WO2008021995A1 - Methods and compositions for detecting an analyte in a biological sample - Google Patents

Abstract

The invention provides compositions and methods for capturing, purifying, and detecting the presence of at least one nucleic acid molecule present in a liquid biological sample. In particular, the invention provides diagnostic methods for the detection of nucleic acid molecules present in urine.

Description

METHODS AND COMPOSITIONS FOR DETECTING AN ANALYTE IN A

BIOLOGICAL SAMPLE

BACKGROUND OF THE INVENTION Procedures for detecting minute quantities of analytes in solution are widely used in the forensic sciences. In forensic laboratories, genomic DNA present in a blood sample is typically analysed using an FT A® Card as a blood-storage medium. The FT A® GeneCard is a chemically-treated filter paper designed for the collection and storage of biological samples for subsequent DNA analysis. Typically the FT A® Card is used to store genomic DNA in the form of dried spots of human whole blood, the cells of which were lysed on the paper. The captured DNA is washed to remove cellular inhibitors of enzymatic reactions and chemical stabilizers added to the blood sample. Once isolated on the card, the genomic DNA is stable at room temperature for at least five to ten years.

In the medical field, analytes of clinical interest are detected in a variety of biological samples for diagnostic purposes or for monitoring the efficacy of a therapeutic regimen.

Nucleic acid molecules or fragments thereof can be released into biological fluids when cells die via apoptosis or necrosis related to pathogen infection, cancer, or other pathology. Nucleic acid molecules present in biological fluids may be isolated and analysed for diagnosis of pathology. Urine is not considered an ideal source of such nucleic acid molecules because it contains only a low concentration of nucleated cells present in urine. Because urine is a biohazardous liquid that may be contaminated with pathogens, precautionary measures must be used when shipping it to laboratories for analysis. Moreover, nucleic acid molecules present in urine are susceptible to degradation by nucleases naturally present in urine or derived from growth of microorganisms in urine. Methods for isolating nucleic acid molecules present in urine are required to facilitate their subsequent analysis. These are needed due to the desirability of urine as a clinical sample, since urine can be obtained through painless, non-invasive methods that are safe for donor and collector.

SUMMARY OF THE INVENTION The invention provides compositions and methods for capturing, purifying, and analyzing nucleic acid molecules in a liquid biological sample.

In one aspect, the invention provides a method for purifying at least one nucleic acid molecule present in a liquid sample, the method involving contacting a matrix with a liquid sample at a sample application site; binding at least one nucleic acid molecule to the matrix at the sample application site; applying a wash buffer and chromatographically purifying the nucleic acid molecule(s) at the sample application site, and absorbing excess liquid (e.g., excess sample or excess wash buffer) from the matrix with an absorbent reservoir. In another aspect, the invention provides a method for purifying at least one nucleic acid molecule present in a biological sample selected from any one or more of urine, spinal fluid, semen, vaginal fluid, sputum, phlegm, stool, cellular lysates, a cell, tissue, products of conception, organ lysate, or any other composition excreted or secreted by a subject, the method involving contacting a matrix with a biological sample at a sample application site; binding at least one nucleic acid molecule to the matrix at the sample application site; applying a wash buffer and chromatographically purifying the nucleic acid molecule(s) at the sample application site, and absorbing excess liquid from the matrix with an absorbent reservoir.

In one embodiment of the previous aspects, the nucleic acid molecule(s) has a length of between about 10 and 3000 base pairs. In another embodiment of the previous aspects, the nucleic acid molecule(s) is a single or double-stranded DNA or RNA molecule. In another embodiment of the previous aspects, the nucleic acid molecule(s) is from a cell that lysed and released its cellular contents into the lumen of the urinary tract. In yet other embodiments of the previous aspects, the nucleic acid molecule(s) is derived from a white blood cell, a urinary tract epithelial cell, or a bladder cell. In yet other embodiments of the previous aspects, the matrix comprises at least one nucleic acid molecule-binding conjugate at the sample application site. In yet other embodiments of the previous aspects, the nucleic acid molecule-binding conjugate(s) is a DNA-binding protein, a non-protein ligand, a polysaccharide, an aptamer, a detectable polypeptide or nucleic acid molecule(s), or an antibody that specifically binds DNA or combinations thereof. In yet other embodiments of the previous aspects, the matrix comprises a cellulose-based material, a metal, a ceramic, a polylysine polymer, a charged polymer that binds DNA, or a plastic material. In yet other embodiments of the previous aspects, the matrix further comprises one or more components from any one or more of a buffer having a weakly basic pH, a chelating agent, a surfactant, uric acid or a urate salt, an enzyme having proteolytic activity, a polyanionic agent, an agent capable of precipitating lipid or hydrophobic matter, and a nuclease inhibitor. In yet other embodiments of the previous aspects, the chelating agent is EDTA. In yet other embodiments of the previous aspects, the surfactant is an anionic surfactant. In yet other embodiments of the previous aspects, the component is adsorbed on or incorporated into the matrix. In yet other embodiments of the previous aspects, the method further involves the step of washing the matrix involving the bound nucleic acid molecule(s). In yet other embodiments of the previous aspects, the matrix binds the nucleic acid molecule(s) via a covalent or non-covalent interaction.

In another aspect, the invention provides a method for detecting at least one nucleic acid molecule having between 10 and 3000 bases in length in a liquid sample, the method involving capturing a nucleic acid molecule(s) containing between 10 and 3000 bases present in a liquid sample on a matrix; contacting the matrix with a wash buffer to purify the nucleic acid molecule(s); and detecting the presence or the absence of the nucleic acid molecule(s) on the matrix.

In another aspect, the invention provides a method for identifying the disease status in a subject, the method involving capturing and purifying at least one nucleic acid molecule containing between 25 and 1000 bases present in urine from a subject according to the method of any previous aspect; and detecting a nucleic acid molecule(s) captured on the matrix, wherein the detection identifies the disease status of the subject.

