US20200354769A1 - Compositions and methods for obtaining nucleic acids from sputum - Google Patents
Compositions and methods for obtaining nucleic acids from sputum Download PDFInfo
- Publication number
- US20200354769A1 US20200354769A1 US16/809,131 US202016809131A US2020354769A1 US 20200354769 A1 US20200354769 A1 US 20200354769A1 US 202016809131 A US202016809131 A US 202016809131A US 2020354769 A1 US2020354769 A1 US 2020354769A1
- Authority
- US
- United States
- Prior art keywords
- dna
- composition
- container
- barrier
- nucleic acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Images
Classifications
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
- B01L3/5082—Test tubes per se
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/04—Closures and closing means
- B01L2300/041—Connecting closures to device or container
- B01L2300/042—Caps; Plugs
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/04—Closures and closing means
- B01L2300/046—Function or devices integrated in the closure
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/04—Closures and closing means
- B01L2300/046—Function or devices integrated in the closure
- B01L2300/047—Additional chamber, reservoir
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0672—Integrated piercing tool
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/08—Geometry, shape and general structure
- B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2400/00—Moving or stopping fluids
- B01L2400/06—Valves, specific forms thereof
- B01L2400/0677—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers
- B01L2400/0683—Valves, specific forms thereof phase change valves; Meltable, freezing, dissolvable plugs; Destructible barriers mechanically breaking a wall or membrane within a channel or chamber
Definitions
- the present invention relates to compositions and methods for preserving nucleic acids at room temperature for extended periods of time and for simplifying the isolation of nucleic acids.
- DNA can be extracted from virtually every type of cell in the human body, with the exception of red blood cells.
- the usual source of bodily samples for extraction of DNA is venous blood, since the number of nucleated white blood cells (principally neutrophils and lymphocytes) is relatively high and quite consistent: the normal range is about 5 to 10 million white blood cells per milliliter of blood.
- the DNA content of human cells is about 6 micrograms per million cells, so 1 milliliter can theoretically yield from 30 to 60 micrograms of DNA.
- red blood cells per milliliter of blood which, since they contain no DNA, must be removed to obtain pure DNA.
- Collection of blood is not a trivial procedure.
- venous blood is not the ideal source of DNA.
- a simpler procedure for obtaining blood is to collect a few drops after a finger prick and blotting it onto a piece of filter paper. Less training of personnel is required. Once dried, the DNA is quite stable. The amount of DNA recovered is small but sufficient for many forensic purposes. However, a finger prick is still an invasive procedure and heme derived from hemoglobin in blood can inhibit some types of DNA analysis.
- Swabbing the inside of the cheek with a brush is another source of cells that contain DNA. It is much less invasive than taking of blood and can be collected by individuals with less training than is required in the collection of blood. Once collected, the time that useable DNA can be recovered can be extended by either drying the swab or wiping onto filter paper and drying it. However, as the inside of the mouth is not a sterile source (as compared to blood) and microbes can degrade the quality of the DNA after a period of time. The number of cells recovered by this procedure is not large and typically less than 1-2 micrograms of DNA can be expected in the entire sample.
- Saliva is a fairly clear, colorless fluid secreted principally by the major salivary glands (parotid, submandibular, and sublingual). Its function is to lubricate and cleanse the oral cavity, as well as to initiate the process of digestion.
- the parotid gland primarily secretes serous (watery) saliva, while the other glands secrete a mixture of serous and mucinous (sticky) saliva.
- Components of saliva include albumin, globulin, mucins, and digestive enzymes. It has long been known that cellular DNA is present in saliva and that this DNA is suitable for forensic purposes.
- DNA samples are now routinely taken from living persons thought to be relatives of unidentified victims of accident or foul play, to aid in identification of the dead.
- military personnel or other individuals who expect to encounter hazardous situations where their lives may be at risk may wish to store DNA samples prior to exposing themselves to these hazards.
- convicted felons in both Canada and the United States are now required to provide DNA samples.
- DNA-based tests are expected to increase in medicine, such as testing for cystic fibrosis, cytochrome P450 isotypes, polymorphisms affecting susceptibility to infectious and autoimmune diseases, HLA typing, paternity issues, to name but a few.
- an example would be to screen populations for colon cancer-predisposing genes or family members of a breast cancer victim for breast cancer predisposing genes.
- Saliva has a further advantage of not requiring specialized personnel thereby reducing cost where mass sample collection is being carried out. The risk of blood-borne infection is likewise decreased.
- Multimeric proteins called mucins are high molecular weight glycosylated proteins that form a major part of a protective biofilm on the surface of epithelial cells, where they can provide a barrier to particulate matter and bind microorganisms. These glycoproteins contribute greatly to the viscoelastic nature of saliva.
- the major high-molecular-weight mucin in salivary secretions is MUC5B, one of four gel-forming mucins that exist as multimeric proteins with molecular weights greater than 20-40 million daltons.
- MUC5B is a large oligomeric mucin composed of disulphide-linked subunits.
- reagents that reduce disulfides also reduce the viscosity of mucin, such as that found in sputum or saliva.
- Reducing agents in particular sulfur-containing chemicals such as ⁇ -mercaptoethanol and dithiothreitol, are widely used in biochemistry.
- many biochemically relevant reducing agents are capable of reacting in solution with dissolved oxygen. This is known are autooxidation (also called autoxidation or auto-oxidation), where 1-electron reduction intermediates of oxygen are formed, viz., superoxide (O 2 ⁇ .), hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH.).
- transitional metal cations function as catalysts and O 2 ⁇ .
- nucleic acids can be conveniently recovered from it, especially after extended periods of time in the presence of oxygen at neutral or mildly alkaline pH.
- the present inventor has developed a composition, which, when mixed with a mucin-containing bodily fluid, preserves the nucleic acids at room temperature under ambient conditions for extended periods of time. There is no requirement for freezing of the samples before nucleic acid recovery and purification.
- the properties of this composition are that it (a) chemically stabilizes nucleic acids, (b) inhibits nucleases that may be present in the saliva, and (c) is compatible with proteolytic enzymes and other reagents used to purify/amplify oligo- or polynucleotides.
- a fourth and novel property of this composition is that it contains an agent that rapidly reduces the viscous properties of mucin, greatly facilitating the extraction of nucleic acids contained within.
- a first aspect of the invention features a composition for preserving nucleic acids that includes a chelating agent, and a denaturing agent, where the pH of the composition is greater than 5.0.
- the composition is an aqueous solution.
- the composition also includes a reducing agent.
- a reducing agent can include one or more of the following: ascorbic acid, dithionite, erythiorbate, N-acetylcysteine, cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, dierythritol, a resin-supported thiol, a resin-supported phosphine, vitamin E, and trolox, or salts thereof.
- the reducing agent is ascorbic acid, erythiorbate, N-acetylcysteine, dithiothreitol, or 2-mercaptoethanol, and most desirably, the reducing agent is ascorbic acid.
- the composition does not contain ascorbic acid.
- the concentration of the reducing agent in the composition is greater than or equal to 50 millimolar.
- Antioxidant free-radical scavengers are also desirable reducing agents for the composition of the present invention.
- examples include antioxidant vitamins, antioxidant hormones, antioxidant enzymes, thiols, and phenols.
- the reducing agent retains reducing activity for at least 46 days in the presence of one or more of the following: oxygen, ambient air, ambient light, and alkaline pH.
- the chelating agent of the composition can be selected from the group consisting of: ethylenediamine tetraacetie acid (EDTA), cyclohexane diaminetetraacetate (CDTA), diethylenetriamine pentaacetic acid (DTPA), tetraazacyclododecanetetraacetic acid (DOTA), tetraazacyclotetradecanetetraacetic acid (TETA), and desferrioximine, or chelator analogs thereof.
- EDTA ethylenediamine tetraacetie acid
- CDTA cyclohexane diaminetetraacetate
- DTPA diethylenetriamine pentaacetic acid
- DOTA tetraazacyclododecanetetraacetic acid
- TETA tetraazacyclotetradecanetetraacetic acid
- desferrioximine or chelator analogs thereof.
- the chelating agent is cyclohexane diaminetetraacetate (CDTA), diethylenetriamine pcntaacctic acid (DTPA), tctraazacyclododecanetetraacetic acid (DOTA), or desferrioximine, and most desirably, the chelating agent is cyclohexane diaminetetraacetate (CDTA).
- the chelating agent of the composition inhibits metal redox cycling.
- metal redox cycling is meant the inhibition of metal-based oxidation/reduction cycles that produce reactive oxygen free-radical species.
- redox ion pairs involved in such cycles include Fe 2+ /Fe 3+ , Cu 1+ /Cu 2+ , and various oxidation states of molybdenum, vanadium, nickel, and cobalt.
- Chelators that bind one or both ions of a redox ion pair can inhibit the production of reactive oxygen species such as, for example, hydroxyl radical (HO.), hydroperoxyl radical (HOO.), superoxide radical (O 2 ⁇ .), nitric oxide radical (NO.), or peroxynitrite radical (ONO 2 ⁇ .).
- reactive oxygen species such as, for example, hydroxyl radical (HO.), hydroperoxyl radical (HOO.), superoxide radical (O 2 ⁇ .), nitric oxide radical (NO.), or peroxynitrite radical (ONO 2 ⁇ .).
- the nucleic acid to be preserved by the composition can be DNA or RNA, including mRNA or viral RNA.
- the pH of the composition can between from about 5.0 and about 11.0, desirably from about 6.5 to about 7.5, and most desirably, about 7.0.
- a pH from about 7.0 to about 10.0 can be used.
- desirable pHs are about 7.5, about 8.0, or a pH range from about 8.0 to about 9.0.
- a buffer, such as HEPES, TRIS, or carbonate buffer can be added to the composition to maintain the pH in a constant range.
- a pH from about 5.0 to about 7.0, desirably from about 6.5 to about 6.8 can be used.
- a buffer such as BES, can be used to maintain the pH in a constant range.
- the denaturing agent of the composition can be selected from the group consisting of: urea, dodecyl sulfate, guanidinium chloride, guanidinium thiocyanate, perchlorate, and an alcohol.
- the denaturing agent is urea, dodecyl sulfate, or an alcohol, wherein the alcohol is 10%-60% of the total composition volume.
- the alcohols can be methanol, ethanol, n-propanol, isopropanol, n-butanol, trifluoroethanol, phenol, or 2,6-di-tert-butyl-4-methylphenol.
- the composition includes an antimicrobial agent.
- antimicrobial agent is meant a substance or group of substances which reduces the rate of growth of an organism compared to the rate of growth of the organism in their absence. A reduction in the rate of growth of an organism may be by at least 5%, more desirably, by at least 10%, even more desirably, by at least 20%, 50%, or 75%, and most desirably, by 90% or more.
- the definition also extends to substances which affect the viability, virulence, or pathogenicity of an organism.
- An antimicrobial agent can be natural (e.g., derived from bacteria), synthetic, or recombinant. An antimicrobial agent can be bacteriostatic, bactericidal or both.
- an antimicrobial agent is bacteriostatic if it inhibits cell division without affecting the viability of the inhibited cell.
- An antimicrobial agent is bactericidal if it causes cell death. Cell death is commonly detected by the absence of cell growth in liquid growth medium (e.g., absence of turbidity) or on a solid surface (e.g., absence of colony formation on agar).
- liquid growth medium e.g., absence of turbidity
- a solid surface e.g., absence of colony formation on agar.
- bacteriostatic substances are not bactericidal at any concentration.
- the composition of the invention includes an alcohol as an antimicrobial agent, and most desirably the composition includes ethanol.
- the composition also includes an inhibitor of ribonuclease.
- Desirable inhibitors are selected from the group consisting of: heparin, heparan sulfate, oligo(vinylsulfonic acid), poly(vinylsulfonic acid), oligo(vinylphosphonic acid), and poly(vinylsulfuric acid), or salts thereof.
- the inclusion of an inhibitor of ribonuclease in the composition of the invention is particularly desirable when the nucleic acid to be preserved is RNA, desirably mRNA, or when the nucleic acid to be preserved is from a virus or a bacterium.
- a second aspect of the invention features a method of reducing the viscosity of a mucin-containing bodily fluid or tissue by reducing disulfide bonds inherent to mucin, wherein the bodily fluid or tissue is mixed with a composition of the invention that includes a reducing agent.
- the bodily fluid is sputum, desirably saliva.
- saliva is meant that mucoid matter contained in or discharged from the nasal or buccal cavity of an animal, including saliva and discharges from the respiratory passages, including the lungs.
- the method includes the recovery of a nucleic acid.
- a third aspect of the invention features a method of preserving a nucleic acid contained in sputum that includes the steps of obtaining sputum from a subject, and contacting the sputum with a composition of the invention, thus preserving the nucleic acid.
- the DNA when the nucleic acid is DNA, the DNA is stable for more than 14 days, desirably more than 30 days, and more desirably more than 60 days. In another embodiment, when the nucleic acid is DNA and the composition does not contain ascorbic acid, the DNA is stable for more than 60 days, and desirably more than 360 days.
- a fourth aspect of the invention features a method of recovering a nucleic acid from sputum that includes the steps of: i) obtaining sputum from a subject, ii) contacting the sputum with a composition of the invention to form a mixture, iii) contacting the mixture with a protease, and iv) recovering the nucleic acid from the mixture.
- the protease is proteinase K or pronase.
- the sputum is saliva.
- the sputum is from a mammal, desirably a human.
- the nucleic acid is DNA or RNA. If the nucleic acid is RNA, desirably it is mRNA or viral RNA.
- the nucleic acid can be from a source foreign to the subject from which the sputum sample is taken.
- the nucleic acid can be from a bacterium or a virus that is residing in the buccal, nasal, or respiratory passages of the subject.
- the invention features a method of preserving and/or recovering a nucleic acid from a bodily fluid that includes, placing the bodily fluid into a first region of a container, placing a composition of the invention into a second region of the container, which is separated from the first region by a barrier, closing the container, and disturbing the integrity of the barrier such that the composition and the bodily fluid are brought into contact.
- the disestablishment of the barrier is coupled to the closing of the container when a lid is placed on it.
- the barrier is punctured.
- the barrier is in the form of a pivoting sealing disc.
- attachment of the lid to the container forces the disc to pivot from its original position of spanning the space between the first region and the second region to a position in which both regions are exposed to each other, thereby forming a mixture between a composition of the invention and the bodily fluid is allowed.
- the bodily fluid is sputum, and most desirably, saliva.
- the invention features a device for preserving and/or isolating a nucleic acid obtained from a biological sample.
- the device includes: a container that has a first region for collecting a biological sample and a second region containing a composition for preserving a nucleic acid, a barrier between the first region the second region that keeps the biological sample and the composition separate, a means for closing the container, and a means for disturbing the integrity of the barrier such that the composition is capable of contacting the biological sample.
- the first region can have an opening of from 2.0 to 7.0 cm, desirably from 2.5 to 3.5 cm, and most desirably 3.0 cm.
- the biological sample is sputum, and most desirably, saliva.
- the nucleic acid-preserving composition is a composition of the present invention.
- the means for closing the container is coupled to the means for disturbing the integrity of the barrier.
- the means for closing the container is an airtight lid.
- the invention features a method of manufacturing a device for preserving a nucleic acid in a biological sample that includes: providing a container that has a first region and a second region, with the first region suitable for containing a composition of the invention and the second region having an opening suitable for the application of a biological sample; placing the composition into the first region; and applying a barrier to the container between the first region and the second region, with the barrier being impermeable to the composition and capable of being disestablished.
- the barrier can be a pivoting disc, where in a first position, the disc spans the compartment and separates the first and second areas. Pivoting the disc to a second position (e.g., by connecting a screw-on lid to a plunger mechanism which contacts the disc, causing it to pivot when the lid is screwed on) disestablishes the barrier and allows the biological sample contained in the first region to contact the composition that is contained in the second region.
- alcohol is meant a water-miscible organic compound containing a hydroxyl group, including water-miscible mixtures of hydroxyl-containing organic compounds.
- antioxidant free-radical scavenger a substance that reduces a reactive oxygen free radical species.
- free radicals include, for example, hydroxyl radical (HO.), hydroperoxyl radical (HOO.), superoxide radical (O 2 ⁇ .), nitric oxide radical (NO.), or peroxynitrite radical (ONO 2 ⁇ .).
- aqueous solution is meant a solution or suspension that contains 30% or more water by volume.
- substantially fluid is meant a naturally occurring fluid from an animal, such as saliva, serum, plasma, blood, urine, mucus, gastric juices, pancreatic juices, semen, products of lactation or menstration, tears, or lymph.
- biological sample any sample containing nucleic acids that has been obtained from or deposited by an animal.
- Non-limiting examples include skin, hair, bodily fluids, fecal matter, and tissue.
- chelator analog is meant a derivative chelator compound with the same backbone structure and having the same general properties as the parent chelator compound.
- denaturing agent is meant a substance that alters the natural state of that to which it is added.
- mucin is meant any mucoprotein that raises the viscosity of the medium surrounding the cells that secrete it.
- nucleic acid is meant a chain of the nucleotides, including deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically found in chromosomes, mitochodria, ribosomes, bacteria, or viruses.
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- nucleic acid-preserving composition any composition of the present invention, unless otherwise specified.
- stable when referring to a nucleic acid, by “stable” is meant that at least about 50% of the initial amount of high molecular weight nucleic acid (DNA, RNA, mRNA, or viral RNA) contained in a sample is still present after storing the sample at ambient temperature (i.e., 20° C. to 25° C.) for the specified time period.
- the amount of high molecular weight DNA in a sample can quantified by densitometry analysis of the high molecular weight DNA band from an agarose gel (see FIG. 1 and Example 4).
- resin-supported phosphine is meant a polymer that contains a multiplicity of covalently-bound phosphine groups.
- resin-supported thiol is meant is meant a polymer that contains a multiplicity of covalently-bound sulfhydryl groups.
- saliva is meant the secretion, or combination of secretions, from any of the salivary glands, including the parotid, submaxillary, and sublingual glands, optionally mixed with the secretion from the buccal glands.
- saliva is meant that mucoid matter contained in or discharged from the nasal or buccal cavity of a mammal, including saliva and discharges from the respiratory passages, including the lungs.
- subject is meant any animal. Desirably, the subject is a mammal that can produce saliva for the purposes of nucleic acid extraction. Most desirably, the subject is a human.
- FIG. 1 is an electrophoresis agarose analysis of DNA isolated from saliva using the capacity of methods of one embodiment of the invention.
- FIG. 2 is a graph illustrating real time PCR of stimulated saliva DNA of Example 5.
- FIG. 3 is a graph illustrating real time PCR of unstimulated saliva DNA of Example 6.
- FIG. 4 is an electrophoresis agarose analysis of the DNA in saliva samples mixed with compositions of the invention, the mixtures having been incubated for various times at various temperatures.
- FIG. 5 shows structures of (oxidized) ascorbate anion, (reduced) dehydroascorbic acid, and a free radical intermediate
- FIG. 6 is a compilation of two spectrophotometric scans of sodium ascorbate (100 ⁇ M) in CB (1 mM CDTA, 10 mM BES, pH 7.4), prepared under aerobic conditions over 30 minutes at room temperature (scan 1) and 3 minutes after addition of a few crystals of MnCl 2 . (scan 2), as per Example 8.
- FIG. 7 is a compilation of spectrophotometric scans, at the indicated times, of the 100 ⁇ M sodium ascorbate prepared in CB of Example 8. The solution was exposed to ambient atmosphere and temperature between scans but was not contacted with MnCl 2 (see Example 9).
- FIG. 8 is a graph of absorbances at 265 nm, obtained at the indicated times, of a solution of sodium ascorbate (250 mM) containing 30 mM Tris-HCl, pH 8.0, 30% ethanol, 3 mM CDTA, mixed with 50 mL of CB, as per Example 10.
- the stock solution was maintained at room temperature and no precaution was taken to exclude ambient atmosphere or ambient light.
- FIG. 9 is a compilation of spectrophotometric scans of the 46 day-old solution prepared in Example 10.
- Scan 2 was taken 2 minutes after the addition MnCl 2 .
- Scan 3 was taken 8 minutes after the addition MnCl 2 .
- Scan 4 was taken 27 minutes after the addition MnCl 2 .
- FIG. 10 is an exploded view of a sample container of the invention. Included in the figure is a cross-sectional top view taken at line 1 - 1 of container 3 showing plunger 4 and plunger channel 5 . Also shown is a cross-sectional top view taken at line 2 - 2 of container 3 , showing supports 6 for sealing disc 7 (not shown in this figure but shown in FIG. 11 ).
- FIG. 11 is a side view of the sample container of FIG. 10 , now showing sealing disc 7 .
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- mRNA messenger RNA
- HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- BES N,N-bis[2-hydroxyethyl]-2-aminoethane-sulfonic acid
- TRIS tris(hydroxymethyl)aminomethane
- CDTA cyclohexane diaminetetraacetate
- DTPA N,N-bis(2-(bis(carboxymethyl)amino)ethyl)glycine
- DOTA 1,4,7,10-tetrazacyclododecanetetraacetic acid
- TETA 1,4,8,11-tetraazacyclotetradecanetetraacetic acid.
- compositions that render sputum as a viable option to the use of blood as a source of nucleic acids.
- the compositions provide the advantageous properties of chemical stabilization of nucleic acids and the inhibition of nucleases, including deoxyribonucleases, and microbial growth. Chemical stabilization of the nucleic acids in a saliva sample is achieved through the use of buffers to maintain an appropriate pH, as well as the use of chelating agents to prevent the phenomenon of metal redox cycling or the binding of metal ions to the phosphate backbone of nucleic acids.
- the chelating agents of the invention also participate in the inhibition of deoxyribonucleases and microbial growth, which can be additionally inhibited by the inclusion of denaturing agents and/or other suitable antimicrobial substances, such as ethanol, into the compositions of the invention.
- the compositions of the invention can also include one or more reducing agents, which can reduce sample viscosity, thereby making nucleic acid recovery an easier process.
- the present invention features a composition for preserving and/or recovering nucleic acids from sputum, desirably saliva, that includes one or more chelators and one or more denaturing agents, wherein the pH of the composition is greater than 5, desirably within a pH range of about 6 to about 11, more desirably within a pH range of about 7.5 to about 10.0, and most desirably, within a pH of about 7.0.
- the chemical backbone and the purine bases of DNA are most stable at slightly alkaline pH, with an optimal stability generally recognized as being within a pH range of about 7-11, and desirably a pH of about 8.
- depurination i.e., spontaneous loss of purine bases from the deoxyribose-phosphate backbone
- a pH of about 10 spontaneous loss of amino groups from cytosine nucleotides may occur, thereby converting cytosine to uracil.
- DNA is denatured, converting it from the double-strand form to the single-strand form.
