a very high effective surface area. The hybrid structure comprising the microporous membrane imbued or coated with the highly electropositive hydrophilic materials allows for increased presentation of the electropositive materials to permit enhanced nucleic acid binding. As indicated herein, such captured nucleic acid molecules may be used as templates for enzymatic amplification. The further incorporation or modification of the membrane of the present invention to include sequence-specific PNA's provides further capabilities in the practice of sequence-specific nucleic acid capture, amplification and/or identification that heretofore possible in the prior art.

[0092] The membrane of the present invention may be placed into microtiter plates (e.g., 96-, 384-, 1536-wells) thereby allowing for capture of individual nucleic acid samples from biological sources and may be placed into a thermal cycler for PCR, or into a constant temperature incubator for isothermal amplification procedures.

[0093] Advantageously, the highly electropositive material capable of irreversibly binding one or more nucleic acids is selected from the group consisting of silicon (Si), boron (B), titanium (Ti), and aluminum (Al). Such material can be rendered sufficiently hydrophilic by methods well known to those of ordinary skill in the art, as for example by the addition of hydroxyl groups. A particularly useful compound of the present disclosure is a composition known as Xtra Bind (Xtrana, Inc. Denver, Colo.), a composition having significant DNA binding affinity and avidity. Suitable electropositive matrices have been disclosed, containing silicon (Si), boron (B), titanium (Ti), or aluminum (Al), which have been rendered sufficiently hydrophilic by hydroxyl (—OH) or other groups, to result in a surface that irreversibly binds DNA (See for example WO98/46797, the disclosure of which is herein incorporated by reference). Examples of such matrices have been demonstrated using aluminum oxide, silica, or titania. Aluminum oxide particles are particularly useful as this matrix including, but not limited to, alpha (α) aluminum oxide in hexagonal crystal form, which can be milled and classified in a variety of particle sizes. Such materials are available from various commercial sources, such as Washington Mills Electro Minerals Corporation, Niagara Falls, N.Y., as Duralum Special White; also from Atlantic Equipment Engineers, Bergenfield, N.J., as fused alpha aluminum oxide high purity powders.

[0094] Combining the highly electropositive material capable of irreversibly binding one or more nucleic acids (Xtra Bind) with single-layer or multi-layer membranes, results in an enabling platform for isolation and capture of nucleic acids from complex biological samples. The nucleic acid, once captured, can then be analyzed using amplification procedures known to those skilled in the art (PCR, NASBA, RCA) thereby enabling the detection of minute quantities of analyte, such as, for example, nucleic acid, from large sample volumes. When a layer in the multi-layer membrane, with or without highly electropositive material, is further modified to comprise sequence-specific PNA's, additional capabilities involving the capture, identification and/or quantification of specific polynucleotide sequences in a biological sample comprising myriad sources of genetic material are made possible that have heretofore not been possible in the prior art.

[0095] The present disclosure encompasses, at least in part, a microporous multi-layer membrane that comprises a highly electropositive material capable of irreversibly binding single- or multiple-strand nucleic acid (non-sequence specific capture). Such a membrane provides a solid phase platform for the essentially simultaneous capture and amplification of nucleic acid that is capable of handling large sample volumes so as to isolate nucleic acid found in low quantity in the sample volume. When utilizing an embodiment comprising sequence-specific PNA's, then the membrane of the present invention makes it possible to not only detect the presence of nucleic acid in a complex sample comprising many other, possibly interfering, components but also to detect the presence, both qualitatively and quantitatively, of specific polynucleotide sequences in a sample comprising multiple sources of genetic material, even when the specific nucleic acid of interest is present in the sample at a low copy number in comparison to the total nucleic acid content of the sample.

[0096] In one representative embodiment, there are provided one or more microporous membranes, such as a microporous polyamide membrane, comprising (on both interior and exterior surfaces) a highly electropositive material having hydrophilic properties which is capable of irreversibly binding nucleic acid (DNA, RNA, etc.). Preferably, the microporous membrane is a microporous phase inversion membrane, such membranes being well known in the art. Microporous phase inversion membranes are porous solids, which contain microporous interconnecting passages that extend from one surface to the other. The passages provide tortuous tunnels or paths through which the liquid that is being filtered must pass. Due to the high effective surface area of such membranes, such constructs provide a much enhanced capture of nucleic acids from a given volume of sample. Such membranes also permit enhanced amplification of bound nucleic acid when used as a solid amplification medium. In addition, such membranes may provide additional advantages in being able to effectively filter out, by size exclusion, solid phase components of complex samples.