In another aspect, the invention provides a method for identifying at least one fetal genetic marker in a biological sample, the method involving capturing and purifying at least one nucleic acid molecule containing between 25 and 1000 bases present in urine from a subject according to the method of any one previous aspect; and detecting a nucleic acid molecule(s) captured on the matrix, wherein the detection identifies a fetal genetic marker in a biological sample. In one embodiment, the fetal genetic marker identifies fetal gender, a fetal genetic defect, a metabolic defect or combinations thereof. In another aspect, the invention provides a method for identifying the rehabilitation status or therapeutic tolerance status of a subject having a history of addiction or medical use of therapeutics, the method involving capturing and purifying at least one nucleic acid molecule containing between 25 and 1000 bases present in urine from the subject according to the method of any previous aspect; and detecting a nucleic acid molecule(s) captured on the matrix, wherein the detection identifies the rehabilitation or therapeutic tolerance status of the subject. In another aspect, the invention provides a device for purifying at least one nucleic acid molecule present in a liquid sample, the device containing a first portion containing a matrix that binds at least one nucleic acid molecule at a sample application site, wherein the matrix is present on a solid support; a second portion containing an absorbent reservoir present on a solid support; and a hinge connecting the first and second portions, wherein the hinge brings the matrix and the reservoir into contact when closed. In one embodiment of the previous aspect, the device further includes a removable container centered over the sample application site that allows a liquid to contact the sample application site. In another embodiment of the previous aspect, the matrix comprises at least one nucleic acid molecule- binding conjugate at the sample application site. In another embodiment of the previous aspect, the nucleic acid molecule-binding conjugate(s) is a DNA-binding protein or an antibody that specifically binds DNA. In another embodiment of the previous aspect, the matrix and the absorbent reservoir are circular, and the reservoir includes a hole centered over the application site when the matrix and the reservoir are brought into capillary communication.

In another aspect, the invention provides a device for purifying a nucleic acid molecule present in a urine sample, the device containing a first portion containing a matrix that binds at least one nucleic acid molecule having a length of less than 1000 base pairs at a sample application site, wherein the matrix is present on a solid support; a second portion containing an absorbent reservoir present on a solid support; and a hinge connecting the first and second portions, wherein the hinge brings the matrix and the reservoir into contact when closed. In one embodiment of the previous aspect, the device further containing a removable container centered over the sample application site that allows a liquid to contact the sample application site. In another embodiment of the previous aspect, the matrix comprises at least one nucleic acid molecule-binding conjugate at the sample application site. In yet another embodiment of the previous aspect, the device has increased sensitivity relative to or compared to a conventional test device. In yet another embodiment of the previous aspect, the sensitivity is increased by at least 10%.

In yet another aspect, the invention provides a kit containing a device of any previous aspect. In one embodiment, the kit further contains instructions for the use of the device for the detection of an analyte. In yet another aspect, the invention provides at least one nucleic acid molecule purified according to the method of any previous aspect.

In yet another aspect, the invention provides a method for storing at least one nucleic acid molecule, containing contacting a liquid biological sample with the test device of any one previous aspect; and drying the matrix and the solid support.

In yet another aspect, the invention provides a method for shipping at least one nucleic acid molecule, the method involving: contacting a liquid biological sample with the test device of previous aspect; drying the matrix and the solid support; and shipping the device. In various embodiments of any previous aspect, the nucleic acid molecule(s) is a single or double-stranded DNA or RNA molecule. In other embodiments of any previous aspect, the nucleic acid molecule(s) is derived from a white blood cell. In various embodiments of any previous aspect, the matrix comprises a nucleic acid molecule-binding conjugate(s) at the sample application site. In other embodiments of any previous aspect, the nucleic acid molecule-binding conjugate(s) is a DNA-binding protein, an antibody that specifically binds DNA or a combination thereof. In other embodiments of any previous aspect, the matrix comprises a cellulose-based material, a metal, a ceramic, or a plastic material. In other embodiments of any previous aspect, the matrix further comprises one or more components selected from any one or more of a buffer having a weakly basic pH, a chelating agent (e.g., EDTA), a surfactant (e.g., an anionic surfactant), uric acid or a urate salt, an enzyme having proteolytic activity, a polyanionic agent, an agent capable of precipitating lipid or hydrophobic matter, and a nuclease inhibitor. In various embodiments of any previous aspect, the component is adsorbed on or incorporated into the matrix. In various embodiments of any previous aspect, detection of the nucleic acid molecule(s) identifies the subject as having a neoplasia, pre-neoplasia, a genetic disorder, an epigenetic alteration, or combinations thereof. In other embodiments of any previous aspect, the epigenetic alteration is in DNA methylation, or DNA adduct formation, a metabolic disorder, or a pathogen infection or combinations thereof. In still other embodiments of the above aspects, wherein the nucleic acid molecule(s) is detected by hybridization, amplification, restriction digestion, restriction fragment length polymorphism, or single nucleotide polymorphism, ligand binding or combinations thereof. In various embodiments of any previous aspect, the nucleic acid molecule(s) is derived from a white blood cell. In still other embodiments of any previous aspect, the matrix comprises a nucleic acid molecule-binding conjugate(s) at the sample application site.

The invention provides compositions and methods for detecting the presence of nucleic acid molecules in urine. Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

Definitions

By "binding" is meant an intermolecular interaction that may be covalent or non- covalent. In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited are not changed by the presence of more than that which is recited, but excludes prior art embodiments.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

By "disease status" is meant the presence or absence of pathology in a subject. By "fetal genetic marker" is meant the detection of a nucleic acid sequence of interest in a polynucleotide derived from a fetus.

By "fragment" is meant a portion of a nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides.

By "hybridize" is meant pairing to form a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507). For example, stringent salt concentration will ordinarily be less than about 750 mM

NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide. Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and more preferably of at least about 42 °C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed. In a preferred: embodiment, hybridization will occur at 30⁰C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA (ssDNA). In another preferred embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.

For most applications, washing steps that follow hybridization will also vary in stringency. Wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include a temperature of at least about 25 °C, more preferably of at least about 42°C, and most preferably of at least about 68°C. In a preferred embodiment, wash steps will occur at 25⁰C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another preferred embodiment, wash steps will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps will occur at 68⁰C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

By "isolated nucleic acid molecule" is meant a nucleic acid (e.g., a DNA, RNA, etc.) that is substantially free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule which is transcribed from a DNA molecule, as well as a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.

By "liquid sample" is meant a fluid containing one or more analytes. Such analytes may naturally occur in the liquid or may be solubilized therein. By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

By "matrix" is meant any liquid permeable material that binds a nucleic acid molecule.

By "nucleic acid molecule" is meant an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid, or analog thereof. Nucleic acid molecules include, but are not limited to, single-stranded or double-stranded DNA, RNA cDNA, mRNA, siRNA, microRNA, shRNA, and analogs thereof. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake and increased stability in the presence of nucleases. Specific examples of some preferred nucleic acids envisioned for this invention may contain phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are those with CH₂-NH-O-CH₂, CH₂~N(CH₃)~O~ CH₂, CH₂-O-N(CH₃)- - CH₂, CH₂~N(CH₃)-N(CH₃)~ CH₂ and 0-N(CH₃)- CH₂- - CH₂ backbones (where phosphodiester is O--P--O— CH₂). Also preferred are oligonucleotides having morpholino backbone structures (Summerton, J. E. and Weller, D. D., U.S. Pat. No: 5,034,506). In other preferred embodiments, such as the protein-nucleic acid (PNA) backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone, the bases being bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone (P. E. Nielsen et al. Science 199: 254, 1997). Other preferred oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one of the following at the 2' position: OH, SH, S CH₃, F, OCN, 0(CH₂) _nNH₂ or O(CH₂)_n CH₃, where n is from 1 to about 10; Ci to Cio lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃; OCF₃; O~, S-, or N-alkyl; O~, S-, or N-alkenyl; SOCH₃; SO₂ CH₃; ONO₂; NO₂; N₃; NH₂; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a conjugate; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Oligonucleotides may also have sugar mimetics such as cyclobutyls in place of the pentofuranosyl group. This term includes oligomers consisting of naturally occurring bases, sugars, and intersugar (backbone) linkages as well as oligomers having non-naturally occurring portions which function similarly.