- RNA is most stable in the pH range of 5.0 to 7.0, desirably a pH of from 6.5 to 6.8.
- the pH of the composition may be adjusted using pH buffers, desirably those that best control the pH within the range of about 5 to about 11.
- pH buffers with desirable properties include, but not limited to, TRIS hydrochloride, HEPES and BES.
- a composition of the invention includes one or more chelators that can form complexes with metal ions to prevent them from binding to DNA, remove metal ions that that have already bound to DNA, or bind to metal ions (e.g., Fe(II)/Fe(III) or Cu(I)/Cu(II)) strongly enough to inhibit their redox cycling, and hence, the formation of reactive oxygen species.
- EDTA a commonly used chelator in biological reagents, can be of some use for either of these purposes.
- the amount or concentration of chelator will depend upon the strength of the chelator, which would need to be determined empirically. For CDTA, concentrations in the 1-20 mM range are sufficient, however other concentrations would work, and the compositions of the invention are not intending to be limited to this range.
- Deoxyribonucleases and ribonucleases are enzymes that breakdown DNA or RNA, respectively. Their main source in the digestive tract is secretions of the pancreas, although lower levels may be present in cells of the salivary gland and buccal mucosa. In addition, microorganisms resident in the mouth or from recently ingested foods may contain deoxyribonucleases or ribonucleases. Pancreatic deoxyribonuclease is known to require divalent metal ions such as Mg(II), Mn(II) and/or Ca(II) for enzymatic activity.
- the strong chelators described above in addition to providing chemical stability to the nucleic acids, will inhibit this class of metal ion-requiring deoxyribonucleases.
- the action of deoxyribonucleases and ribonucleases can also be inhibited by denaturing agents that will destroy the complex structures of these enzymes (proteins). Hence, denaturing agents are included in the nucleic acid preserving composition of the invention.
- denaturing agents examples include, but not limited to, urea, soluble salts of dodecyl sulfate and other strong detergents, guanidinium chloride, guanidinium thiocyanate, soluble salts of perchlorate, alcohols, such as ethanol, above 10%.
- Other reagents such as heparin, heparan sulfate, or oligo(vinylsulfonic acid) (Smith, et al., J. Biol. Chem. Mar. 20, 2003; [epub ahead of print]) are known to inhibit the action of deoxynucleases and/or ribonucleases.
- proteases such as proteinase K are frequently used in the purification of nucleic acids. Since proteases are themselves proteins, their action can be inhibited by denaturing agents. Thus, a balance must be struck between the concentration of denaturing agents that will, on the one hand, inhibit deoxyribonucleases or ribonucleases and denature other proteins in saliva and, on the other hand, not significantly inhibit the proteolytic enzymes.
- the DNA is often concentrated by precipitation with alcohol.
- salts, buffers, chelators and other components of the nucleic acid preserving/recovery solution must be chosen so as not to precipitate when concentrations of alcohol over 50% are added to precipitate the DNA.
- a composition of the invention further includes one or more reducing agents.
- the reducing agents are desirably at high concentration (greater than 0.05 M). While not wishing to be limited by theory, it is presumed that the reducing agent reduces the viscosity of the saliva by breaking disulfide bonds that hold together chains of mucin, and that any reducing agent that has the appropriate redox potential to reduce disulfide bonds in proteins would be suitable.
- the reducing agent is selected from the group consisting of: ascorbic acid, dithionite, erythiorbate, N-acetylcysteine, cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, dierythritol, a resin-supported thiol, a resin-supported phosphine, vitamin E, and trolox, or salts thereof.
- a composition of the invention that includes a reducing agent maintains reducing capacity at room temperature in a sealed container in the presence of ambient oxygen, and/or in the presence of ambient light for more than a week, desirably for up to about 46 days, and most desirably for at least 46 days.
- This embodiment combines the nucleic acid stabilization provided by a strong chelator a denaturing agent, and a reducing agent in a composition with a pH within the range of about 6 to about 11, and desirably a pH of about 8.0.
- a particularly desirable reducing agent is sodium ascorbate.
- ascorbic acid vitamin C
- vitamin C is a non-thiol reducing agent and is inexpensive, non-toxic, and stable in the presence of the chelators and denaturing agents that are included in the compositions of the invention.
- the structures of (oxidized) ascorbate anion, (reduced) dehydroascorbic acid, and a free radical intermediate are shown in FIG. 5 .
- the most thoroughly studied oxidation reaction of ascorbate is its oxidation by oxygen.
- trace amounts of transitional metals such as iron or copper can promote autooxidation (Buettner, Free Radic. Res. Commun.
- Metal cation-catalyzed oxidation of ascorbate can be conveniently monitored as a decrease in absorbance at 265 nm (Buettner Free Radic. Res. Commun. 10:5-9, 1990), as described in Example 8 and shown in FIGS. 5, 6 , and 8 .
- Certain chelating agents can appreciably slow down autooxidation of ascorbate at pH 7.0 or lower (Buettner J. Biochem. Biophys. Methods 16:27-40, 1988), as described in Example 10 and shown in FIG. 8 .
- a composition of the present invention includes one or more chelators, one or more denaturing agents, and one or more antimicrobial agents, wherein the pH of the composition is within a pH range of about 6.0 to about 11.0, desirably at a pH of about 8.0. Microbial growth may also be inhibited by the strong chelators and denaturing agents, for example, ethanol, described above. Therefore, in a further embodiment of the present invention, a composition for preserving and/or recovering DNA from sputum includes one or more chelators and one or more denaturing agents, wherein at least one or more of the denaturing agents and/or chelating agents is present in amounts to act as an antimicrobial agent.
- Reagents that indicate when a biological sample has been contacted with a composition of the invention can also be included as part of the composition. Desirable are those reagents that result in a visual color change of the composition solution upon mixing with the added sample. These reagents can function by reacting with any number of functional groups that are contained in biological samples, including, for example, amines, thiols, or glycosyl groups. Such colorimetric reagents are known to those skilled in the art and are chosen in such a manner that other components of the composition do not interfere with their effective usage.
- the present invention features methods of collecting, preserving, and recovering nucleic acids from sputum using a composition of the invention.
- the methods of the invention involve contacting a sputum sample from a subject with a composition of the invention and optionally mixing the resulting solution with a protease, such as pronase or proteinase K.
- a protease such as pronase or proteinase K.
- some compositions of the invention feature a reducing agent that can facilitate the recovery of nucleic acids from composition/sample mixtures by decreasing the viscosity of these mixtures.
- one aspect of the invention features a method of preserving a nucleic acid contained in sputum that includes the steps of obtaining sputum from a subject, and contacting the sputum with a composition of the invention, thus preserving the nucleic acid.
- Examples 1 and 2 describe the collection of saliva, both from subjects that can follow instructions and from those that can not.
- the sputum is typically contacted with a composition of the invention upon collection or immediately after it is collected, and preferably not much later than about 1 hour after collection. This time can vary depending on storage conditions of the sputum after collection. For example, it could be indefinite if stored frozen or perhaps 1-2 days if stored at 4° C.
- a reducing agent can be in the preserving composition used, or added at a later time prior to nucleic acid isolation. Desirable reducing agent-containing compositions are those that are stable and retain a reducing capacity for more than a week, desirably for up to about 46 days, and most desirably for at least 46 days.
- Example 5 saliva was collected and mixed with approximately an equal volume of a composition of the invention (see Example 3 for preparation), and analyzed for DNA content by PCR analysis at later timepoints.
- the mouth be rinsed before sampling.
- Food particles can introduce foreign DNA and saliva transferred by kissing can be a source of foreign human DNA.
- the mouth can be rinsed with about 50 mL of tepid water by vigorous swishing or by brushing with a tooth brush without tooth paste.
- Unstimulated saliva is usually of the mucinous type and is secreted at a slow rate.
- Stimulated saliva anticipation of tasty food, sweet or sour candy
- Stimulated saliva is of the serous (watery) type and secreted at a faster rate. It has been found (see Table 2) that there is more DNA in 2 mL of unstimulated saliva than 2 mL of stimulated saliva.
- the donor may spit a volume (for example, about 2 mL) of “unstimulated” saliva into the receiving tube. If this proves to be difficult, saliva flow can conveniently be stimulated with a cube of table sugar, or any other such saliva-stimulatory substance that does not interfere with DNA recovery or purification.
- Another aspect of the invention features a method of reducing the viscosity of a mucin-containing bodily fluid or tissue by reducing disulfide bonds inherent to mucin, wherein the bodily fluid or tissue is mixed with a composition of the invention that includes a reducing agent.
- the bodily fluid is sputum, desirably saliva.
- Yet another aspect of the invention features a method of recovering a nucleic acid from sputum that includes the steps of: i) obtaining sputum from a subject, ii) contacting the sputum with a composition of the invention to form a mixture, iii) contacting the mixture with a protease, and iv) recovering the nucleic acid from the mixture.
- Suitable proteases include, for example, proteinase K or pronase.
- the protease may suitably be in a dry form that would become activated once mixed with sputum and a composition of the invention.
- the protease is deposited onto an interior surface of the collection device. This can be accomplished by dissolving the protease in a solution made up of equal volumes of 5% sucrose in water and 5% glycerol in ethanol and then, after placing the solution on the surface, removing the volatiles under a controlled vacuum to leave the protease bound to the surface as a sticky residue.
- composition does not contain a reducing agent (or even if it does), a reducing agent can be added at any time prior to isolation of the nucleic from the sample, desirably prior to or concurrently with contacting the sample with a suitable protease.
- a composition of the present invention When sputum is mixed with a composition of the present invention, cells are disrupted, nucleic acids are liberated from the cells, membranous material is solubilized, proteins are stripped from the nucleic acids, and protein digestion begins. If present, a reducing agent in the composition reduces the viscosity of the gel-forming mucin. Incubation can be at room temperature over a relatively long period of time (days or weeks) while samples are being shipped to a laboratory for analysis. If transferred to a laboratory soon after collection, incubation at 55° C. for 4 to 16 hours is sufficient to allow the activated protease to digest the majority of protein to small peptides or amino acids. Under such conditions, nucleic acids and polysaccharides remain relatively intact.
- nucleic acid isolation can be performed using any technique known in the art ( Short Protocols in Molecular Biology, 5 th Edition Frederick M. Ausubel, Roger Brent, Robert E. Scientific, David D. Moore, J. G. Seidman, John A. Smith (Editor), Kevin Struhl (Editors). ISBN: 0-471-25092-9. 2002. John Wiley and Sons).
- SDS Short Protocols in Molecular Biology, 5 th Edition Frederick M. Ausubel, Roger Brent, Robert E. Scientific, David D. Moore, J. G. Seidman, John A. Smith (Editor), Kevin Struhl (Editors). ISBN: 0-471-25092-9. 2002. John Wiley and Sons).
- SDS sodium chloride
- a “precipitation solution” consisting of, for example, potassium chloride may be added to a portion of the sputum-composition mixture resulting in the precipitation of potassium dodecyl sulfate, after standing on ice to cool the solution.
- the supernatant is collected.
- the supernatant is expected to contain as much as 10-30 nanograms per microliter of DNA.
- the sample can be diluted.
- the DNA in the supernatant can be precipitated by the addition of alcohol and redissolved in any suitable buffer. This step has the effect of removing inhibitory components of the composition, which are present to preserve the nucleic acids during transport to the laboratory.
- the methods of the present invention can be used to isolate nucleic acids from sputum for any application requiring a nucleic acid sample.
- some specific applications of the methods of the present invention include, but are not limited to, forensic applications, medical applications (including genetic screening and disease typing), and paternity testing.
- Another aspect of the invention features a method of preserving and/or recovering a nucleic acid from a bodily fluid that includes, placing the bodily fluid into a first region of a container, placing a composition of the invention into a second region of the container, which is separated from the first region by a barrier, closing the container, and disturbing the integrity of the barrier such that the composition and the bodily fluid are brought into contact.
- Collection devices of the invention which also can serve as containers for bring the compositions and nucleic acid-containing bodily fluids together are described below.
- the invention also provides a novel collection device useful for collecting a biological sample from a subject, and subsequently mixing the collected sample with a composition intended to stabilize, preserve, or facilitate the recovery of components of the sample.
- Such components may include, without limiting the invention, nucleic acids, proteins, peptides, toxins, chitins, fatty acids, and glycogens.
- Non-limiting examples of biological samples are skin, hair, fecal matter, bodily fluids, and tissue.
- the invention features a device for preserving and/or recovering a nucleic acid obtained from a biological sample.
- the device includes: a container that has a first region for collecting a biological sample and a second region containing a composition for preserving a nucleic acid, a barrier between a first region and a second region that keeps the sample and composition separate, a means for closing the container, and a means for disturbing the integrity of the barrier, such that the composition is capable of contacting the bodily sample.
- the composition is a composition of the present invention.
- the sample is a biological fluid.
- the collection device of the invention simultaneously serves several functions. Some of the desirable features of this collection vessel include one or more of the following:
- the container may be constructed of a sturdy breakage-resistant plastic, desirably a biocompatible plastic.
- the container would be constructed from a material that would not leach chemicals into the container's contents;
- the bottom part of the container would be narrow to reduce the overall volume of the container to make it easier to collect the small volume (1-2 milliliters) of fluid that would be expected from a routine sampling, in particular, when the sample is an expectorate.
- the device would contain markings to allow for an estimate of the sample volume collected;
- the means for closing the container may be a cap that is designed to lock once tightened to become tamper-resistant;
- the means for closing the container may be a cap that is designed to provide a liquid-tight and/or airtight seal for the container once the cap is fixed into place;
- the barrier may be a septum or plastic bag compact lent that would separate the composition from the fluid until the septum or bag compartment is pierced or the contents otherwise released;
- the barrier may be in the form of a pivoting partition.
- attachment of the lid to the container forces the partition to pivot from its original position of spanning the space between the first region and the second region to a position in which both regions are exposed to each other and contact between the composition contained in one space and the bodily fluid contained in the other space is allowed;
- the barrier can be press fit, glued, or heat fit into place
- the means for closing the container may be coupled to the disestablishment of the barrier
- FIGS. 10 and 11 A device of the invention is shown in FIGS. 10 and 11 .
- a biological sample (not shown) is applied to a first region 8 of container 3 , which is separated from a second region 9 by sealing disc 7 .
- cap 1 is placed onto the device and secured via a screw thread mechanism to a tight fit, thereby sealing container 3 .
- ram 2 which is attached to cap 1 , moves downward as shown by dotted line arrow 11 .
- This downward movement forces plunger 4 , which is contained in plunger barrel 5 , downward as indicated by dotted line and arrow 12 .
- kits for performing the methods of the invention that include a device of the invention containing a composition of the invention, with instructions for stabilizing, preserving, or facilitating the recovery of nucleic acids from a biological sample by using the device to bring a biological sample into contact with the composition.
- the subject is instructed to wait for a period of 20-30 minutes before last eating.
- the subject will brush his teeth without using toothpaste, if possible.
- the subject will rinse his mouth vigorously with 50 mL of cool or tepid water.
- the subject will then spit saliva into the special collection tube until the level of saliva reaches the 2 mL mark. This may take several minutes. If the subject finds that he is unable to deliver sufficient saliva, he will be given a cube of table sugar to chew, and told not to be concerned if some of the sugar is spit into the tube.
- nucleic acid-preserving composition When the required amount of saliva is collected, it is mixed with 2 mL of a nucleic acid-preserving composition. The precise way this will be introduced will depend upon the container design.
- the cap is attached to the container and tightened to seal it securely.
- the container is then vigorously shaken and the process is complete.
- the DNA is now in an intermediate preserved state. It can be maintained in a frozen state or at any temperature up to about 60° C.
- the container can be mailed back to the testing lab at room temperature.
- a rubber or plastic tube or nipple will be introduced into the mouth, attached to a sponge, suction bulb or small syringe, and kept in the mouth for several minutes until visible drooling occurs.
- a bit of sugar cube will be placed in the mouth to stimulate saliva if necessary.
- the responsible adult will wear disposable gloves provided for the purpose to avoid contamination with his/her DNA.
- the responsible adult will draw saliva into the bulb or syringe and transfer it into the collection container.
- the DNA preserving/extraction composition is introduced and the container is capped and sealed. The tube is vigorously shaken for 1 minute.
- composition of the nucleic acid-preserving solution used in Examples 4-6 is 33 mM TRIS-HCl, 0.67 M urea, 0.67 M LiCl, 0.6% sodium dodecyl sulfate, 3.3 mM CDTA, 30% ethanol, and 0.25 M sodium ascorbate, all adjusted to a final pH of 8.0.
- the composition is mixed with an equal volume of saliva.
- FIG. 3 is a graph showing saliva DNA samples collected on 2002 Mar. 25 (see Table 1) and analyzed on 30 days later in accordance with FIG. 1 .
- Minimally purified DNA was used Polymerase chain reaction and other conditions as described in Examples 4 and 5 except saliva collection was done under unstimulated conditions. Numbers refer to individual donors.
- C is control DNA, a highly purified sample of DNA purified from blood.
- Tables 1 and 2 show estimates of DNA recovered from saliva samples. In all cases, the individual donor has been identified by a unique number. These data show that the amount of DNA that can be recovered from this group of donors ranges from 16 micrograms per milliliter of saliva and higher. Estimation of the amount of DNA by chemical methods such as DABA presents some problems and the DNase method provides most reliable results.
- Saliva was mixed with an equal volume of the indicated composition and the mixture was incubated for the indicated time period at the indicated temperature (see Table 3). After incubation, approximately 40 ⁇ L of mixture was digested briefly with ribonuclease to remove the majority of the RNA present in the sample, then applied to the indicated lane of a 0.8% agarose gel. Following electrophoresis, the gel was stained with ethidium bromide as in Example 4.
- transition metal manganous chloride
- Example 10 Stability of Sodium Ascorbate in a Nucleic Acid-Preserving Composition
- a stock solution of sodium ascorbate (250 mM) was prepared in a solution containing 30 mM Tris-HCl, pH 8.0, 30% ethanol, 3 mM CDTA. 20 ⁇ L was removed at the indicated times, mixed with 50 mL of CB (see Example 8) and the absorbance at 265 nm was read immediately. The stock solution was maintained at room temperature. The results are shown in FIG. 8 .
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Hematology (AREA)
- Clinical Laboratory Science (AREA)
- Immunology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Saccharide Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Cosmetics (AREA)
Abstract
The present invention relates to compositions and methods for preserving and extracting nucleic acids from saliva. The compositions include a chelating agent, a denaturing agent, buffers to maintain the pH of the composition within ranges desirable for DNA and/or RNA. The compositions may also include a reducing agent and/or antimicrobial agent. The invention extends to methods of using the compositions of the invention to preserve and isolate nucleic acids from saliva as well as to containers for the compositions of the invention.
Description
- This application is a Continuation of U.S. patent application Ser. No. 10/455,680, entitled, “Compositions and Methods for Obtaining Nucleic Acids from Sputum”, filed Jun. 5, 2003, which claims the benefit of U.S. Application No. 60/386,397, filed Jun. 7, 2002, U.S. Application No. 60/386,398, filed Jun. 7, 2002, and U.S. Application No. 60/386,399, filed Jun. 7, 2002, each of which is hereby incorporated by reference.
- The present invention relates to compositions and methods for preserving nucleic acids at room temperature for extended periods of time and for simplifying the isolation of nucleic acids.
- DNA can be extracted from virtually every type of cell in the human body, with the exception of red blood cells. The usual source of bodily samples for extraction of DNA is venous blood, since the number of nucleated white blood cells (principally neutrophils and lymphocytes) is relatively high and quite consistent: the normal range is about 5 to 10 million white blood cells per milliliter of blood. The DNA content of human cells is about 6 micrograms per million cells, so 1 milliliter can theoretically yield from 30 to 60 micrograms of DNA. However, there are about 5 billion red blood cells per milliliter of blood, which, since they contain no DNA, must be removed to obtain pure DNA. Furthermore, the use of blood as a source of DNA has many other disadvantages. Collection of blood is not a trivial procedure. Taking of venous blood requires trained personnel. It is an invasive procedure, which frequently causes some distress and pain to the donor. Precautions are needed to minimize exposure of personnel to blood-borne pathogens. Once collected, the blood sample must be either frozen or quickly transported to a laboratory for extraction of DNA. For these reasons, venous blood is not the ideal source of DNA. A simpler procedure for obtaining blood is to collect a few drops after a finger prick and blotting it onto a piece of filter paper. Less training of personnel is required. Once dried, the DNA is quite stable. The amount of DNA recovered is small but sufficient for many forensic purposes. However, a finger prick is still an invasive procedure and heme derived from hemoglobin in blood can inhibit some types of DNA analysis.
- Swabbing the inside of the cheek with a brush (a buccal swab) is another source of cells that contain DNA. It is much less invasive than taking of blood and can be collected by individuals with less training than is required in the collection of blood. Once collected, the time that useable DNA can be recovered can be extended by either drying the swab or wiping onto filter paper and drying it. However, as the inside of the mouth is not a sterile source (as compared to blood) and microbes can degrade the quality of the DNA after a period of time. The number of cells recovered by this procedure is not large and typically less than 1-2 micrograms of DNA can be expected in the entire sample.
- Saliva is a fairly clear, colorless fluid secreted principally by the major salivary glands (parotid, submandibular, and sublingual). Its function is to lubricate and cleanse the oral cavity, as well as to initiate the process of digestion. The parotid gland primarily secretes serous (watery) saliva, while the other glands secrete a mixture of serous and mucinous (sticky) saliva. Components of saliva include albumin, globulin, mucins, and digestive enzymes. It has long been known that cellular DNA is present in saliva and that this DNA is suitable for forensic purposes. Forensic use is typically limited to victim or suspect identification, using the tiny amounts of DNA from saliva that may recovered at a crime scene or from the back of a postage stamp. The notion that saliva may be a reliable source of genomic DNA and a rival to venous blood samples for this purpose has been investigated more recently in a scientific publication (van Schie, et al., J. Immunol. Methods 208:91-101, 1997). The authors used freshly collected or frozen saliva samples and purified the DNA by a fairly complex extraction procedure. Estimates of the quantity of DNA recovered were based upon light absorption at 260 nm, a procedure known to be an unreliable method since other common biological macromolecules, such as RNA, have essentially the same ultraviolet light absorption spectrum. Nevertheless, these authors showed that quality genomic DNA was indeed present by gel electrophoretic analysis and polymerase chain reaction analysis for certain allelic polymorphisms. Another communication (Terasaki, et al., Hum. Immunol. 59:597-598, 1998) reported similar results about the suitability of saliva as a source of DNA for HLA typing by polymerase chain reaction analysis. Although the amount of DNA recovered was reported, the method used to measure DNA was not. These authors provided 3 examples where saliva dried on filter paper yielded DNA suitable for analysis.