[0097] Such membranes may function in sample preparation wherein one captures nucleic acid from any number of sources (bacteria, fungi, blood samples, etc.) on the membrane, and the captured nucleic acids are amplified and identified using specific probe molecules, which probe molecules may be PNA's specifically synthesized to be complementary to polynucleotides of particular interest.

[0098] By “phase inversion support” it is meant a polymeric support that is formed by the gelation or precipitation of a polymer membrane structure from a “phase inversion dope.” A “phase inversion dope” consists of a continuous phase of dissolved polymer in a solvent, coexisting with a discrete phase of one or more non-solvent(s) dispersed within the continuous phase. The formation of the polymer membrane structure generally includes the steps of casting and quenching a thin layer of the dope under controlled conditions to effect precipitation of the polymer and transition of discrete (non-solvent phase) into a continuous interconnected pore structure. This transition from discrete phase of non-solvent (sometimes referred to as a “pore former”) into a continuum of interconnected pores is generally known as “phase inversion.” Such membranes are well known in the art. Particular attention is drawn to the '441 patent the disclosure of which teaches the preparation of a supported, multi-layer microporous membrane such as used in the present invention. Typically, a phase inversion support is formed by dissolving the polymer(s) of choice in a mixture of miscible solvent(s) and non-solvent(s), casting a support pre-form, and then placing the surface of the support preform in contact with a non-solvent (liquid or atmosphere) diluent miscible with the solvent(s) (thereby precipitating or gelling the porous structure.

[0099] Advantageously, the electropositive material capable of irreversibly binding one or more nucleic acids is highly electropositive and is selected from the group consisting of silicon (Si), boron (B) and aluminum (Al). Such material can be rendered sufficiently hydrophilic by methods well known to those of ordinary skill in the art, as for example by the addition of hydroxyl groups or by formation of an oxide.

[0100] A presently preferred phase inversion support comprises polyamides—organic polymers formed by the creation of amide bonds between monomers of one or more types. Particularly useful polyamides in the present disclosure are nylons. Nylons comprise aliphatic carbon chains, usually alkylene groups, between amide groups. The amide groups in nylons are very polar and can hydrogen bond with each other, and are essentially planar due to the partial double-bond character of the C—N bond. Nylons are polymers of intermediate crystallinity; crystallinity being due to the ability of the NH group to form strong hydrogen bonds with the C═O group. Nylon typically consists of crystallites of different size and perfection. By way of example, nylon 6,6 is typically synthesized by reacting adipic acid with hexamethylene diamine, and is a particularly presently preferred nylon useful with the practice of the present invention as disclosed herein.

[0101] The present inventors have discovered that hydrophilic electropositive materials may be dispersed into polyamide materials so as to be operatively positioned therein to produce superior nucleic acid binding matrices. Particularly useful matrices are microporous in nature, more particularly microporous membranes having asymmetric pores. Such microporous membranes facilitate capture of nucleic acids contained in relatively very low concentration in relatively large volume of sample fluid and allow the relatively large volumes of sample fluid to be filtered due to the high effective surface areas thereof. Further modification of the membranes to include sequence-specific PNA's renders the membranes of the present invention useful for the capture and identification of specific nucleotides in samples with complex mixtures of nucleic acid.

[0102] The nucleic acid of interest, once irreversibly bound to the membrane, can function as a template for enzymatic amplification procedures, including, but not limited to, PCR, NASBA, RCA and other isothermic amplification methods, as presently known in the art, or as may become known. Such use of the microporous membrane comprising the highly electropositive material capable of irreversibly binding one or more nucleic acids, as disclosed herein, enables the detection of minute quantities of analyte, such as, for example, nucleic acid, from large sample volumes, for example allowing detection of a single organism from a large input volume. As is known to those skilled in the art, such detection is not easily performed using currently available technologies, such as those described above. Furthermore, the incorporation of sequence-specific PNA into the membrane makes possible the capture and detection of specific polynucleotides at low copy number even from samples comprising multiple sources of nucleic acid.