By "nucleic acid molecule-binding conjugate" is meant any polypeptide, polynucleotide or other molecule that specifically binds a nucleic acid molecule.

By "pathogen" is meant any bacteria, viruses, fungi, parasite, or protozoans. Expressly included are nematodes, schistosomes, and capable of interfering with the normal function of a cell.

Exemplary bacterial pathogens include, but are not limited to, Aerobacter, Aeromonas, Acinetobacter, Actinomyces israelii, Agrobacterium, Bacillus, Bacillus antracis, Bacteroides, Bartonella, Bordetella, Bortella, Borrelia, Brucella, Burkholderia, Calymmatobacterium, Campylobacter, Citrobacter, Clostridium, Clostridium perfringers, Clostridium tetani, Cornyebacterium, corynebacterium diphtheriae, corynebacterium sp. , Enterobacter, Enterobacter aerogenes, Enterococcus, Erysipelothrix rhusiopathiae, Escherichia, Francisella, Fusobacterium nucleatum, Gardnerella, Haemophilus, Hafnia, Helicobacter, Klebsiella, Klebsiella pneumoniae, Lactobacillus, Legionella, Leptospira, Listeria, Morganella, Moraxella, Mycobacterium, Neisseria, Pasteurella, Pasturella multocida, Proteus, Providencia, Pseudomonas, Rickettsia, Salmonella, Serratia, Shigella, Staphylococcus, Stentorophomonas, Streptococcus, Streptobacillus moniliformis, Treponema, Treponema pallidium, Treponema pertenue, Xanthomonas, Vibrio, and Yersinia. The term "parasite" as used herein refers to protozoa, helminths, and ectoparasitic arthropods (e.g., ticks, mites, etc.). Protozoa are single celled organisms which can replicate both intracellularly and extracellularly, particularly in the blood, intestinal tract or the extracellular matrix of tissues. Helminths are multicellular organisms which almost always are extracellular (the exception being Trichinella). Helminths normally require exit from a primary host and transmission into a secondary host in order to replicate. In contrast to these aforementioned classes, ectoparasitic arthropods form a parasitic relationship with the external surface of the host body. Parasites can be classified based on whether they are intracellular or extracellular. An "intracellular parasite" as used herein is a parasite whose entire life cycle is intracellular. Examples of human intracellular parasites include Leishmania, Plasmodium, Trypanosoma cruzi, Toxoplasma gondii, Babesia, and Trichinella spiralis. An "extracellular parasite" as used herein is a parasite whose entire life cycle is extracellular. Extracellular parasites capable of infecting humans include Entamoeba histolytica, Giardia lamblia, Enterocytozoon bieneusi, Naegleria and Acanthamoeba as well as most helminths. Yet another class of parasites is defined as being mainly extracellular but with an obligate intracellular existence at a critical stage in their life cycles. Such parasites are referred to herein as "obligate intracellular parasites". These parasites may exist most of their lives or only a small portion of their lives in an extracellular environment, but they all have at lest one obligate intracellular stage in their life cycles. This latter category of parasites includes Trypanosoma rhodesiense and Trypanosoma gambiense, Isospora, Cryptosporidium, Eimeria, Neospora, Sarcocystis, and Schistosoma.

Examples of pathogenic fungi include, without limitation, Alternaria, Aspergillus, Basidiobolus, Bipolaris, Blastoschizomyces, Candida, Candida albicans, Candida krusei, Candida glabrata (formerly called Torulopsis glabrata), Candida parapsilosis, Candida tropicalis, Candida pseudotropicalis, Candida guilliermondii, Candida dubliniensis, and Candida lusitaniae, Coccidioides, Cladophialophora, Cryptococcus, Cunninghamella, Curvularia, Exophiala, Fonsecaea, Histoplasma, Madurella, Malassezia, Plastomyces, Rhodotorula, Scedosporium, Scopulariopsis, Sporobolomyces, Tinea, and Trichosporon. Examples of viruses that have been found in humans include but are not limited to:

Retroviridae (e.g. human immunodeficiency viruses, such as HIV-I (also referred to as HDTV-III, LAVE or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. strains that cause gastroenteritis); Togaviridae (e.g. equine encephalitis viruses, rubella viruses); Flaviridae (e.g. dengue viruses, encephalitis viruses, yellow fever viruses); Coronoviridae (e.g. coronaviruses); Rhabdoviridae (e.g. vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. ebola viruses); Paramyxoviridae (e.g. parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g. influenza viruses); Bungaviridae (e.g. Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);

Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g. the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1 = internally transmitted; class 2 = parenterally transmitted (i.e. Hepatitis C); Norwalk and related viruses, and astroviruses).

By "rehabilitation status" is meant the presence or absence of molecular alterations related to addictive compounds or their metabolites. By "reference" is meant a standard or control condition.

By "sample application site" is meant the portion of the device that contacts a liquid under analysis.

By "specifically binds" is meant binds a target analyte without substantially binding to other non-target compounds present in the sample. By "subject" is meant a mammal, including, but not limited to, a human or non-human organism, such as an avian,bovine, equine, canine, ovine, feline, fish, or insect.

By "test device" is meant a device used in the detection of an analyte in a sample.

Brief Description of the Drawings Figures 1-3 shows the results of a genotyping procedure carried out using DNA isolated from urine as described in the examples. Called alleles matched the donor's FBI Laboratory's Combined DNA Index System (CODIS) Database profile. DETAILED DESCRIPTION OF THE INVENTION

The invention generally provides compositions and methods for capturing, purifying, stabilizing for storage and detecting the presence of nucleic acid molecules present in a liquid biological sample. In particular, the invention provides diagnostic methods for the detection of small nucleic acid molecules present in urine. The invention is based, at least in part, on the observation that nucleic acid molecules can be isolated and chromatographically purified using an FTA card facilitating subsequent analysis, shipment, or storage. As reported in more detail below, urine was applied to the FTA card, chromatographed directly on the card by application of buffer to the sample origin, and the purified nucleic acid molecules were amplified by PCR. Amplicons were then analysed for selected genetic loci by capillary electrophoresis. Nucleic acid molecules isolated and purified in this manner showed an increase in the number of alleles that could be identified relative to nucleic acid molecules isolated and analyzed in the same manner, but not subjected to chromatographic purification on the card by buffer application.