- With the increasing use of DNA-based analysis in forensics, law enforcement, military, human medicine, veterinary medicine, and research, there is a need for a product that would allow saliva to become a standard reliable source of DNA from an individual (to replace blood, the current standard). In forensic, military and mass disaster situations, for example, DNA samples are now routinely taken from living persons thought to be relatives of unidentified victims of accident or foul play, to aid in identification of the dead. Military personnel or other individuals who expect to encounter hazardous situations where their lives may be at risk may wish to store DNA samples prior to exposing themselves to these hazards. In the law enforcement area, convicted felons in both Canada and the United States are now required to provide DNA samples. DNA-based tests are expected to increase in medicine, such as testing for cystic fibrosis, cytochrome P450 isotypes, polymorphisms affecting susceptibility to infectious and autoimmune diseases, HLA typing, paternity issues, to name but a few. In clinical studies, an example would be to screen populations for colon cancer-predisposing genes or family members of a breast cancer victim for breast cancer predisposing genes. In all of these cases, there are significant advantages to providing a saliva sample rather than providing a blood sample as a source of DNA. All donors would prefer donating saliva rather than blood because of the discomfort, pain, or apprehension associated with phlebotomy or pin-pricks. Saliva has a further advantage of not requiring specialized personnel thereby reducing cost where mass sample collection is being carried out. The risk of blood-borne infection is likewise decreased.
- In addition to the problem of developing a standard collection and preservation method for DNA in saliva, there remains an ongoing need to improve methods of overcoming problems specific to the recovery of nucleic acids from saliva. The problem of extraction of high molecular weight DNA and RNA from mammalian cells has been partially addressed by Birnboim in Methods of Enzymology 216:154-160, 1993, but this work was not extended to the recovery of nucleic acids from mucin-containing bodily fluids.
- Multimeric proteins called mucins are high molecular weight glycosylated proteins that form a major part of a protective biofilm on the surface of epithelial cells, where they can provide a barrier to particulate matter and bind microorganisms. These glycoproteins contribute greatly to the viscoelastic nature of saliva. The major high-molecular-weight mucin in salivary secretions is MUC5B, one of four gel-forming mucins that exist as multimeric proteins with molecular weights greater than 20-40 million daltons. MUC5B is a large oligomeric mucin composed of disulphide-linked subunits.
- It is known that reagents that reduce disulfides also reduce the viscosity of mucin, such as that found in sputum or saliva. Reducing agents, in particular sulfur-containing chemicals such as β-mercaptoethanol and dithiothreitol, are widely used in biochemistry. However, many biochemically relevant reducing agents are capable of reacting in solution with dissolved oxygen. This is known are autooxidation (also called autoxidation or auto-oxidation), where 1-electron reduction intermediates of oxygen are formed, viz., superoxide (O2 −.), hydrogen peroxide (H2O2) and hydroxyl radical (OH.). In addition, transitional metal cations function as catalysts and O2 −. has been demonstrated to be an intermediate. Unfortunately, reducing agents and reducing compositions of the prior art have a relatively short shelf life, especially in basic solutions, and stock solutions that contain reducing agents cannot be prepared and stored under ambient conditions for an extended period time, usually not more than a day or two.
- Therefore, in addition to a need for a means to collect sputum or saliva, and subsequently preserving the nucleic acids contained therein by contacting them with a stabilizing composition, there is a need for the inclusion of a stable reducing agent into the composition, such that nucleic acids can be conveniently recovered from it, especially after extended periods of time in the presence of oxygen at neutral or mildly alkaline pH.
- The present inventor has developed a composition, which, when mixed with a mucin-containing bodily fluid, preserves the nucleic acids at room temperature under ambient conditions for extended periods of time. There is no requirement for freezing of the samples before nucleic acid recovery and purification. The properties of this composition are that it (a) chemically stabilizes nucleic acids, (b) inhibits nucleases that may be present in the saliva, and (c) is compatible with proteolytic enzymes and other reagents used to purify/amplify oligo- or polynucleotides. A fourth and novel property of this composition is that it contains an agent that rapidly reduces the viscous properties of mucin, greatly facilitating the extraction of nucleic acids contained within.
- Accordingly, a first aspect of the invention features a composition for preserving nucleic acids that includes a chelating agent, and a denaturing agent, where the pH of the composition is greater than 5.0. In one embodiment, the composition is an aqueous solution.
- In another embodiment, the composition also includes a reducing agent. For example, it can include one or more of the following: ascorbic acid, dithionite, erythiorbate, N-acetylcysteine, cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, dierythritol, a resin-supported thiol, a resin-supported phosphine, vitamin E, and trolox, or salts thereof. Desirably, the reducing agent is ascorbic acid, erythiorbate, N-acetylcysteine, dithiothreitol, or 2-mercaptoethanol, and most desirably, the reducing agent is ascorbic acid. In another embodiment, the composition does not contain ascorbic acid. In yet another embodiment, the concentration of the reducing agent in the composition is greater than or equal to 50 millimolar.
- Antioxidant free-radical scavengers are also desirable reducing agents for the composition of the present invention. Examples include antioxidant vitamins, antioxidant hormones, antioxidant enzymes, thiols, and phenols.
- Desirably, the reducing agent retains reducing activity for at least 46 days in the presence of one or more of the following: oxygen, ambient air, ambient light, and alkaline pH.
- The chelating agent of the composition can be selected from the group consisting of: ethylenediamine tetraacetie acid (EDTA), cyclohexane diaminetetraacetate (CDTA), diethylenetriamine pentaacetic acid (DTPA), tetraazacyclododecanetetraacetic acid (DOTA), tetraazacyclotetradecanetetraacetic acid (TETA), and desferrioximine, or chelator analogs thereof. Desirably, the chelating agent is cyclohexane diaminetetraacetate (CDTA), diethylenetriamine pcntaacctic acid (DTPA), tctraazacyclododecanetetraacetic acid (DOTA), or desferrioximine, and most desirably, the chelating agent is cyclohexane diaminetetraacetate (CDTA).
- In another embodiment, the chelating agent of the composition inhibits metal redox cycling. By “inhibits metal redox cycling” is meant the inhibition of metal-based oxidation/reduction cycles that produce reactive oxygen free-radical species. Examples of redox ion pairs involved in such cycles include Fe2+/Fe3+, Cu1+/Cu2+, and various oxidation states of molybdenum, vanadium, nickel, and cobalt. Chelators that bind one or both ions of a redox ion pair can inhibit the production of reactive oxygen species such as, for example, hydroxyl radical (HO.), hydroperoxyl radical (HOO.), superoxide radical (O2 −.), nitric oxide radical (NO.), or peroxynitrite radical (ONO2 −.).
- The nucleic acid to be preserved by the composition can be DNA or RNA, including mRNA or viral RNA.
- The pH of the composition can between from about 5.0 and about 11.0, desirably from about 6.5 to about 7.5, and most desirably, about 7.0. For the preservation of DNA, a pH from about 7.0 to about 10.0 can be used. Depending on other components of the compositions, desirable pHs are about 7.5, about 8.0, or a pH range from about 8.0 to about 9.0. A buffer, such as HEPES, TRIS, or carbonate buffer can be added to the composition to maintain the pH in a constant range. For the preservation of RNA, a pH from about 5.0 to about 7.0, desirably from about 6.5 to about 6.8 can be used. Again, a buffer, such as BES, can be used to maintain the pH in a constant range.
- The denaturing agent of the composition can be selected from the group consisting of: urea, dodecyl sulfate, guanidinium chloride, guanidinium thiocyanate, perchlorate, and an alcohol. Desirably, the denaturing agent is urea, dodecyl sulfate, or an alcohol, wherein the alcohol is 10%-60% of the total composition volume. The alcohols can be methanol, ethanol, n-propanol, isopropanol, n-butanol, trifluoroethanol, phenol, or 2,6-di-tert-butyl-4-methylphenol.
- In another embodiment, the composition includes an antimicrobial agent. By “antimicrobial agent” is meant a substance or group of substances which reduces the rate of growth of an organism compared to the rate of growth of the organism in their absence. A reduction in the rate of growth of an organism may be by at least 5%, more desirably, by at least 10%, even more desirably, by at least 20%, 50%, or 75%, and most desirably, by 90% or more. The definition also extends to substances which affect the viability, virulence, or pathogenicity of an organism. An antimicrobial agent can be natural (e.g., derived from bacteria), synthetic, or recombinant. An antimicrobial agent can be bacteriostatic, bactericidal or both. An antimicrobial agent is bacteriostatic if it inhibits cell division without affecting the viability of the inhibited cell. An antimicrobial agent is bactericidal if it causes cell death. Cell death is commonly detected by the absence of cell growth in liquid growth medium (e.g., absence of turbidity) or on a solid surface (e.g., absence of colony formation on agar). Those of skill in the art know that a substance or group of substances which is bacteriostatic at a given concentration may be bactericidal at a higher concentration. Certain bacteriostatic substances are not bactericidal at any concentration. Desirably, the composition of the invention includes an alcohol as an antimicrobial agent, and most desirably the composition includes ethanol.
- In another embodiment, the composition also includes an inhibitor of ribonuclease. Desirable inhibitors are selected from the group consisting of: heparin, heparan sulfate, oligo(vinylsulfonic acid), poly(vinylsulfonic acid), oligo(vinylphosphonic acid), and poly(vinylsulfuric acid), or salts thereof. The inclusion of an inhibitor of ribonuclease in the composition of the invention is particularly desirable when the nucleic acid to be preserved is RNA, desirably mRNA, or when the nucleic acid to be preserved is from a virus or a bacterium.
- A second aspect of the invention features a method of reducing the viscosity of a mucin-containing bodily fluid or tissue by reducing disulfide bonds inherent to mucin, wherein the bodily fluid or tissue is mixed with a composition of the invention that includes a reducing agent. In one embodiment, the bodily fluid is sputum, desirably saliva. By “sputum” is meant that mucoid matter contained in or discharged from the nasal or buccal cavity of an animal, including saliva and discharges from the respiratory passages, including the lungs. In another embodiment, the method includes the recovery of a nucleic acid.
- A third aspect of the invention features a method of preserving a nucleic acid contained in sputum that includes the steps of obtaining sputum from a subject, and contacting the sputum with a composition of the invention, thus preserving the nucleic acid.
- In one embodiment, when the nucleic acid is DNA, the DNA is stable for more than 14 days, desirably more than 30 days, and more desirably more than 60 days. In another embodiment, when the nucleic acid is DNA and the composition does not contain ascorbic acid, the DNA is stable for more than 60 days, and desirably more than 360 days.
- A fourth aspect of the invention features a method of recovering a nucleic acid from sputum that includes the steps of: i) obtaining sputum from a subject, ii) contacting the sputum with a composition of the invention to form a mixture, iii) contacting the mixture with a protease, and iv) recovering the nucleic acid from the mixture. Desirably, the protease is proteinase K or pronase.
- In one embodiment of any of the second, third, or fourth aspects, the sputum is saliva. In another embodiment, the sputum is from a mammal, desirably a human. In yet another embodiment, the nucleic acid is DNA or RNA. If the nucleic acid is RNA, desirably it is mRNA or viral RNA. The nucleic acid can be from a source foreign to the subject from which the sputum sample is taken. For example, the nucleic acid can be from a bacterium or a virus that is residing in the buccal, nasal, or respiratory passages of the subject.
- In a fifth aspect, the invention features a method of preserving and/or recovering a nucleic acid from a bodily fluid that includes, placing the bodily fluid into a first region of a container, placing a composition of the invention into a second region of the container, which is separated from the first region by a barrier, closing the container, and disturbing the integrity of the barrier such that the composition and the bodily fluid are brought into contact.
- In one embodiment, the disestablishment of the barrier is coupled to the closing of the container when a lid is placed on it. In one example, the barrier is punctured. In a desirable example, the barrier is in the form of a pivoting sealing disc. In this example, attachment of the lid to the container forces the disc to pivot from its original position of spanning the space between the first region and the second region to a position in which both regions are exposed to each other, thereby forming a mixture between a composition of the invention and the bodily fluid is allowed. Desirably, the bodily fluid is sputum, and most desirably, saliva.
- In a sixth aspect, the invention features a device for preserving and/or isolating a nucleic acid obtained from a biological sample. The device includes: a container that has a first region for collecting a biological sample and a second region containing a composition for preserving a nucleic acid, a barrier between the first region the second region that keeps the biological sample and the composition separate, a means for closing the container, and a means for disturbing the integrity of the barrier such that the composition is capable of contacting the biological sample. The first region can have an opening of from 2.0 to 7.0 cm, desirably from 2.5 to 3.5 cm, and most desirably 3.0 cm. Desirably, the biological sample is sputum, and most desirably, saliva.
- In one embodiment of the sixth aspect, the nucleic acid-preserving composition is a composition of the present invention. In another embodiment, the means for closing the container is coupled to the means for disturbing the integrity of the barrier. In yet another embodiment, the means for closing the container is an airtight lid.
- In a seventh aspect, the invention features a method of manufacturing a device for preserving a nucleic acid in a biological sample that includes: providing a container that has a first region and a second region, with the first region suitable for containing a composition of the invention and the second region having an opening suitable for the application of a biological sample; placing the composition into the first region; and applying a barrier to the container between the first region and the second region, with the barrier being impermeable to the composition and capable of being disestablished.
- In an embodiment of either the sixth or seventh aspect, the barrier can be a pivoting disc, where in a first position, the disc spans the compartment and separates the first and second areas. Pivoting the disc to a second position (e.g., by connecting a screw-on lid to a plunger mechanism which contacts the disc, causing it to pivot when the lid is screwed on) disestablishes the barrier and allows the biological sample contained in the first region to contact the composition that is contained in the second region.
- By “about” is meant +/−10% of the stated value or a chemical or obvious equivalent thereof.
- By “alcohol” is meant a water-miscible organic compound containing a hydroxyl group, including water-miscible mixtures of hydroxyl-containing organic compounds.
- By “antioxidant free-radical scavenger” is meant a substance that reduces a reactive oxygen free radical species. Such free radicals include, for example, hydroxyl radical (HO.), hydroperoxyl radical (HOO.), superoxide radical (O2 −.), nitric oxide radical (NO.), or peroxynitrite radical (ONO2 −.).
- By “aqueous solution” is meant a solution or suspension that contains 30% or more water by volume.
- By “bodily fluid” is meant a naturally occurring fluid from an animal, such as saliva, serum, plasma, blood, urine, mucus, gastric juices, pancreatic juices, semen, products of lactation or menstration, tears, or lymph.
- By “biological sample” is meant any sample containing nucleic acids that has been obtained from or deposited by an animal. Non-limiting examples include skin, hair, bodily fluids, fecal matter, and tissue.
- By “chelator analog” is meant a derivative chelator compound with the same backbone structure and having the same general properties as the parent chelator compound.
- By “denaturing agent” is meant a substance that alters the natural state of that to which it is added.
- By “mucin” is meant any mucoprotein that raises the viscosity of the medium surrounding the cells that secrete it.
- By “mucoid” is meant any bodily fluid containing mucin By “nucleic acid” is meant a chain of the nucleotides, including deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), typically found in chromosomes, mitochodria, ribosomes, bacteria, or viruses.
- By “nucleic acid-preserving composition” is meant any composition of the present invention, unless otherwise specified.
- When referring to a nucleic acid, by “stable” is meant that at least about 50% of the initial amount of high molecular weight nucleic acid (DNA, RNA, mRNA, or viral RNA) contained in a sample is still present after storing the sample at ambient temperature (i.e., 20° C. to 25° C.) for the specified time period. The amount of high molecular weight DNA in a sample can quantified by densitometry analysis of the high molecular weight DNA band from an agarose gel (see
FIG. 1 and Example 4). - By “resin-supported phosphine” is meant a polymer that contains a multiplicity of covalently-bound phosphine groups.
- By “resin-supported thiol” is meant is meant a polymer that contains a multiplicity of covalently-bound sulfhydryl groups.
- By “saliva” is meant the secretion, or combination of secretions, from any of the salivary glands, including the parotid, submaxillary, and sublingual glands, optionally mixed with the secretion from the buccal glands.
- By “sputum” is meant that mucoid matter contained in or discharged from the nasal or buccal cavity of a mammal, including saliva and discharges from the respiratory passages, including the lungs.
- By “subject” is meant any animal. Desirably, the subject is a mammal that can produce saliva for the purposes of nucleic acid extraction. Most desirably, the subject is a human.
- Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
-
FIG. 1 is an electrophoresis agarose analysis of DNA isolated from saliva using the capacity of methods of one embodiment of the invention. -
FIG. 2 is a graph illustrating real time PCR of stimulated saliva DNA of Example 5. -
FIG. 3 is a graph illustrating real time PCR of unstimulated saliva DNA of Example 6. -
FIG. 4 is an electrophoresis agarose analysis of the DNA in saliva samples mixed with compositions of the invention, the mixtures having been incubated for various times at various temperatures. -
FIG. 5 shows structures of (oxidized) ascorbate anion, (reduced) dehydroascorbic acid, and a free radical intermediate -
FIG. 6 is a compilation of two spectrophotometric scans of sodium ascorbate (100 μM) in CB (1 mM CDTA, 10 mM BES, pH 7.4), prepared under aerobic conditions over 30 minutes at room temperature (scan 1) and 3 minutes after addition of a few crystals of MnCl2. (scan 2), as per Example 8. -
FIG. 7 is a compilation of spectrophotometric scans, at the indicated times, of the 100 μM sodium ascorbate prepared in CB of Example 8. The solution was exposed to ambient atmosphere and temperature between scans but was not contacted with MnCl2 (see Example 9). -
FIG. 8 is a graph of absorbances at 265 nm, obtained at the indicated times, of a solution of sodium ascorbate (250 mM) containing 30 mM Tris-HCl, pH 8.0, 30% ethanol, 3 mM CDTA, mixed with 50 mL of CB, as per Example 10. The stock solution was maintained at room temperature and no precaution was taken to exclude ambient atmosphere or ambient light. -
FIG. 9 is a compilation of spectrophotometric scans of the 46 day-old solution prepared in Example 10. Scan 1 (t=46 days) was taken before the addition of MnCl2.Scan 2 was taken 2 minutes after the addition MnCl2.Scan 3 was taken 8 minutes after the addition MnCl2.Scan 4 was taken 27 minutes after the addition MnCl2. -
FIG. 10 is an exploded view of a sample container of the invention. Included in the figure is a cross-sectional top view taken at line 1-1 ofcontainer 3showing plunger 4 andplunger channel 5. Also shown is a cross-sectional top view taken at line 2-2 ofcontainer 3, showing supports 6 for sealing disc 7 (not shown in this figure but shown inFIG. 11 ). -
FIG. 11 is a side view of the sample container ofFIG. 10 , now showingsealing disc 7. - The following standard abbreviations are used herein: DNA, deoxyribonucleic acid; RNA, ribonucleic acid; mRNA, messenger RNA; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; BES, N,N-bis[2-hydroxyethyl]-2-aminoethane-sulfonic acid; TRIS, tris(hydroxymethyl)aminomethane, CDTA, cyclohexane diaminetetraacetate; DTPA, N,N-bis(2-(bis(carboxymethyl)amino)ethyl)glycine; DOTA, 1,4,7,10-tetrazacyclododecanetetraacetic acid; and TETA, 1,4,8,11-tetraazacyclotetradecanetetraacetic acid.
- The present inventors have developed compositions that render sputum as a viable option to the use of blood as a source of nucleic acids. The compositions provide the advantageous properties of chemical stabilization of nucleic acids and the inhibition of nucleases, including deoxyribonucleases, and microbial growth. Chemical stabilization of the nucleic acids in a saliva sample is achieved through the use of buffers to maintain an appropriate pH, as well as the use of chelating agents to prevent the phenomenon of metal redox cycling or the binding of metal ions to the phosphate backbone of nucleic acids. The chelating agents of the invention also participate in the inhibition of deoxyribonucleases and microbial growth, which can be additionally inhibited by the inclusion of denaturing agents and/or other suitable antimicrobial substances, such as ethanol, into the compositions of the invention. The compositions of the invention can also include one or more reducing agents, which can reduce sample viscosity, thereby making nucleic acid recovery an easier process.
- Accordingly, the present invention features a composition for preserving and/or recovering nucleic acids from sputum, desirably saliva, that includes one or more chelators and one or more denaturing agents, wherein the pH of the composition is greater than 5, desirably within a pH range of about 6 to about 11, more desirably within a pH range of about 7.5 to about 10.0, and most desirably, within a pH of about 7.0.
- The chemical backbone and the purine bases of DNA are most stable at slightly alkaline pH, with an optimal stability generally recognized as being within a pH range of about 7-11, and desirably a pH of about 8. Below a pH of about 6, depurination (i.e., spontaneous loss of purine bases from the deoxyribose-phosphate backbone) can occur. Above a pH of about 10, spontaneous loss of amino groups from cytosine nucleotides may occur, thereby converting cytosine to uracil. Above a pH of about 12, DNA is denatured, converting it from the double-strand form to the single-strand form. In contrast, RNA is most stable in the pH range of 5.0 to 7.0, desirably a pH of from 6.5 to 6.8. Accordingly, the pH of the composition may be adjusted using pH buffers, desirably those that best control the pH within the range of about 5 to about 11. Examples of pH buffers with desirable properties include, but not limited to, TRIS hydrochloride, HEPES and BES.
- DNA has a strong affinity for metal ions, some of which, such as the common transition metals iron or copper, can catalyze the formation of reactive oxygen species. Therefore, a composition of the invention includes one or more chelators that can form complexes with metal ions to prevent them from binding to DNA, remove metal ions that that have already bound to DNA, or bind to metal ions (e.g., Fe(II)/Fe(III) or Cu(I)/Cu(II)) strongly enough to inhibit their redox cycling, and hence, the formation of reactive oxygen species. EDTA, a commonly used chelator in biological reagents, can be of some use for either of these purposes. More desirable are stronger chelators (i.e., chelators with a higher dissociation constant than EDTA when bound to a metal), used alone or in combination, that include, but are not limited to, CDTA, DTPA, DOTA, TETA, and desferioximine, or chelator analogs thereof. The amount or concentration of chelator will depend upon the strength of the chelator, which would need to be determined empirically. For CDTA, concentrations in the 1-20 mM range are sufficient, however other concentrations would work, and the compositions of the invention are not intending to be limited to this range.