[0103] The membranes of the present disclosure may also be placed into a container or other structure to optimize sample flow and handling, as well as for amplification and detection. Such structures may include, but are not limited to, a microcentrifuge or centrifuge tube, a multiwell plate, a filter housing, or a manifold, or other devices as would be known to those skilled in the art.

[0104] In another representative embodiment, there is disclosed a multi-layer membrane having one or more layers that do not include any significant amounts of highly electropositive hydrophilic material(s) capable of irreversibly binding nucleic acids in conjunction, with one or more additional layers that include the electropositive materials, optionally modified by the presence of PNA's. The membrane layers that do not include any electropositive material can be used to remove debris from the sample prior to exposing the nucleic acid fraction with the membrane layer comprising the electropositive material and/or PNA. Discrete layers in the membrane may be produced that include the electropositive material. The problem of isolating small quantities of a nucleic acid molecules from a large sample volume can be greatly reduced by incorporating the electropositive material in a membrane layer downstream of a membrane layer without the propensity for binding nucleic acids, by removing debris that might interfere with nucleic acid binding. A multi-layer microporous membrane that might be used for such purposes may be produced, for example, by the methods described in the '441 patent to Vining, et al. A presently preferred multi-layer membrane comprises one or more microporous polyamide layers, more preferably one or more microporous nylon layers.

[0105] In yet another representative embodiment, there is disclosed a multi-layer membrane having one or more layers individually functionalized to facilitate the capture of specific nucleic acid molecules. Such individually functionalized layers optionally may comprise highly electropositive hydrophilic material(s) capable of irreversibly binding nucleic acids and/or sequence-specific PNA's. Furthermore, it is possible to modify the individual layers of the multi-layer membrane to facilitate binding, either specific or non-specific, of nucleic acid. Such modification can include both physical modification, such as the incorporation of highly electropositive hydrophilic material, as well as chemical modification, such as derivitization of exterior or interior surfaces, or charge-transfer treatment, such as with nylon membrane materials.

[0106] In a presently preferred embodiment, the individually functionalized layers are used to remove nucleic acids that are not desired to be detected in a subsequent membrane layer or layers. For example, a multi-layer membrane of such representative embodiment may comprise a first or outer layer individually functionalized so as to be capable of removing bacterial nucleic acid from a sample containing human nucleic acid, the human nucleic acid being desired to be enriched in an inner membrane layer of the multi-layer membrane. That is, the layers can be arranged with respect to each other such that undesired nucleic acid can be removed upstream of a membrane layer in which a particular analyte of interest (such as nucleic acid) is desired to be collected. A multi-layer microporous membrane of such embodiment may be produced, for example, by the methods described in the '441 patent referenced above.

[0107] In yet another representative embodiment, there is provided a nucleic acid archival substrate comprising a microporous membrane imbued or coated with a highly electropositive hydrophilic material capable of irreversibly binding one or more nucleic acid types, and optionally modified to comprise sequence-specific PNA's. Nucleic acid bound to such substrate can be stored for long periods of time. Nucleic acid storage can be particularly useful, for example, when samples may need to be compared to known samples obtained in the future, such as when biological material is isolated at a crime scene without a suspect being immediately identifiable.

[0108] In still another representative embodiment, there is provided one or more microporous membranes, such as, a microporous polyamide membrane, comprising a highly electropositive material with hydrophilic properties that is capable of irreversibly binding nucleic acid and further comprising another nucleic binding material, e.g., anion exchange resin, intercalating dye, PNA, etc.

[0109] In a presently preferred embodiment, the highly electropositive hydrophilic material capable of irreversibly binding one or more nucleic acid types has a particle size in the range of about one nanometer (1 nm) to about one thousand microns (1000μ). Such particle sizes have been found to provide enhanced efficacy with respect to nucleic acid binding per unit area of the membrane, which membrane, being microporous, comprises significant interior as well as exterior surface area.

[0110] Methods for preparing such microporous membranes are also disclosed. In a presently preferred representative method, a dope is prepared with the highly electropositive hydrophilic material capable of irreversibly binding one or more nucleic acid types operatively dispersed therein, and the dope is used in the production of microporous membrane by methods well known in the art. In another method, the highly electropositive hydrophilic material is placed in a polymer that is coated onto, or saturated into, a preformed microporous membrane.