The invention provides compositions suitable for the capture and purification of a nucleic acid molecule. Such compositions comprise a matrix, an absorbent reservoir and/or a support.

Matrix

Compositions of the invention contain a matrix that comprises a liquid permeable or absorbent material suitable for DNA capture (e.g., glass fibers, polyester, nitrocellulose, fibers of cellulose or derivatives thereof, non-cellulose hydrocarbon materials, ceramics, metals, biological polymers eg polylysine coated supports). Suitable materials for the matrix include, but are not limited to, nitrocellulose, cellulose, diazocellulose, carboxymethylcellulose, hydrophilic polymers (e.g., polyester, polyamide, carbohydrate polymers), polytetra- fluro-ethylene, fiberglass, porous ceramics, polystyrene, polyvinylchloride, polypropylene, polyethylene, dextran, Sepharose, agar, starch, nylon, synthetic plastic micromesh, latex beads, magnetic beads, or glass beads. Matrices suitable for the capture of a nucleic acid molecule are known in the art and are commercially available; for example, FTA® paper is available from Life Technologies, Inc.; GenPrep™ and GenSpin™ are available from Whatman; and IsoCode™ is available from Schleicher and Schuell. In one embodiment, materials are wettable and exhibit binding of nucleic acid molecules (e.g., single or double stranded DNA, RNA, or fragments, variants, or analogs thereof). Such binding may be non-covalent (e.g., via an ionic interaction, van der Waal's forces, hydrogen bonding) or covalent in nature. In one embodiment, the matrix is capable of chromatographically purifying the sample to remove materials that naturally accompany the nucleic acid molecule.

The matrix may also comprise at least one or more of the following components: a buffer having a weakly basic pH (e.g. a pH between 7.5 and 8.5), a chelating agent (e.g., EDTA), a surfactant or detergent (e.g., an anionic surfactant or detergent), and uric acid or a urate salt, where the composition is adsorbed on or incorporated into the matrix. If desired, the matrix may contain a nuclease inhibitor (e.g., a DNAse or RNAse inhibitor). For certain applications, it may be useful to use higher or lower pH's. For example, volatile buffers comprising ammonium acetate (pK 4.0) may be employed.

The matrix includes a site for the application of a biological sample. If desired, the application site is indicated by a marking that defines the boundaries of the sample application site. Optionally, the matrix includes at least one nucleic acid molecule-binding conjugate present at the sample application site, such as an aptamer, a DNA-binding polypeptide, such as polylysine or polypeptide fragments that include and preserve function of the ligand binding site of nucleic acid binding proteins, or an antibody that specifically binds a DNA molecule. If desired, the sample application site includes or can have applied to it during sample processing an indicator that detects the presence of a deposited nucleic acid molecule. Exemplary indicators include luminol, SYBR Green, ethidium bromide, propidium iodide, or DAPI.

Adsorbent Reservoir

Where the concentration of analyte in a liquid sample is particularly low, it may be desirable to apply large volumes of a liquid sample to the application site. It may desirable to remove excess liquid sample applied at the sample application site. This can be accomplished using an adsorbent reservoir that sorbs excess liquid from the application site. Optionally, the matrix is in capillary communication with an absorbent reservoir that contains sorbent material capable of absorbing or adsorbing excess liquid present in a liquid sample. Suitable sorbent materials include virtually any commercial material (e.g., synthetic or natural materials, such as cotton) capable of absorbing many times its weight in water. Such materials are widely available in commerce. Where minute quantities of analyte are present in a large volume of a liquid sample, the absorbent reservoir may concentrate the analyte at the application site. For some applications, it may be useful to provide the matrix and absorbent reservoir together with a support.

Solid Supports

The matrix and the absorbent reservoir may be held in contact or brought into contact using a solid support. The physical shape of the support is not critical, although some shapes may be more convenient than others. Accordingly, the solid support may be a circle, paper strip, dipstick, membrane (e.g. a nylon membrane or a cellulose filter), a plate (e.g. a microtiter plate), solid particle (e.g. latex beads), or slide. For some applications, it may be desirable to have the matrix enclosed in a solid support that protects the matrix. The enclosure may take the form of a box or shell. The solid support may be made of any suitable material, including but not limited to a plastic (e.g., polyethylene, polypropylene, polystyrene, latex, polyvinylchloride, polyurethane, polyacrylamide, polyvinylalcohol, nylon, polyvinyl acetate, or any suitable copolymers thereof), cellulose (e.g. various types of paper, such as nitrocellulose paper and the like), a silicon polymer (e.g. siloxane), or any other non- reactive support. In one embodiment, the matrix or absorbent reservoir is fixed to the support in a permanent or semi-permanent manner. In another embodiment, the matrix or absorbent reservoir rests on or is enclosed within the support, and can be easily removed from the support for subsequent manipulation.

Device for Sample Preparation, Storage and Shipment The invention further provides a device that facilitates analyte collection, preservation of analyte integrity (stabilization), purification, storage and shipment. The device includes a matrix having a sample application site; an absorbent reservoir; and a solid support. Optionally, the device includes a container for holding a liquid in contact with the sample application site. The liquid held by the container may be a liquid biological sample that is held in contact with the sample application site to facilitate the capture of a nucleic acid molecule. In one embodiment, the liquid biological sample includes a solid material containing an analyte of interest to which liquid can be added to solubilize the analyte and facilitate it's delivery onto the matrix. Alternatively, the container holds a wash buffer in contact with the sample application site. The device further comprises a funnel that facilitates loading of the sample into the container. The container is generally between about 1 and 5 mm in diameter (e.g., 1.0, 1.5, 1.8, 2.0, 3.0, 4.0, and 5.0) and may be of a height suitable to the amount of sample being applied. Such methods can be adapted for automation, for example, by applying the sample with a syringe to an FTA card held in a flange.

In one approach, the device includes a solid support having a first portion that contains a matrix with a sample application site; a second portion that contains an absorbent reservoir; and a hinge connecting the first and second portions. The hinge brings the first and second portions into communication, such that the absorbent reservoir contacts the matrix and absorbs excess liquid. Preferably, the absorbent reservoir does not contact the application site. In one embodiment, the device resembles a hinged clam shell, where the first half of the shell comprises a circular matrix; the second half of the shell comprises a donut-shaped absorbent reservoir; and a hinge connects the two shells and allows the donut- shaped absorbent reservoir to be brought into contact with the circular matrix. Preferably, the hole within the reservoir is centered over the sample application site, such that the absorbent reservoir does not contact the application site.

