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WO2008021995A1 - Procédés et compositions de détection d'un analyte dans un échantillon biologique - Google Patents

Procédés et compositions de détection d'un analyte dans un échantillon biologique Download PDF

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Publication number
WO2008021995A1
WO2008021995A1 PCT/US2007/075641 US2007075641W WO2008021995A1 WO 2008021995 A1 WO2008021995 A1 WO 2008021995A1 US 2007075641 W US2007075641 W US 2007075641W WO 2008021995 A1 WO2008021995 A1 WO 2008021995A1
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WIPO (PCT)
Prior art keywords
nucleic acid
acid molecule
matrix
application site
sample
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PCT/US2007/075641
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English (en)
Inventor
Ariel B. Cohen
Original Assignee
Cohen Ariel B
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Publication of WO2008021995A1 publication Critical patent/WO2008021995A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Definitions

  • 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.
  • 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.
  • the genomic DNA is stable at room temperature for at least five to ten years.
  • 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.
  • the invention provides compositions and methods for capturing, purifying, and analyzing nucleic acid molecules in a liquid biological sample.
  • 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.
  • excess liquid e.g., excess sample or excess wash buffer
  • 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.
  • 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.
  • 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.
  • the matrix comprises a cellulose-based material, a metal, a ceramic, a polylysine polymer, a charged polymer that binds DNA, or a plastic material.
  • 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.
  • the chelating agent is EDTA.
  • the surfactant is an anionic surfactant.
  • the component is adsorbed on or incorporated into the matrix.
  • the method further involves the step of washing the matrix involving the bound nucleic acid molecule(s).
  • the matrix binds the nucleic acid molecule(s) via a covalent or non-covalent interaction.
  • 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.
  • 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.
  • 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.
  • the fetal genetic marker identifies fetal gender, a fetal genetic defect, a metabolic defect or combinations thereof.
  • 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.
  • 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.
  • the device further includes a removable container centered over the sample application site that allows a liquid to contact the sample application site.
  • the matrix comprises at least one nucleic acid molecule- binding conjugate at the sample application site.
  • the nucleic acid molecule-binding conjugate(s) is a DNA-binding protein or an antibody that specifically binds DNA.
  • 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.
  • 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.
  • the device further containing a removable container centered over the sample application site that allows a liquid to contact the sample application site.
  • the matrix comprises at least one nucleic acid molecule-binding conjugate at the sample application site.
  • 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%.
  • the invention provides a kit containing a device of any previous aspect.
  • the kit further contains instructions for the use of the device for the detection of an analyte.
  • the invention provides at least one nucleic acid molecule purified according to the method of any previous 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.
  • 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.
  • the nucleic acid molecule(s) is a single or double-stranded DNA or RNA molecule.
  • the nucleic acid molecule(s) is derived from a white blood cell.
  • the matrix comprises a nucleic acid molecule-binding conjugate(s) at the sample application site.
  • the nucleic acid molecule-binding conjugate(s) is a DNA-binding protein, an antibody that specifically binds DNA or a combination thereof.
  • the matrix comprises a cellulose-based material, a metal, a ceramic, or a plastic material.
  • 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.
  • the component is adsorbed on or incorporated into the matrix.
  • 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.
  • the epigenetic alteration is in DNA methylation, or DNA adduct formation, a metabolic disorder, or a pathogen infection or combinations thereof.
  • 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.
  • the nucleic acid molecule(s) is derived from a white blood cell.
  • 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.
  • binding is meant an intermolecular interaction that may be covalent or non- covalent.
  • “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.
  • disease is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.
  • disease status is meant the presence or absence of pathology in a subject.
  • fetal genetic marker is meant the detection of a nucleic acid sequence of interest in a polynucleotide derived from a fetus.
  • 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.
  • hybridize is meant pairing to form a double-stranded molecule between complementary polynucleotide sequences, or portions thereof, under various conditions of stringency.
  • 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.
  • hybridization time the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA
  • concentration of detergent e.g., sodium dodecyl sulfate (SDS)
  • SDS sodium dodecyl sulfate
  • Various levels of stringency are accomplished by combining these various conditions as needed.
  • hybridization will occur at 30 0 C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS.
  • hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 ⁇ g/ml denatured salmon sperm DNA (ssDNA).
  • ssDNA denatured salmon sperm DNA
  • hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 ⁇ g/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
  • 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.
  • 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.
  • wash steps will occur at 25 0 C 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 68 0 C 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.
  • 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.
  • 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.
  • liquid sample is meant a fluid containing one or more analytes. Such analytes may naturally occur in the liquid or may be solubilized therein.
  • marker is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.
  • matrix any liquid permeable material that binds a nucleic acid molecule.
  • nucleic acid molecule 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.
  • 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.
  • 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 CH 2 -NH-O-CH 2 , CH 2 ⁇ N(CH 3 ) ⁇ O ⁇ CH 2 , CH 2 -O-N(CH 3 )- - CH 2 , CH 2 ⁇ N(CH 3 )-N(CH 3 ) ⁇ CH 2 and 0-N(CH 3 )- CH 2 - - CH 2 backbones (where phosphodiester is O--P--O— CH 2 ).
  • oligonucleotides having morpholino backbone structures are also preferred.
  • 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).
  • oligonucleotides may contain alkyl and halogen-substituted sugar moieties comprising one of the following at the 2' position: OH, SH, S CH 3 , F, OCN, 0(CH 2 ) n NH 2 or O(CH 2 ) n CH 3 , where n is from 1 to about 10; Ci to Cio lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF 3 ; OCF 3 ; O ⁇ , S-, or N-alkyl; O ⁇ , S-, or N-alkenyl; SOCH 3 ; SO 2 CH 3 ; ONO 2 ; NO 2 ; N 3 ; NH 2 ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a conjugate; a reporter group; an intercalator; a group for improving the
  • 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.
  • nucleic acid molecule-binding conjugate any polypeptide, polynucleotide or other molecule that specifically binds a nucleic acid molecule.
  • pathogen 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 peramba, Xanthomonas, Vibrio, and Yersinia.