[0111] By dispersing the highly electropositive hydrophilic material capable of irreversibly binding one or more nucleic acid types into the material to be used in the formation of a microporous membrane, a composite membrane is formed which permits high surface area for the capture and/or removal of nucleic acids. Alternatively, but less desirably (due to the difficulty in providing a uniform coating throughout the microporous structure), the microporous membrane may be coated with material, such as a resin, comprising the highly electropositive hydrophilic material.

[0112] As would be recognized by one of skill in the appropriate art, it is possible to synthesize PNA's with complementarity to specific nucleotide sequences of interest on solid supports. These art-recognized methods of PNA synthesis can be adapted to prepare sequence-specific PNA's directly on the highly electropositive hydrophilic materials prior to the incorporation of such material into a layer or layers of the membranes of the present invention. Preferably, the synthesis of such PNA's on the solid phase material renders the highly electropositive hydrophilic material capable of binding to only the specific nucleic acid of interest and not to all sources of nucleic acid present in the sample, at least within a specific layer of the multi-layer membrane.

[0113] As referenced above, the composite membrane may be used not only to capture the nucleic acid, but may be used as a platform for amplifying the bound nucleic acid, and detecting the same. The captured nucleic acid associated with the microporous membrane may function as a solid phase template for amplification, enabling detection of minute quantities of nucleic acid in a large sample volume. The microporous membrane having the captured nucleic acid may also be saved for archival purposes, with amplification and detection being performed at a later date, either on the membrane of after removal of the nucleic acid from the archival membrane.

[0114] Turning to FIG. 1, there is shown an illustrative, representative, method for amplifying nucleic acids using the composite microporous membranes of the present disclosure. In step A, the microporous membrane comprising the highly electropositive hydrophilic material capable of irreversibly binding the nucleic acid of interest is exposed to a complex biological sample containing cellular debris and nucleic acid. Nucleic acid is irreversibly captured on the membrane that is washed several times to remove non-bound proteins and cellular debris. In step B, the bound nucleic acid is amplified by known techniques with the addition of, for example, primers, deoxynucleotide triphosphate molecules (dNTPs), buffer, etc., producing amplified product (step C). The membrane having the bound nucleic acid can be used as a template for further amplification cycles, or be stored for archival purposes.

[0115] In a presently preferred representative embodiment, a multi-layer microporous membrane is employed, having at least one layer incorporating the highly electropositive hydrophilic material capable of binding irreversibly to one or more nucleic acid types and one or more layers of the membrane being void of any of the highly electropositive hydrophilic material capable of binding irreversibly to nucleic acid. Those layers that do not incorporate the electropositive materials can be used to remove debris from the sample prior to exposure of the nucleic acid fraction to a layer including the electropositive material. A further variation of the membrane structure, and one that is preferred for certain unique application of the use of the membranes of the present invention, incorporates a layer, separate from the layer comprising highly electropositive material alone, wherein the layer is modified to comprise PNA's. These embodiments of the membrane of the present invention make possible the solution of problems associated with the isolation of very small quantities of a nucleic acid molecule from a large sample volume in a manner not possible in the prior art. These ends can be more readily reached by incorporating the electropositive material in a layer downstream of a layer without the capacity to bind nucleic acid, whereby the upstream layer functions to remove debris that might interfere with nucleic acid binding, and/or amplification, and/or detection in the layer comprising the electropositive material.

[0116] In another presently preferred representative embodiment, there is disclosed a multi-layer membrane having at least one layer functionalized for the capture of specific nucleic acid molecules and at least one layer comprising highly electropositive hydrophilic material(s) capable of irreversibly binding nucleic acids. The functionalized layers optionally may comprise highly electropositive hydrophilic material(s) capable of irreversibly binding nucleic acids. In a presently preferred type of such representative embodiment, the functionalized layers are used to remove nucleic acids that are not desired to be detected in a subsequent layer of the multi-layer membrane. In a more preferred embodiment, one of the layers is modified to comprise sequence-specific PNA's capable of selectively binding to a specific polynucleotide of interest from a sample comprising multiple sources of nucleic acid.