In another embodiment, the device comprises a matrix strip, an absorbent reservoir and a solid support. The device comprises an opening at the leading edge of the matrix that allows a sample to be brought into contact with the sample application site. The opening may be walled by the device material to create a well into which a volume of sample may be placed. The device further comprises an absorbent reservoir at the opposite end that absorbs excess liquid. In one embodiment, the device encases the strip and the opening is positioned directly over the sample application site. In another embodiment, the device comprises a first portion that comprises the matrix; a second portion that comprises an opening that provides access to the sample application site; and a fastener that allows the first and second portions to be fixed together to encase the strip. If desired, the two portions are brought together by means of a fastener, such as for example, a hinge or a snap that fastens the two portions together.

The matrix may be permanently or semi-permanently fixed to the solid support. Alternatively, the matrix is inserted into or rests on the solid support. Sample Application

The invention provides methods for the capture and purification of a nucleic acid molecule present in a liquid sample. In one example, the liquid sample is brought into contact with the matrix by applying a liquid sample to the matrix in a drop-wise fashion. Where the matrix is circular, the liquid sample is typically applied at the center of the circle. Where it is desirable to apply a large volume of liquid, the matrix may be in capillary communication with an adsorbent reservoir or may be brought into capillary communication with the adsorbent reservoir, or may be walled at the site of sample application so that a larger volume of sample may be applied to the site.

In another example, the assay is conducted by placing the leading edge of a matrix in contact with a liquid sample or by applying a liquid sample to the leading edge of a matrix in a drop- wise fashion. If desired, an absorbent reservoir is positioned at the opposite end of the device from the application site, such that the analyte is captured at the application site and excess liquid or contaminating materials are wicked away from the leading edge of the matrix.

Antibodies

As described herein, compositions of the invention provide for the capture and purification of a nucleic acid molecule present in a biological sample using a matrix. The matrix optionally contains at least one conjugate that binds the analyte. In one approach, the conjugate is an antibody capable of binding a nucleic acid molecule. Any antibody, antibody conjugate, or fragment thereof that binds an antigen of interest may be used in the present invention. Such antibodies or antibody conjugates are adsorbed on or incorporated into a matrix. In various embodiments, different conjugates are present at discrete spatial locations on the matrix thus providing for the identification of specific targets by the detection of the target at that spatial location. Suitable antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, or fragments thereof. Antibodies and their fragments that bind nucleic acid molecules are well known in the art. As used herein, the term "antibody" means not only intact immunoglobulin molecules but also the well-known active fragments F(ab')₂, and Fab. In one embodiment, antibodies comprising hypervariable regions having random mutations are generated and prepared in vitro. Antibodies having a desired binding profile are then selected using standard methods. In one embodiment, the selected antibody binds a nucleic acid molecule with high affinity. Many such antibodies are in the public domain or are commercially available. See, for example, Alpha Diagnostic International (San Antonio, TX), Autogen Bioclear UK Ltd (Wiltshire, UK), Roche Molecular Biochemicals (Indianapolis, IN). The invention further employs antibodies and other reagents that detect a DNA molecule, a DNA adduct, a methylated, or epigenetically modified DNA, a histone modification, or an association of DNA with proteins that modify the DNA; methods of using such reagents are described, for example, in U.S. Patent Publication No. 20040023282. Antibodies having a desired binding characteristic may be identified using methods known to the skilled artisan. One method of obtaining antibodies is to immunize a suitable host animal with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production. The desired antibodies are then purified from the host. Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column preferably run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and antiimmunoglobulin.

In one approach, antibodies are produced from hybridoma cells engineered to express a desired antibody. Methods of making hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source. Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition (e.g., Pristane).

Methods of Using the Nucleic Acid Molecules Nucleic acid molecules isolated from a biological sample as described herein are useful for virtually any type of genetic analysis known in the art. According to the present invention, a biological sample may be a biological fluid (e.g., urine, spinal fluid, semen, vaginal fluid, sputum, phlegm, stool, or cellular lysates) or a biological sample that can be solubilized (e.g., a cell, tissue, products of conception (e.g., aborted fetus) organ lysate, or any other composition excreted or secreted by a subject that contains one or more nucleic acid molecules (e.g., single or double-stranded RNA, DNA, or fragments thereof or combinations thereof). Depending on the health of the subject from which the biological sample is derived, the sample may also contain molecular or cellular components, including proteins, glucose, hormones, salts, red or white blood cells, /or cellular debris cells (epithelial cells, white blood cells, red blood cells), pathogens, such as viruses (e.g., AIDS, HIV, HTLV, herpes, hepatitis and the like), bacteria, fungi, yeast, parasites, or cells affected by a genetic or biochemical pathology (e.g., neoplasia, pathogen infection) In other embodiments, the invention provides for the detection of an analyte of interest in an agricultural product, such as an environmental contaminant, including but not limited to dirt, vegetation, biological material from another person or animalln one embodiment, the invention provides methods for detecting the contamination of a meat product intended for human consumption (e.g., meat from a cow, pig, chicken, sheep, lamb, or other animal bred for meat production) with the meat of an animal that has not been raised for consumption or with a type of meat that is not listed on the packaging of the meat product, including but not limited to kangaroo, horse, or cat meat In still other embodiments, the invention provides compositions and methods for detecting adulterated food products with food products not listed on the label as an ingredient. For example, the invention provides for the detection of ground corn in ground coffee. The invention further provides for the detection of adulterants in drugs or for an indicator doped into a pill by a pharmaceutical manufacturer. The invention can be used in combination with methods known in the art for the detection of such molecular or cellular components. The combined methods are useful for the diagnosis of pathological conditions as described herein.