- Deoxyribonucleases and ribonucleases are enzymes that breakdown DNA or RNA, respectively. Their main source in the digestive tract is secretions of the pancreas, although lower levels may be present in cells of the salivary gland and buccal mucosa. In addition, microorganisms resident in the mouth or from recently ingested foods may contain deoxyribonucleases or ribonucleases. Pancreatic deoxyribonuclease is known to require divalent metal ions such as Mg(II), Mn(II) and/or Ca(II) for enzymatic activity. The strong chelators described above, in addition to providing chemical stability to the nucleic acids, will inhibit this class of metal ion-requiring deoxyribonucleases. The action of deoxyribonucleases and ribonucleases can also be inhibited by denaturing agents that will destroy the complex structures of these enzymes (proteins). Hence, denaturing agents are included in the nucleic acid preserving composition of the invention. Examples of denaturing agents that may be used (alone or in combination) include, but not limited to, urea, soluble salts of dodecyl sulfate and other strong detergents, guanidinium chloride, guanidinium thiocyanate, soluble salts of perchlorate, alcohols, such as ethanol, above 10%. Other reagents, such as heparin, heparan sulfate, or oligo(vinylsulfonic acid) (Smith, et al., J. Biol. Chem. Mar. 20, 2003; [epub ahead of print]) are known to inhibit the action of deoxynucleases and/or ribonucleases.
- Low specificity proteases such as proteinase K are frequently used in the purification of nucleic acids. Since proteases are themselves proteins, their action can be inhibited by denaturing agents. Thus, a balance must be struck between the concentration of denaturing agents that will, on the one hand, inhibit deoxyribonucleases or ribonucleases and denature other proteins in saliva and, on the other hand, not significantly inhibit the proteolytic enzymes. At later stages in DNA purification, the DNA is often concentrated by precipitation with alcohol. Thus, salts, buffers, chelators and other components of the nucleic acid preserving/recovery solution must be chosen so as not to precipitate when concentrations of alcohol over 50% are added to precipitate the DNA.
- The viscosity of sputum and saliva depends upon the presence of very high molecular weight glycoproteins complexes called mucins, particular the gel-forming mucins (Offner, et al., Adv. Dent. Res. 14:69-75, 2000; Seregni, et al., Tumori 83:625-632, 1997). It has been found that the inclusion of a reducing agent into a composition of the invention has the effect of markedly reducing the viscosity of saliva, especially “unstimulated” saliva, thereby facilitating the recovery of nucleic acids. Accordingly, in one embodiment, a composition of the invention further includes one or more reducing agents. The reducing agents are desirably at high concentration (greater than 0.05 M). While not wishing to be limited by theory, it is presumed that the reducing agent reduces the viscosity of the saliva by breaking disulfide bonds that hold together chains of mucin, and that any reducing agent that has the appropriate redox potential to reduce disulfide bonds in proteins would be suitable. Desirably, the reducing agent is selected from the group consisting of: ascorbic acid, dithionite, erythiorbate, N-acetylcysteine, cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, dierythritol, a resin-supported thiol, a resin-supported phosphine, vitamin E, and trolox, or salts thereof.
- In another embodiment, a composition of the invention that includes a reducing agent maintains reducing capacity at room temperature in a sealed container in the presence of ambient oxygen, and/or in the presence of ambient light for more than a week, desirably for up to about 46 days, and most desirably for at least 46 days. This embodiment combines the nucleic acid stabilization provided by a strong chelator a denaturing agent, and a reducing agent in a composition with a pH within the range of about 6 to about 11, and desirably a pH of about 8.0.
- A particularly desirable reducing agent is sodium ascorbate. As well as an important dietary antioxidant micronutrient, ascorbic acid (vitamin C) is a non-thiol reducing agent and is inexpensive, non-toxic, and stable in the presence of the chelators and denaturing agents that are included in the compositions of the invention. The structures of (oxidized) ascorbate anion, (reduced) dehydroascorbic acid, and a free radical intermediate are shown in
FIG. 5 . The most thoroughly studied oxidation reaction of ascorbate is its oxidation by oxygen. As with many other reducing agents, trace amounts of transitional metals such as iron or copper can promote autooxidation (Buettner, Free Radic. Res. Commun. 1:349-53, 1986; Buettner and Jurkiewicz Radiat. Res. 145:532-41, 1996; Miller, et al., Free Radic. Biol. Med. 8:95-108, 1990). Metal cation-catalyzed oxidation of ascorbate can be conveniently monitored as a decrease in absorbance at 265 nm (Buettner Free Radic. Res. Commun. 10:5-9, 1990), as described in Example 8 and shown inFIGS. 5, 6 , and 8. Certain chelating agents can appreciably slow down autooxidation of ascorbate at pH 7.0 or lower (Buettner J. Biochem. Biophys. Methods 16:27-40, 1988), as described in Example 10 and shown inFIG. 8 . - In another embodiment, a composition of the present invention includes one or more chelators, one or more denaturing agents, and one or more antimicrobial agents, wherein the pH of the composition is within a pH range of about 6.0 to about 11.0, desirably at a pH of about 8.0. Microbial growth may also be inhibited by the strong chelators and denaturing agents, for example, ethanol, described above. Therefore, in a further embodiment of the present invention, a composition for preserving and/or recovering DNA from sputum includes one or more chelators and one or more denaturing agents, wherein at least one or more of the denaturing agents and/or chelating agents is present in amounts to act as an antimicrobial agent.
- Reagents that indicate when a biological sample has been contacted with a composition of the invention can also be included as part of the composition. Desirable are those reagents that result in a visual color change of the composition solution upon mixing with the added sample. These reagents can function by reacting with any number of functional groups that are contained in biological samples, including, for example, amines, thiols, or glycosyl groups. Such colorimetric reagents are known to those skilled in the art and are chosen in such a manner that other components of the composition do not interfere with their effective usage.
- The present invention features methods of collecting, preserving, and recovering nucleic acids from sputum using a composition of the invention. The methods of the invention involve contacting a sputum sample from a subject with a composition of the invention and optionally mixing the resulting solution with a protease, such as pronase or proteinase K. Furthermore, some compositions of the invention feature a reducing agent that can facilitate the recovery of nucleic acids from composition/sample mixtures by decreasing the viscosity of these mixtures.
- Accordingly, one aspect of the invention features a method of preserving a nucleic acid contained in sputum that includes the steps of obtaining sputum from a subject, and contacting the sputum with a composition of the invention, thus preserving the nucleic acid. Examples 1 and 2 describe the collection of saliva, both from subjects that can follow instructions and from those that can not.
- The sputum is typically contacted with a composition of the invention upon collection or immediately after it is collected, and preferably not much later than about 1 hour after collection. This time can vary depending on storage conditions of the sputum after collection. For example, it could be indefinite if stored frozen or perhaps 1-2 days if stored at 4° C. A reducing agent can be in the preserving composition used, or added at a later time prior to nucleic acid isolation. Desirable reducing agent-containing compositions are those that are stable and retain a reducing capacity for more than a week, desirably for up to about 46 days, and most desirably for at least 46 days.
- In an example (see Example 5), the results of which are presented in Table 1, saliva was collected and mixed with approximately an equal volume of a composition of the invention (see Example 3 for preparation), and analyzed for DNA content by PCR analysis at later timepoints.
-
TABLE 1 Estimated amounts of DNA in saliva samples* Donor # 1 2 3 4 5 6 7 8 9 10 11 Stim. saliva collected on 2002 Feb. 26 analyzed 64 days by the DNase method 21.2 21.4 16.6 16.0 28.8 44.8 22.2 16.6 Unstim. saliva collected on 2002 Mar. 25 analyzed 15 days later by DNase method 64.2 80.6 24.4 27.2 69.0 *DNA content in nanograms per microliter - To collect the sputum from the subject it is preferred that the mouth be rinsed before sampling. Food particles can introduce foreign DNA and saliva transferred by kissing can be a source of foreign human DNA. The mouth can be rinsed with about 50 mL of tepid water by vigorous swishing or by brushing with a tooth brush without tooth paste. Unstimulated saliva is usually of the mucinous type and is secreted at a slow rate. Stimulated saliva (anticipation of tasty food, sweet or sour candy) is of the serous (watery) type and secreted at a faster rate. It has been found (see Table 2) that there is more DNA in 2 mL of unstimulated saliva than 2 mL of stimulated saliva. After rinsing of the mouth and waiting about two or three minutes, the donor may spit a volume (for example, about 2 mL) of “unstimulated” saliva into the receiving tube. If this proves to be difficult, saliva flow can conveniently be stimulated with a cube of table sugar, or any other such saliva-stimulatory substance that does not interfere with DNA recovery or purification.
-
TABLE 2 Comparison of DNA content of unstimulated and stimulated saliva Donor # 7 unstimulated stimulated Collected on 2002 Apr. 6, analyzed 36.2* 21.8* 2 days later by the DNase method *Estimated amount of DNA in ng per μL of original undiluted saliva sample - Another aspect of the invention features a method of reducing the viscosity of a mucin-containing bodily fluid or tissue by reducing disulfide bonds inherent to mucin, wherein the bodily fluid or tissue is mixed with a composition of the invention that includes a reducing agent. In one embodiment, the bodily fluid is sputum, desirably saliva.
- Yet another aspect of the invention features a method of recovering a nucleic acid from sputum that includes the steps of: i) obtaining sputum from a subject, ii) contacting the sputum with a composition of the invention to form a mixture, iii) contacting the mixture with a protease, and iv) recovering the nucleic acid from the mixture.
- Suitable proteases include, for example, proteinase K or pronase. The protease may suitably be in a dry form that would become activated once mixed with sputum and a composition of the invention. In one embodiment, the protease is deposited onto an interior surface of the collection device. This can be accomplished by dissolving the protease in a solution made up of equal volumes of 5% sucrose in water and 5% glycerol in ethanol and then, after placing the solution on the surface, removing the volatiles under a controlled vacuum to leave the protease bound to the surface as a sticky residue. If the composition does not contain a reducing agent (or even if it does), a reducing agent can be added at any time prior to isolation of the nucleic from the sample, desirably prior to or concurrently with contacting the sample with a suitable protease.
- When sputum is mixed with a composition of the present invention, cells are disrupted, nucleic acids are liberated from the cells, membranous material is solubilized, proteins are stripped from the nucleic acids, and protein digestion begins. If present, a reducing agent in the composition reduces the viscosity of the gel-forming mucin. Incubation can be at room temperature over a relatively long period of time (days or weeks) while samples are being shipped to a laboratory for analysis. If transferred to a laboratory soon after collection, incubation at 55° C. for 4 to 16 hours is sufficient to allow the activated protease to digest the majority of protein to small peptides or amino acids. Under such conditions, nucleic acids and polysaccharides remain relatively intact.
- Once digestion is complete, nucleic acid isolation can be performed using any technique known in the art (Short Protocols in Molecular Biology, 5th Edition Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J. G. Seidman, John A. Smith (Editor), Kevin Struhl (Editors). ISBN: 0-471-25092-9. 2002. John Wiley and Sons). In one example, in which SDS is used as a denaturant component of the composition, a “precipitation solution” consisting of, for example, potassium chloride may be added to a portion of the sputum-composition mixture resulting in the precipitation of potassium dodecyl sulfate, after standing on ice to cool the solution. Following a short period of centrifugation to remove the precipitate and any residual insoluble material, the supernatant is collected. At this stage, the supernatant is expected to contain as much as 10-30 nanograms per microliter of DNA. For analyses where as little as 1 nanogram of DNA is sufficient, the sample can be diluted.
- When larger amounts of DNA are required, the DNA in the supernatant can be precipitated by the addition of alcohol and redissolved in any suitable buffer. This step has the effect of removing inhibitory components of the composition, which are present to preserve the nucleic acids during transport to the laboratory.
- If more highly purified DNA is required, then other known purification steps can be used (Short Protocols in Molecular Biology, 5th Edition Frederick M. Ausubel, Roger Brent, Robert E. Kingston, David D. Moore, J. G. Seidman, John A. Smith (Editor), Kevin Struhl (Editors). ISBN: 0-471-25092-9. 2002. John Wiley and Sons), such as extraction with phenol or solid-phase extraction. It should be noted that, because the DNA is in a relatively pure state using the procedures described above, any additional purification steps are made easier when compared to analogous purifications of DNA originating from a blood sample.
- The methods of the present invention can be used to isolate nucleic acids from sputum for any application requiring a nucleic acid sample. For example, some specific applications of the methods of the present invention include, but are not limited to, forensic applications, medical applications (including genetic screening and disease typing), and paternity testing.
- Another aspect of the invention features a method of preserving and/or recovering a nucleic acid from a bodily fluid that includes, placing the bodily fluid into a first region of a container, placing a composition of the invention into a second region of the container, which is separated from the first region by a barrier, closing the container, and disturbing the integrity of the barrier such that the composition and the bodily fluid are brought into contact. Collection devices of the invention, which also can serve as containers for bring the compositions and nucleic acid-containing bodily fluids together are described below.
- The invention also provides a novel collection device useful for collecting a biological sample from a subject, and subsequently mixing the collected sample with a composition intended to stabilize, preserve, or facilitate the recovery of components of the sample. Such components may include, without limiting the invention, nucleic acids, proteins, peptides, toxins, chitins, fatty acids, and glycogens. Non-limiting examples of biological samples are skin, hair, fecal matter, bodily fluids, and tissue.
- Desirably, the invention features a device for preserving and/or recovering a nucleic acid obtained from a biological sample. The device includes: a container that has a first region for collecting a biological sample and a second region containing a composition for preserving a nucleic acid, a barrier between a first region and a second region that keeps the sample and composition separate, a means for closing the container, and a means for disturbing the integrity of the barrier, such that the composition is capable of contacting the bodily sample. In one embodiment, the composition is a composition of the present invention. In another embodiment, the sample is a biological fluid.
- The collection device of the invention simultaneously serves several functions. Some of the desirable features of this collection vessel include one or more of the following:
- a) it may be constructed of a sturdy breakage-resistant plastic, desirably a biocompatible plastic. Desirably, the container would be constructed from a material that would not leach chemicals into the container's contents;
- b) it would have a broad mouth that would make it relatively simple for a subject to place the required volume of fluid sample, desirably expectorated sputum, and most desirably expectorated saliva, into the device's container;
- c) the bottom part of the container would be narrow to reduce the overall volume of the container to make it easier to collect the small volume (1-2 milliliters) of fluid that would be expected from a routine sampling, in particular, when the sample is an expectorate. Optionally, the device would contain markings to allow for an estimate of the sample volume collected;
- d) the means for closing the container may be a cap that is designed to lock once tightened to become tamper-resistant;
- e) the means for closing the container may be a cap that is designed to provide a liquid-tight and/or airtight seal for the container once the cap is fixed into place;
- f) the barrier may be a septum or plastic bag compact lent that would separate the composition from the fluid until the septum or bag compartment is pierced or the contents otherwise released;
- g) the barrier may be in the form of a pivoting partition. In this embodiment, attachment of the lid to the container forces the partition to pivot from its original position of spanning the space between the first region and the second region to a position in which both regions are exposed to each other and contact between the composition contained in one space and the bodily fluid contained in the other space is allowed;
- h) the barrier can be press fit, glued, or heat fit into place;
- i) the means for closing the container may be coupled to the disestablishment of the barrier; and
- j) an antimicrobial agent that coats the outside of the device.
- A device of the invention is shown in
FIGS. 10 and 11 . Withcap 1 not attached to the device, a biological sample (not shown) is applied to afirst region 8 ofcontainer 3, which is separated from asecond region 9 by sealingdisc 7. After sample application,cap 1 is placed onto the device and secured via a screw thread mechanism to a tight fit, thereby sealingcontainer 3. As the cap is twisted on (shown by dotted line andarrow 10,ram 2, which is attached to cap 1, moves downward as shown by dottedline arrow 11. This downwardmovement forces plunger 4, which is contained inplunger barrel 5, downward as indicated by dotted line andarrow 12. The downward movement ofplunger 4forces sealing disc 7 to pivot, as shown by dotted line andarrow 13. Pivoting ofdisc 7 disestablishes the barrier betweenregions region 9. - The present invention also features kits for performing the methods of the invention that include a device of the invention containing a composition of the invention, with instructions for stabilizing, preserving, or facilitating the recovery of nucleic acids from a biological sample by using the device to bring a biological sample into contact with the composition.
- The subject is instructed to wait for a period of 20-30 minutes before last eating. The subject will brush his teeth without using toothpaste, if possible. The subject will rinse his mouth vigorously with 50 mL of cool or tepid water. The subject will then spit saliva into the special collection tube until the level of saliva reaches the 2 mL mark. This may take several minutes. If the subject finds that he is unable to deliver sufficient saliva, he will be given a cube of table sugar to chew, and told not to be concerned if some of the sugar is spit into the tube.
- When the required amount of saliva is collected, it is mixed with 2 mL of a nucleic acid-preserving composition. The precise way this will be introduced will depend upon the container design.
- Once the composition is introduced, the cap is attached to the container and tightened to seal it securely. The container is then vigorously shaken and the process is complete. The DNA is now in an intermediate preserved state. It can be maintained in a frozen state or at any temperature up to about 60° C.
- The container can be mailed back to the testing lab at room temperature.
- A rubber or plastic tube or nipple will be introduced into the mouth, attached to a sponge, suction bulb or small syringe, and kept in the mouth for several minutes until visible drooling occurs. A bit of sugar cube will be placed in the mouth to stimulate saliva if necessary. The responsible adult will wear disposable gloves provided for the purpose to avoid contamination with his/her DNA. The responsible adult will draw saliva into the bulb or syringe and transfer it into the collection container. The DNA preserving/extraction composition is introduced and the container is capped and sealed. The tube is vigorously shaken for 1 minute.
- The composition of the nucleic acid-preserving solution used in Examples 4-6 is 33 mM TRIS-HCl, 0.67 M urea, 0.67 M LiCl, 0.6% sodium dodecyl sulfate, 3.3 mM CDTA, 30% ethanol, and 0.25 M sodium ascorbate, all adjusted to a final pH of 8.0. In the examples, the composition is mixed with an equal volume of saliva. Subsequent to these experiments, it has been found that a composition which is 0.3 M TRIS-HCl, 0.67 M urea, 0.67 M NaOAc, 0.6% sodium dodecyl sulfate, 3.3 mM CDTA, 30% ethanol, and 0.1 M sodium ascorbate, all adjusted to a final pH of 8.0, stabilizes DNA for longer periods of time.
- After collection of saliva in an equal volume of the composition as noted in Example 3, followed by 14 days storage at room temperature, a 0.25 mL portion of each donor's sample was treated with proteinase K, centrifuged briefly to remove insoluble material and the DNA therein was precipitated with 2 volumes of ethanol. The precipitate was dissolved in 0.05 mL of water, and an 8 μL aliquot (equivalent to about 20 μL of undiluted saliva) was analyzed by electrophoresis on a 0.8% agarose gel, stained with ethidium bromide to visualize the DNA (see
FIG. 1 ). Of note is the characteristic band of chromosomal DNA present in all samples at the position of the arrow, that corresponds to the position of chromosomal DNA extracted from white blood cells (data not shown). - Stimulated saliva samples collected on 26 Feb. 2002 (see Table 1) and stored at room temperature were analyzed 62 days later. Minimally purified DNA was prepared as follows: an aliquot was centrifuged to remove insoluble material; to the clarified supernatant was added 2 volumes of ethanol; the precipitate containing DNA was collected by centrifugation and redissolved in water. A volume of the redissolved DNA equivalent to 0.05 microliters of each of the original saliva samples was used for analysis. Real time PCR was carried out using a Roche Light Cycler instrument, where the fluorescent dye SYBR green I was added to follow the reaction (see results of
FIG. 2 ). The primers were designed to detect the human Clotting Factor IX gene (Grant, et al., J. Immunol. Methods 225:61-6, 1999). C=control, highly purifed white blood cell DNA. Each curve represents results using saliva DNA from different donors, represented by a number. These results using real time PCR demonstrate the suitability of minimally purified saliva DNA from different donors for PCR analysis. -
FIG. 3 is a graph showing saliva DNA samples collected on 2002 Mar. 25 (see Table 1) and analyzed on 30 days later in accordance withFIG. 1 . Minimally purified DNA was used Polymerase chain reaction and other conditions as described in Examples 4 and 5 except saliva collection was done under unstimulated conditions. Numbers refer to individual donors. C is control DNA, a highly purified sample of DNA purified from blood. - Tables 1 and 2 show estimates of DNA recovered from saliva samples. In all cases, the individual donor has been identified by a unique number. These data show that the amount of DNA that can be recovered from this group of donors ranges from 16 micrograms per milliliter of saliva and higher. Estimation of the amount of DNA by chemical methods such as DABA presents some problems and the DNase method provides most reliable results.
- Saliva was mixed with an equal volume of the indicated composition and the mixture was incubated for the indicated time period at the indicated temperature (see Table 3). After incubation, approximately 40 μL of mixture was digested briefly with ribonuclease to remove the majority of the RNA present in the sample, then applied to the indicated lane of a 0.8% agarose gel. Following electrophoresis, the gel was stained with ethidium bromide as in Example 4.
-
TABLE 3 Lane Incubation No. Composition Conditions 1 0.5M NaOAc, 0.2M TRIS-HCl, 0.15M 70° C. for 3 days, Na ascorbate, 10 mM CDTA, 1% SDS, then 30% (v/v) ethanol, pH = 9.5 50° C. for 16 days 2 0.5M NaOAc, 0.2M TRIS-HCl, 50° C. for 21 days 10 mM CDTA, 1% SDS, 30% (v/v) ethanol, pH = 9.5 3 0.5M NaOAc, 0.2M TRIS-HCl, 70° C. for 3 days, 10 mM CDTA, 1% SDS, 30% (v/v) then ethanol, pH = 9.5 50° C. for 31 days 4 0.67M LiCl, 33 mM TRIS-HCl, 0.67M 20-25° C. for 15 urea, 0.6% SDS, 3.3 mM CDTA, months 30% (v/v) ethanol, pH = 8.0 5 0.67M LiCl, 33 mM TRIS-HCl, 0.67M 20-25° C. for 15 urea, 0.6% SDS, 3.3 mM CDTA, months 30% (v/v) ethanol, pH = 8.0 6 Control chromosomal DNA prepared from white blood cells - A solution of sodium ascorbate (100 μM) in CB (10 mM BES, pH 7.4, containing 1 mM CDTA) was freshly prepared under aerobic (equilibrated with ambient air) conditions. Several spectrophotometric scans over 30 minutes at room temperature showed no change in the absorbance profile (all similar to scan (1)). Scan (2) was taken 3 minutes after addition of a few crystals of MnCl2. The results can be seen in
FIG. 6 . As shown, 100 μM ascorbate at neutral pH has an absorbance (λmax=265 nm) of about 1.25 (corresponding to the expected molar extinction coefficient (AM) of about 12,500. Upon addition, the transition metal, manganous chloride, catalyzed the autooxidation of ascorbate, which can conveniently be monitored by a decrease in absorbance at λ=265 nm (Buettner, Free Radic. Res. Commun. 10:5-9, 1990). - Repeated scans at the indicated time points were taken of an aliquot of the 100 μM sodium ascorbate solution prepared in Example 8, before the addition of MnCl2. The sample was exposed to air and maintained at room temperature between scans. The results are illustrated in
FIG. 7 , and indicate that autooxidation of ascorbate occurs at pH 7.4 can occur over an extended period of time in the presence of low concentrations (1 mM) of CDTA, a “strong” chelator. - A stock solution of sodium ascorbate (250 mM) was prepared in a solution containing 30 mM Tris-HCl, pH 8.0, 30% ethanol, 3 mM CDTA. 20 μL was removed at the indicated times, mixed with 50 mL of CB (see Example 8) and the absorbance at 265 nm was read immediately. The stock solution was maintained at room temperature. The results are shown in
FIG. 8 . - While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
- All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
Claims (29)
1. A device for storage of nucleic acid from a biological sample, said device comprising:
(a) a container having a barrier capable of disestablishment, said barrier separating a reagent compartment from a sample receiving compartment; and
(b) a nucleic acid storage composition within said reagent compartment, wherein upon disestablishment of said barrier there is fluid communication between said sample receiving compartment and said reagent compartment to facilitate mixing of said biological sample with said nucleic acid storage composition.