[0117] It is presently preferred that the membrane in which the highly electropositive hydrophilic material is incorporated has pore sizes in the range of about 0.04 microns to about 20 microns. Preferably, the membrane is a phase inversion microporous membrane. Such membrane preferably comprises nylon (such as nylon 6,6), but may comprise other materials used in the fabrication of single-layer and multiple-layer phase inversion microporous membranes as would be known to those of ordinary skill in the art.

EXAMPLE 1

Preparation of Nylon Membrane Comprising Highly Electropositive Hydrophilic Material

[0118] A dope formulation comprising approximately 16.1% by weight Nylon 6,6 (Monsanto® Vydyne™ 66Z), 77.1% by weight formic acid, and about 6.8% by weight methanol, was produced using the methods disclosed in U.S. Pat. Nos. 3,876,738 and 4,645,602, the disclosure of each of which is herein incorporated specifically by reference. This is the standard formulation and method used to produce the control (white) membrane.

[0119] To produce the multi-layer composite microporous membrane comprising a highly electropositive hydrophilic material (Xtra Bind) of the present invention, the method is similar to the above, but with the additional step of adding the Xtra Bind prior to the addition of Nylon, and changing the mixing apparatus to facilitate uniform dispersion and uniform suspension of the heterogeneous Xtra Bind material in the dope. Briefly, the altered method consists of the following steps: a dope formulation comprising approximately 75.1% by weight formic acid and 6.3% by weight methanol (to final weight of dope) was mixed in a Silverson® Model #L4SRT\SU (Sealed Unit) one-half liter sealed vessel with high dispersion mixing head for about 15 minutes at about 400 rpm. To this mixture, 3.1% Xtra Bind material (500 mesh Xtra Bind Matrix) at an intended ratio of about a 1:5 parts by weight of Xtra Bind:Nylon was added. The resultant composition was mixed for about 10 minutes using the same mixing apparatus at about 2000 rpm. The resultant was then dispensed into a 16 oz. glass jar. To this resultant about 15.5% by weight Nylon-66 (Monsanto® Vydyne™ 66Z) (to final weight of dope) was added. The resulting composition was mixed with a 1.25 inch diameter three-blade propeller mounted on a T-line® Model #134-1 laboratory mixer. A cap with a sealing arrangement for the propeller shaft was fabricated to minimize volatile losses. Mixing occurred at ambient temperatures. The mix cycle began with an initial mix at about 350 rpm for about one-half hour; then the mixer was slowed to about 70 rpm for about another two hours to homogenize the dope. After the resultant was mixed, the glass jar was removed from the mixer, and sealed with a cap. The sealed vessel and it's contents were rolled on a rolling mill jar mixer, submerged in a waterbath at about 34° C. for several hours to ensure a uniform thermal history (maximum mix temperature) of the dope, and maintain the suspension of Xtra Bind material in the mix. The rolling mill was then removed from the water bath. The jar and its contents were allowed to cool to room temperature while rolling (again, to maintain the suspension). Gentle rolling continued until the dope was used to form a microporous membrane.

[0120] To gain an appreciation for the pore size of a microporous nylon membrane with Xtra Bind cast directly from this dope, a small portion (˜20 cc) of the dope was cast and quenched in a laboratory apparatus which simulates the casting process described in U.S. Pat. No. 3,876,738, to Marinaccio and Knight, to produce a single layer, nominally 5 mil thick, wet, non-reinforced layer of microporous nylon membrane. This membrane was washed in deionized water, then folded over onto itself (about 10 mils, wet) and dried under conditions of restraint to prevent shrinkage in either the machine direction (x-direction) or cross direction (y-direction). This produced a small sample of dried double layer non-reinforced microporous nylon membrane having a combined thickness of about eight (8) mils after shrinkage in thickness (z-direction) of the collapsing wet pore structure was complete (actual thickness shown in Table 1, below). An Initial Bubble-Point and Foam-All-Over-Point test was performed, as described in U.S. Pat. No. 4,645,602, using deionized water as a wetting fluid.

[0121] A second casting was also produced via cast, quench, and wash. It was not folded over onto itself, but dried under conditions of restraint as a single layer, to produce a small sample of dried single layer non-reinforced microporous nylon membrane. This sample was produced for Scanning Electron Microscopy (SEM) analysis.

[0122] The control (white) membrane was similarly cast, quenched, washed, and dried in both single and double layer samples, and tested. The results of such testing are provided in Table 1, below.

1TABLE 1For SEM analysisDouble layerSingle layerXtra Bind: NylonDryDryContentIBP psigFAOP psigThickness (mils)Thickness (mils)0:10030.534.08.54.5Control(White)1:539448.24.3

[0123] The dry single-layer versions of the control (white) membrane and the Xtra Bind-containing membrane were submitted for SEM analysis in cross section. The results are shown in FIGS. 2A, 3A and 4A. From a review of the SEMs, it is evident that the Xtra Bind matrix is embedded within the pore structure of the nylon membrane, in such a way that the surfaces of the heterogeneous Xtra Bind particles are accessible to fluids within the pores; therefore, the binding functionality of the Xtra Bind is maintained.

[0124] As can be clearly seen from the SEMs of FIGS. 2B, 3B and 4B, the Control sample contains no irregularly shaped objects/particles in the passages or tortuous tunnels or paths formed in the final membrane, while the Test sample clearly shows non-membrane material, in this case the highly electropositive hydrophilic material capable of irreversibly binding one or more nucleic acids, positioned in with the passages, tortuous tunnels or paths formed in the final membrane.

[0125] A sample similar to the second casting was also produced via cast, quench, and wash. This casting was not folded over onto itself, but dried under conditions of restraint as a single layer, to produce a small sample of dried single layer non-reinforced microporous nylon membrane. This sample was utilized for determining if the non-reinforced microporous nylon membrane having the highly electropositive hydrophilic material is capable of irreversibly binding one or more nucleic acid types. The results of these tests are reported in Example 2 below.

EXAMPLE 2

Binding of Nucleic Acid to Membranes of Example 1

[0126] To assess whether microporous nylon membrane containing the highly electropositive hydrophilic material (Xtra Bind™) irreversibly binds nucleic acid and if the captured nucleic acid is capable of functioning as a template for PCR, the following experiment was performed.

[0127] Known amounts of K562 cells were lysed and diluted in water. Either 10.0-ng or 1.0-ng samples of genomic DNA was contacted with an Xtra Bind-containing microporous nylon membrane or unmodified nylon microporous membrane (without Xtra Bind) and incubated in the lysis/binding buffer for an appropriate time in microcentrifuge tubes. The first two lanes of a data set are duplicates of 10.0-ng samples; the second two are duplicates of 1.0-ng samples. The membranes were then washed with buffer and the membranes were combined with appropriate components to support DNA amplification using the polymerase chain reaction (PCR). Forward and reverse primers directed against the human leukocyte antigen DRβ (HLA-DRβ) were used to amplify the product of interest.

[0128]
FIG. 5 is a photograph of an agarose gel stained with ethidium bromide. The first four lanes (PCR Controls) are controls indicating that the PCR is functional for the production of the product of interest. The next four lanes contain the samples of reaction product when the genomic DNA is incubated in the presence of the unmodified nylon, washed and then amplified by the PCR. It can be readily seen that no product is detected. The last four lanes are negative controls—PCR's that lack DNA template, indicating that any product seen is not the result of contaminating DNA in any of the buffer components used in the reaction.

[0129]
FIG. 6 is a photograph of the lower portion of the gel in FIG. 5. This sample is one where the genomic DNA was incubated in the presence of membrane containing the Xtra Bind material, washed and then amplified by the PCR. PCR product is readily seen in these lanes indicating that K562 genomic DNA was retained by the membrane and that this genomic DNA is functional as a template for enzymatic amplification.

[0130] Clearly a difference is seen between the Xtra Bind-containing microporous nylon membrane and the unmodified microporous nylon membrane demonstrating the superior performance of the Xtra Bind-containing microporous nylon membrane in retaining nucleic acid as a functional template for the PCR.

[0131] Thus, it should be apparent from the above example that the microporous membranes disclosed herein and the methods of making and using same provides improved membranes and methods for separating nucleic acids from liquid biological samples and amplifying the same.