In one example, the biological sample is a urine sample that comprises nucleic acid molecules derived from a neoplastic cell, a fetus (i.e., where the urine is obtained from a pregnant subject), an epithelial cell (e.g., a cell lining the urinary tract), a white blood cell, an aging cell, a cell exposed to a toxin (e.g., aflatoxin), a pathogen or pathogen infected cell. In one embodiment, transrenal nucleic acid molecules are not included in the class of polynucleotides detected by the methods of the invention. By "transrenal nucleic acid molecule" is meant a naked polynucleotide that has crossed the kidney barrier. In preferred embodiments, the invention provides methods for the detection of nucleic acid molecules released from cells that have lysed in the bladder prior to sample processing, such that their nucleic acid molecules are present in urine. In one preferred embodiment, the invention provides methods for the detection of DNA present in whole or lysed white blood cells present in urine. In another example, the nucleic acid molecule is a trans-renal nucleic acid molecule that has crossed the kidney barrier. See, for example, U.S. Patent Nos. 6,251,638, 6,287,820, 6,492,144, or U.S. Patent Publication No. 2002/0119478, each of which is hereby incorporated by reference in their entirety. In one approach, a clean-catch urine sample is used to contact the sample application site. In another approach, a urine sample is centrifuged and the urine supernatant is reserved for analysis. In yet another approach, the urine pellet is used for analysis. Centrifugation reduces or eliminates cellular debris present in the urine. In other embodiments, the methods of the invention are combined with known diagnostic methods for the detection of proteins, glucose, hormones, salts, red or white blood cells, /or cellular debris cells (epithelial cells, white blood cells, red blood cells), pathogens, such as viruses (e.g., AIDS, HIV, HTLV, herpes, hepatitis and the like), bacteria, fungi, yeast, parasites, or cells affected by a genetic or biochemical pathology in urine. The combination provides for the diagnosis or detection of maternal exposure to a teratogen, consanguinity, population-based cancer screening, infertility diagnosis, repeated miscarriage, still birth/ fetal or baby death, and genetic conditions. In other embodiments, the combination provides for the detection of metabolic or epigenetic profile changes (e.g., changes in DNA methylation or content of DNA adduct). One epigenetic change associated with drug abuse would be DNA methylation or DNA adduct associated with drug rehabilitation, recovery, exposure to noxious agents, or relapse. In still other embodiments, the method detects cytochrome p450 status using HPLC. In still other embodiments, the method detects a genetic or epigenetic change in a neoplastic cell that confers drug resistance to chemotherapy. The liquid sample is contacted with a matrix, where the nucleic acid molecules are captured by binding to a charged moiety on the surface of the matrix, by binding to an antibody that specifically binds nucleic acid molecules, or by any other method known in the art. The captured nucleic acid molecules are purified in that they are separated from the other components of urine. Preferably, the captured nucleic acid molecules are at least 1%, 5%, 10%, 15%, 25%50%, 65%, 75%, 85%, 90%, 95%, or even 100% pure. Purification is sufficient so long as it reduces the concentration of a nuclease or a highly inhibitory component that interferes with further analysis. If desired, the captured nucleic acid molecules on the matrix are washed with a buffer to remove residual impurities. Nucleic acid molecules captured using the methods of the invention are maintained in a stable form for analysis, storage, shipment, or other manipulation.

Such analysis includes the detection of particular nucleic acid fragments or sequences using standard methods for the diagnosis of fetal gender, neoplasia (e.g., by identifying a mutation in an oncogene, such as K-ras), metabolic or genetic disorders, or pathogen infections (e.g., an infection by P. mirabilis, K. pneumoniae, E. faecalis, or P. aeruginosa). In other embodiments, the presence of fetal nucleic acid fragments is used to non-invasively determine fetal sex and or the presence of a genetic or metabolic disorder in a fetus (e.g., achondroplasia, cystic fibrosis, Down's syndrome, Marfan's syndrom, Tay-sachs disease, Congenital Hypothyroidism, Phenylketonuria (PKU), Hemoglobin Disorders (e.g., Sickle Cell Disease, Congenital Toxoplasmosis, Biotinidase Deficiency, Galactosemia, "Maple Syrup" Urine Disease, Homocystinuria, Congenital Adrenal Hyperplasia, or Medium-chain acyl Co-A dehydrogenase deficiency). Nucleic acid molecules present on the matrix are manipulated or analysed in one or more standard molecular biology techniques, such as digestion, sequencing, amplification, synthesis and transformation/transfection reactions. Nucleic acid molecules present on a matrix of the invention may, optionally, be isolated from the matrix using any method known in the art. In one embodiment, a nucleic acid molecule harboring a mutation is compared to a reference sequence by amplifying the molecule using primers that bind to identical sites in wild-type and mutant DNA targets. The presence or absence of a mutation in the molecule is identified using methods known in the art (e.g., sequencing, altered mobility in electrophoresis, restriction fragment length polymorphisms, single nucleotide polymorphism and other routine detection methods). The diagnostic methods of the invention include methods for identifying a subject having or having a propensity to develop a disease or disorder. In general, the invention provides for the identification of an analyte of interest in a sample obtained from a subject (e.g., animal, human), including a mammal, particularly a human, insect, bird, or fish. Such methods will be suitably used as diagnostics for the identification of subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).

In one embodiment, the invention provides a method of monitoring treatment progress. The method includes the step of determining the presence or absence of a diagnostic marker (Marker) (e.g., any target modulated by a compound, a protein or indicator thereof, etc.) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with a genetic defect or pathogen infection, in which the subject has been administered a therapeutic amount of a compound sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment.

Nucleic Acid Molecules

Nucleic acid molecules present in a biological sample may be derived from a cell, tissue, or organ of a subject or may be derived from a pathogen infecting the subject. Nucleic acid molecules are not typically released at high levels into biological fluids, such as urine or cerebrospinal fluid, in the absence of some pathology. Nucleic acid molecules may be present where white blood cells, urinary epithelial cells, bladder cells, or other related cells have lysed. Many different cellular pathologies result in apoptotic or necrotic cell death that releases nucleic acid molecules into the extracellular environment. Such pathologies include, but are not limited to, neoplasia, neurodegenerative disease, viral, bacterial, fungal, yeast, or parasite infection, ischemic injury (e.g., stroke or heart attack, thrombosis, pulmonary embolism), or gall bladder stones. Once released from the nucleus of the cell (e.g., host cell or pathogen cell), the nucleic acid molecules are broken up into fragments that are shed in the urine. The invention provides methods for assaying DNA present in a genome or in fragments generated by apoptosis, due to nuclease activity (e.g., nucleases present in urine) or fragments generated by restriction digestion or shearing. Polynucleotides and fragments of virtually any length can be analysed using the methods described herein. In particular embodiments, the polynucleotides or fragments thereof are between 10 base pairs in length and 10,000 base pairs in length (e.g.,1 kb, 5kb, 7.5 kb,10 k). Such fragments can be captured, purifed, and analysed as described herein. In general, the fragments are between about 10 base pairs and 3,000 base pairs in length (e.g., 10, 25, 50, 100, 200, 250, 500, 750, 1000,

1500, 2000, 2500, 3000) where the bottom of the range is any integer between 10 and 2,999 and the top of the range is any integer between 26 and 1,000. In other embodiments, the nucleic acid molecules are about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 300, 325, 350, 375, 400, 450, or 500 base pairs in length. Many biological samples contain enzymes having nuclease activity (e.g., DNAse or RNAse activity). The biological sample may be treated to inhibit undesirable nuclease activity. Methods of inhibiting DNase activity include, but are not limited to, the use of ethylenediaminetetraacetic acid (EDTA), guanidine-HCl, GITC (Guanidine isothiocyanate), N-lauroylsarcosine, Na-dodecylsulphate (SDS), high salt concentration and heat inactivation of DNase. Methods of inhibiting RNAse activity include the use of commercially available RNAse inhibitorsln other embodiments, chromatography is used to remove nucleases.