2. The device according to claim 1 , which additionally comprises means for closing said container, means for disestablishment of the barrier, or both.
3. The device according to claim 1 , wherein the storage composition comprises a denaturing agent, a chelator and has a pH of greater than 5.0.
4. (canceled)
5. (canceled)
6. The device according to claim 1 , wherein the nucleic acid is DNA.
7. (canceled)
8. The device according to claim 1 , wherein the biological sample is tissue or a bodily fluid.
9. The device according to claim 8 , wherein the bodily fluid is sputum.
10. The device according to claim 2 , wherein the displaceable barrier is a pivoting disk.
11. The device of claim 2 , wherein the means for closing said container includes means for disestablishment of said barrier.
12. The device of claim 2 , wherein the means for closing said container is an airtight lid.
13. A method of preserving a nucleic acid from a biological sample comprising:
(a) placing a nucleic acid storage composition into a reagent compartment of a storage container;
(b) placing a biological sample into a sample receiving compartment of said storage device, said sample receiving compartment separated from said reagent compartment by a barrier;
(c) closing said storage container; and
(d) disestablishing said barrier such that said composition contacts said biological sample to form a mixture thereby preserving the nucleic acid from said biological sample.
14. The method according to claim 13 , wherein the storage composition comprises a denaturing agent, a chelator and has a pH of greater than 5.0.
15. The method according to claim 14 , additionally comprising the step of storing said mixture for at least one day at room temperature.
16. (canceled)
17. (canceled)
18. The method according to claim 13 , wherein the nucleic acid is DNA.
19. (canceled)
20. The method according to claim 13 , wherein the biological sample is tissue or a bodily fluid.
21. The method according to claim 20 , wherein the bodily fluid is sputum.
22. (canceled)
23. The method of claim 13 , wherein the closing of said container is coupled to the disestablishing of said barrier.
24. A sample receiving container comprising:
(a) a sample receiving compartment;
(b) a reagent storage compartment;
(c) a barrier separating said sample receiving compartment from said reagent storage compartment and capable of disestablishment; and
(d) means for closing said container that comprises means for disestablishing said barrier to allow fluid communication between said sample receiving compartment and said reagent storage compartment.
25. The container of claim 24 , which additionally comprises a nucleic acid storage composition within said reagent storage compartment.
26. The container of claim 25 , wherein the storage composition comprises a denaturing agent, a chelator and has a pH of greater than 5.0.
27. (canceled)
28. (canceled)
29. The container of claim 24 , wherein said barrier is a disc pivotable about a pivot axis from a closed position to an open position, such that when said barrier is in the open position there is fluid communication between sample receiving compartment from said reagent storage compartment.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16/809,131 US20200354769A1 (en) | 2002-06-07 | 2020-03-04 | Compositions and methods for obtaining nucleic acids from sputum |
US16/986,765 US11572581B2 (en) | 2002-06-07 | 2020-08-06 | Compositions and methods for obtaining nucleic acids from sputum |
Applications Claiming Priority (8)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US38639802P | 2002-06-07 | 2002-06-07 | |
US38639902P | 2002-06-07 | 2002-06-07 | |
US38639702P | 2002-06-07 | 2002-06-07 | |
US10/455,680 US7482116B2 (en) | 2002-06-07 | 2003-06-05 | Compositions and methods for obtaining nucleic acids from sputum |
US12/338,873 US20090162866A1 (en) | 2002-06-07 | 2008-12-18 | Compositions and methods for obtaining nucleic acids from sputum |
US14/549,344 US9523115B2 (en) | 2002-06-07 | 2014-11-20 | Compositions and methods for obtaining nucleic acids from sputum |
US15/345,420 US10619187B2 (en) | 2002-06-07 | 2016-11-07 | Compositions and methods for obtaining nucleic acids from sputum |
US16/809,131 US20200354769A1 (en) | 2002-06-07 | 2020-03-04 | Compositions and methods for obtaining nucleic acids from sputum |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/345,420 Continuation US10619187B2 (en) | 2002-06-07 | 2016-11-07 | Compositions and methods for obtaining nucleic acids from sputum |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/986,765 Division US11572581B2 (en) | 2002-06-07 | 2020-08-06 | Compositions and methods for obtaining nucleic acids from sputum |
Publications (1)
Publication Number | Publication Date |
---|---|
US20200354769A1 true US20200354769A1 (en) | 2020-11-12 |
Family
ID=29740816
Family Applications (7)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/455,680 Expired - Lifetime US7482116B2 (en) | 2002-06-07 | 2003-06-05 | Compositions and methods for obtaining nucleic acids from sputum |
US12/338,873 Abandoned US20090162866A1 (en) | 2002-06-07 | 2008-12-18 | Compositions and methods for obtaining nucleic acids from sputum |
US12/338,848 Abandoned US20090162924A1 (en) | 2002-06-07 | 2008-12-18 | Compositions and methods for obtaining nucleic acids from sputum |
US14/549,344 Expired - Lifetime US9523115B2 (en) | 2002-06-07 | 2014-11-20 | Compositions and methods for obtaining nucleic acids from sputum |
US15/345,420 Expired - Fee Related US10619187B2 (en) | 2002-06-07 | 2016-11-07 | Compositions and methods for obtaining nucleic acids from sputum |
US16/809,131 Abandoned US20200354769A1 (en) | 2002-06-07 | 2020-03-04 | Compositions and methods for obtaining nucleic acids from sputum |
US16/986,765 Expired - Lifetime US11572581B2 (en) | 2002-06-07 | 2020-08-06 | Compositions and methods for obtaining nucleic acids from sputum |
Family Applications Before (5)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US10/455,680 Expired - Lifetime US7482116B2 (en) | 2002-06-07 | 2003-06-05 | Compositions and methods for obtaining nucleic acids from sputum |
US12/338,873 Abandoned US20090162866A1 (en) | 2002-06-07 | 2008-12-18 | Compositions and methods for obtaining nucleic acids from sputum |
US12/338,848 Abandoned US20090162924A1 (en) | 2002-06-07 | 2008-12-18 | Compositions and methods for obtaining nucleic acids from sputum |
US14/549,344 Expired - Lifetime US9523115B2 (en) | 2002-06-07 | 2014-11-20 | Compositions and methods for obtaining nucleic acids from sputum |
US15/345,420 Expired - Fee Related US10619187B2 (en) | 2002-06-07 | 2016-11-07 | Compositions and methods for obtaining nucleic acids from sputum |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/986,765 Expired - Lifetime US11572581B2 (en) | 2002-06-07 | 2020-08-06 | Compositions and methods for obtaining nucleic acids from sputum |
Country Status (8)
Country | Link |
---|---|
US (7) | US7482116B2 (en) |
EP (1) | EP1513952B1 (en) |
AT (1) | ATE493506T1 (en) |
AU (1) | AU2003240327A1 (en) |
CA (1) | CA2488769C (en) |
DE (1) | DE60335543D1 (en) |
DK (1) | DK1513952T3 (en) |
WO (1) | WO2003104251A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11002646B2 (en) | 2011-06-19 | 2021-05-11 | DNA Genotek, Inc. | Devices, solutions and methods for sample collection |
WO2022195531A1 (en) * | 2021-03-18 | 2022-09-22 | Vimal Soomnath Pumposh | A single time use device to identify sputum |
US11572581B2 (en) | 2002-06-07 | 2023-02-07 | DNA Genotek, Inc. | Compositions and methods for obtaining nucleic acids from sputum |
Families Citing this family (69)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP1585974B1 (en) | 2003-01-24 | 2013-02-27 | University of Utah | Methods of predicting mortality risk by determining telomere length |
JP4662932B2 (en) | 2003-08-15 | 2011-03-30 | ユニバーシティー オブ サウス フロリダ | Materials and methods for capturing pathogens from samples and removing aurintricarboxylic acid |
WO2005064010A1 (en) * | 2003-12-19 | 2005-07-14 | Preanalytix Gmbh | Composition for binding a nucleic acid to a solid phase |
EP1859039A4 (en) * | 2005-03-16 | 2009-06-03 | Dna Genotek Inc | Compositions and method for storage of nucleic acid from bodily fluids |
CN100478671C (en) * | 2005-10-25 | 2009-04-15 | 艾康生物技术(杭州)有限公司 | Detector and method for liquid sampler |
PL1956969T3 (en) * | 2005-12-09 | 2012-04-30 | Dna Genotek Inc | Container system for releasably storing a substance |
WO2007134254A2 (en) * | 2006-05-12 | 2007-11-22 | San Diego State University Research Foundation | Surveying and monitoring respiratory tract viral communities using uncultured human sputum samples |
AU2013206564B2 (en) * | 2006-10-06 | 2016-02-25 | Dna Genotek Inc. | Stabilizing compositions and methods for extraction of ribonucleic acid |
US20100099149A1 (en) * | 2006-10-06 | 2010-04-22 | Dna Genotek Inc. | Stabilizing compositions and methods for extraction of ribonucleic acid |
US7807359B2 (en) * | 2006-12-01 | 2010-10-05 | Quest Diagnostics Investments Incorporated | Methods of detecting TPMT mutations |
US7521213B2 (en) * | 2006-12-01 | 2009-04-21 | Quest Diagnostics Investments Incorporated | Sample processing for nucleic acid amplification |
EP2042595A1 (en) | 2007-09-27 | 2009-04-01 | Qiagen GmbH | Permanent inactivation of nucleases |
CA2702841C (en) * | 2007-10-23 | 2016-01-19 | C. Mark Newby | Container system for tissue stabilization for molecular and histopathology diagnostics |
WO2009055595A1 (en) | 2007-10-23 | 2009-04-30 | Becton, Dickinson And Company | Closed kit for tissue containment and stabilization for molecular and histopathology diagnostics |
EP2214561B1 (en) | 2007-10-23 | 2020-04-29 | Becton, Dickinson and Company | Tissue container for molecular and histology diagnostics incorporating a breakable membrane |
EP2532424B1 (en) * | 2007-10-23 | 2020-11-25 | Becton, Dickinson and Company | Fluid displacement tissue container for molecular and histology diagnostics |
CA2716337C (en) * | 2008-02-20 | 2017-11-14 | Streck, Inc. | Thermocycler and sample vessel for rapid amplification of dna |
CA2734736C (en) | 2008-08-21 | 2017-02-28 | Dna Genotek Inc. | Sample receiving device |
US8039794B2 (en) * | 2008-12-16 | 2011-10-18 | Quest Diagnostics Investments Incorporated | Mass spectrometry assay for thiopurine-S-methyl transferase activity and products generated thereby |
CN102439171B (en) | 2008-12-22 | 2017-07-14 | 犹他大学研究基金会 | Monochromatic multiple quantitative PCR |
EP2429704A2 (en) * | 2009-05-14 | 2012-03-21 | Streck Inc. | Sample processing cassette, system, and method |
EP2388312A1 (en) | 2010-05-17 | 2011-11-23 | Curetis AG | Universally applicable lysis buffer and processing methods for the lysis of bodily samples |
WO2012166913A1 (en) | 2011-06-01 | 2012-12-06 | Streck, Inc. | Rapid thermocycler system for rapid amplification of nucleic acids and related methods |
CA2779850C (en) * | 2011-06-17 | 2016-08-09 | Norgen Biotek Corporation | Methods, reagents and kits for preservation of nucleic acids in biological samples |
US20130164738A1 (en) * | 2011-12-21 | 2013-06-27 | Pathway Genomics | Genetic Sample Collection Systems |
US9044738B2 (en) | 2012-04-30 | 2015-06-02 | General Electric Company | Methods and compositions for extraction and storage of nucleic acids |
US9040675B2 (en) | 2012-04-30 | 2015-05-26 | General Electric Company | Formulations for nucleic acid stabilization on solid substrates |
US9040679B2 (en) | 2012-04-30 | 2015-05-26 | General Electric Company | Methods and compositions for extraction and storage of nucleic acids |
US9480966B2 (en) | 2012-04-30 | 2016-11-01 | General Electric Company | Substrates and methods for collection, stabilization and elution of biomolecules |
EP2883039A1 (en) | 2012-08-10 | 2015-06-17 | Streck Inc. | Real-time optical system for polymerase chain reaction |
CN103196729A (en) * | 2013-04-10 | 2013-07-10 | 吴鹏 | Reagent for processing mucus |
ES2762860T3 (en) | 2013-05-22 | 2020-05-26 | Telomere Diagnostics Inc | Measures of abundance of short telomeres |
WO2014210593A1 (en) | 2013-06-28 | 2014-12-31 | Streck, Inc. | Devices for real-time polymerase chain reaction |
NZ629537A (en) | 2013-08-01 | 2017-12-22 | Ancestry Com Dna Llc | Sample collection device |
WO2015031994A1 (en) * | 2013-09-03 | 2015-03-12 | Dna Genotek Inc. | Method and composition for nucleic acid storage from blood fractions |
SG10201807736SA (en) * | 2014-03-07 | 2018-10-30 | Dna Genotek Inc | Composition and method for stabilizing nucleic acids in biological samples |
RU2016141811A (en) | 2014-04-10 | 2018-05-10 | ДиЭнЭй ГЕНОТЕК ИНК. | METHOD AND SYSTEM FOR LYSIS OF MICRO-ORGANISMS WITH APPLICATION OF PERIODATES |
AU2015268067A1 (en) | 2014-05-27 | 2016-12-22 | Dna Genotek Inc. | Composition and method for stabilizing and maintaining the viability of hardy microorganisms |
US9528105B2 (en) | 2014-09-04 | 2016-12-27 | Techlab, Inc. | Nucleic acid extraction using organic solvents to remove inhibitors |
EP3237910B1 (en) | 2014-12-23 | 2019-04-17 | Siemens Healthcare Diagnostics Inc. | Proteolytic digestion and determination of cardiac troponin |
BR112017014116A2 (en) | 2014-12-30 | 2018-03-06 | Telomere Diagnostics Inc | quantitative multiplex pcr |
CN114965989A (en) | 2015-02-09 | 2022-08-30 | Dna吉诺特克股份有限公司 | Apparatus, solution and method for collecting samples for related applications, analysis and diagnosis |
EP3259065A1 (en) | 2015-02-20 | 2017-12-27 | Ventana Medical Systems, Inc. | Assembly for storing and transporting tissue samples immersed in a fluid |
EP4289356A3 (en) | 2015-09-09 | 2024-02-28 | Drawbridge Health, Inc. | Devices for sample collection, stabilization and preservation |
US10905113B2 (en) | 2015-11-12 | 2021-02-02 | Regents Of The University Of Minnesota | Compositions and method for storing liquid biospecimens |
US10064606B1 (en) | 2015-11-25 | 2018-09-04 | Spectrum Solutions L.L.C. | Specimen collection and preservation apparatus |
AU2017269608C1 (en) | 2016-05-27 | 2023-12-07 | Norgen Biotek Corp. | Preservation of cell-free nucleic acids in biological samples |
US10525473B2 (en) | 2016-08-03 | 2020-01-07 | Spectrum Solutions, L.L.C. | Sample collection kit including twist and tear solution cap |
CN113801876A (en) * | 2017-01-16 | 2021-12-17 | 光谱解决方案有限责任公司 | Nucleic acid preservation solution and method for producing and using same |
MX2019010800A (en) | 2017-03-15 | 2020-07-28 | Ancestry Com Dna Llc | Sample collection device and method. |
US10799422B2 (en) | 2017-05-30 | 2020-10-13 | Spectrum Solutions L.L.C. | Sample collection kit including removable stopper |
NZ764035A (en) | 2017-10-06 | 2020-05-29 | Ancestry Com Dna Llc | Systems, devices, and methods for sample collection |
CN107748095A (en) * | 2017-11-15 | 2018-03-02 | 湖南省天骑医学新技术股份有限公司 | A kind of multipurpose sputum specimen processing unit and its method |
EP3713498A4 (en) | 2017-11-22 | 2021-08-04 | Ancestry.com DNA, LLC | Sample collection kit including cap having selectively movable sleeve |
US11426734B2 (en) | 2017-11-22 | 2022-08-30 | Ancestry.Com Dna, Llc | Sample collection kit including cap having selectively movable sleeve |
AU2019221318A1 (en) | 2018-02-14 | 2020-09-24 | Salignostics Ltd. | Methods and apparatus for detecting analytes |
EP3826646A4 (en) * | 2018-07-25 | 2022-07-27 | Convergent Genomics, Inc. | Urinary microbiomic profiling |
US20220003777A1 (en) * | 2018-11-08 | 2022-01-06 | The Board Of Trustees Of The Leland Stanford Junior University | Methods Employing Mucin-Specific Proteases |
AU2019384801A1 (en) | 2018-11-20 | 2021-06-10 | Spectrum Solutions, Llc | Sample collection system including sealing cap and valve |
AU2020227306A1 (en) | 2019-02-27 | 2021-10-07 | Ancestry.Com Dna, Llc | Graphical user interface displaying relatedness based on shared DNA |
US11968975B2 (en) | 2019-04-30 | 2024-04-30 | Regents Of The University Of Minnesota | Compositions and methods for storing liquid biospecimens |
US11701094B2 (en) | 2019-06-20 | 2023-07-18 | Spectrum Solutions L.L.C. | Sample collection system including valve and plug assemblies |
EP4072432A1 (en) * | 2019-12-12 | 2022-10-19 | Viome Life Sciences, Inc. | Sample collection device with reagent |
CN111218444B (en) * | 2020-04-24 | 2020-09-01 | 广州安必平医药科技股份有限公司 | Sputum preserving fluid |
CN113956979A (en) * | 2020-07-20 | 2022-01-21 | 袁梁 | Clinical sputum specimen liquefaction treatment method |
US20220064709A1 (en) * | 2020-09-01 | 2022-03-03 | Longhorn Vaccines And Diagnostics, Llc | Compositions and Methods for Screening Biological Samples |
CN113234793A (en) * | 2021-05-18 | 2021-08-10 | 江苏迅睿生物技术有限公司 | Novel method for quickly processing saliva sample applied to saliva virus nucleic acid detection |
WO2023150879A1 (en) * | 2022-02-10 | 2023-08-17 | Dna Genotek Inc. | Methods and systems for defining test sample read count limits for a range of microbial sequence reads |
EP4230149A1 (en) | 2022-02-18 | 2023-08-23 | MolGen B.V. | Device for collecting a sample, a kit comprising such device and a method using such device or kit |
Family Cites Families (448)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2631521A (en) | 1953-03-17 | Beverage mixing container | ||
US718127A (en) | 1902-05-15 | 1903-01-13 | Gus Holmgren | Meat-tenderer. |
US2275567A (en) | 1933-11-03 | 1942-03-10 | Arthur E Smith | Container closure |
US2653611A (en) | 1950-11-24 | 1953-09-29 | Arthur E Smith | Closure |
US2773591A (en) | 1952-10-22 | 1956-12-11 | Novo Terapeutisk Labor As | Vessel with sterile closure for separate storage of at least two substances |
US2764983A (en) | 1953-03-20 | 1956-10-02 | Barasch Hans Pius | Dual compartment mixing vial |
GB725784A (en) | 1953-04-01 | 1955-03-09 | James Garrick Sutherland Irons | Improved dispensing device |
US2793776A (en) | 1956-05-23 | 1957-05-28 | Lipari Michael | Container attachment for providing a compartmental dispensing receptacle |
US2958439A (en) | 1959-04-29 | 1960-11-01 | Donald E Yochem | Container and closure |
US3464414A (en) | 1963-07-31 | 1969-09-02 | Upjohn Co | Mixing vial construction |
US3199704A (en) | 1963-10-22 | 1965-08-10 | Guild Molders | Closure and sealing assembly for bottles |
US3340873A (en) | 1966-05-13 | 1967-09-12 | Solowey Ida | Compartmented medical container having a rupturable diaphragm between compartments |
US3321097A (en) | 1966-05-13 | 1967-05-23 | Solowey Ida | Bottle with two or more separate compartments |
US3347410A (en) | 1966-08-31 | 1967-10-17 | Schwartzman Gilbert | Mixing assemblies for applicators |
US3518164A (en) | 1967-04-11 | 1970-06-30 | B D Lab Inc | Diagnostic sputum collection system |
US3441179A (en) | 1967-05-29 | 1969-04-29 | Ways & Means Inc | Mixing container |
US3419179A (en) | 1967-06-07 | 1968-12-31 | Brunswick Corp | Captive cap specimen vial |
GB1235668A (en) | 1967-08-03 | 1971-06-16 | Amalgamated Dental Co Ltd | Containers for two-part mixtures |
US3537606A (en) | 1968-06-17 | 1970-11-03 | Ida Solowey | Compartment bottle |
US3603484A (en) | 1969-02-28 | 1971-09-07 | Mix O Matic Corp | A two-compartment mixing and dispensing device |