[0132] It should be pointed out that the capture of nucleic acids using highly electropositive hydrophilic material capable of irreversibly binding one or more nucleic acids, such as, for example, Xtra Bind, is the irreversible binding of the nucleic acids to the highly electropositive hydrophilic material. This enables the membrane containing the highly electropositive hydrophilic material of the present disclosure to be used, among other uses, as an archiving system. Additionally, it should be clear that a large volume of sample can be processed using the, presently preferred, nylon microporous membrane containing the, presently preferred, Xtra Bind material.

EXAMPLE 3

Capture and Identification of Single Nucleotide Polymorphisms (SNPs) in a Target Sequence

[0133] As the results from Example 2, above, indicate, microporous nylon membranes modified to comprise a highly electropositive, hydrophilic material such as Xtra Bind are capable of the capture and subsequent amplification of target nucleic acid. The results of example 2, coupled with the teachings of the '441 patent, illustrate that multi-layer composite membranes of the present invention can provide a unique combination of characteristics that makes possible a powerful set of applications heretofore unavailable in the prior art. Example 3, and the examples that follow, illustrate a representative selection of these applications.

[0134] A rapid and accurate detection of genetic variants, including single-base mismatches, is essential for the detection of genetic diseases. Even a single-base substitution in a human gene can result in deleterious effects in humans. Thus, there is a need for a sensitive nucleic acid-based diagnostic technique for the detection of genetic diseases which will include the ability to ascertain whether an individual is heterozygous or homozygous allelic for a genetic disease.

[0135] Single nucleotide polymorphisms (SNPs) are the most common type of genetic variation found in the human genome, occurring approximately once per 1000 bases. Since they are single base changes in the primary DNA sequence they are easily detected by sequencing analysis. They can be useful as genetic markers for navigating the genome. As unrelated entities, the value of SNP's in disease manifestation is questionable. However, it is believed that if a SNP linkage map can be built, SNP's that travel together may be indicators of disease states or predispositions for developing a particular disease. In addition, it is hypothesized that SNP's may be useful in designing patient-specific pharmaceutical therapies to optimize the efficacy of the drug and to avoid adverse drug reactions (ADR's).

[0136] Nucleic acid sample (DNA or RNA) is filtered through the membrane as it appears in FIG. 9A. The sample can be lysed prior to filtration or on the membrane itself. Buffer is functional for lysis and does not affect nucleic acid binding to the PNA. Nucleic acid is bound in a sequence-specific fashion in Layer 1 (FIG. 9B) using membrane-bound PNA's directed against the sequence(s) of interest. Buffer for binding in Layer 1 is designed so as to select against binding in Layer 3. Should strand invasion be required, PNA clamps can be employed in Layer 1, as well.

[0137] PNA-nucleic acid hybrids have a much higher thermal stability than DNA-DNA DNA-RNA, or RNA-RNA species. In addition, PNA duplexes are not affected by ionic strength. This feature allows for formation of duplexes under conditions that do not favor formation of standard nucleic acid duplex molecules (e.g., cell extracts and serum). PNA's also exhibit greater specificity in binding to a complementary DNA or RNA molecule, since a PNA-DNA mismatch is significantly more destabilizing than a mismatch in a DNA duplex species. Mismatches, which correlate to SNP's in the complementary region between the PNA and target nucleic acid, can be dissociated from sequences lacking the nucleotide variants at temperatures ˜10° C. below the temperature that denatures a perfect hybrid molecule. This feature will allow for the specific release of the bound SNP-containing variant DNA while leaving the wild-type sequence attached to the PNA in Layer 1 of the membrane (FIG. 9C).

[0138] A buffer at the appropriate temperature is passed through the membrane that releases the DNA/mRNA molecule containing a nucleotide variant from the PNA tethers in Layer 1. This buffer also functions to optimize binding of the released nucleic acid to the Xtra Bind material in Layer 3.

[0139] Once freed from Layer 1, the SNP-containing sequence variants pass through Layer 2 and are bound irreversibly in Layer 3 of the membrane on the Xtra Bind material. The bound DNA can then be amplified or analyzed as desired, utilizing techniques well known in the art.