Manipulation and Analysis of Nucleic Acid Molecules

Nucleic acid molecules isolated as described herein may be analysed using any method known in the art. Methods include, but are not limited to, techniques for hybridization, amplification and detection of nucleic acids. In one embodiment, a nucleic acid molecule is analysed using restriction fragment length polymorphism-based PCR ("PCR-RFLP"). In this technique nucleic acid sequences are amplified, treated with one or more restriction enzymes, and separated by electrophoresis, allowing for the detection of nucleic acids containing small alterations, such as point mutations.

Specific DNA sequences can be amplified using a cycling probe reaction (Bekkaoui, F. et al, BioTechniques 20,240-248 (1996), polymerase chain reaction (PCR), nested PCR, PCR- single strand conformation polymorphism, ligase chain reaction (F. Barany Proc. Natl. Acad. Sci USA 88:189-93 (1991)), cloning, strand displacement amplification (G. K. Terrance Walker et al., Nucleic Acids Res., 22:2670-77 (1994), and variations thereof, such as allele—specific amplification. Another method for the detection of a specific nucleic acid sequence is hybridization of a nucleic acid cleavage structure with the specific sequence, followed by cleavage of the cleavage structure in a site-specific manner. This method is referred to as cleavage product detection and is described in U.S. Pat. Nos. 5,541,331 and 5,614,402, and PCT publication Nos. WO 94/29482 and WO 97/27214. This method provides for the detection of small amounts of specific nucleic acid sequences without amplifying the DNA sequence of interest. The nucleic acid molecules isolated as described herein may also be characterized by cloning and sequencing, using a northern blot, or using hybridization probes. Other methods useful in the methods of the invention include, but are not limited to, those set forth in Current Protocols in Molecular Biology, eds. Ausubel et al., Greene Publishing and Wiley- Interscience: New York (1987).

Kits

The invention provides kits that include a device for the detection of a nucleic acid molecule in a liquid biological sample. In one embodiment, the kit includes a test device described herein. In some embodiments, the kit comprises a container, which contains a test device comprising a matrix, an absorbent reservoir, and a support; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister packs, or other suitable container forms known in the art. In one embodiment, such containers may be sterile. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding elements for detecting nucleic acid molecules in a sample.

If desired the device is provided together with instructions for using it to identify the presence or absence of an analyte (e.g., nucleic acid molecules) in a sample. The instructions will generally include information about the use of the device for the identification of a particular analyte, such as a nucleic acid molecule in a liquid sample (e.g., a biological sample, a biological fluid, such as urine, sputum, phlegm, stool, cerebrospinal fluid), cell, tissue or organ lysate, biopsy sample, environmental sample, water sample or liquid sample extracted from an agricultural commodity). The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container. If desired, the kit may also include a standard measure pipet, a test vial, and/or a liquid (e.g., ethanol, methanol, organic solvent, suitable buffer, such as phosphate buffered saline, or water) to be used in the extraction of a sample.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989);

"Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991) including subsequent editions thereof. These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Samples of freshly voided urine were applied to FTA cards. The cards were treated with wash buffer (100 μl Tris/EDTA buffer). This provided for the chromatographic separation of DNA, which remained fixed at the sample application site, from some of the other materials that naturally occur in urine. The FTA cards were dried and punched at the sample application site to obtain DNA. The punches from the card, containing DNA was analysed by punch-in amplification using PCR to amplify specific allelic sequences present in the human genome using the Identifiler kit (Applied Biosystems). Amplicons generated were then analysed by capillary electrophoresis and by fluorescence properties using an Applied Biosystems 3130 genetic analyzer. The number of alleles called, based on electrophoretic migration and signal intensity, was evaluated. An increased number of alleles were detected using chromatographically purified DNA relative to unpurified DNA (See Figures 1-3).

Table 1: Use of a Chromatographic Step Improves Genetic Profiling of Urinary DNA Affixed to Solid Matrix

Urine was applied to a solid matrix that binds DNA. Bound DNA was untreated or purified chromatographically in situ, by application of buffer to the site of sample application. Punch-in amplification of core CODIS loci (Identifiler kit, Applied Biosystems) generated amplicon that was analyzed by capillary electrophoresis. Positive and negative controls (not shown) produced expected results. Called alleles all matched the donor's CODIS profile, n.d., not done.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof. All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

Claims

What is claimed is:

1. A method for purifying at least one nucleic acid molecule present in a liquid sample, the method comprising a) contacting a matrix with a liquid sample at a sample application site; b) binding said at least one nucleic acid molecule to the matrix at the sample application site; c) applying a wash buffer and chromatographically purifying said at least one nucleic acid molecule at the sample application site; and d) absorbing excess liquid from the matrix with an absorbent reservoir.

2. A method for purifying at least one nucleic acid molecule present in a biological sample selected from the group consisting of urine, spinal fluid, semen, vaginal fluid, sputum, phlegm, stool, cellular lysates, a cell, tissue, products of conception, organ lysate, or any other composition excreted or secreted by a subject, and combinations thereof, the method comprising a) contacting a matrix with a liquid sample at a sample application site; b) binding said at least one nucleic acid molecule to the matrix at the sample application site; c) applying a wash buffer and chromatographically purifying said at least one nucleic acid molecule at the sample application site; and d) absorbing excess liquid from the matrix with an absorbent reservoir.

3. The method of claim 1 or 2, wherein the at least one nucleic acid molecule has a length of between about 10 and 3000 base pairs.

4. The method of claim 1 or 2, wherein the at least one nucleic acid molecule is a single or double-stranded DNA or RNA molecule.

5. The method of claim 1 or 2, wherein the at least one nucleic acid molecule is from a cell that lysed and released its cellular contents into the lumen of the urinary tract.

6. The method of claim 5, wherein the at least one nucleic acid molecule is derived from a white blood cell, a urinary tract epithelial cell, or a bladder cell.

7. The method of claim 1 or 2, wherein the matrix comprises at least one nucleic acid molecule-binding conjugate at the sample application site.

8. The method of claim -7, wherein the at least one nucleic acid molecule-binding conjugate is a DNA-binding protein, a non-protein ligand, a polysaccharide, an aptamer, a detectable polypeptide or nucleic acid molecule,an antibody that specifically binds DNA or combinations thereof.

9. The method of claim 1, wherein the matrix comprises a cellulose-based material, a metal, a ceramic, a polylysine polymer, a charged polymer that binds DNA, or a plastic material.

10. The method of claim 1, wherein the matrix further comprises one or more components selected from the group consisting of a buffer having a weakly basic pH, a chelating agent, a surfactant, uric acid or a urate salt, an enzyme having proteolytic activity, a polyanionic agent, an agent capable of precipitating lipid or hydrophobic matter, and a nuclease inhibitor.

11. The method of claim 10, wherein the chelating agent is EDTA.

12. The method of claim 10, wherein the surfactant is an anionic surfactant.

13. The method of claim 10, wherein the component is adsorbed on or incorporated into the matrix.

14. The method of claim 1, further comprising the step of washing the matrix comprising the bound at least one nucleic acid molecule.

15. The method of claim 1, wherein the matrix binds at least one nucleic acid molecule via a covalent or non-covalent interaction.

16. The method of claim 1 or 2, wherein the wash buffer comprises Tris/EDTA.

17. A method for detecting at least one nucleic acid molecule having between 10 and 3000 bases in length in a liquid sample, the method comprising: a) contacting a matrix with a liquid sample at a sample application site; b) binding said at least one nucleic acid molecule to the matrix at the sample application site; c) applying a wash buffer and chromatographically purifying said at least one nucleic acid molecule at the sample application site; and d) absorbing excess liquid from the matrix with an absorbent reservoir.

18. The method of claim 17, wherein the at least one nucleic acid molecule is a single or double-stranded DNA or RNA molecule.

19. The method of claim 17, wherein the at least one nucleic acid molecule is derived from a white blood cell.

20. The method of claim 17, wherein the matrix comprises at least one nucleic acid molecule-binding conjugate at the sample application site.

21. The method of claim 20, wherein the at least one nucleic acid molecule-binding conjugate is a DNA-binding protein, an antibody that specifically binds DNA or combinations thereof.

22. The method of claim 17, wherein the matrix comprises a cellulose-based material or a plastic material.

23. The method of claim 17, wherein the matrix further comprises one or more components selected from the group consisting of a buffer having a weakly basic pH, a chelating agent, a surfactant, uric acid or a urate salt, and a nuclease inhibitor.

24. The method of claim 23, wherein the chelating agent is EDTA.

25. The method of claim 23 wherein the surfactant is an anionic surfactant.

26. The method of claim 23, wherein the component is adsorbed on or incorporated into the matrix.

27. A method for identifying the disease status in a subject, the method comprising: a) capturing and purifying at least one nucleic acid molecule comprising between 25 and 1000 bases present in urine from said subject according to the method of any one of claims 1-6; and b) detecting said at least one nucleic acid molecule captured on the matrix, wherein the detection identifies the disease status of the subject.

28. The method of claim 27, wherein detection of the at least one nucleic acid molecule identifies the subject as having a neoplasia, pre-neoplasia, a genetic disorder, an epigenetic alteration, or combination thereof.

29. The method of claim 28, wherein the epigenetic alteration is in DNA methylation, or DNA adduct formation, a metabolic disorder, or a pathogen infection.

30. A method for identifying a fetal genetic marker in a subject, the method comprising: a) capturing and purifying at least one nucleic acid molecule comprising between 25 and 1000 bases present in urine from said subject according to the method of any one of claims 1-16; and b) detecting said at least one nucleic acid molecule captured on the matrix, wherein the detection identifies a fetal genetic marker in a subject.

31. The method of claim 30, wherein the fetal genetic marker identifies fetal gender, a fetal genetic defect or a fetal metabolic defect or combinations thereof.

32. A method for identifying the rehabilitation status or therapeutic tolerance status of a subject having a history of addiction or medical use of therapeutics, the method comprising: a) capturing and purifying at least one nucleic acid molecule comprising between 25 and 1000 bases present in urine from said subject according to the method of any one of claims 1-16; and b) detecting said at least one nucleic acid molecule captured on the matrix, wherein the detection identifies the rehabilitation or therapeutic tolerance status of the subject.

33. The method of any one of claims 17-32, wherein the at least one nucleic acid molecule is detected by hybridization, amplification, restriction digestion, restriction fragment length polymorphism, single nucleotide polymorphism, ligand binding or combinations thereof.

34. The method of any one of claims 17-32, wherein the at least one nucleic acid molecule is derived from a white blood cell.

35. The method of any one of claims 17-32, wherein the matrix comprises at least one nucleic acid molecule-binding conjugate at the sample application site.

36. A device for purifying at least one nucleic acid molecule present in a liquid sample, the device comprising a) a first portion comprising a matrix that binds said at least one nucleic acid molecule at a sample application site, wherein the matrix is present on a solid support; b) a second portion comprising an absorbent reservoir present on a solid support; and c) a hinge connecting the first and second portions, wherein the hinge brings the matrix and the reservoir into contact when closed.

37. The device of claim 36, further comprising a removable container centered over the sample application site that allows a liquid to contact the sample application site.

38. The device of claim 36, wherein the matrix comprises at least one nucleic acid molecule-binding conjugate at the sample application site.

39. The device of claim 38, wherein the at least one nucleic acid molecule-binding conjugate is a DNA-binding protein, or an antibody that specifically binds DNA or combinations thereof.

40. The device of claim 36, wherein the matrix and the absorbent reservoir are circular, and wherein the reservoir includes a hole centered over the application site when the matrix and the reservoir are brought into capillary communication.

41. A device for purifying at least one nucleic acid molecule present in a urine sample, the device comprising a) a first portion comprising a matrix that binds said at least one nucleic acid molecule having a length of less than 1000 base pairs at a sample application site, wherein the matrix is present on a solid support; b) a second portion comprising an absorbent reservoir present on a solid support; and c) a hinge connecting the first and second portions, wherein the hinge brings the matrix and the reservoir into contact when closed.

42. The device of claim 41, further comprising a removable container centered over the sample application site that allows a liquid to contact the sample application site.

43. The device of claim 41 , wherein the matrix comprises said at least one nucleic acid molecule-binding conjugate at the sample application site.

44. The device of claim 41, wherein the device has increased sensitivity when compared to a conventional test device.

45. The device of claim 44, wherein the sensitivity is increased by at least 10%.

46. A kit comprising a test device of any one of claims 36-45.

47. The kit of claim 46, further comprising instructions for the use of the device for the detection of an analyte.

48. A nucleic acid molecule purified according to the method of any one of claims 1-16.

49. A method for storing at least one nucleic acid molecule, comprising:

(a) contacting a liquid biological sample with the test device of any one of claims 36-45; and

(b) drying the matrix and the solid support.

50. A method for shipping at least one nucleic acid molecule, comprising:

(a) contacting a liquid biological sample with the test device of any one of claims 36-45;

(b) drying the matrix and the solid support; and (c ) shipping the device.