US3985608A (en) | 1969-05-03 | 1976-10-12 | Rolf Saxholm | Supporting element for use in microbiological, serological, immunological, clinical-chemical and similar laboratory work |
US3674028A (en) | 1969-06-04 | 1972-07-04 | Ims Ltd | Multi-mix |
DE1939315B2 (en) | 1969-08-01 | 1973-03-15 | Etablissement Dentaire Ivoclar, Schaan (Liechtenstein) | MULTI-CHAMBER CONTAINER FOR THE ACCOMMODATION OF REACTIVE SUBSTANCES FOR THE PRODUCTION OF READY-TO-USE DENTAL PREPARATIONS |
US3651990A (en) | 1969-10-23 | 1972-03-28 | Edward J Cernei | Container for keeping liquids in separate condition and commingling and dispensing the same |
US3694455A (en) | 1970-11-19 | 1972-09-26 | Smith Kline French Lab | Acyloxyalkyl amidobenzimidazoles |
BE794915A (en) | 1972-02-03 | 1973-08-02 | Inge Spa | CLOSING DEVICE FOR BOTTLES AND ANALOGUES, ALLOWING SEPARATE STORAGE OF INGREDIENTS TO BE MIXED AT THE TIME OF USE |
US3799426A (en) | 1972-04-12 | 1974-03-26 | D Lacy | Container |
US3815580A (en) | 1972-08-31 | 1974-06-11 | C Oster | Apparatus for and method of collecting and preserving cytologic samples |
US3846077A (en) | 1972-09-18 | 1974-11-05 | P Ohringer | Liquid sample collection tube |
US3831742A (en) | 1972-10-16 | 1974-08-27 | Pennwalt Corp | Dental mixing capsule |
US4184483A (en) | 1973-05-08 | 1980-01-22 | U.S. Medical Research & Development, Inc. | Method of and apparatus for collecting cultures |
IT994127B (en) | 1973-08-03 | 1975-10-20 | Sigma Tau Ind Farmaceuti | DEVICE FOR THE HERMETIC CLOSING OF CONTAINERS EQUIPPED WITH PERFORATOR AND GOCCIMETER |
FR2279378A1 (en) * | 1974-07-22 | 1976-02-20 | Chibret Laboratoires | Flask for mixing powders and solvents esp for pharmaceutical compsns - has screw-cap with perforated tube which pierces seal between two containers |
DE2554379A1 (en) | 1974-12-04 | 1976-06-10 | William Wardock Feaster | DEVICE FOR THE PREPARATION OF BLOOD AND SIMILAR SAMPLES |
USD244555S (en) | 1975-03-25 | 1977-05-31 | Greiner Electronic Ag | Covered test tube |
USD246600S (en) | 1975-05-06 | 1977-12-06 | Japan Medical Supply Co., Ltd. | Test tube |
FR2342914A1 (en) | 1976-03-02 | 1977-09-30 | Oreal | PACKAGING DEVICE FOR TWO INSULATED PRODUCTS ONE OF THE OTHER BEFORE DISTRIBUTION |
USD246698S (en) | 1976-05-28 | 1977-12-20 | Morris Glenn H | Reversible safety cap and screw cap for containers |
US4081356A (en) | 1976-09-24 | 1978-03-28 | The United States Of America As Represented By The Department Of Health, Education And Welfare | Fecalator, an apparatus and method for concentration of parasite eggs and larvae |
USD255092S (en) | 1976-11-18 | 1980-05-27 | Wong Johnson N S | Combined vial and supporting base |
USD252612S (en) | 1976-12-08 | 1979-08-14 | Starplex Inc. | Container for blood samples or the like |
US4140489A (en) | 1977-02-07 | 1979-02-20 | Lee Sun Y | Test tube for easy enumeration and cultivation of anaerobic and facultatively anaerobic microorganisms |
US4170798A (en) | 1977-05-05 | 1979-10-16 | University Of Alabama In Birmingham | Apparatus for homogenizing and handling biowaste and other materials in isolation |
US4089432A (en) | 1977-05-06 | 1978-05-16 | The Upjohn Company | Vial and closure |
US4102451A (en) | 1977-05-25 | 1978-07-25 | Eli Lilly And Company | Mixing vial |
US4150950A (en) | 1977-09-28 | 1979-04-24 | Corning Glass Works | Transport system for clinical specimens |
US4131016A (en) | 1977-11-09 | 1978-12-26 | The Kendall Company | Peak flow measuring device |
US4200100A (en) | 1978-04-20 | 1980-04-29 | Aluminum Company Of America | Additive transfer unit with piercing member having a penetratable protective tip |
USD256053S (en) | 1978-04-20 | 1980-07-22 | The Kendall Company | Specimen device |
US4195730A (en) | 1978-06-20 | 1980-04-01 | General Foods Corporation | Container having separate storage facilities for two materials |
US4221291A (en) | 1978-06-20 | 1980-09-09 | General Foods Corporation | Container having separate storage facilities for two materials |
US4175008A (en) | 1978-06-26 | 1979-11-20 | Bio-Pharmaceutical Packaging Corp. | Culture specimen collection and transport package |
US4217798A (en) | 1979-04-30 | 1980-08-19 | The United States Of America As Represented By The Department Of Health, Education And Welfare | Automated test tube stopper remover |
US4301812A (en) | 1979-12-12 | 1981-11-24 | The Kendall Company | Midstream sampling device |
US4312950A (en) | 1980-03-31 | 1982-01-26 | Hillwood Corporation | Disposable swab and culture unit |
US4340147A (en) | 1980-11-03 | 1982-07-20 | Mack-Wayne Plastics Company | Cap with built in piercing device |
US4418702A (en) | 1981-01-12 | 1983-12-06 | Metpath Inc. | Method and apparatus for collecting saliva |
JPS5896365U (en) | 1981-12-22 | 1983-06-30 | 日本電気株式会社 | Carrier sheet for facsimile document transmission |
USD274132S (en) | 1982-01-25 | 1984-06-05 | Pacer Technology And Resources, Inc. | Container cap |
US4465183A (en) | 1982-07-19 | 1984-08-14 | Kao Soap Co., Ltd. | Two-part liquid container with breakable partition |
USD277736S (en) | 1982-09-20 | 1985-02-26 | Pacer Technology & Resources, Inc. | Container cap |
US4505433A (en) | 1982-09-28 | 1985-03-19 | Selenke William M | Tissue grinding and transporting device |
USD287570S (en) | 1983-05-26 | 1987-01-06 | A/S Nunc | Combined sample tube, screw cap and marking plug |
JPS604433U (en) | 1983-06-21 | 1985-01-12 | 株式会社吉野工業所 | Two-component mixing container |
DE3426739A1 (en) | 1984-07-20 | 1986-01-30 | Robert Finke KG, 5950 Finnentrop | TWO-COMPONENT PACK |
DE3327615C2 (en) | 1983-07-30 | 1985-08-14 | Robert Finke KG, 5950 Finnentrop | Two-component pack |
USD285115S (en) | 1983-12-05 | 1986-08-12 | Terumo Medical Corporation | Collector for capillary blood |
USD286546S (en) | 1984-01-14 | 1986-11-04 | Shachihata Industrial Co., Ltd. | Felt tip pen |
US4583971A (en) | 1984-02-10 | 1986-04-22 | Travenol European Research And Development Centre (Teradec) | Closed drug delivery system |
US4832917A (en) | 1984-04-19 | 1989-05-23 | California State University Fullerton Foundation | Grommetted system for liquid scintillation vials |
US4678559A (en) | 1984-07-23 | 1987-07-07 | Andreas Szabados | Test specimen container for pasty specimen material |
US4761379A (en) | 1984-08-09 | 1988-08-02 | Becton, Dickinson And Company | Biological specimen collection device |
US4634003A (en) | 1984-08-22 | 1987-01-06 | Suntory Limited | Container for accommodating two kinds of liquids |
US4589548A (en) | 1984-12-06 | 1986-05-20 | Biomedical Polymers, Inc. | Sputum collection apparatus |
USD296241S (en) | 1985-04-15 | 1988-06-14 | Miskinis Robert J | Laboratory glass joint |
US4663161A (en) | 1985-04-22 | 1987-05-05 | Mannino Raphael J | Liposome methods and compositions |
IT1185850B (en) | 1985-08-02 | 1987-11-18 | Zambon Spa | DROP TANK CAP FOR BOTTLES |
ATE53671T1 (en) | 1985-09-05 | 1990-06-15 | Graub Ag Ernst | VETERINARY REAGENT SET FOR RAPID TEST FOR DETERMINING BLOOD CALCIUM. |
JPH0672837B2 (en) | 1985-12-25 | 1994-09-14 | アンドレアス・スツアバドス | Inspection container for pasty sample material |
US4727985A (en) | 1986-02-24 | 1988-03-01 | The Boc Group, Inc. | Mixing and dispensing apparatus |
US5030559A (en) | 1986-04-01 | 1991-07-09 | Board Of Regents, The University Of Texas System | Methods and compositions for the identification of metastatic human tumors |
ATE127923T1 (en) | 1986-04-30 | 1995-09-15 | Igen Inc | ELECTROCHEMILUMINESCENCE TEST METHODS. |
JPH0338704Y2 (en) | 1986-04-30 | 1991-08-15 | ||
GB8614084D0 (en) | 1986-06-10 | 1986-07-16 | Serono Diagnostics Ltd | Immunoassay |
US4741346A (en) | 1986-06-16 | 1988-05-03 | Evergreen Industries, Inc. | Speciman collector |
JPH0627578Y2 (en) | 1986-10-28 | 1994-07-27 | ア−ス製薬株式会社 | Cap for container containing substances to be mixed |
US4935342A (en) | 1986-12-01 | 1990-06-19 | Syngene, Inc. | Method of isolating and purifying nucleic acids from biological samples |
EP0273015A3 (en) | 1986-12-24 | 1988-10-05 | Vifor S.A. | Container with a receiving device for a vial |
USD303710S (en) | 1986-12-29 | 1989-09-26 | Kontes Glass Company | Connector for joining laboratory glassware |
US4753358A (en) | 1987-03-02 | 1988-06-28 | Promega Corporation | Vial cap coupling device |
USD310264S (en) | 1987-03-04 | 1990-08-28 | Nalge Company | Cryogenic vial |
CA1317860C (en) | 1987-04-01 | 1993-05-18 | Daniel Louis Kacian | Techniques for preparing specimens for bacterial assays |
US4927605A (en) | 1987-04-22 | 1990-05-22 | Wadley Technologies, Inc. | Specimen collection and sampling container |
US5128104A (en) | 1987-04-27 | 1992-07-07 | Murphy Harold R | Cuvette for automated testing machine |
US4785931A (en) | 1987-09-24 | 1988-11-22 | Letica Corporation | Molded plastic closure having integral stacking support ribs and rupturable mix compartments |
US4918178A (en) | 1987-10-22 | 1990-04-17 | Wisconsin Alumni Research Foundation | Test for Johne's disease |
US4999288A (en) | 1987-10-28 | 1991-03-12 | Gds Technology, Inc. | Test composition and method for the determination of anilides |
DE3882040T2 (en) | 1987-12-01 | 1993-09-30 | Biotope Inc | METHOD AND DEVICES FOR CARRYING OUT EXAMINATIONS. |
JP2733700B2 (en) | 1988-01-11 | 1998-03-30 | マイクロプローブ・コーポレーシヨン | Oligonucleotide probes for detecting periodontal pathogens |
JP2791367B2 (en) | 1988-04-21 | 1998-08-27 | マイクロプローブ・コーポレーション | Nucleic acid extraction method |
CA1340208C (en) | 1988-06-09 | 1998-12-15 | James F. Monthony | Biological sample collection and transport device |
US5091316A (en) | 1988-06-09 | 1992-02-25 | Becton, Dickinson And Company | Biological sample collection and transport device |
USD330011S (en) | 1988-06-13 | 1992-10-06 | Marr Electric Limited | Twist-on electrical connector |
DE68902025T2 (en) | 1988-09-01 | 1992-12-10 | Capsulit Srl | CLOSURE FOR BOTTLES AND THE LIKE THAT CONTAINS A CONTAINER WITH A PUSHABLE BOTTOM. |
US5496562A (en) * | 1988-10-05 | 1996-03-05 | Flinders Technologies Pty Ltd | Solid medium and method for DNA storage |
US5756126A (en) | 1991-05-29 | 1998-05-26 | Flinders Technologies Pty. Ltd. | Dry solid medium for storage and analysis of genetic material |
US5807527A (en) * | 1991-05-29 | 1998-09-15 | Flinders Technologies Pty. Ltd. | Solid medium and method for DNA storage |
CA1326809C (en) | 1988-11-01 | 1994-02-08 | C. Albert Kezes | Swab retaining vial cap |
USD318727S (en) | 1988-11-04 | 1991-07-30 | Sun Brokers, Inc. | Vial |
USD325444S (en) | 1988-12-13 | 1992-04-14 | Terumo Kabushiki Kaisha | Blood collecting tube |
US5139031A (en) | 1989-09-18 | 1992-08-18 | La Mina Ltd. | Method and device for cytology and microbiological testing |
US5234809A (en) | 1989-03-23 | 1993-08-10 | Akzo N.V. | Process for isolating nucleic acid |
US5140043A (en) * | 1989-04-17 | 1992-08-18 | Duke University | Stable ascorbic acid compositions |
US5152965A (en) | 1989-06-02 | 1992-10-06 | Abbott Laboratories | Two-piece reagent container assembly |
US4932081A (en) | 1989-08-11 | 1990-06-12 | Becton, Dickinson And Company | Sputum cup |
US5128247A (en) | 1989-08-14 | 1992-07-07 | Board Of Regents, The University Of Texas System | Methods for isolation of nucleic acids from eukaryotic and prokaryotic sources |
DE69004949T2 (en) | 1989-09-14 | 1994-07-07 | Becton Dickinson France | ELONGATED CONTAINER WITH TWO SEPARATE CHAMBERS ON THE SAME LONG AXIS. |
US5335673A (en) | 1989-09-21 | 1994-08-09 | Epitope, Inc. | Oral collection device and method for immunoassay |
US5479937A (en) | 1989-09-21 | 1996-01-02 | Epitope, Inc. | Oral collection device |
US5849890A (en) | 1990-06-11 | 1998-12-15 | Nexstar Pharmaceuticals, Inc. | High affinity oligonucleotide ligands to chorionic gonadotropin hormone and related glycoprotein hormones |
JPH0492141A (en) | 1990-08-03 | 1992-03-25 | Bridgestone Corp | Torsional damper |
US5066463A (en) | 1990-10-01 | 1991-11-19 | Chang Maw Guay | Multiple-purpose fecal examination apparatus |
DE4034036C2 (en) | 1990-10-26 | 1994-03-03 | Diagen Inst Molekularbio | Device and method for isolating nucleic acids from cell suspensions |
JPH04187077A (en) | 1990-11-22 | 1992-07-03 | Shimadzu Corp | Apparatus for extraction and purification of nucleic acid |
US5268148A (en) | 1990-12-18 | 1993-12-07 | Saliva Diagnostic Systems, Inc. | Saliva sampling device and sample adequacy system |
US5393496A (en) | 1990-12-18 | 1995-02-28 | Saliva Diagnostic Systems, Inc. | Saliva sampling device and sample adequacy system |
US5380492A (en) | 1990-12-18 | 1995-01-10 | Seymour; Eugene H. | Sampling device and sample adequacy system |
US5283038A (en) | 1990-12-18 | 1994-02-01 | Saliva Diagnostic Systems, Inc. | Fluid sampling and testing device |
US5478722A (en) | 1991-02-17 | 1995-12-26 | The Curators Of The University Of Missouri | Preserved cell preparations for flow cytometry and immunology |
US5196182A (en) | 1991-05-08 | 1993-03-23 | Streck Laboratories, Inc. | Tissue fixative |
SK192592A3 (en) | 1991-06-25 | 1995-04-12 | Saliva Diagnostic Systems Inc | Sample device |
USD338956S (en) | 1991-10-04 | 1993-08-31 | Abbott Laboratories | Protective overcap for a stick holder for medicament |
JPH0599923A (en) | 1991-10-11 | 1993-04-23 | Nitto Denko Corp | Human-hemoglobin detecting method and feces melting buffer solution used therefor |
AU665376B2 (en) | 1991-11-01 | 1996-01-04 | University Of Birmingham, The | Assay device |
US5512440A (en) | 1991-12-18 | 1996-04-30 | Becton Dickinson And Company | Process for lysing Mycobacteria |
AU664050B2 (en) | 1991-12-18 | 1995-11-02 | Becton Dickinson & Company | Process for lysing mycobacteria |
DE4204012A1 (en) | 1992-02-12 | 1993-08-19 | Ulrich Prof Dr Zimmermann | MITOGEN-FREE SUBSTANCE, THEIR PRODUCTION AND USE |
USD344804S (en) | 1992-02-20 | 1994-03-01 | Ralph Muniz | Vial for holding an expandable liquid |
ES2210239T3 (en) | 1992-04-01 | 2004-07-01 | The Johns Hopkins University School Of Medicine | METHOD FOR DETECTING NUCLEIC ACIDS OF MAMMALS ISOLATED IN MAKES AND REAGENTS FOR THE SAME. |
JP2579257Y2 (en) | 1992-05-30 | 1998-08-20 | 有限会社佐藤化成工業所 | Stool collection container |
WO1994004929A1 (en) | 1992-08-24 | 1994-03-03 | Baxter Diagnostics Inc. | Sealable vessel for containing and processing analytical samples |
WO1994012657A1 (en) | 1992-11-20 | 1994-06-09 | Katcher Harold L | Direct-phenol pcr, rt and rt-pcr methods |
WO1994012881A2 (en) | 1992-12-02 | 1994-06-09 | Hochstrasser Denis F | A METHOD FOR DETECTING GROWING CELLS USING TRANSLATIONALLY CONTROLLED TUMOR PROTEIN p21 |
US5843654A (en) | 1992-12-07 | 1998-12-01 | Third Wave Technologies, Inc. | Rapid detection of mutations in the p53 gene |
USD362184S (en) | 1993-02-17 | 1995-09-12 | Unilever Patent Holdings B.V. | Overcap for a microbiological culture bottle |
US5422273A (en) | 1993-03-23 | 1995-06-06 | Baal Medical Products, Inc. | Cell collection apparatus |
US5477863A (en) | 1993-04-14 | 1995-12-26 | Grant; Michael A. | Collection kit with a sample collector |
US5494646A (en) | 1993-04-14 | 1996-02-27 | Seymour; Eugene H. | Sampling device and sample adequacy system |
JP2599763Y2 (en) | 1993-04-20 | 1999-09-20 | 株式会社資生堂 | Container for mixing two kinds of storage items |
US5384096A (en) | 1993-05-12 | 1995-01-24 | Becton, Dickinson And Company | Microcollection tube assembly |
USD357985S (en) | 1993-05-27 | 1995-05-02 | Becton Dickinson And Company | Microcollection tube |
US5409826A (en) | 1993-06-08 | 1995-04-25 | Coulter Corporation | Preserved, non-infectious control cells prepared by the modulation or modification of normal cells |
US5396986A (en) | 1993-06-16 | 1995-03-14 | Special Metals Corporation | Mixing capsule having three tubular members |
DE4321904B4 (en) | 1993-07-01 | 2013-05-16 | Qiagen Gmbh | Method for chromatographic purification and separation of nucleic acid mixtures |
US5330048A (en) | 1993-07-09 | 1994-07-19 | Habley Medical Technology Corporation | Controlled access mixing vial |
USD355606S (en) | 1993-08-10 | 1995-02-21 | Comar, Inc. | Neck of a glass vial |
USD383214S (en) | 1993-08-18 | 1997-09-02 | Brennan V Jack | Combined cap and vial |
USD375160S (en) | 1993-09-10 | 1996-10-29 | Sampson Richard K | Tubing connector handle |
USD372093S (en) | 1993-09-10 | 1996-07-23 | Sampson Richard K | Circular tubing connector handle |
US5624554A (en) | 1993-11-22 | 1997-04-29 | Biomedical Polymers, Inc. | Collection and transfer device |
JP3866762B2 (en) | 1993-11-29 | 2007-01-10 | ジェン−プローブ・インコーポレイテッド | Nucleic acid extraction methods from a wide range of organisms |
US5567309A (en) | 1994-02-14 | 1996-10-22 | Alcott Chromatography, Inc. | Self-filtration cap |
US5714341A (en) | 1994-03-30 | 1998-02-03 | Epitope, Inc. | Saliva assay method and device |
GB2288384B (en) | 1994-04-07 | 1997-06-25 | Johnson & Johnson Medical | Two-component packages |
CA2147593C (en) | 1994-04-22 | 2008-07-29 | Hyman C. Birnboim | Dual purpose tissue fixative |
US5643767A (en) | 1994-05-02 | 1997-07-01 | The Rockefeller University | Process for isolating cellular components |
US5814442A (en) | 1994-06-10 | 1998-09-29 | Georgetown University | Internally controlled virion nucleic acid amplification reaction for quantitation of virion and virion nucleic acid |
WO1996000228A1 (en) | 1994-06-23 | 1996-01-04 | Dade International Inc. | Method for the rapid isolation of nucleic acid |
USD362623S (en) | 1994-07-13 | 1995-09-26 | Inter-Continental Nail Products | Fingernail glue bottle cap |
FR2722765B1 (en) | 1994-07-25 | 1996-08-23 | Oreal | CONTAINER ALLOWING THE STORAGE OF AT LEAST TWO PRODUCTS, THE MIXTURE OF THESE PRODUCTS AND THE DISTRIBUTION OF THE MIXTURE THUS OBTAINED |
USD367114S (en) | 1994-09-07 | 1996-02-13 | Pasteur Sanofi Diagnostics | Specimen sample cup for use in an automatic analyzer |
USD379663S (en) | 1994-09-23 | 1997-06-03 | Unipath Limited | Testing instrument |
US5490971A (en) | 1994-10-25 | 1996-02-13 | Sippican, Inc. | Chemical detector |
DE9417612U1 (en) | 1994-11-03 | 1995-01-05 | Kloth, Bernd, 22399 Hamburg | Sampling device |
DE69526593D1 (en) | 1994-12-23 | 2002-06-06 | Univ Newcastle Res Ass | Evaporators |
AU4606196A (en) | 1994-12-23 | 1996-07-19 | Ludwig Institute For Cancer Research | Assay, receptor proteins and ligands |
USD383851S (en) | 1994-12-30 | 1997-09-16 | Evergreen Industries, Inc. | Leak proof vial for microscope slides |
RU2101354C1 (en) | 1995-01-31 | 1998-01-10 | Ставропольский научно-исследовательский противочумный институт | Method of bacillus anthracis nucleic acid preparing |
US6423550B1 (en) | 1995-03-30 | 2002-07-23 | Ortho Pharmaceutical Corporation | Home oral fluid sample collection device and package for mailing of such device |
USD392187S (en) | 1995-05-18 | 1998-03-17 | Beeson And Sons Limited | Container closure |
US5786228A (en) * | 1995-06-07 | 1998-07-28 | Biex, Inc. | Fluid collection kit and method |
EP1007935B1 (en) | 1995-06-07 | 2006-08-30 | Adeza Biomedical Corporation | Fluid collection kit and method |
US5736322A (en) | 1995-06-07 | 1998-04-07 | Epitope, Inc. | Synthetic oral fluid standard |
US7422902B1 (en) | 1995-06-07 | 2008-09-09 | The University Of British Columbia | Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer |
CA2224360A1 (en) | 1995-06-14 | 1997-01-03 | Paul Anthony Byrne | A closure containing a fluid for mixture with a beverage |