EXAMPLE 4

Separation of Victim's Vaginal/Cervical Epithelia From Perpetrator's Sperm Cells

[0140] A problem frequently encountered in forensic analyses of genetic material, such as in identifying the attacker in a rape case, is that the epithelial cells collected during a vaginal swab often overwhelm the PCR and prevent the detection of the perpetrator's DNA. A means for enriching for Y-chromosome-specific sequences by achieving the separation of epithelia from sperm would significantly reduce the amount of interfering DNA in the sample.

[0141] In this Example, an asymmetric, three-layered membrane is employed for separating epithelial cells from sperm in Layer 1, capture of sperm cells in Layer 2, and sequence-specific capture of Y-chromosome sequences using PNA incorporated in Layer 3. The PNA's used for capture of Y-chromosome-specific sequences are derived from nucleotide sequence databases in the public domain. The PNA capture probes are designed to recognize short tandem repeats (STR's) unique to the male Y-chromosome.

[0142] A swab from victim is placed into PBS to release the cells. The sample is then filtered through the membrane where the epithelial cells are captured as described above. Alternatively, as would be recognized by one of skill in the relevant art, the membrane can be contained in a “collection tube” or other similar apparatus, and effect the same result. It is also possible to utilize an integral construction and reduce concerns of cross contamination. The sperm cells are washed through the first membrane layer by either subsequent ‘forward’ washes or repeated cycles of reverse and forward PBS washes. Sperm cells are lysed in Layer 2, and the released DNA is captured on the PNA contained in Layer 3. Contaminating DNA from Layer 1-captured cells would not bind and can be washed away using stringency washes. The captured sperm DNA can then be released from the membrane or analyzed in situ by PCR or other method(s).

EXAMPLE 5

Capture and Identification of the Presence of Genetically Modified Organisms (GMOs)

[0143] The presence of GMOs is not only a concern for uncontrolled proliferation of artificially modified genetic material, with an accompanying potential for loss of native species by GM species with greater robustness, but also a concern for possible allergic reactions to gene products not usually present in a particular food (e.g., peanut proteins in potatoes). In this application, an asymmetric membrane containing PNA's specific for the transgene contained in the GMO of interest are contained in Layer 3. DNA is prepared from organisms to be evaluated for the presence of sequences indicating incorporation of genetically-modified loci, for example, the presence of genes encoding virus resistance proteins and genes improving resistance to herbicides in food plants. A concern, particularly in the European Union (EU), is gene transfer among species by cross pollination.

[0144] Use of a membrane similar to that described in Example 3 above would improve the ability of researchers and agricultural agencies to monitor more rapidly and efficiently the extent of gene transfer and identify plants/animals harboring the exogenous gene(s).

[0145] Samples are prepared, and then contacted with membranes containing PNA's to capture the sequence of interest by placing the PNA-containing membrane in a multi-well plate (96-, 384-, 1536-well), creating, in essence, a two-dimensional, semi-micro array. In this fashion, a large number of samples could be screened rapidly for one or several exogenous sequences.

[0146] The process and membrane is as depicted in FIG. 10. DNA samples are introduced to the membrane and captured in Layer 1, containing PNA's directed toward the transgenic locus. Non-specific DNA is easily washed away using appropriately stringent conditions. DNA captured on the PNA's located in Layer 1 are released and captured irreversibly in the Xtra Bind-modified membrane of Layer 3 downstream. Again, Layer 2 functions, in effect, as a wash layer as described in Example 3 and in FIG. 8.

[0147] The DNAs bound in the Xtra Bind matrix optionally can be amplified for analysis. As would be recognized by one of skill in the art, the combined capture and amplification of the target DNA would be desirable in those situations where the transgene would be present in total nucleic acid at very low levels. In addition, the PNA-bound DNA can be examined for sequence variation and provide insight into how these modifications affect the expression of the transgene.

[0148] While the disclosure has been described with respect to presently preferred embodiments, those skilled in the art will readily appreciate that various changes and/or modifications can be made to the disclosure without departing from the spirit or scope of the disclosure as defined by the appended claims. All references cited in this specification are herein incorporated by reference to the same extent as if each individual reference was specifically and individually indicated to be incorporated by reference.

	Number	Date	Country
Parent	09873675	Jun 2001	US
Child	10162131	Jun 2002	US

Modified microporous membrane for non-specific and sequence-specific nucleic acid capture and methods of use

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