  • parasite 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.
  • 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.
  • intracellular parasite is a parasite whose entire life cycle is intracellular.
  • human intracellular parasites include Leishmania, Plasmodium, Trypanosoma cruzi, Toxoplasma gondii, Babesia, and Trichinella spiralis.
  • 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.
  • parasites 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.
  • 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.
  • 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 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
  • Herpesviridae variola viruses, vaccinia viruses, pox viruses
  • Iridoviridae e.g. African swine fever virus
  • unclassified viruses e.g.
  • rehabilitation status is meant the presence or absence of molecular alterations related to addictive compounds or their metabolites.
  • reference is meant a standard or control condition.
  • sample application site is meant the portion of the device that contacts a liquid under analysis.
  • binds By “specifically binds” is meant binds a target analyte without substantially binding to other non-target compounds present in the sample.
  • 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.
  • test device is meant a device used in the detection of an analyte in a sample.
  • 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.
  • 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.
  • 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.
  • compositions suitable for the capture and purification of a nucleic acid molecule comprise a matrix, an absorbent reservoir and/or a support.
  • 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).
  • 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.
  • hydrophilic polymers e.g., polyester, polyamide, carbohydrate polymers
  • 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.; GenPrepTM and GenSpinTM are available from Whatman; and IsoCodeTM is available from Schleicher and Schuell.
  • 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.
  • 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.
  • 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
  • uric acid or a urate salt where the composition is adsorbed on or incorporated into the matrix.
  • the matrix may contain a nuclease inhibitor (e.g., a DNAse or RNAse inhibitor).
  • pK 4.0 volatile buffers comprising ammonium acetate
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • membrane e.g. a nylon membrane or a cellulose filter
  • a plate e.g. a microtiter plate
  • solid particle e.g. latex beads
  • 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.
  • the matrix or absorbent reservoir is fixed to the support in a permanent or semi-permanent manner.
  • the matrix or absorbent reservoir rests on or is enclosed within the support, and can be easily removed from the support for subsequent manipulation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the absorbent reservoir does not contact the application site.
  • 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.
  • the hole within the reservoir is centered over the sample application site, such that the absorbent reservoir does not contact the application site.
  • 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.
  • the device encases the strip and the opening is positioned directly over the sample application site.
  • 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.
  • the invention provides methods for the capture and purification of a nucleic acid molecule present in a liquid sample.
  • the liquid sample is brought into contact with the matrix by applying a liquid sample to the matrix in a drop-wise fashion.
  • the liquid sample is typically applied at the center of the circle.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the term "antibody” means not only intact immunoglobulin molecules but also the well-known active fragments F(ab') 2 , and Fab.
  • 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.
  • 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.
  • 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
  • affinity resins such as protein A, protein G, hydroxyapatite, and antiimmunoglobulin.
  • 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.
  • 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).
  • 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).
  • a biological fluid e.g., urine, spinal fluid, semen, vaginal fluid, sputum, phlegm, stool, or cellular lysates
  • 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
  • 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)
  • 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)
  • 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
  • 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.
  • 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.
  • transrenal nucleic acid molecules are not included in the class of polynucleotides detected by the methods of the invention.
  • transrenal nucleic acid molecule is meant a naked polynucleotide that has crossed the kidney barrier.
  • 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.
  • the invention provides methods for the detection of DNA present in whole or lysed white blood cells present in urine.
  • 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.
  • a clean-catch urine sample is used to contact the sample application site.
  • a urine sample is centrifuged and the urine supernatant is reserved for analysis.
  • the urine pellet is used for analysis. Centrifugation reduces or eliminates cellular debris present in the urine.
  • 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.
  • 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.
  • 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.
  • the method detects cytochrome p450 status using HPLC.
  • 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.
  • the captured nucleic acid molecules are at least 1%, 5%, 10%, 15%, 25%50%, 65%, 75%, 85%, 90%, 95%, or even 100% pure.
  • 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).
  • an infection by P. mirabilis, K. pneumoniae, E. faecalis, or P. aeruginosa e.g., an infection by P. mirabilis, K. pneumoniae, E. faecalis, or P. aeruginosa.
  • 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).
  • a genetic or metabolic disorder in a fetus e.g., achondroplasia, cystic fibrosis, Down's syndrome, Marfan's synd
  • 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.
  • 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.
  • 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.
  • a subject e.g., animal, human
  • 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).
  • 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.
  • a diagnostic marker e.g., any target modulated by a compound, a protein or indicator thereof, etc.
  • 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.
  • 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.
  • 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 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.
  • ischemic injury e.g., stroke or heart attack, thrombosis, pulmonary embolism
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • a nucleic acid molecule is analysed using restriction fragment length polymorphism-based PCR ("PCR-RFLP").
  • PCR-RFLP restriction fragment length polymorphism-based PCR
  • 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.
  • a cycling probe reaction Bekkaoui, F. et al, BioTechniques 20,240-248 (1996)
  • PCR polymerase chain reaction
  • nested PCR PCR- single strand conformation polymorphism
  • ligase chain reaction F. Barany Proc. Natl. Acad. Sci USA 88:189-93 (1991)
  • cloning
  • 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 that include a device for the detection of a nucleic acid molecule in a liquid biological sample.
  • the kit includes a test device described herein.
  • 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.
  • 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.
  • 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.
  • 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.
  • a liquid e.g., ethanol, methanol, organic solvent, suitable buffer, such as phosphate buffered saline, or water
  • 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.

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Abstract

La présente invention concerne des compositions et des procédés permettant la capture, la purification et la détection de la présence d'au moins une molécule d'acide nucléique présente dans un échantillon biologique liquide. En particulier, l'invention concerne des procédés de diagnostic pour la détection de molécules d'acides nucléiques présentes dans l'urine.
PCT/US2007/075641 2006-08-10 2007-08-09 Procédés et compositions de détection d'un analyte dans un échantillon biologique WO2008021995A1 (fr)

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WO2011095888A2 (fr) 2010-01-29 2011-08-11 Selfdiagnostics OÜ Procédé et dispositif d'essai rapide pour la détection d'une molécule cible
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WO2008155549A3 (fr) * 2007-06-19 2009-02-26 Oncomethylome Sciences Sa Collecte et traitement perfectionnés d'échantillons d'urine
WO2011095888A2 (fr) 2010-01-29 2011-08-11 Selfdiagnostics OÜ Procédé et dispositif d'essai rapide pour la détection d'une molécule cible
WO2011153244A1 (fr) * 2010-06-01 2011-12-08 Streck, Inc. Procédé et dispositif de traitement d'échantillon
US11002646B2 (en) 2011-06-19 2021-05-11 DNA Genotek, Inc. Devices, solutions and methods for sample collection
US11536632B2 (en) 2011-06-19 2022-12-27 DNA Genotek, Inc. Biological collection system
US11549870B2 (en) 2011-06-19 2023-01-10 DNA Genotek, Inc. Cell preserving solution
US11592368B2 (en) 2011-06-19 2023-02-28 DNA Genotek, Inc. Method for collecting and preserving a biological sample
US10435735B2 (en) 2014-03-07 2019-10-08 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples
US11198899B2 (en) 2014-03-07 2021-12-14 Dna Genotek Inc. Composition and method for stabilizing nucleic acids in biological samples

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