US5827675A (en) * | 1995-07-12 | 1998-10-27 | Charm Sciences, Inc. | Test apparatus, system and method for the detection of test samples |
US5945515A (en) | 1995-07-31 | 1999-08-31 | Chomczynski; Piotr | Product and process for isolating DNA, RNA and proteins |
DE19530132C2 (en) | 1995-08-16 | 1998-07-16 | Max Planck Gesellschaft | Process for the purification, stabilization or isolation of nucleic acids from biological materials |
US6815541B1 (en) | 1995-08-21 | 2004-11-09 | Palma Bee'z Research Institute Co., Ltd. | Coprecipitant and method for extracting nucleic acids |
GB9518156D0 (en) | 1995-09-06 | 1995-11-08 | Medical Res Council | Method of isolating cells |
US5556544A (en) | 1995-09-08 | 1996-09-17 | Didier; Emmanuel R. | Concentrator & filter |
NO954667D0 (en) | 1995-11-17 | 1995-11-17 | Dagfinn Oegreid | Method for detecting Ki-ras mutations |
EP0877940A1 (en) | 1995-12-05 | 1998-11-18 | Donald R. Branch | Methods for the early detection of hiv infection |
US6133036A (en) * | 1995-12-12 | 2000-10-17 | The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration | Preservation of liquid biological samples |
GB9525414D0 (en) | 1995-12-13 | 1996-02-14 | Rocep Lusol Holdings | A device for releasing a fluid into a liquid in a container |
US6551791B1 (en) * | 1995-12-21 | 2003-04-22 | University Of Florida | Rapid diagnostic method for distinguishing allergies and infections and nasal secretion collection unit |
EP0880840A4 (en) | 1996-01-11 | 2002-10-23 | Mrj Inc | System for controlling access and distribution of digital property |
US5830154A (en) | 1996-01-11 | 1998-11-03 | Epitope, Inc. | Device for collecting substances for testing |
JPH09193977A (en) | 1996-01-19 | 1997-07-29 | Anest Iwata Corp | Pressurized air feeding mechanism for pressurized container of low pressure spray gun |
GB9602025D0 (en) | 1996-02-01 | 1996-04-03 | Amersham Int Plc | Nucleoside analogues |
US6667053B1 (en) | 1996-02-16 | 2003-12-23 | Elan Pharmaceuticals, Inc. | D and L etherlipid stereoisomers and liposomes |
US5707860A (en) | 1996-03-12 | 1998-01-13 | Becton Dickinson And Company | Vehicle for delivery of particles to a sample |
USD388519S (en) | 1996-04-02 | 1997-12-30 | Charm Sciences, Inc. | Pocket-size sanitation test kit |
CA2251186A1 (en) | 1996-04-05 | 1997-10-16 | The Johns Hopkins University | A method of enriching rare cells |
WO1997042344A1 (en) | 1996-05-03 | 1997-11-13 | Institut Für Medizinische Molekulardiagnostik Gbrmbh | Process for dna analysis of blood, and means for carrying out said process |
US6020196A (en) | 1996-05-09 | 2000-02-01 | Baxter International Inc. | Devices for harvesting and homogenizing adipose tissue containing autologous endothelial cells |
US5736355A (en) | 1996-05-13 | 1998-04-07 | Steris Corporation | Self contained biological indicator |
FI102642B (en) | 1996-06-19 | 1999-01-15 | Orion Diagnostica Oy | Plug for a reaction vessel or equivalent |
US5939262A (en) | 1996-07-03 | 1999-08-17 | Ambion, Inc. | Ribonuclease resistant RNA preparation and utilization |
WO1998003265A1 (en) | 1996-07-18 | 1998-01-29 | Kyoritsu Chemical-Check Lab., Corp. | Cap-shape reagent container for analysis reagents |
US5980834A (en) | 1996-07-25 | 1999-11-09 | The United States Of America As Represented By The Secretary Of Commerce | Sample storage devices |
US6194204B1 (en) | 1996-08-02 | 2001-02-27 | Center For Blood Research, Inc. | Enrichment of dendritic cells from blood |
USD385793S (en) | 1996-08-05 | 1997-11-04 | Revlon Consumer Products Corporation | Combined container and cap |
US5735320A (en) | 1996-08-21 | 1998-04-07 | The Sherwin-Williams Company | Dispenser for a two-part composition |
EP0920535B1 (en) | 1996-08-26 | 2007-11-14 | Invitek Gesellschaft für Biotechnik & Biodesign mbH. | Method to detect clinically relevant mutations of the dna sequence of ki -ras oncogene, its use and a testkit for early diagnosis of tumours |
US5871905A (en) | 1996-09-04 | 1999-02-16 | Epitope, Inc. | Reduction of false positives in oral-fluid based immunoassays |
DE19635833C2 (en) | 1996-09-04 | 1998-08-06 | Henkel Kgaa | Two-component container |
AU4775497A (en) | 1996-09-19 | 1998-04-14 | W. Kurt Roth | Method for purifying and eventually analyzing nucleic acids from biological test samples |
USD412990S (en) | 1996-10-07 | 1999-08-17 | Cortecs (Uk) Limited | Medical test device |
US5935864A (en) | 1996-10-07 | 1999-08-10 | Saliva Diagnostic Systems Inc. | Method and kit for collecting samples of liquid specimens for analytical testing |
JPH10132824A (en) | 1996-10-28 | 1998-05-22 | Eiken Chem Co Ltd | Hemoglobin stabilizing method |
US6228323B1 (en) | 1996-12-13 | 2001-05-08 | Alcon Laboratories, Inc. | Multi-purpose compositions containing an alkyl-trypsin and methods of use in contact lens cleaning and disinfecting |
US6113257A (en) | 1996-12-26 | 2000-09-05 | M.L.I.S. Projects Ltd. | Two-compartment container |
US5921396A (en) | 1997-01-21 | 1999-07-13 | Brown, Jr.; Jacob T. | Specimen collection kit for mailing and method of using same |
IT1292677B1 (en) | 1997-02-28 | 1999-02-11 | Bormioli Metalplast Spa | PACKAGING TO KEEP THE PRODUCTS SEPARATE BEFORE USE. |
WO1998038917A1 (en) | 1997-03-05 | 1998-09-11 | Point Of Care Technologies, Inc. | Fluid specimen collecting and testing apparatus |
US6277646B1 (en) | 1997-05-05 | 2001-08-21 | Dade Behring Inc. | Fluid specimen collecting and testing apparatus |
US6720141B1 (en) | 1999-11-01 | 2004-04-13 | Interleukin Genetics, Inc. | Diagnostics and therapeutics for restenosis |
US6524795B1 (en) | 1997-03-10 | 2003-02-25 | Interleukin Genetics, Inc. | Diagnostics for cardiovascular disorders |
US5817630A (en) * | 1997-03-18 | 1998-10-06 | Austin Nutriceutical Corporation | Glutathione antioxidant eye drops |
US5935804A (en) | 1997-03-21 | 1999-08-10 | Laine; Roger A. | Method for detecting eubacteria in biological samples with catalytically inactive murein binding enzymes |
US5788652A (en) | 1997-03-24 | 1998-08-04 | S&H Diagnostics, Inc. | Blood sample collection device |
JPH10273161A (en) | 1997-03-26 | 1998-10-13 | Mitsubishi Materials Corp | Container cap and container |
WO1998044158A1 (en) | 1997-03-28 | 1998-10-08 | Epitope, Inc. | Simultaneous collection of dna and non-nucleic analytes from oral fluids |
WO1998044932A1 (en) | 1997-04-07 | 1998-10-15 | The Iams Company | Process for improving glucose metabolism, satiety, and nutrient absorption in companion animals |
US5939259A (en) | 1997-04-09 | 1999-08-17 | Schleicher & Schuell, Inc. | Methods and devices for collecting and storing clinical samples for genetic analysis |
CA2289543C (en) | 1997-05-15 | 2005-08-09 | Rc Development Ag | Dual-component container system |
US6613881B1 (en) | 1997-05-20 | 2003-09-02 | Corixa Corporation | Compounds for immunotherapy and diagnosis of tuberculosis and methods of their use |
USD401697S (en) | 1997-05-21 | 1998-11-24 | Abbott Laboratories | Container |
MX203911B (en) | 1997-05-21 | 2001-08-24 | Pablo Andres Robles Gonzalez | Fluid means-soluble substances container apparatus, and method for mixtures preparation in situ. |
USD412107S (en) | 1997-05-27 | 1999-07-20 | Cebal S.A. | Combined tube and cap |
JPH10332734A (en) | 1997-05-29 | 1998-12-18 | Japan Aviation Electron Ind Ltd | Accelerometer |
US6268136B1 (en) | 1997-06-16 | 2001-07-31 | Exact Science Corporation | Methods for stool sample preparation |
WO1998058081A1 (en) | 1997-06-16 | 1998-12-23 | Exact Laboratories, Inc. | Methods for stool sample preparation |
US6310195B1 (en) | 1997-06-24 | 2001-10-30 | Imclone Systems Incorporated | Nucleic acid encoding a lectin-derived progenitor cell preservation factor |
US6071745A (en) | 1997-06-27 | 2000-06-06 | Bio-Rad Laboratories | Method and formulation for lyophilizing cultured human cells to preserve RNA and DNA contained in cells for use in molecular biology experiments |
FR2765859B1 (en) | 1997-07-08 | 1999-09-24 | Oreal | DEVICE FOR PACKAGING TWO COMPONENTS |
US5869328A (en) | 1997-08-08 | 1999-02-09 | Cdc Technologies, Inc. | Cuvette for performing a diagnostic test on a specimen |
US5829696A (en) | 1997-08-27 | 1998-11-03 | Michelle S. DeStefano | Sealed grinding and homogenizing apparatus |
US6190875B1 (en) | 1997-09-02 | 2001-02-20 | Insight Strategy & Marketing Ltd. | Method of screening for potential anti-metastatic and anti-inflammatory agents using mammalian heparanase as a probe |
JP2000146957A (en) * | 1997-10-13 | 2000-05-26 | Kikkoman Corp | Specimen extracting tool and instrument for smear test |
US6786330B2 (en) | 1997-10-14 | 2004-09-07 | Biogaia Ab | Two-compartment container |
US6120985A (en) | 1997-10-31 | 2000-09-19 | Bbi Bioseq, Inc. | Pressure-enhanced extraction and purification |
US6875617B2 (en) | 1997-11-07 | 2005-04-05 | Geno Technology, Inc. | Agent for protein precipitation, a method of protein precipitation, a method of protein assay using protein precipitation agent, and a kit for protein assay |
USD402766S (en) | 1997-11-13 | 1998-12-15 | Bayer Corporation | Puck |
US5968746A (en) | 1997-11-26 | 1999-10-19 | Schneider; David R. | Method and apparatus for preserving human saliva for testing |
WO1999029904A2 (en) | 1997-12-10 | 1999-06-17 | Sierra Diagnostics, Inc. | Methods and reagents for preservation of dna in bodily fluids |
WO1999032616A2 (en) | 1997-12-18 | 1999-07-01 | Invitek Gmbh | Method for isolating short and long-chain nucleic acids |
JP3719572B2 (en) | 1997-12-19 | 2005-11-24 | 栄研器材株式会社 | Sample collection container for environmental health inspection |
US5941380A (en) | 1998-01-10 | 1999-08-24 | Rothman; David | Device for dispensing flowable material |
EP0939118A1 (en) | 1998-02-20 | 1999-09-01 | Universiteit Maastricht | Method for isolating DNA and RNA from faeces |
US5927549A (en) | 1998-03-20 | 1999-07-27 | Aptargroup, Inc. | Dispensing structure with frangible membrane for separating two products |
DE19812657A1 (en) | 1998-03-23 | 1998-12-24 | Wella Ag | Two-component container for the temporary storage of peroxide and hair dye |
US6303081B1 (en) | 1998-03-30 | 2001-10-16 | Orasure Technologies, Inc. | Device for collection and assay of oral fluids |
SE511864C2 (en) | 1998-04-01 | 1999-12-06 | Medscand Medical Ab | Spatula for sampling including perforations |
US5950819A (en) | 1998-05-08 | 1999-09-14 | Sellars; Andy | Storage, admixing, and dispensing system |
US5984141A (en) | 1998-05-20 | 1999-11-16 | Gibler; Gregory A. | Beverage storage and mixing device |
FR2781500B1 (en) | 1998-07-23 | 2000-09-08 | Bio Merieux | IMPROVED DEVICE AND METHOD FOR LYSIS OF MICROORGANISMS |
US6204375B1 (en) | 1998-07-31 | 2001-03-20 | Ambion, Inc. | Methods and reagents for preserving RNA in cell and tissue samples |
WO2000008136A1 (en) | 1998-08-04 | 2000-02-17 | Center For Advanced Science And Technology Incubation, Ltd. | Method for enzymatic amplification of nucleic acid |
AU5231599A (en) | 1998-08-19 | 2000-03-14 | David S. Soane | Container cap for release of contents contained therein |
US6562300B2 (en) | 1998-08-28 | 2003-05-13 | Becton, Dickinson And Company | Collection assembly |
US6022339A (en) | 1998-09-15 | 2000-02-08 | Baxter International Inc. | Sliding reconstitution device for a diluent container |
US6777210B1 (en) * | 1998-09-24 | 2004-08-17 | Ambion, Inc. | Method and reagents for inactivating ribonucleases RNase A, RNase I and RNase T1 |
US6176836B1 (en) * | 1998-09-25 | 2001-01-23 | David Trudil | Biological sample collection kit |
USD437786S1 (en) | 1998-10-09 | 2001-02-20 | Henkel Kommanditgesellschaft Auf Aktien | Combined bottle and cap |
US6003728A (en) | 1998-10-22 | 1999-12-21 | Aptargroup, Inc. | Dispensing structure with an openable member for separating two products |
US6152296A (en) | 1998-11-06 | 2000-11-28 | Shih; Kuang-Sheng | Additive holder for a pet bottle |
AU1716500A (en) | 1998-11-12 | 2000-06-05 | University Of Virginia Patent Foundation | Non-invasive detection of helicobacter pylori infection |
US20010008614A1 (en) * | 1998-11-16 | 2001-07-19 | Jack L. Aronowitz | Sample collection system and method of use thereof |
US6039198A (en) | 1998-11-24 | 2000-03-21 | Owens-Illinois Closure Inc. | Pierce and cut closure |
US7666609B1 (en) | 1998-12-01 | 2010-02-23 | Shanghai Cp Guojian Pharmaceutical Co. Ltd. | Method and composition for diagnosis of melanocytic lesions |
DE19856064C2 (en) | 1998-12-04 | 2000-11-30 | Invitek Gmbh | Universal method for the isolation of DNA from any starting material |
DE19900638C2 (en) | 1999-01-11 | 2002-12-19 | Max Planck Gesellschaft | Method of isolating DNA from biological materials |
GB9903767D0 (en) | 1999-02-18 | 1999-04-14 | Univ Glasgow | Receptor assay |
FR2790005B1 (en) | 1999-02-22 | 2004-01-30 | Commissariat Energie Atomique | PROCESS FOR PRODUCING MORPHOLINO-NUCLEOTIDES, AND USE THEREOF FOR THE ANALYSIS AND MARKING OF NUCLEIC ACID SEQUENCES |
US6551777B1 (en) * | 1999-02-25 | 2003-04-22 | Exact Sciences Corporation | Methods for preserving DNA integrity |
US6299842B1 (en) | 1999-03-05 | 2001-10-09 | Meridian Bioscience, Inc. | Biological sampling and storage container utilizing a desiccant |
US20020004206A1 (en) | 1999-04-09 | 2002-01-10 | Berger Barry M. | Methods of screening for disease |
WO2000066606A1 (en) | 1999-04-30 | 2000-11-09 | Whatman, Inc. | Substrate including anionic detergent for purifying nucleic acid |
US6270970B1 (en) | 1999-05-14 | 2001-08-07 | Promega Corporation | Mixed-bed solid phase and its use in the isolation of nucleic acids |
US6716396B1 (en) | 1999-05-14 | 2004-04-06 | Gen-Probe Incorporated | Penetrable cap |
WO2000078150A1 (en) | 1999-06-21 | 2000-12-28 | Symbollon Corporation | Iodine germicides that continuously generate free molecular iodine |
US6350578B1 (en) | 1999-06-25 | 2002-02-26 | The Regents Of The University Of California | Method of quantitating dsDNA |
DE19933458B4 (en) | 1999-07-15 | 2015-08-20 | Eppendorf Ag | Equipment and systems for handling liquid samples |
USD432245S (en) | 1999-07-27 | 2000-10-17 | Becton Dickinson And Company | Collection assembly with a specimen label |
US6242188B1 (en) * | 1999-07-30 | 2001-06-05 | Applied Gene Technologies, Inc. | Sample processing to release nucleic acids for direct detection |
US6170719B1 (en) | 1999-08-06 | 2001-01-09 | Becton Dickinson And Company | Medical safety closure |
USD425625S (en) | 1999-08-06 | 2000-05-23 | Becton, Dickinson And Company | Specimen sampling tube |
US6235010B1 (en) | 1999-08-06 | 2001-05-22 | Becton Dickinson And Company | Closed system specimen collection container |
USD424440S (en) | 1999-08-06 | 2000-05-09 | Becton, Dickinson And Company | Tube cap |
USD445908S1 (en) | 1999-08-06 | 2001-07-31 | Becton, Dickinson And Company | Stackable tube assembly |
USD425618S (en) | 1999-08-06 | 2000-05-23 | Becton, Dickinson And Company | Specimen collection device |
US6586177B1 (en) | 1999-09-08 | 2003-07-01 | Exact Sciences Corporation | Methods for disease detection |
US6849403B1 (en) | 1999-09-08 | 2005-02-01 | Exact Sciences Corporation | Apparatus and method for drug screening |
FR2798291B1 (en) | 1999-09-10 | 2005-01-14 | Pasteur Institut | IMMUNOGENIC ACELLULAR COMPOSITIONS AND VACCINE ACELLULAR COMPOSITIONS AGAINST BACILLUS ANTHRACIS |
US6664379B1 (en) | 1999-09-24 | 2003-12-16 | Ambion, Inc. | Nuclease inhibitor cocktail |
DE19950884A1 (en) | 1999-10-22 | 2001-04-26 | Wella Ag | Package for liquids mixed before use has bottle containing one and screw cap containing other, foil in screw cap being pierced by pin on bottle stopper and line of weakness in stopper being ruptured when cap and bottle are screwed together |
US7041484B1 (en) | 1999-10-29 | 2006-05-09 | National Research Council Of Canada | Starch branching enzymes |
WO2001032886A2 (en) | 1999-10-29 | 2001-05-10 | National Research Council Of Canada | Starch branching enzymes |
WO2001034844A1 (en) | 1999-11-10 | 2001-05-17 | Ligochem, Inc. | Method for isolating dna from a proteinaceous medium and kit for performing method |
AU6066799A (en) | 1999-11-26 | 2001-05-31 | Lily Hsu | Container device for separately enclosing two different substances |
US6471069B2 (en) | 1999-12-03 | 2002-10-29 | Becton Dickinson And Company | Device for separating components of a fluid sample |
US6409528B1 (en) | 1999-12-06 | 2002-06-25 | Becton, Dickinson And Company | Device and method for collecting, preparation and stabilizing a sample |
AU2064201A (en) | 1999-12-06 | 2001-06-12 | Eukarion, Inc. | Carbohydrate-aminated glycoproteins |
EP1238106A2 (en) | 1999-12-07 | 2002-09-11 | Exact Sciences Corporation | Apparatus and methods for drug screening based 0on nucleic acid analysis |
US6919174B1 (en) | 1999-12-07 | 2005-07-19 | Exact Sciences Corporation | Methods for disease detection |
US6224922B1 (en) | 1999-12-16 | 2001-05-01 | Mark J. Fonte | Drink colorizer |
US6489172B1 (en) | 2000-01-05 | 2002-12-03 | Varian, Inc. | Saliva sampling device |
US6832994B2 (en) | 2000-01-24 | 2004-12-21 | Bracco Diagnostics Inc. | Table top drug dispensing vial access adapter |
DE50113146D1 (en) | 2000-02-04 | 2007-11-29 | Qiagen Gmbh | NUCLEIC ACID INSULATION FROM CHAIR SAMPLES AND OTHER BIOLOGICAL MATERIALS RICH IN INHIBITORS |
BR0108198A (en) | 2000-02-09 | 2003-03-25 | Basf Ag | isolated nucleic acid, gene construction, amino acid sequence, vector, organism, antibody, antisense nucleic acid molecule, process for the preparation of puffs, oil, lipid or fatty acid or a fraction thereof, composition, use of oleaginous composition , lipid or fatty acid kit, and process for identifying an elongase antagonist |
DE10006662A1 (en) | 2000-02-15 | 2001-08-23 | Antigen Produktions Gmbh | Sample vessel for stabilizing and isolating nucleic acid, contains a lytic solution that stabilizes nucleic acid and a solid phase that binds it, especially for sampling whole blood |
AU2001241738A1 (en) | 2000-02-25 | 2001-09-03 | Corixa Corporation | Compounds and methods for diagnosis and immunotherapy of tuberculosis |
USD438800S1 (en) | 2000-03-10 | 2001-03-13 | Alcoa Closure Systems International | Closure |
USD442090S1 (en) | 2000-03-29 | 2001-05-15 | Pechiney Plastic Packaging, Inc. | Closure |
FR2808276B1 (en) | 2000-04-26 | 2004-04-02 | Renaud Nalin | METHOD FOR INDIRECT EXTRACTION OF DNA FROM NON-CULTIVABLE ORGANISMS AND DNA LIKELY TO BE OBTAINED BY SAID PROCESS |
AU5574801A (en) | 2000-04-28 | 2001-11-12 | Sangamo Biosciences Inc | Methods for binding an exogenous molecule to cellular chromatin |
US6627152B1 (en) | 2000-04-29 | 2003-09-30 | Branan Medical Corp. | Fluid testing apparatus |
USD457247S1 (en) | 2000-05-12 | 2002-05-14 | Gen-Probe Incorporated | Cap |
US6495534B2 (en) | 2000-05-15 | 2002-12-17 | Pharmacia & Upjohn Spa | Stabilized aqueous suspensions for parenteral use |
DE10031236A1 (en) | 2000-06-27 | 2002-01-10 | Qiagen Gmbh | Use of carboxylic acids and other additives in combination with cationic compounds to stabilize nucleic acids in biological materials |
US6548256B2 (en) | 2000-07-14 | 2003-04-15 | Eppendorf 5 Prime, Inc. | DNA isolation method and kit |
JP4599684B2 (en) | 2000-07-26 | 2010-12-15 | 株式会社島津製作所 | Nucleic acid purification method from feces |
USD447812S1 (en) | 2000-08-24 | 2001-09-11 | Becton Dickinson And Company | Collection tube assembly |
AUPR038300A0 (en) | 2000-09-26 | 2000-10-19 | Cryovac Australia Pty Ltd | Reclosable container |
CU23095A1 (en) | 2000-11-07 | 2005-11-18 | Cnic Ct Nac Investigaciones | PROCESS FOR QUICK TYPIFICATION OF MICROORGANISMS AND REAGENT GAME EMPLOYED |
CN100386441C (en) | 2000-11-08 | 2008-05-07 | 贝克顿迪肯森公司 | Method and device for collecting and stabilizing biological sample |
US6602718B1 (en) | 2000-11-08 | 2003-08-05 | Becton, Dickinson And Company | Method and device for collecting and stabilizing a biological sample |
EP1207208A3 (en) | 2000-11-15 | 2003-12-10 | Becton Dickinson and Company | Method for preservation of cells and nucleic acid targets |
US7029840B2 (en) | 2000-11-15 | 2006-04-18 | Becton, Dickinson And Company | Method for preservation of cells and nucleic acid targets |
JP3639897B2 (en) | 2000-11-17 | 2005-04-20 | 幸孝 古川 | Medical specimen storage container |
DE60018431T2 (en) | 2000-11-24 | 2005-12-29 | Chemunex S.A. | Process for trapping airborne microorganisms by means of water-soluble polymers |
US6503716B1 (en) * | 2000-11-28 | 2003-01-07 | Pe Corporation (Ny) | Compositions and methods for extracting a nucleic acid |
WO2002044691A2 (en) | 2000-11-28 | 2002-06-06 | Mcmaster University | Sputum fixative and methods and uses therefor |
US7001724B1 (en) | 2000-11-28 | 2006-02-21 | Applera Corporation | Compositions, methods, and kits for isolating nucleic acids using surfactants and proteases |
US6533113B2 (en) | 2000-12-01 | 2003-03-18 | Brett Moscovitz | System, devices and methods for storing and mixing substances |
US6527110B2 (en) | 2000-12-01 | 2003-03-04 | Brett Moscovitz | Device for storing and dispensing a substance by mating with a container and associated methods |
US6911308B2 (en) | 2001-01-05 | 2005-06-28 | Exact Sciences Corporation | Methods for detecting, grading or monitoring an H. pylori infection |
WO2002055739A2 (en) | 2001-01-15 | 2002-07-18 | Cytyc Corporation | Nucleic acid extraction solution and use thereof |
DE10102338A1 (en) | 2001-01-19 | 2002-07-25 | Basf Plant Science Gmbh | New expression cassette for plant genes, useful for preparing transgenic plants that have increased production of polyunsaturated fatty acids |
US6634234B1 (en) | 2001-02-10 | 2003-10-21 | Vega Grieshaber Kg | Adjustable measurement head and a level measurement device and method employing it |
US6428962B1 (en) * | 2001-02-12 | 2002-08-06 | Dna Analysis, Inc. | Nucleic acid collection barrier method and apparatus |
DE10109354A1 (en) | 2001-02-27 | 2002-09-05 | Icon Genetics Ag | Recombinant viral switch systems |
ES2271232T3 (en) | 2001-03-09 | 2007-04-16 | Gen-Probe Incorporated | PERFORABLE CAPERUZA. |
US6833259B2 (en) | 2001-03-19 | 2004-12-21 | Council Of Scientific And Industrial Research | ‘Pseudomonas stutzeri’ strain and process for preparation of xylanase |
US7957907B2 (en) | 2001-03-30 | 2011-06-07 | Sorenson Molecular Genealogy Foundation | Method for molecular genealogical research |
EP1390466A4 (en) * | 2001-04-26 | 2010-07-28 | Pressure Biosciences Inc | Multichamber device and uses thereof for processing of biological samples |
US6543612B2 (en) | 2001-05-21 | 2003-04-08 | 3M Innovative Properties Company | Container for compositions made of two or more components |
USD455908S1 (en) | 2001-07-31 | 2002-04-23 | Lausan Chung-Hsin Liu | Single-user swing chair |
CA2484062A1 (en) | 2001-08-20 | 2003-02-27 | James C. Davis | Dna purification and recovery from high particulate and solids samples |
USD470240S1 (en) | 2001-09-28 | 2003-02-11 | Orasure Technologies, Inc. | Handle for a sample collector |
US7879293B2 (en) | 2001-09-28 | 2011-02-01 | Orasure Technologies, Inc. | Sample collector and test device |
USD467349S1 (en) | 2001-09-28 | 2002-12-17 | Orasure Technologies, Inc. | Analyzer |
USD467665S1 (en) | 2001-09-28 | 2002-12-24 | Orasure Technologies, Inc. | Test device |
US7148343B2 (en) | 2001-10-12 | 2006-12-12 | Gentra Systems, Inc. | Compositions and methods for using a solid support to purify RNA |
EP1438426A1 (en) | 2001-10-12 | 2004-07-21 | Gentra Systems Inc. | Compositions and methods for using a solid support to purify rna |
US7893228B2 (en) | 2001-10-12 | 2011-02-22 | Qiagen North American Holdings, Inc. | Compositions and methods for using a solid support to purify RNA |
EP3112025B1 (en) | 2001-10-12 | 2019-11-27 | Becton, Dickinson and Company | Apparatus for transporting biological samples |
US6617137B2 (en) | 2001-10-15 | 2003-09-09 | Molecular Staging Inc. | Method of amplifying whole genomes without subjecting the genome to denaturing conditions |
US7297485B2 (en) | 2001-10-15 | 2007-11-20 | Qiagen Gmbh | Method for nucleic acid amplification that results in low amplification bias |
AUPR848001A0 (en) | 2001-10-29 | 2001-11-15 | Dixon, Brodie | Container/bottle cap with a storage compartment |
BR0105971A (en) | 2001-11-09 | 2003-08-05 | Gustavo Senna Chelles | Multicomponent Packaging |
US20030109548A1 (en) | 2001-11-09 | 2003-06-12 | Royt Paulette W. | Compositions and methods of treating iron excess |
ES2337665T3 (en) * | 2001-11-15 | 2010-04-28 | Whatman, Inc. | PROCEDURES AND MATERIALS TO DETECT GENETIC MATERIAL. |
US20030162308A1 (en) | 2001-12-04 | 2003-08-28 | Dave Smith | Orthogonal read assembly |
BRPI0214680B1 (en) | 2001-12-06 | 2018-09-18 | Biocontrol Systems Inc | instrument for use in monitoring a product, ingredient, environment or process, instrument for detecting light emission from a sample and method for monitoring a sample of a product, ingredient, process or environment |
USD471639S1 (en) | 2001-12-17 | 2003-03-11 | Kimberly-Clark Worldwide, Inc. | Tool for handling a specimen |
US20030170694A1 (en) | 2001-12-21 | 2003-09-11 | Daniel Wall | Stabilized nucleic acids in gene and drug discovery and methods of use |
US6634243B1 (en) | 2002-01-14 | 2003-10-21 | Rapid Medical Diagnostics Corporation | Sample testing device |
DE10202147A1 (en) | 2002-01-17 | 2003-09-18 | Birkmayer Gesundheitsprodukte | Plastic bottle cap |
US6880771B2 (en) | 2002-02-01 | 2005-04-19 | Monsanto Technology Llc | Axially reciprocating tubular ball mill grinding device and method |
JP3848201B2 (en) | 2002-03-18 | 2006-11-22 | 株式会社エルメックス | Wiping inspection kit |
DE10219117C1 (en) | 2002-04-29 | 2003-10-30 | Adnagen Ag | Use of lithium dodecyl sulfate for stabilizing RNA in solution, particularly during purification of RNA from cell lysate |
EP1527172B1 (en) | 2002-05-17 | 2008-11-26 | Molzym GmbH & Co. KG | Method for nucleic acid extraction and nucleic acid purification |
DE10222133A1 (en) | 2002-05-17 | 2003-12-04 | Gl Biotech Gmbh | Process for nucleic acid extraction and nucleic acid purification |
US20030215954A1 (en) | 2002-05-17 | 2003-11-20 | Cockerill Franklin R. | Nucleic acid recovery reagents and methods |
WO2003097816A1 (en) | 2002-05-21 | 2003-11-27 | Arkray, Inc. | Method of effecting lysis of acid-fast bacteria and method of performing gene amplification or detection therewith |
JP4092141B2 (en) | 2002-06-07 | 2008-05-28 | 株式会社島津製作所 | Nucleic acid extraction simultaneous purification method |
JP4092139B2 (en) | 2002-06-06 | 2008-05-28 | 株式会社島津製作所 | Nucleic acid extraction method |
US6852495B2 (en) | 2002-06-06 | 2005-02-08 | Shimadzu Corporation | Process of extracting nucleic acid and process of simultaneously carrying out extraction and purification of nucleic acid |
US7482116B2 (en) * | 2002-06-07 | 2009-01-27 | Dna Genotek Inc. | Compositions and methods for obtaining nucleic acids from sputum |
USD471234S1 (en) | 2002-06-25 | 2003-03-04 | Easthill Industry Ltd. | Pen cap |
US20040018575A1 (en) | 2002-07-29 | 2004-01-29 | Craig Rappin | Sample preparation device and method |
US20040018120A1 (en) | 2002-07-29 | 2004-01-29 | Craig Rappin | Sample preparation device and method |
EP2311978A1 (en) | 2002-08-20 | 2011-04-20 | Millennium Pharmaceuticals, Inc. | Compositions, kits, and methods for identification, assessment, prevention, and therapy of cervical cancer |
EP1391520A1 (en) | 2002-08-20 | 2004-02-25 | Becton Dickinson and Company | Method for preservation of cells and nucleic acid targets |
US6913932B2 (en) | 2002-08-23 | 2005-07-05 | Beckman Coulter, Inc. | Formaldehyde-ammonium salt complexes for the stabilization of blood cells |
US20040197845A1 (en) | 2002-08-30 | 2004-10-07 | Arjang Hassibi | Methods and apparatus for pathogen detection, identification and/or quantification |
US7537778B2 (en) | 2002-09-26 | 2009-05-26 | W. Neudorff Gmbh Kg | Pesticidal compositions and methods |
JP5441290B2 (en) | 2002-10-04 | 2014-03-12 | ジーイー・ヘルスケア・バイオサイエンス・コーポレイション | Methods and materials using chemicals as tools for storing nucleic acids on nucleic acid purification system media |
AU2002951977A0 (en) | 2002-10-10 | 2002-10-24 | Leo Engineering Pty Ltd | Improvements to two-part vessels |
IL152905A0 (en) | 2002-11-17 | 2003-06-24 | Univ Ramot | Dopaminergic markers induction in neuronal-like cells isolated from adult human bone marrow stromal cells: implications for novel gene therapy strategy for parkinsons disease |
CA101498S (en) | 2002-12-06 | 2004-01-20 | Dna Genotek Inc | Saliva collection tube |
US7214484B2 (en) | 2002-12-17 | 2007-05-08 | Sigma-Aldrich Co. | Compositions and methods for nucleic acid extraction from biological samples |
CN2598551Y (en) | 2002-12-27 | 2004-01-14 | 珠海亿胜生物制药有限公司 | Connector used for sealing and mixing two kinds or more than two kinds of different substances |
US7560272B2 (en) | 2003-01-04 | 2009-07-14 | Inverness Medical Switzerland Gmbh | Specimen collection and assay container |
JP2004222795A (en) | 2003-01-20 | 2004-08-12 | Naigai Kasei Kk | Instrument for mixed injection of medicine and method for manufacturing the same |
US7718421B2 (en) | 2003-02-05 | 2010-05-18 | Iquum, Inc. | Sample processing |
US20040157219A1 (en) | 2003-02-06 | 2004-08-12 | Jianrong Lou | Chemical treatment of biological samples for nucleic acid extraction and kits therefor |
TW587693U (en) | 2003-03-14 | 2004-05-11 | Mau-Guei Jang | Attaching and stirring type quantitative excrements inspection device |
US7267980B1 (en) | 2003-04-04 | 2007-09-11 | Research & Diagnostic Systems, Inc. | Stabilizing solution for cells and tissues |
US6935493B2 (en) | 2003-04-12 | 2005-08-30 | Young Kook Cho | Cap device for mixing different kinds of materials separately contained therein and in bottle |
US6994211B2 (en) | 2003-04-12 | 2006-02-07 | Young Kook Cho | Cap device for mixing different kinds of materials separately contained therein and in bottle |
US6974024B2 (en) | 2003-04-12 | 2005-12-13 | Young Kook Cho | Cap device for mixing different kinds of materials separately contained therein and in bottle |
CA2465804A1 (en) | 2003-04-28 | 2004-10-28 | Elena Trkulja | Aerosol collection cap and container |
JP2004338751A (en) | 2003-05-15 | 2004-12-02 | Nippon Tansan Gas Co Ltd | Sealing mechanism and sealing method for container for separately storing material |
DE10324913A1 (en) | 2003-05-30 | 2004-12-30 | Sarstedt Ag & Co. | Method and device for stool sampling |
EP1631345B1 (en) | 2003-06-02 | 2011-10-19 | Becton Dickinson and Company | Medicament microdevice delivery system with a cartridge |
WO2006036163A2 (en) * | 2003-11-05 | 2006-04-06 | Greg Liang | Oral fluid sampling device and method |
US20050239045A1 (en) | 2003-12-08 | 2005-10-27 | Arkray Inc. | Microorganism or cell collecting method, and microorganism or cell collecting implement used for the method |
EP1859039A4 (en) | 2005-03-16 | 2009-06-03 | Dna Genotek Inc | Compositions and method for storage of nucleic acid from bodily fluids |
CA118249S (en) | 2005-12-09 | 2007-08-22 | Dna Genotek Inc | Vial |
PL1956969T3 (en) | 2005-12-09 | 2012-04-30 | Dna Genotek Inc | Container system for releasably storing a substance |
CA113861S (en) | 2005-12-09 | 2007-08-22 | Dna Genotek Inc | Vial |
WO2008020013A2 (en) * | 2006-08-17 | 2008-02-21 | F. Hoffmann La-Roche Ag | Methods and means for determining platelet function and diagnosing platelet- related and cardiovascular disorders |
US20100099149A1 (en) | 2006-10-06 | 2010-04-22 | Dna Genotek Inc. | Stabilizing compositions and methods for extraction of ribonucleic acid |
CA2734736C (en) | 2008-08-21 | 2017-02-28 | Dna Genotek Inc. | Sample receiving device |
CA127470S (en) | 2008-08-21 | 2010-06-21 | Dna Genotek Inc | Sample collector |
EP2379128A4 (en) * | 2008-12-23 | 2014-02-05 | Us Gov Health & Human Serv | Lung aerosol collection device |
US20100241091A1 (en) * | 2009-03-20 | 2010-09-23 | Mr. Tan Wu (Owners in common 1/2) | Sputum collecting device |
PL2430420T3 (en) * | 2009-05-14 | 2020-07-27 | Dna Genotek Inc. | Closure, containing apparatus, and method of using same |
USD631350S1 (en) | 2009-09-01 | 2011-01-25 | Dna Genotek Inc. | Double-ended closure |
WO2011116481A1 (en) | 2010-03-25 | 2011-09-29 | Dna Genotek Inc. | Sample collection tool |
USD640794S1 (en) | 2010-03-26 | 2011-06-28 | Dna Genotek Inc. | Sample collection tool |
EP3150702B1 (en) | 2011-06-19 | 2021-05-19 | DNA Genotek, Inc. | Devices, solutions and methods for sample collection |
WO2015112496A2 (en) | 2014-01-20 | 2015-07-30 | Abogen, Inc. | Devices, solutions and methods for sample collection |
SG10201807736SA (en) | 2014-03-07 | 2018-10-30 | Dna Genotek Inc | Composition and method for stabilizing nucleic acids in biological samples |
RU2016141811A (en) | 2014-04-10 | 2018-05-10 | ДиЭнЭй ГЕНОТЕК ИНК. | METHOD AND SYSTEM FOR LYSIS OF MICRO-ORGANISMS WITH APPLICATION OF PERIODATES |
WO2015172250A1 (en) | 2014-05-14 | 2015-11-19 | Dna Genotek Inc. | Device for collecting, transporting and storing biomolecules from a biological sample |
USD743571S1 (en) | 2014-05-27 | 2015-11-17 | Dna Genotek Inc. | Sample collection device |
AU2015268067A1 (en) | 2014-05-27 | 2016-12-22 | Dna Genotek Inc. | Composition and method for stabilizing and maintaining the viability of hardy microorganisms |
USD743044S1 (en) | 2015-01-16 | 2015-11-10 | Dna Genotek Inc. | Tube restrictor for expressing fluid from a swab |
CA3026134A1 (en) | 2016-05-31 | 2017-12-07 | Dna Genotek Inc. | A composition, system and method for removal of detergents from aqueous solutions |
USD850647S1 (en) | 2016-08-19 | 2019-06-04 | Dna Genotek Inc. | False bottom tube with cap and plug |
US20180235206A1 (en) * | 2017-02-17 | 2018-08-23 | Oasis Diagnostics Corporation | Compositions and methods for stabilizing dna in saliva samples |
MX2019010800A (en) * | 2017-03-15 | 2020-07-28 | Ancestry Com Dna Llc | Sample collection device and method. |
US20170182196A1 (en) * | 2017-03-17 | 2017-06-29 | Nilin Patel | Apparatus configured for collection and sterilization of expectorates |
-
2003
- 2003-06-05 US US10/455,680 patent/US7482116B2/en not_active Expired - Lifetime
- 2003-06-06 WO PCT/CA2003/000869 patent/WO2003104251A2/en not_active Application Discontinuation
- 2003-06-06 CA CA2488769A patent/CA2488769C/en not_active Expired - Lifetime
- 2003-06-06 DK DK03729743.9T patent/DK1513952T3/en active
- 2003-06-06 DE DE60335543T patent/DE60335543D1/en not_active Expired - Lifetime
- 2003-06-06 AT AT03729743T patent/ATE493506T1/en not_active IP Right Cessation
- 2003-06-06 EP EP03729743A patent/EP1513952B1/en not_active Expired - Lifetime
- 2003-06-06 AU AU2003240327A patent/AU2003240327A1/en not_active Abandoned
-
2008
- 2008-12-18 US US12/338,873 patent/US20090162866A1/en not_active Abandoned
- 2008-12-18 US US12/338,848 patent/US20090162924A1/en not_active Abandoned
-
2014
- 2014-11-20 US US14/549,344 patent/US9523115B2/en not_active Expired - Lifetime
-
2016
- 2016-11-07 US US15/345,420 patent/US10619187B2/en not_active Expired - Fee Related
-
2020
- 2020-03-04 US US16/809,131 patent/US20200354769A1/en not_active Abandoned
- 2020-08-06 US US16/986,765 patent/US11572581B2/en not_active Expired - Lifetime
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11572581B2 (en) | 2002-06-07 | 2023-02-07 | DNA Genotek, Inc. | Compositions and methods for obtaining nucleic acids from sputum |
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 |
WO2022195531A1 (en) * | 2021-03-18 | 2022-09-22 | Vimal Soomnath Pumposh | A single time use device to identify sputum |
Also Published As
Publication number | Publication date |
---|---|
AU2003240327A8 (en) | 2003-12-22 |
AU2003240327A1 (en) | 2003-12-22 |
DE60335543D1 (en) | 2011-02-10 |
DK1513952T3 (en) | 2011-02-28 |
CA2488769A1 (en) | 2003-12-18 |
US20170152545A1 (en) | 2017-06-01 |
US20090162866A1 (en) | 2009-06-25 |
ATE493506T1 (en) | 2011-01-15 |
US11572581B2 (en) | 2023-02-07 |
EP1513952B1 (en) | 2010-12-29 |
WO2003104251A3 (en) | 2004-07-15 |
US20150104803A1 (en) | 2015-04-16 |
US20200362395A1 (en) | 2020-11-19 |
US9523115B2 (en) | 2016-12-20 |
WO2003104251A2 (en) | 2003-12-18 |
US7482116B2 (en) | 2009-01-27 |
CA2488769C (en) | 2013-01-29 |
US20090162924A1 (en) | 2009-06-25 |
US10619187B2 (en) | 2020-04-14 |
WO2003104251A9 (en) | 2004-03-11 |
US20040038269A1 (en) | 2004-02-26 |
EP1513952A2 (en) | 2005-03-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11572581B2 (en) | Compositions and methods for obtaining nucleic acids from sputum | |
US8470536B2 (en) | Compositions and method for storage of nucleic acid from bodily fluids | |
EP2958498B1 (en) | Sample recovery and collection device | |
CA2310616C (en) | Method and apparatus for preserving human saliva for testing | |
CN111549101A (en) | Preservation solution for biological sample nucleic acid detection and application | |
US20180235206A1 (en) | Compositions and methods for stabilizing dna in saliva samples | |
CN112322697A (en) | DNA sample preservation solution and preparation method and application thereof | |
Thomas et al. | Hypothiocyanite ion: detection of the antimicrobial agent in human saliva | |
CN111378719B (en) | Reagent compositions and methods for preserving nucleic acid integrity in human saliva | |
Shostak et al. | Cultured rat mesothelial cells generate hydrogen peroxide: a new player in peritoneal defense? | |
CA3123402A1 (en) | Matrices and methods for storage and stabilization of biological samples comprising viral rna | |
ES2356734T3 (en) | COMPOSITION AND PROCEDURES TO OBTAIN NUCLEIC ACIDS FROM SPUT. | |
US20230272368A1 (en) | Products and methods for detection of viral nucleic acid | |
CN113150961A (en) | Integrated device for sampling, storing and rapidly extracting nucleic acid sample | |
CN117165580A (en) | Composition for stabilizing nucleic acid in sample, preparation method and application thereof | |
Ahmed et al. | Inspection of some causes of renal calculi in children under the age of 15 years | |
CN115704022A (en) | Viscous biological sample liquefaction composition and uses thereof | |
Mahavir et al. | Dr. Apoorva V. Shivankar | |
赵晓岚 et al. | Application of Short Tandem Repeat in Prenatal Diagnosis for Phenmylketonuria during the First Trimester |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: DOCKETED NEW CASE - READY FOR EXAMINATION |
|
AS | Assignment |
Owner name: DNA GENOTEK INC., CANADA Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:BIRNBOIM, H. CHAIM;REEL/FRAME:055756/0947 Effective date: 20041213 |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |