WO1999039010A1 - Reactifs d'elution, procedes et trousses permettant d'isoler un adn - Google Patents

Reactifs d'elution, procedes et trousses permettant d'isoler un adn Download PDF

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Publication number
WO1999039010A1
WO1999039010A1 PCT/US1999/002190 US9902190W WO9939010A1 WO 1999039010 A1 WO1999039010 A1 WO 1999039010A1 US 9902190 W US9902190 W US 9902190W WO 9939010 A1 WO9939010 A1 WO 9939010A1
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Prior art keywords
dna
reagent
solid support
buffer
eluting reagent
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PCT/US1999/002190
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English (en)
Inventor
Ellen M. Heath
Ruth M. Shuman
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Gentra Systems, Inc.
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Application filed by Gentra Systems, Inc. filed Critical Gentra Systems, Inc.
Priority to JP2000529467A priority Critical patent/JP2004500002A/ja
Priority to CA2319691A priority patent/CA2319691C/fr
Priority to AU25742/99A priority patent/AU2574299A/en
Priority to EP99905618A priority patent/EP1053353A1/fr
Publication of WO1999039010A1 publication Critical patent/WO1999039010A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • 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
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers

Definitions

  • Nucleic acids such as DNA are used extensively in the field of molecular biology for research and clinical analyses. Common methods for analyzing DNA are Southern blotting, amplification through methods such as polymerase chain reaction (PCR), and sequencing. Using these methods, differences in DNA sequence are determined to aid in gene identification, population screening, pathogen identification and diagnostic testing. All of these analyses require purified DNA samples as the basis for consistent and valid results.
  • PCR polymerase chain reaction
  • liquid phase purification the DNA remains in the liquid phase while impurities are removed by precipitation and/or centrifugation.
  • solid phase purification the DNA is bound to a solid support while impurities are selectively eluted. Both purification strategies utilize conventional methods, which require many steps and often hazardous reagents, as well as more rapid methods, which require fewer steps and usually less hazardous reagents.
  • DNA is most commonly isolated using density gradient centrifugation, organic solvent extraction, or salt precipitation. Protocols describing these purification methods are given in Sambrook et al., Molecular
  • liquid phase purification methods of density gradient centrifugation, phenol-chloroform extraction, and salt precipitation generally require four main steps: lysing the cells to release the DNA from cellular and nuclear membranes; removing impurities (such as proteins, lipids and carbohydrates); concentrating by alcohol precipitation; and then rehydrating the DNA in a hydration solution.
  • impurities such as proteins, lipids and carbohydrates
  • concentrating by alcohol precipitation and then rehydrating the DNA in a hydration solution.
  • the major differences among these three methods occur during the second step, where impurities are removed from the DNA by density differentiation, organic-aqueous phase partitioning, or selective salt precipitation. 2
  • a conventional liquid phase purification method for purifying blood dried on specimen collection cards is described by McCabe et al., Human Genetics, 75, 213-216 (1987). The method follows closely a procedure for liquid blood described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nd ed., 9.16-9.19, Cold Spring Harbor Press, Cold Spring Harbor, NY (1989).
  • dried white cells are removed from the collection paper by rehydrating with a saline solution. The white cells are incubated in a buffer to lyse the cells. Then, three phenol extractions are performed to remove protein impurities followed by three ether extractions to remove the phenol.
  • the DNA is concentrated by sodium acetate-ethanol precipitation, washed with 70% ethanol and then rehydrated in a standard DNA hydration solution.
  • Several reagents ten, two of which are hazardous (phenol and ether), are typically required for this method. Although these conventional methods typically yield highly purified DNA preparations, they are laborious and hazardous.
  • WO 95/01359 Cold et al.
  • U.S. Pat. No. 5,405,951 Wiodard
  • International Publication No. WO 95/02049 Jones
  • WO 92/07863 Qiagen GmbH
  • the method disclosed in U.S. Pat. No. 5,234,809 uses a high concentration chaotropic solution to bind DNA to silica particles and requires six centrifugation steps and five reagents to purify DNA from whole blood.
  • Disadvantages of this method are the use of a paniculate suspension, the use of many centrifugation steps, and the use of hazardous reagents, such as guanidinium isothiocyanate and acetone. 3
  • One means for simplifying the conventional solid phase purification procedures is to eliminate the elution step and use the DNA while it is bound to a solid support for subsequent analyses, such as amplification.
  • immobilized DNA usually at least one reagent and one step is omitted.
  • U.S. Pat. No. 5,234,809 (Boom et al.) describes such a method for purifying DNA, although it is not present in a complex mixture such as blood. Using the method described above, but omitting the elution step, reduces the number of reagents and steps by one.
  • U.S. Pat. No. 5,496,562 (Burgoyne) describes a method of purifying cellulose filter paper containing dried blood that uses four reagents during four phenol washes and five isopropanol washes. After drying, a small piece of the filter paper is cut from the square and used directly as a substrate for PCR amplification. Despite the use of bound DNA for analysis, these methods still require many steps and hazardous reagents. Recently, there has been a trend toward developing more rapid and simple methods for both liquid and solid phase purification. This has been driven in part by the development of DNA amplification assays which reduce the time necessary for analysis.
  • One rapid liquid phase method for DNA purification uses a chelating resin to remove metal impurities from liquid blood or blood stains (Walsh et ⁇ l, BioTechniques. 10, 506-513 (1991)). Using this method, blood cells are first washed with deionized water and then incubated with a suspension of the chelating resin and deionized water at 56°C for 15-30 minutes. This incubation is followed by vortexing, incubating at 100°C for 8 minutes, vortexing again, and removing the impurities by centrifugation. This method is rapid
  • Tris(hydroxymethyl)aminomethanehydrochloric acid Tris-HCl buffer (pH 8.0) and boiled for 3 minutes prior to PCR amplification.
  • Tris-HCl Tris(hydroxymethyl)aminomethanehydrochloric acid
  • W.O. Pat. No. 96/18731 (Deggerdal) describes a method of purifying DNA from cells by mixing the cells with a detergent and a solid phase made up of magnetic beads.
  • cells may be pre-lysed by the detergent to release the DNA which is subsequently bound to the solid phase.
  • detergent may be added to a suspension made up of the cells and the solid phase, or the cells, detergent, and solid phase may be suspended together to allow the detergent to lyse the cells in the liquid phase and subsequently bind the DNA to the solid phase.
  • this method involves multiple steps of adding or removing the liquid phase (i.e., detergent or the cell debris-detergent suspension) from the solid phase.
  • Nucleic Acids may be detected and quantitated by several means. Commonly, UV absorbance at a wavelength of 260 nm is used. A wavelength of 320 nm is used to determine background absorbance. Also, fluorimetry in the presence of Hoechst 33258 dye (e.g., Hoefer, DyNA Quant Fluorimeter, Pharmacia Biotech, Piscataway, NJ), antibody detection strips (DNA Dipstick, Invitrogen, Carlsbad, CA), branched signal amplification
  • Hoechst 33258 dye e.g., Hoefer, DyNA Quant Fluorimeter, Pharmacia Biotech, Piscataway, NJ
  • antibody detection strips DNA Dipstick, Invitrogen, Carlsbad, CA
  • Applied Biosystems 7700 Perkin Elmer Applied Biosystems Division, Foster City, CA
  • quantitative PCR amplification e.g., Applied Biosystems 7700, Perkin Elmer Applied Biosystems Division, Foster City, CA
  • the purity of the DNA may be ascertained by measuring the absorbance at various wavelengths.
  • impurities such as 6
  • PCR other target amplification technologies include Ligase Chain Reaction (LCR), Nucleic Acid Sequence Based Amplification (NASBA), Self-sustained Sequence Replication (SSR or 3SR), Strand Displacement Amplification (SDA), and Transcription Mediated Amplification (TMA).
  • LCR Ligase Chain Reaction
  • NASBA Nucleic Acid Sequence Based Amplification
  • SSR or 3SR Self-sustained Sequence Replication
  • SDA Strand Displacement Amplification
  • TMA Transcription Mediated Amplification
  • PCR is used routinely to amplify one or more targeted nucleic acid sequences within a sample or mixture of nucleic acids. This process is disclosed in U.S. Pat. No. 4,965,188 (Mullis). For each target nucleic acid sequence to be amplified in this process, separate complementary strands of nucleic acid are treated with two primers selected to be substantially complementary to portions of the target nucleic acid within the two strands.
  • thermostable enzyme (a polymerase) is generally used to extend the primers to form complementary primer extension products. When these are separated into their complementary strands, they serve as templates to extend the complementary primer into the target nucleic acid sequence. When separated, these in turn act as templates for synthesis of additional nucleic acid sequences.
  • the PCR amplification process involves a series of simple steps. These include temperature cycling to cause hybridization of primers and templates, polymerase mediated synthesis of the primer extension products, and separation and subsequent annealing of the strands of template strands and the synthesized target nucleic acid sequences. Thus, there is an exponential increase in the amount of targeted nucleic acid sequences synthesized.
  • PCR amplification is a very sensitive process. Therefore, a very high purity of starting sample is necessary.
  • LCR is another diagnostic technique that is often utilized in conjunction with a primary PCR amplification.
  • LCR employs a thermostable ligase and allows the discrimination of DNA sequences differing in only a single base pair.
  • LCR depends on highly pure NA templates due to its sensitivity. 7
  • DNA fragments from gels can be further analyzed by Southern hybridization, or Southern blotting as it is more commonly known.
  • Southern blotting is the capillary transfer of DNA fragments from gels to various types of filter paper. It allows the researcher to detect rare sequences in a complex population of restriction fragments and is useful in gene cloning, reverse genetics, and the analysis of restriction-fragment-length-polymorphisms (RFLP's) for human genetic disease diagnosis.
  • Southern blotting involves the digestion of DNA with one or more restriction enzymes, followed by a size separation by electrophoresis on an agarose gel. The DNA is then denatured in situ .and transferred from the gel to a membrane (e.g., nitrocellulose or nylon).
  • a membrane e.g., nitrocellulose or nylon
  • DNA attached to the membrane is then hybridized to radiolabelled DNA or RNA, and autoradiography is used to locate the positions of bands complementary to the probe.
  • Southern blotting is highly sensitive. A sequence of 1000 base pairs (bp) that occurs only once in the mammalian genome (i.e., 1 part in 3 million) can be detected in an overnight exposure if 10 ⁇ g of genomic DNA is transferred to the filter and hybridized to a probe several hundred nucleotides in length.
  • bp base pairs
  • reagents that are of generally low concentration, stable at room temperature (i.e., 20-25°C), less hazardous (i.e., less corrosive, flammable or toxic), nonparticulate to eliminate the need for mixing, and protective of DNA quality.
  • methods with few steps that can be performed using a variety of biological starting materials, whether hydrated or dried, especially as applied to routine testing as found in clinical laboratories.
  • the reagents must not inhibit subsequent DNA analysis procedures by interfering with the buffering capacity of PCR buffers, or cause degradation of polymerase, primers or oligonucleotides used in DNA amplification.
  • methods with few steps that can be performed using a variety of biological starting materials, whether hydrated or dried, especially as applied to routine testing as found in clinical laboratories.
  • PCR Polymerase Chain Reaction
  • LCR Ligase Chain Reaction
  • NASBA Nucleic Acid Sequence Based Amplification
  • SSR or 3SR Self-sustained Sequence Replication
  • SDA Strand Displacement Amplification
  • TMA Transcription Mediated Amplification
  • the present invention provides reagents, methods, and kits that incorporate a solid support for purifying, amplifying, and characterizing DNA from liquid and dried biological samples.
  • the purified DNA is suitable for use in subsequent widely used techniques such as amplification and restriction enzyme digestion.
  • the reagents of the present invention generally contain low concentrations of buffers, salts, acids, bases, chelating agents, and/or detergents so that they are not significantly inhibitory to subsequent DNA analyses.
  • reagents containing high concentrations of one or more of these components are typically used for
  • DNA purification By using these low concentration reagents, the number of steps required for DNA purification is reduced making the method more rapid and simple. These reagents are also generally less hazardous than those used for conventional DNA purification.
  • the solid phase purification methods described typically require only two main steps (e.g., washing and drying). If removal of DNA from a solid support (or solid support matrix) is required, another step (elution) is used.
  • a commercially available DNA purifying reagent is used in the present invention to solubilize and/or rupture cell or protein coat membranes facilitating the release of DNA and/or to solubilize impurities facilitating their removal.
  • the composition of the DNA purifying reagent should render it compatible with (i.e., not significantly inhibitory to) subsequent DNA analyses, such as PCR amplification.
  • the molarity of the DNA purifying reagent should be low.
  • a DNA eluting reagent may be used to remove purified DNA from a solid support following solid phase purification.
  • the DNA eluting reagent includes: a buffer to maintain the pH at least about 7 (preferably, at least about 8, more preferably, at least about 9, 9
  • the buffer preferably has a pKa of at least about 8.
  • a preferred buffer is Tris.
  • the base is preferably one that can raise the pH of the reagent to no less than 7.
  • the base is preferably an alkaline metal hydroxide. Such alkaline metal hydroxides include sodium hydroxide, potassium hydroxide, and lithium hydroxide.
  • the chelating agent is preferably ethylenediamine-tetraacetatic acid (EDTA) or cyclohexanediamine-tetraacetatic acid (CDTA).
  • EDTA ethylenediamine-tetraacetatic acid
  • CDTA cyclohexanediamine-tetraacetatic acid
  • any chelating agent capable of reducing nuclease activity is suitable for use.
  • the combined amount of buffer, base, and chelating agent is of low concentration (typically, no greater than about 20 mM), rendering it generally compatible with (i.e., not significantly inhibitory to) subsequent DNA analyses, such as PCR amplification or restriction enzyme digestion.
  • Suitable solid supports include cellulose, cellulose acetate, glass fiber, nitrocellulose, nylon, polyester, polyethersulfone, polyolefin, polyvinylidene fluoride, and combinations thereof.
  • a preferred solid support is composed of cellulose such as that used commonly for specimen collection.
  • the solid support may be treated with a lysing reagent to assist in lysis and subsequent purification.
  • this lysing step is performed typically prior to contacting the biological material with the solid support.
  • a lysing reagent is added to the solid support, a step is eliminated and the method is simplified.
  • the lysing reagent is applied to the solid support and then dried on the solid support before contacting the biological material with the treated solid support, although this is not a necessary requirement.
  • the lysing reagent is used preferably for the purification of DNA and can be composed of an amount of a detergent effective to lyse cells or protein coats sufficiently to release DNA; a chelating agent to reduce DNA damage; water; and optionally, a buffer effective to provide a pH of greater than about 2.
  • the detergent is preferably anionic. Examples of anionic detergents include N-lauroyl sarcosine or a dodecylsulfate salt. Sodium dodecylsulfate is a particularly preferred anionic detergent.
  • the buffer is preferably effective 10
  • Tris is a particularly preferred buffer.
  • the chelating agent is preferably EDTA or CDTA.
  • the lysing reagent can include an RNA digesting enzyme, such as RNase A, for the purpose of degrading RNA present in the biological sample.
  • an RNA digesting enzyme such as RNase A
  • Including an RNA digesting enzyme eliminates the need for a separate RNase digestion step, as is typically required in conventional methods.
  • This invention also provides methods for purifying DNA from biological samples.
  • the biological samples include, for example, cell or virus suspensions, body fluids, whole blood, bone marrow, buffy coat, plasma, cultured cells, all suspensions (e.g., bacteria, tissue homogenates) and environmental samples.
  • the environmental samples include, for example, air, water or soil.
  • the methods of the present invention involve contacting the biological sample with a solid support.
  • a DNA purifying reagent is added to the solid support containing the biological sample to facilitate solubilization of impurities, lysis of cell walls, release of DNA from the cells and DNA binding to the solid support. Washing of the solid support with a DNA purifying reagent (preferably, at least twice) causes impurities to be removed from the solid support.
  • the solid support containing the bound and purified DNA may be used directly in amplification or other analyses.
  • the DNA may be removed using the DNA eluting reagent.
  • the DNA eluting reagent is contacted with the solid support, incubated, and then removed.
  • Another aspect of this invention involves the combination of a DNA purifying reagent and/or the DNA eluting reagent with one or more optional ancillary reagents.
  • the first ancillary reagent an RBC lysis reagent, is used to lyse red blood cells and facilitate subsequent purification of NAs from the white blood cells contained in mammalian whole blood.
  • the second and third ancillary reagents are used together to digest cell walls in yeast and Gram-positive bacteria prior to DNA purification.
  • the fourth ancillary reagent, a protein digesting reagent is used to digest contaminating proteins.
  • a fifth .ancillary reagent, an isotonic solution is used to suspend DNA and/or cells as needed. 11
  • a further embodiment of the present invention is a method for purifying DNA from yeast and Gram-positive bacteria.
  • the method involves combining the biological sample with a cell suspension reagent.
  • the cell suspension reagent includes a buffer, a chelating agent, and a cell suspending agent to form a cell suspension.
  • a lytic enzyme reagent includes an enzyme to digest cell walls, a buffer, an acid to adjust the pH of the reagent, and two stabilizing agents.
  • the digested cells may be used for liquid or solid phase purification described above.
  • kits for purifying DNA comprising instruction means for preparing substantially pure DNA from a biological sample and one or all of the following: a DNA purifying reagent, a DNA eluting reagent, a lysing reagent, an
  • the kit can also include a solid support, either untreated or treated treated with a lysing reagent.
  • the kit can include a vessel to contain the solid support.
  • Substantially pure nucleic acids are those that are suitable for use in subsequent analyses known to those with skill in the art, for example, DNA amplification, reverse transcription, and restriction enzyme digestion.
  • the present invention provides reagents, methods and kits for incorporating a solid support purifying, amplifying, and characterizing DNA from biological samples.
  • biological samples include biological material, typically in an aqueous mixture or dried, that contains nucleic acids (NAs) like DNA, including complex biological mixtures of prokaryotic or eukaryotic cells.
  • NAs nucleic acids
  • the biological material also contains carbohydrates, proteins, and lipids.
  • Biological materials include the following: body fluids such as whole blood, bone marrow, blood spots, blood serum, blood plasma, buffy coat preparations, saliva and cerebrospinal fluid, buccal swabs, cultured cells, cell suspensions of bacteria or tissue homogenates, solid animal tissues such as heart, liver and brain, body waste products, such as feces and urine, environmental samples taken from air, water, sediment or soil, plant tissues, yeasts, bacteria, viruses, mycoplasmas, fungi, protozoa, rickettsia, and other small microbial cells. Lysates or homogenates of these biological materials may also be used. 12
  • the reagents, methods and kits incorporating a solid support of the present invention provide substantially pure DNA in any form.
  • the DNA may be composed of, for example, chromosomal or genomic DNA, extrachromosomal DNA (such as mitochondrial and plasmid DNA), single stranded DNA, and viral DNA.
  • extrachromosomal DNA such as mitochondrial and plasmid DNA
  • single stranded DNA and viral DNA.
  • purified means substantially free of carbohydrate, protein, and lipid impurities, such that the purified DNA can be used in subsequent analyses known to those with skill in the art.
  • the isolated and purified DNA obtained according to the present invention is suitable for use in subsequent analyses.
  • the methods and kits of the present invention purify a wide range of DNA, all of which can be recovered over a wide molecular weight range.
  • This invention describes methods for using a low concentration reagent for purification of DNA. These methods are generally more rapid and simple than those used typically for DNA purification.
  • the purified DNA obtained from these purification steps can be evaluated for purity, yield, size, amplification ability, etc.
  • the biological samples include, for example, cell or virus suspensions, body fluids, and tissue homogenates. If the biological sample consists of cells or viruses, the cells or viruses may be enumerated prior to this step. The enumeration may be conducted using standard cell counting methods such as an electronic cell counter (e.g., CBC5 Coulter Counter, Coulter Corp., Hialeah, FL) or a visual counter (e.g., a hemacytometer, Bright Line, American Optical, Buffalo, NY).
  • an electronic cell counter e.g., CBC5 Coulter Counter, Coulter Corp., Hialeah, FL
  • a visual counter e.g., a hemacytometer, Bright Line, American Optical, Buffalo, NY.
  • the process for solid phase DNA purification consists of applying a biological sample to a solid support which may be treated with a lysing reagent. Contact with the solid support causes the cell and nuclear membranes to solubilize and/or rupture, thereby releasing the DNA which then binds to the solid support.
  • the solid support may be heated to facilitate solubilization and rupture of the cell and nuclear membranes.
  • the released DNA binds to the solid support allowing impurities to be removed by the addition of a first reagent.
  • This first reagent may be a commercially available purifying reagent for DNA. Impurities are 13
  • solubilized in the first reagent and are removed by suitable means such as centrifugation, pipetting, pressure, or vacuum leaving DNA is left bound to the solid support.
  • suitable means such as centrifugation, pipetting, pressure, or vacuum leaving DNA is left bound to the solid support.
  • the process uses only one reagent and two main steps (i.e., washing and the removal of excess aqueous solution). If DNA is removed from the solid support, an additional reagent, a DNA eluting reagent, is added and another step (eluting) is carried out.
  • the present invention also includes a low concentration DNA elution reagent for eluting DNA from a solid support.
  • the composition of the low concentration DNA elution reagent may vary, but the total concentration is typically less than 20 mM for all compositions.
  • the low concentration DNA elution reagent herein referred to as the "DNA Eluting Reagent,” is capable of removing DNA from a solid support. It contains a base, a buffer, a chelating agent, and water in combination to maintain a pH of at least about 7, preferably, at least about 8, more preferably, at least about 9, and most preferably, at least about 10.
  • the DNA Eluting Reagent contains a buffer to maintain the pH at least about 7 and preferably has a pKa of at least about 8.
  • Suitable buffers include, but are not limited to, N,N-bis[2-hydroxyethyl]glycine (available under the trade designation "BICINE” from Sigma Chemical Company, St. Louis, MO), 3-[cyclohexylamino]-2-hydroxy-l- propanesulfonic acid (available under the trade designation "CAPSO” from Sigma Chemical Company), and Tris.
  • the buffer is used in an amount that is not significantly inhibitory to subsequent DNA analyses, such as PCR amplification. Thus, it is typically used in an amount of no greater than about 20 mM.
  • the buffer is Tris and is used in an amount of about 0.001-20 mM, more preferably, about 0.01-15 mM and most preferably, about 1-10 mM, based on the total volume of the reagent.
  • the DNA Eluting Reagent contains a base to adjust the reagent pH.
  • the base is used in an amount that is not significantly inhibitory to subsequent DNA analyses, such as PCR amplification.
  • the base is typically used in an amount of no greater than about 20 mM.
  • bases include, but are not limited to, potassium hydroxide and sodium hydroxide.
  • the base is an alkaline metal 14
  • hydroxide like sodium hydroxide, potassium hydroxide, or lithium hydroxide and is used in an amount of about 0.2-20 mM, more preferably, about 0.5-15 mM, and most preferably, about 1-5 mM, based on the total volume of the reagent.
  • the DNA Eluting Reagent includes a chelating agent.
  • a chelating agent is used in an amount effective to reduce DNA damage
  • Suitable chelating agents are those capable of chelating divalent cations in aqueous media.
  • Such chelating agents include, but are not limited to, ethylene diamine tetraacetate (EDTA) and cyclohexane diamine tetraacetate (CDTA).
  • EDTA ethylene diamine tetraacetate
  • CDTA cyclohexane diamine tetraacetate
  • the chelating agent is EDTA.
  • the chelating agent is used in an amount that is not significantly inhibitory to subsequent DNA analyses, such as PCR amplification. Thus, it is typically used in an amount of no greater than about 0.1 mM.
  • the chelating agent is used in an amount of about 0.0001-0.1 mM, more preferably, about 0.005-0.05 mM, and most preferably, about 0.0015-0.015 mM, based on the total volume of the reagent.
  • the buffer, base and chelating agent are combined with water to form the DNA Eluting Reagent.
  • the water is preferably deionized and nuclease free.
  • the combined amount of buffer, base, and chelating agent is of low concentration (typically, no greater than about 20 mM), rendering it generally compatible with (i.e., not significantly inhibitory to) subsequent DNA analyses, such as PCR amplification or restriction enzyme digestion.
  • reagents used in purifying DNA from the source biological material have been formulated to be compatible with amplification and other analyses.
  • the reagents have low concentrations of buffer, salt, detergent, and chelating agents that make them compatible with amplification analyses.
  • the final purified nucleic acids are suspended in the DNA Eluting Reagent for solid phase nucleic acid extractions. Therefore, these reagents have been optimized for use in downstream DNA analyses.
  • DNA analyses might include, but are not limited to, RT-PCR and PCR.
  • Tris buffer, salt, and nonionic detergent at a basic pH between 8-10 as shown in Table 1.
  • the lysing reagents and eluting reagents have Tris buffer concentrations that are either of the same order of magnitude or significantly lower than the concentrations found in PCR systems. Concentrations of Tris buffer in the eluting reagent typically range from 1- 10 mM and will not inhibit or significantly interfere with the amplification process.
  • Another aspect of this invention involves the combination of the DNA purifying reagent and/or the DNA Eluting Reagent with one or more optional ancillary reagents.
  • ancillary reagents include reagents known to one of skill in the art for nucleic acid purification.
  • the methods and kits of the present invention are not limited to the use of these specific ancillary reagents, as one of skill in the art may use other reagents and/or techniques to achieve the same purpose.
  • the DNA purifying reagent and/or the DNA Eluting Reagent can be used with other reagents and/or techniques if desired.
  • the first ancillary reagent is a red blood cell lysing reagent used to lyse red blood cells and facilitate subsequent purification of DNA from the white blood cells contained in mammalian whole blood. This reagent is referred to herein as the "RBC Lysis
  • the RBC Lysis Reagent contains ammonium chloride, sodium bicarbonate, and EDTA.
  • the ammonium chloride is used in the RBC Lysis Reagent at a concentration of about 140-150 mM, and more preferably, about 142-146 mM, based on the total volume of the reagent. 16
  • the sodium bicarbonate is used at a concentration of about 0.5-5 mM, and more preferably, about 0.5-2 mM, based on the total volume of the reagent.
  • the EDTA is used at a concentration of about 0.5-10 mM, and more preferably, about 0.75-1.25, based on the total volume of the reagent.
  • RBC Lysis Reagent contains water, preferably of the purity described above.
  • RBC Lysis Reagent is contacted with mammalian whole blood in an amount of 3 volumes of RBC Lysis Reagent to 1 volume of blood. The sample incubates about 1-30 minutes, preferably about 10 minutes, and white cells are separated from the sample by centrifuging at 15,000 for 20 seconds.
  • the RBC Lysis Reagent When combined with mammalian whole blood, the RBC Lysis Reagent forms a red cell lysate containing substantially intact white blood cells. It can also contain viruses with substantially intact protein coats. The white blood cells (and any cell-associated viruses that may be present) are then separated from the red cell lysate.
  • the white blood cells can be combined with a nucleic acid purifying reagent directly or following application to a solid support.
  • the second and third ancillary reagents are used together to digest cell walls from yeast and Gram-positive bacteria prior to DNA purification.
  • the reagents are referred to herein as "Cell Suspension Reagent” and “Lytic Enzyme Reagent.” They are used in the first steps of the DNA purification procedure to digest cell walls that may be resistant to lysis by a nucleic acid purifying reagent.
  • the Cell Suspension Reagent is combined with a biological sample to form a cell suspension.
  • the Lytic Enzyme Reagent is combined with the cell suspension to form a mixture containing digested cells.
  • the Cell Suspension Reagent keeps cells intact while their cell walls are being digested by lytic enzymes.
  • This reagent contains a buffer, preferably Tris, to maintain the reagent pH at about 7-8.5, and more preferably, about 7.5-8.0.
  • the buffer is used preferably at a concentration of about 0.05-0.15 M, and more preferably, about 0.08-0.12 M, based on the total volume of the reagent.
  • the Cell Suspension Reagent also contains a chelating agent, preferably EDTA, to reduce DNA damage.
  • the chelating agent is used, preferably at a 17
  • the buffer, chelating agent and cell suspending agent are combined with water.
  • the water is preferably deionized and substantially nuclease-free.
  • the Lytic Enzyme Reagent contains a lytic enzyme that digests beta- 1,3- glucose polymers that are contained in yeast cell walls.
  • a purified form of this enzyme is readily available from commercial sources, such as Sigma Chemical Company, St. Louis,
  • the Lytic Enzyme Reagent contains a buffer, preferably Tris, to maintain the reagent pH. Tris is used preferably at a concentration of about 1-20 mM, more preferably, about 5-15 mM, and most preferably, about 8-12 mM, based on the total volume of the reagent.
  • the pH of the lytic enzyme reagent is adjusted to a pH of about 7.5- 8.2 using an acid, such as hydrochloric acid.
  • the Lytic Enzyme Reagent contains two stabilizing agents.
  • the first is preferably glycerol.
  • Glycerol is used preferably in an amount of about 20-50% glycerol (volume/volume), more preferably, about 24-40% glycerol, and most preferably, about 28-32% glycerol.
  • the second stabilizing agent is preferably calcium chloride. Calcium chloride is used preferably at a concentration of about 0.5-5 mM, and more preferably, at about 0.75-1.25 mM, based on the total volume of the reagent.
  • the enzyme, buffer, acid, and two stabilizing agents are combined with water.
  • the water is preferably d.I. water.
  • the reagent is purified by passing through a filter of about 0.2 ⁇ M pore size.
  • a fourth ancillary reagent is used to digest contaminating protein, especially in solid tissue samples.
  • the Proteinase K is readily available from commercial sources such as Sigma Chemical Company and is used at a concentration of about 0.1 mg/mL. Heating at greater than 36°C accelerates the activity of this enzyme.
  • a fifth ancillary reagent is typically used to make cell or DNA suspensions.
  • Suitable isotonic solutions are salt-based, often buffered with tris, citrate or phosphate.
  • An example is phosphate buffered saline (PBS).
  • suitable solid supports include, but are not limited to, cellulose, cellulose acetate, glass fiber, nitrocellulose, nylon, polyethersulfone, polyester, polyolefin, polyvinylidene fluoride, and combinations thereof.
  • a preferable solid support is composed of cellulose fibers such as found in the specimen collection paper 903 available from Schleicher and Schuell (Keene, NH) or BFC 180 available from Whatman International Ltd. (Springfield Mill, Kent, England).
  • Another preferable solid support is polyolefin.
  • Polyolefin is herein defined as any olefin based copolymer or homopolymer including modified polymers such as graft copolymers.
  • Acceptable polyolefins include low, medium, and high density polyolefins and linear, low density polyethylene, polypropylene and polybutylene.
  • the polyolefin solid support is hydrophilic and composed of a mixture of low density polyethylene and polypropylene fibers such as those found in the Filtrona ® polyolefin available from American Filtrona, Inc. (Richmond, VA).
  • the solid support is directionally porous giving uniform flow characteristics and low back pressure, is composed of fibers that are interlaced and bonded to each other, is resilient to allow for easy packing into a spin tube, well, cartridge, or another vessel, has a void volume of about 50-90% and is composed of fibers having a diameter of about 20-30 ⁇ m.
  • the size of the solid support suitable for use with the reagents of this invention may vary according to the volume of biological material. For example, when Schleicher and Schuell 903 paper, which has a thickness of 0.5 mm, is used for the solid support, a 3 mm diameter disk will hold about 3 ⁇ l blood and an 8 mm diameter disk will hold about 25 ⁇ l 19
  • the thickness and/or diameter may increase accordingly.
  • the shape of the solid support suitable for use with the reagents of this invention may vary according to the type of biological material.
  • a swab is an appropriate collection device.
  • body fluids such as blood or saliva samples are obtained
  • the solid support may be, for example, a sheet, a precut disk or a cylinder.
  • the solid support is contained in an appropriate vessel, e.g., a paper form (such as a Guthrie card), a microcentrifuge tube, a spin tube, a 96-well plate, a chamber, or a cartridge.
  • the solid support may be treated with a lysing reagent to assist in lysis and subsequent purification.
  • the volume of the lysing reagent used to treat the solid support is at least one-tenth of the total volume of the solid support. More preferably, the volume of the lysing reagent is at least half the total volume of the solid support, and most preferably, the volume of the lysing reagent corresponds to the total volume of the solid support.
  • the total volume of the solid support refers to the volume defined by the external boundaries of the solid support.
  • the resulting product is a lysing matrix for isolation of nucleic acids such as DNA, herein referred to as the "Lysing Matrix.”
  • the DNA purification method is simplified by removing a separate lysing step.
  • the lysing reagent is applied to the solid support and then dried on the solid support before contact with the biological material.
  • conventional systems typically contact the biological material with the lysing reagent as a step prior to contact with the solid support, or the biological material is suspended with the solid support after which the lysis reagent is added to the resulting suspension.
  • the lysing reagent can include an RNA digesting enzyme if it is necessary to digest RNA present in a biological sample.
  • an RNA digesting enzyme is RNase A.
  • a purified form of this enzyme is readily available from commercial sources such as Sigma Chemical Company, St. Louis, MO.
  • RNase A is added to the Lysing Reagent in an amount of about 0.005-1 mg per ml and more preferably, about 0.01-0.1 mg per ml. The activity of this 20
  • enzyme is preferably at least about 50 units per mg and more preferably, at least about 100 units per mg.
  • the lysing reagent includes the anionic detergent and the RNA digesting enzyme, but does not include a chelating agent or a buffer.
  • Suitable anionic detergents are capable of lysing cells and/or solubilizing proteins .and lipids.
  • Such anionic detergents include, but are not limited to, salts (e.g., sodium, potassium, lithium salts) of dodecyl sulfate as well as N-lauroyl sarcosine or a dodecylsulfate salt.
  • the anionic detergent is a dodecyl sulfate salt.
  • RNA digesting enzyme is necessary for biological samples with a high RNA content, such as cell culture suspensions.
  • a preferred RNA digesting enzyme is RNase A.
  • RNase A for biological samples with a high RNA content, commercially available RNase A, (e.g., Puregene ® RNase A available at a concentration of 4 mg/mL, Gentra Systems Inc., Minneapolis, MN) is added to the anionic detergent solution in an amount of about 0.005 - 1 mg per mL and more preferably, about 0.01-0.1 mg per mL.
  • the activity of this enzyme is preferably at least about 50 units per mg and more preferably, at least about 100 units per mg.
  • the lysing reagent includes only the anionic detergent, but does not include a chelating agent, buffer, or the RNA digesting enzyme.
  • Anionic detergents are used as described above.
  • This invention also provides methods for purifying DNA from biological samples.
  • the methods of the present invention typically use only one reagent and two main steps (e.g., washing and drying).
  • the method involves contacting the biological sample with a solid support to lyse the cells thereby releasing the DNA which then binds to the solid support.
  • a commercially available DNA purifying reagent is added to the solid support to facilitate solubilization and removal of impurities. Sequential washing of the solid support with this reagent causes impurities to be removed from the solid support.
  • excess aqueous solution may be removed from the solid support containing thepurified DNA by methods such as evaporation or centrifugation as described in examples 1, 2, and 3. 21
  • the DNA may be removed from the solid support.
  • An additional reagent and an additional step (eluting) are used if the DNA is removed from the solid support.
  • the DNA Eluting Reagent is used to remove DNA from the solid support. This is illustrated in Examples 4, 5, 7, 8, 9, 10, 11, and 13.
  • the method involves contacting the solid support containing the DNA with the DNA Eluting Reagent and incubating.
  • the amount of DNA Eluting Reagent is about 0.25 volumes DNA Eluting Reagent to about 1 volume solid support, more preferably, the volume is about 1 volume reagent to about 1 volume solid support, and most preferably, the volume is about 4 volumes reagent to about 1 volume solid support.
  • the temperature of the incubation is, preferably, at least about 30 °C, more preferably, at least about 80°C, and most preferably, at least about 100°C.
  • the duration of the incubation is preferably, at least about 2 minutes, more preferably, at least about 5 minutes and most preferably, at least about 10 minutes.
  • the DNA is removed from the solid support by standard methods, such as centrifugation, vacuum or pressure.
  • the solid support is treated with the Lysing Reagent such that the Lysing Reagent is bound to the solid support.
  • the Lysing Reagent may be bound covalently, non-covalently, by being trapped within the interstitial spaces of the solid support, or by being deposited on the material (e.g., fibers, beads, etc.) of the solid support.
  • the resulting product is a Lysing Matrix.
  • the Lysing Reagent is allowed to dry on the solid support.
  • the Lysing Reagent is added to the solid suppport, preferably at a volume corresponding to at least one-tenth of the total volume of the solid support, more preferably at a volume corresponding to at least half of the total volume of the solid support, and most preferably at a volume corresponding to at least the total volume of the solid support.
  • the Lysing Reagent may be added directly to the material (e.g., fibers, beads, etc.) used in making the solid support and preferably allowed to dry before it is made into the final user-ready form (e.g., paper, swab, disk, plug, column, etc.).
  • the solid support may be treated with a crystalline or powder form of the lysing reagent and allowed to bind to the solid support. 22
  • DNA is isolated by allowing the sample of biological material (cultured cells, whole blood, etc.) to contact the Lysing Matrix.
  • the sample may be treated with a lysing reagent before contact with the Lysing Matrix, the efficiency of purification and the DNA yield are greatly improved when the biological material is not pre-lysed.
  • the biological material is added directly to the Lysing Matrix which lyses the cells and solubilizes protein coats and lipids.
  • the efficiency of lysis may be improved by heating at greater than at least 30 °C, more preferably at greater than at least 50 °C, and most preferably at greater than at least 80 °C.
  • the amount of DNA purifying reagent used for DNA purification is about 0.5 volume DNA purifying reagent to about 1 volume of biological material, more preferably, about 2 volumes of DNA purifying reagent to about 1 volume of biological material, and most preferably, about 5 volumes of DNA purifying reagent to about 1 volume of biological material.
  • the number of washes with the DNA purifying reagent is at least two and more preferably, at least 3. This method is illustrated in Examples 7, 8, 9, 10, 12, and 13.
  • a further embodiment of the present invention is a method for purifying nucleic acids, like DNA, from yeast and Gram-positive bacteria. These biological materials are typically more resistant to lysis.
  • the method involves combining the biological sample with a first ancillary reagent, i.e., a Cell Suspension Reagent (e.g., Cell Suspension Solution, Gentra Systems, Inc., Minneapolis, MN).
  • a Cell Suspension Reagent e.g., Cell Suspension Solution, Gentra Systems, Inc., Minneapolis, MN.
  • the Cell Suspension Reagent includes a buffer, a chelating agent and a cell suspending agent to form a cell suspension.
  • a Lytic Enzyme Reagent e.g., Lytic Enzyme Solution, Gentra Systems, Inc., Minneapolis, MN.
  • the Lytic Enzyme Reagent includes an enzyme to digest cell walls, a buffer, an acid to adjust the pH of the reagent and two stabilizing agents.
  • the digested cells may be used for liquid or solid phase purification described above.
  • kits that includes specific protocols, which in combination with the reagents and optionally the solid supports described 23
  • the kit includes instruction means. Depending on the application, the kit may also include any combination of a DNA purifying reagent, DNA Eluting Reagent, RBC Lysis Reagent, Cell Suspension Reagent, Lytic Enzyme Reagent, Protein Digesting Reagent, Isotonic Solution, solid supports, solid supports treated with Lysing Reagent and/or RNA digesting enzyme, vessels to contain the solid supports, vessels to contain the waste liquids, and vessels to contain any eluted DNA. Two preferred DNA Purification Kits are described below.
  • a kit for purifying DNA using solid phase purification contains: a DNA purifying reagent (e.g., GENERATION ® DNA Purification Solution, Gentra Systems, Inc.,
  • a kit for purifying DNA using solid phase purification and subsequent elution contains: a DNA purifying reagent, instruction means, the DNA Eluting Reagent, a solid support or a Lysing Matrix, a vessel to hold the solid support or the Lysing Matrix, one or more vessels to collect the waste, and a vessel to collect the purified DNA. Methods to illustrate the use of this kit are given in Examples 4, 5, 7, 8, 9, 10, and 25.
  • the vessel is a cartridge equipped with two inlet ports at the top.
  • the inlet ports are attached to vessels upstream containing the sample or reagents through a connector, such as a female Luer-Lock TM .
  • One inlet, the sample port is used for the application of the biological sample to the solid support.
  • An optional feature on the sample port is a self-sealing mechanism that seals the sample port after sample has been transferred through it.
  • the second inlet serves as a reagent port.
  • An optional feature on both inlet ports is a protective breakaway seal.
  • the inlet ports, breakaway seals and diffuser may be housed in an optional screw-cap.
  • At the bottom of the solid support is an optional diffuser with a pore size suitable for the passage of cellular debris, proteins and lipid molecules. The diffuser allows for a uniform traversal of biological 24
  • the outlet of the cartridge comes equipped with a protective cap that fits neatly over the tapered barrel.
  • the purified DNA is collected in a collection tube that consists of a conical tube with a snap cap for easy and contamination-free storage.
  • the entire vessel can be scaled in size depending on the size of the samples to be processed and the yields needed for subsequent analysis.
  • the vessel consists of a spin tube designed to hold an insert into which the solid support is packed.
  • the solid support may be cellulose, cellulose acetate, glass fiber, nitrocellulose, nylon, polyester, polyethersulfone, polyolefin, polyvinylidene fluoride, and combinations thereof.
  • the insert consists of a flanged top to hold it in the spin tube and a perforated bottom to allow fluids to pass through while supporting the solid support.
  • a cap tethered to the spin tube was used to cover the insert. Examples of a commercially available spin tube are given in Examples 2, 4, 7, 8, 9, and 10.
  • the biological material passes through the perforated bottom and is collected at the bottom of the spin tube. When used, the biological material is applied to the solid support.
  • the vessel may be multiple well plates, for example, 6, 12, 24, 48 or 96 well plates where a solid support is packed into each well. The bottom of each well has an exit port through which waste and debris can pass.
  • the solid supports could be dried by evaporation at room temperature to about 80 °C, an optional alcohol washing step was used to accelerate the drying process.
  • a volume of 200 ⁇ l 100% ethanol was pipetted onto each solid support, incubated for 1 minute and then removed.
  • Isopropanol (2-propanol) at 100% was also found to be a suitable alcohol to accelerate drying.
  • the ethanol rinse was repeated once more for a total of two rinses and then the disks were allowed to dry at room temperature for at least two hours.
  • each was transferred to a 0.6 ml tube.
  • Each amplification reaction contained IX amplification buffer (Promega, Madison, Wl), 1.5 mM MgCl 2 , 200 ⁇ M each deoxynucleotide, 2.5 units Taq DNA Polymerase (Promega, Madison, Wl), and primers each at 1 ⁇ M specific to the D1S80 locus.
  • the primers were shortened from those given by Budowle et al., Am. J. Hum. Genet.. 48. 137-144 (1991) with the oligonucleotide sequences as follows: sense 5' GAA-ACT-GGC-
  • the DNA purifying reagent was removed by centrifugation at 15,000 x g for 10 seconds to collect the eluted impurities in the spin tube. A second and third wash with a DNA purifying reagent was performed in the same way for a total of three washes. Each disk containing purified DNA was transferred to a 0.6 ml siliconized tube for amplification analysis. The waste collected in the spin tube was discarded.
  • each amplification reaction contained IX amplification buffer (Promega, Madison, Wl), 1.5 mM MgCl 2 , 200 ⁇ M each 28
  • the gel and running buffer contained 0.125 ⁇ g per ml ethidium bromide to allow visualization of the amplified DNA on a transiluminator.
  • a factor V amplification product of 223 base pairs was observed for each of the six samples following gel electrophoresis.
  • the amplification results showed that the rapid solid phase DNA purification method, which was performed in about 5 minutes, gave substantially pure DNA from fresh or frozen whole blood.
  • Example 3 Solid Phase Purification of DNA from Several Biological Samples At least two DNA samples were prepared from the following biological materials (human source except where noted): whole blood, bone marrow, saliva, buccal cells, cultured K562 lymphoblast cells, Drosophila melanogaster (D. melanogaster) fruit flies, alfalfa leaves and Escherichia coli (E. coli) bacteria. Samples of whole blood, bone marrow, saliva and buccal cell scrapes were applied to S&S 903 paper, dried and then sampled by punching a 3 mm diameter disk with a hole punch. Samples of plant or animal tissues were prepared by pressing adult D.
  • biological materials human source except where noted
  • PCR solution was as described in Example 2 above except for the primers, which are given below.
  • Whole blood, bone marrow, saliva, buccal cells, D. melanogaster tissue, and K562 cells were amplified using primers specific to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene sequences given in the mRNA Capture Kit (United States Biochemical Corporation, Cleveland, OH).
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • GAPDH was 30 cycles of 94°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute.
  • DNA samples from whole blood were also tested for the presence of extrachromosomal DNA by using the mitochondrial primers MT-1 and MT-2 described by Wang et al, BioTechniques 17, 76-82 (1994) and the amplification program described above for GAPDH.
  • Alfalfa and E. coli DNA samples were tested by using primers specific for 16s-like ribosomal DNA described by Schmidt et al., BioTechniques, 11, 176-177 (1991).
  • the .amplification program was 30 cycles of 94°C for 1 minute, 50°C for 1 minute and 72°C for 2 minutes.
  • DNA purifying reagent Gentra Systems, Inc., Minneapolis, MN
  • the DNA purifying reagent was removed by centrifugation at 15,000 x g for 10 seconds to collect the eluted impurities in the 2 ml spin tube.
  • a second and third wash with DNA purifying reagent was performed in the same way for a total of three washes.
  • each insert was transferred to a clean 2 ml receiver tube.
  • DNA Eluting Reagent 100 ⁇ l DNA Eluting Reagent were pipetted into the insert containing the solid support and heated for 15 minutes at 80° C in a dry block heater (e.g., VWR Scientific Products Catalog No. 13259-007) fitted with an aluminium block containing 12 mm diameter wells.
  • the DNA Eluting Reagent contained 10 mM Tris, 1 mM NaOH and 0.1 mM EDTA, pH 10.9. After heating, each sample was centrifuged for 20 seconds at 1 ,000 x g to collect the purified DNA.
  • a cartridge was constructed using a standard 1 ml polypropylene syringe (Catalog Number 309602, Beckton Dickinson, Franklin Lakes, NJ) into which was inserted a solid support.
  • the solid support was comprised of cellulose acetate (Filtrona ® , American Filtrona, Richmond, VA) of dimensions about 5 mm diameter x 27 mm long.
  • the solid support had been treated previously with 500 ⁇ l Lysing Reagent and RNase A and allowed to 31
  • the Lysing Reagent contained 0.5% SDS, 0.1 M Tris, 0.1 M EDTA to which was added 0.04 mg/ml RNase A (about 100 units RNase A per mg).
  • RNase A about 100 units RNase A per mg.
  • Two whole blood samples and two K562 cultured cells samples each containing about 2 million cells in a 300 ⁇ l volume were each pipetted into a cartridge supported in a vertical position. After incubating for 15 minutes at room temperature to allow the cells to lyse and the RNase to digest RNA present in the samples, a 300 ⁇ l volume of DNA purifying reagent (GENERATION ® DNA Purification Solution, Gentra Systems, Inc., Minneapolis, MN) was introduced via 2.5 mm i.d.
  • GENEATION ® DNA Purification Solution Gentra Systems, Inc., Minneapolis, MN
  • 300 ⁇ l volume of DNA purification reagent was pumped into the cartridge and incubated 1 minute. This washing step was repeated once more for a total of three washes with DNA purifying reagent.
  • the solid support in the cartridge was rinsed by pumping 300 ⁇ l of DNA Eluting Reagent through it. To remove the purified DNA from the solid support, 300 ⁇ l of DNA Eluting Reagent was pumped into the cartridge.
  • the cartridge was plugged at both ends and incubated at 60°C for 30 minutes in a gravity convection oven. Alternately, an appropriate microwave oven may be used. The cartridge may be heated for 25 minutes at 30% power in a 1100 W Sharp microwave oven.
  • the DNA Eluting Reagent which contained the purified DNA, was pumped out of the cartridge and into a 1.5 ml microcentrifuge tube.
  • the DNA eluting reagent can be heated to a temperature greater than 60 °C and then pumped onto the cartridge.
  • DNA purifying reagent Gentra Systems, Inc., Minneapolis, MN
  • seven restriction enzymes were used to test their ability to digest the purified DNA.
  • a volume of 300 ⁇ l whole blood from a single individual was pipetted onto S&S 903 paper and dried at room temperature. Then seven 5 mm diameter disks were punched out and each placed into a 0.6 ml tube.
  • a volume of 200 ⁇ l DNA purifying reagent was added to each tube and incubated for 15 minutes. After pipetting up and down three times to mix, the DNA purifying reagent was discarded. This washing procedure was repeated twice for a total of three washes.
  • each restriction enzyme solution contained the appropriate buffer supplied by the manufacturer (New England Biolabs, Beverly, MA) and 2.5 mM spermidine (Sigma Chemical Company). The following enzymes were tested with the units added to each reaction specified: Pst 1 (12 units), Hind Ifl (20 units), Eco RI (20 units), Msp I (20 units), Bam HI (20 units), Hpa I (5 units), and H ⁇ e DI (10 units). The samples were digested at 37 °C for 4 hours during which time the digested DNA fragments were released from the solid support into the digestion solution.
  • each sample tube was pierced with a 27 gauge needle and placed into a clean 0.6 ml tube.
  • the digestion solution was collected by centrifuging at 2,000 x g for 2 minutes.
  • a 20 ⁇ l volume was removed from each sample and electrophoresed through a 1% agarose gel for 12 hours at 22 volts.
  • Example 7 Evaluation of Detergents on Solid Support Treatment Different detergents were tested to determine the best type of detergent needed to optimize DNA yields. The following types of detergents were tested:
  • Controls were set up with no detergent and with no added sample.
  • Each of the polysulfone solid supports used had a circumference of 25.31 mm and a height of 9.73 mm (Filtrona ® , Lot # 18475, American Filtrona, Richmond, VA). A volume of 360 ⁇ l of a 1% detergent solution was applied to each solid support to saturate it at room temperature for at least 16 hours. Duplicate solid supports were prepared for each treatment. The solid supports were placed into the insert of a 2 ml spin tube (Spin-X, Catalog
  • a whole blood sample containing about 2 million cells in 300 ⁇ l was applied to each solid support and allowed to incubate for at least 1 minute at room temperature to allow the cells to lyse.
  • a 200 ⁇ l volume of DNA purifying reagent (GENERATION ® DNA Purification Solution, Gentra Systems, Inc., Minneapolis, MN) was then added. After a 1 minute incubation, the DNA purifying reagent was removed by centrifugation at 15,000 x g for 10 seconds to collect impurities in the 2 ml collection tube. This was repeated twice for a total of 3 washes. The waste solution was removed from the receiving tube in between the second and the third wash.
  • the solid support was then rinsed by adding 200 ⁇ l of DNA Eluting Reagent (1 mM Tris, 0.001 mM EDTA, 5mM NaOH) and centrifuging at 15,000 x g for 10 seconds. To remove the purified DNA from the support, the insert containing the solid support was transferred to a clean receiver tube and 200 ⁇ l of DNA Eluting Reagent was added to it. The solid support was then incubated at 80°C for 10 minutes in a dry block heater (e.g., VWR Scientific Products Catalog No. 13259-007) and the 34
  • DNA Eluting Reagent which contained the purified DNA was removed by centrifugation at 15,000 x g for 20 seconds.
  • Genomic DNA yields and PCR amplification yields were measured to evaluate the purified DNA.
  • a volume of 10 ⁇ l purified DNA was mixed with 1 ⁇ l 10X standard tracking dye and loaded into a 0.7% agarose gel containing 0.125 ⁇ g ml ethidium bromide in the gel and running buffer.
  • the DNA was electrophoresed for 15 minutes at 80 volts and the bands examined visually for band intensity on a UV transilluminator.
  • the PCR amplification assays was conducted by adding 2.5 ⁇ l purified DNA sample directly to a 22.5 PCR amplification mix for a total amplification volume of 25 ⁇ l.
  • Each amplification reaction contained IX amplification buffer (Promega, Madison, Wl), 1.5 mM MgCl 2 , 200 ⁇ M each deoxynucleotide, 1.25 units Taq DNA Polymerase (Promega, Madison, Wl), and 1 ⁇ M each primer.
  • Primers were sequences specific to the human betaglobin gene: sense 5' CCT-GGC-TCA-CCT-GGA-CAA-CCT-CAA 3' (SEQ ID NO:3) and antisense 5'TAG-CCA-CAC-CAG-CCA-CCA-CTT-TCT 3' (SEQ ID NO:4).
  • the samples were amplified using 35 cycles of 94°C for 1 minute, 70°C for 1 minute, and 72°C for 2 minutes. Then 10 ⁇ l of the amplified DNA were loaded into a 2% agarose gel containing 0.125 ⁇ g/ml ethidium bromide in the gel and running buffer. The samples were electrophoresed at 80 volts for 45 minutes and the 1.1 kb DNA bands were visualized on a UV transilluminator. The detergents were evaluated by visual ranking of the band intensity for both genomic and amplified DNA as shown in Table 2.
  • Non-ionic detergents showed no substantial improvement in DNA yield over the controls.
  • the cationic detergent used showed approximately a two fold increase in genomic DNA yield.
  • the two anionic detergents gave the best yield for genomic and amplified DNA.
  • Example 8 Effects of NaOH concentration. Detergent Type and Solid Support Density on DNA Amplification
  • a 300 ⁇ l whole blood sample was added to each solid support, washed and eluted as described in Example 7, except using a 150 ⁇ l volume of DNA purifying reagent (GENERATION ® DNA Purification Solution, Gentra Systems, Inc., Minneapolis, MN).
  • the purified DNA was analyzed by PCR amplification and UV spectrophotometry.
  • the PCR protocol used was identical to that described in Example 7. Genomic DNA yields were examined by UV spectrophotometry and by 0.7% agarose gel electrophoresis. To quantitate DNA by UV spectrophotometry, 50 ⁇ l of purified DNA was first added to 950 ⁇ l deionized water.
  • the UV absorbance was then measured at wavelengths of 320 (background) nm, 260 nm and 280 nm.
  • the yield was calculated as A 260 x 50 x dilution factor x elution volume.
  • the results showed that there was no detectable difference in DNA yields for solid support treatments carried out using no NaOH and 5 mM NaOH. There was a 2.8 fold increase in DNA yields using SDS over CHAPS. A 75% increase using the high density solid support over the low density solid support was observed.
  • the polyolefin solid supports were treated with 8 solutions containing 0.5% or 2.0% sodium dodecyl sulfate, 0 or 50 mM EDTA, and 0 or 100 mM NaCI.
  • a 200 ⁇ l whole blood sample was added to each solid support, washed, and eluted as described in Example 7.
  • the purified DNA was collected and the genomic DNA yield examined by UV spectrophotometry as described in Example 8. The relative yields were further visualized by agarose gel electrophoresing the purified DNA samples on a 0.7% agarose gel, as described in Example 7. No differences in DNA yields were observed between 0 and 50 mM NaCI.
  • Optimal anionic detergent concentrations were estimated by treating the polyolefin solid support with solutions of sodium dodecyl sulfate (SDS) ranging from 0.2 - 1.6%. Solid supports were treated with 8 solutions containing 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6% SDS as described in Example 7. A 200 ⁇ l whole blood sample was added to each solid support, washed, and eluted as described in Example 7. The purified DNA was collected, and the genomic DNA yield visualized by agarose gel electrophoreses as described in Example 7. Visual estimation of the band intensity on the 0.7% agarose gel showed that all concentrations of SDS showed good genomic DNA yields although the two highest yields were observed at SDS concentrations of 1.0 and 1.2%.
  • SDS sodium dodecyl sulfate
  • DNA from 200 ⁇ l blood samples was purified using two 400 ⁇ l washes with DNA purification reagent (GENERATION ® DNA Purification Solution, Gentra Systems, Inc., Minneapolis, MN) and one wash with DNA Eluting Reagent. 37
  • a cartridge was constructed using a standard 1 ml polypropylene syringe (Catalog Number 309602, Beckton Dickinson, Franklin Lakes, NJ) into which was inserted a solid support.
  • the solid support was comprised of cellulose acetate (Filtrona ® , American Filtrona, Richmond, VA) of dimensions about 5 mm diameter x 27 mm long. 38
  • the cartridge is equipped with two inlet ports at the top.
  • One inlet, the sample port is used for the application of the biological sample to the solid support.
  • An optional feature on the sample port is a self-sealing mechanism that seals the sample port off after sample has been tr.ansferred through it.
  • the second inlet serves as a reagent port.
  • An optional feature on both inlet ports is a protective breakaway seal.
  • At the bottom of the solid support is an optional diffuser with a pore size suitable for the passage of cellular debris, proteins and lipid molecules. The diffuser allows for an equitable dispersal of biological material across the cross section of the cartridge, and prevents unequal buildup of biological material anywhere below the solid support.
  • the outlet of the cartridge comes equipped with a protective cap.
  • the purified DNA is collected in a collection tube that consists of a 0.5 ml conical tube with a snap cap for easy and contamination free storage.
  • a spin tube (Spin-X, Corning Costar No. 9424, Cambridge, MA) was used with an insert into which was placed a solid support.
  • the solid support used was a polyolefin plug (American Filtrona, Richmond, VA) which was placed into the insert.
  • the insert consists of a flanged top to hold it in the spin tube and a perforated bottom to allow fluids to pass through while supporting the solid support.
  • a cap tethered to the spin tube was used to cover the insert.
  • An example of a commercially available spin tube is given in Example 1.
  • the biological material passes through the perforated bottom and is collected at the bottom of the spin tube. When used, the biological material is applied to the solid support.
  • Eluting Reagent is then added to the solid support.
  • the spin tube is then placed in a centrifuge and subjected to centrifugal forces that draw out the biological material, the purifying reagent and the purified DNA through the solid support during the purification process.
  • Example 13 Testing the use of Polyolefin Solid Supports in a 96-well Plate System.
  • hydrophilic polyolefin solid supports (R- 18495, American Filtrona Company, Richmond, VA) were inserted into wells in a 96-well plate with a well capacity of 800 ⁇ l (Unifilter plate manufactured without a filter by Polyfiltronics, Rockland, MA).
  • the solid supports were 39
  • the aforementioned 96-well sample processing plate was placed on top of another 96 well plate with a 2 ml well capacity which served as a waste collection plate.
  • a volume of 100 ⁇ l whole blood was applied to each solid support and allowed to incubate for 1 minute.
  • the solid supports were then subsequently washed twice by adding a volume of 200 ⁇ l DNA purifying reagent (GENERATION ® DNA Purification Solution, Gentra Systems, Inc., Minneapolis, MN) to the well and incubating for 1 minute.
  • the waste material was subsequently removed by centrifuging for 1 minute at 1500 x g in a Jouan C412 centrifuge with an M4 rotor fitted with a microplate carrier (Jouan, Winchester, VA).
  • the solid supports were then washed with 100 ⁇ l DNA Eluting Reagent and centrifuged as above without incubation.
  • the processing plate was transferred to a clean standard polystyrene 96-well plate and a volume of 100 ⁇ l of DNA Eluting Reagent was added to each well.
  • the stacked processing plate and sample collection plate were placed into a convection oven (BioOven I, St.
  • Example 14 The Effect of treating a Solid Support with RNase A
  • Lysing Reagent (0.5% SDS, 0.1 M Tris base, 0.1 M EDTA disodium salt) was applied to a cellulose acetate solid support (American Filtrona, Richmond, VA) and allowed to dry at room temperature.
  • a second solid support was treated with Lysing Reagent which also contained 0.04 mg/ml RNase A (at 4 mg/ml from Gentra).
  • the solid supports had a diameter of 8 mm and a length of 6.75 mm.
  • the two treated solid supports were then placed into the insert of a spin tube (as described in Example 8), and a volume of 150 ⁇ l of E. coli overnight bacterial culture added directly to each solid support.
  • E. coli bacterial cultures contain large amounts of RNA and serve as a useful model to test the efficacy of immobilized 40
  • RNA Digesting Enzyme The s-amples were then incubated at 37°C for 12 minutes to enable RNA digestion. They were subsequently washed three times with a volume of 150 ⁇ l DNA purifying reagent (GENERATION ® DNA Purification Reagent, Gentra Systems, Inc., Minneapolis, MN). Then 150 ⁇ l of Basic Eluting Reagent (10 mM Tris, 0.1 M EDTA and 1 mM NaOH) was added to the solid support for 20 minutes at room temperature. The nucleic acids were eluted by centrifuging for 10 seconds at 15,000 x g.
  • DNA purifying reagent GenERATION ® DNA Purification Reagent, Gentra Systems, Inc., Minneapolis, MN
  • Basic Eluting Reagent 10 mM Tris, 0.1 M EDTA and 1 mM NaOH
  • a volume of 10 ⁇ l was analyzed for the presence of DNA by electrophoreses through a 1% agarose gel for 60 minutes at 80 V. Examination of the gel on a UV transilluminator showed clearly the presence of a prominent low molecular weight smear (approximately 0.1 - 1.4 kb) corresponding to RNA in the sample purified using the solid support with the solid support treated with Lysing Reagent. In contrast, the sample purified with the Lysing Reagent plus RNase lacked the low molecular weight RNA smear showing that the presence of RNase was effective in removing contaminating RNA.
  • the eluting reagent was optimized so as to offer the best DNA yields from the solid support and produce high PCR yields without interfering with the PCR buffering system.
  • Optimal concentrations of base either NaOH or KOH, Tris buffer and chelating agent (EDTA) were tested on DNA amplification yields using PCR.
  • Conditions were tested by adding a 25 ⁇ l blood sample to a cellulose solid support contained in a spin tube. The cellulose solid support was subsequently washed 3 times with 200 ⁇ l DNA purifying reagent (GENERATION ® DNA Purification Solution, Gentra Systems, Inc., Minneapolis, MN) and 2 times with DNA Eluting Reagent. The samples were all treated identically except for the concentration of base in the DNA Eluting Reagent. Concentrations of 1-8 mM NaOH in the DNA Eluting Reagent were tested.
  • a TaqMan 7700 Quantitative PCR system, using a ⁇ -actin amplification target was used for DNA amplification, as per the manufacturer's recommendation (Perkin Elmer, Applied Biosystems Division, Foster City, CA).
  • a dilution series was prepared for each test sample and the starting quantity of DNA computed for each. All test samples were compared to the DNA sample at the highest dilution since 41
  • inhibitors would also be maximally diluted at the highest dilution. If a diluted DNA test sample gave a similar yield to the DNA sample at maximum dilution, (after adjusting by the dilution factor), then no amplification inhibition in that test sample was assumed.
  • Tris buffer concentrations and concentrations of EDTA were tested to determine which concentrations optimized high PCR yields and low amplification inhibition. It was observed that lowering the concentration of Tris buffer from 1 mM to 0.1 mM, and lowering the concentration of EDTA from 0.1 mM to 0.001 mM significantly reduced amplification inhibition.
  • a volume of 25 ⁇ l of blood was applied to a cellulose solid support contained in a spin tube (Spin-X, Corning Costar No. 9424, Cambridge, MA) and allowed to absorb for
  • Cellulose solid supports were treated with Lysing Reagents having different compositions for comparison to the untreated solid support.
  • the first composition was composed of 0.5% SDS, 0.1 M Tris and 0.1 mM EDTA, while the second composition was composed of 1% SDS, 10 mM Tris and 0.1 mM EDTA.
  • Cellulose paper was inserted into a spin tube (Spin-X, Corning Costar No. 9424, Cambridge, MA) and treated with the two aforementioned compositions. The treated cellulose papers were then allowed to dry for at least 16 hours at room temperature. Untreated cellulose paper was also used as a comparison to the treated samples. A volume of 25 ⁇ l of blood was applied to each cellulose solid support and allowed to incubate for 5 minutes.
  • the lysing matrix comprised of a cylindrical polyolefin solid support matrix with a diameter of 8 mm, and a height of 8 mm (Filtrona ® , Catalog No. 18475, American Filtrona, Richmond, VA).
  • the polyolefin solid support matrix had been previously saturated with a solution containing 1% SDS and 20 ⁇ g/ml RNase A and was subsequently dried.
  • DNA purifying reagent (GENERATION ® DNA Purification Solution, Gentra Systems, Inc., Minneapolis, MN) were added to the sample and allowed to incubate for 1 minute at room temperature. Impurities were collected in the 2 ml spin tube by centrifuging the spin tube at 12,000 x g for 10 minutes. The insert containing the solid support was transferred to a second spin tube and the first spin tube was discarded. Another 400 ⁇ l DNA purifying reagent was added to the solid support, incubated for 1 minute at room temperature, and the spin tube centrifuged at 12,000 x g for 10 minutes. A 200 ⁇ l volume of DNA Eluting
  • the seven purified samples from example 18 were further analyzed for concentration and yield.
  • a 1 :50 dilution of each sample was prepared in deionized water along with a blank containing DNA Eluting Reagent. Absorbances at 320 nm (background), 260 nm, and 280 nm were read using a Beckman DU64 Spectrophotometer (Beckman
  • the DNA concentration was calculated as follows: (A 260 - A 320 ) X 50 (DNA Extinction Coefficient) X 50 (Dilution Factor) The average for the seven samples was found to be 41 ⁇ g/ml. This concentration was then multiplied by the volume of the sample (200 ⁇ l) to give an average yield of 8.2 ⁇ g for each of the seven samples.
  • the DNA concentration may be adjusted by dilution or concentration as needed. If the DNA is too concentrated, it may be diluted in a diluent such as deionized water. If the DNA is too dilute, it may be concentrated by using a standard 45
  • alcohol salt precipitation method sodium chloride is added to 100 mM with two volumes (relative to DNA sample volume) of 100% ethanol.
  • the sample is mixed by inverting the tube and is centrifuged at 15,000 x g for 5 minutes to pellet out the DNA.
  • the DNA pellet is washed by adding 3 volumes of 70% ethanol, inverting the tube and centrifuging at 15,000 x g for 1 minute. After discarding the supernatant, the pellet is allowed to air dry for 15 minutes. Then a hydration solution such as deionized water is added to prepare the desired, more concentrated solution.
  • DNA size for each of the seven samples of Example 18 was determined by comparison to the 23.1 kb band of lambda DNA digested with Hind HI. A volume of 10 ⁇ l from each of the seven 200 ⁇ l DNA samples was mixed with tracking dye and loaded into a 1% agarose gel. The samples were electrophoresed at 80 volts for 1 hour in IX TAE running buffer. Both gel and running buffer contained 0.125 ⁇ g/ml ethidium bromide so that DNA could be visualized on a transilluminator. Comparison of DNA samples with the marker lanes showed that greater than 95% of the DNA exceeded the 23.1 kb marker indicating that the DNA was of substantially high molecular weight.
  • Example 21 Testing the Suitability of Purified DNA for PCR Amplification and Subsequent Restriction Enzyme Digestion.
  • Each of the seven samples from Example 17 were tested to see if they were suitable for analysis by PCR. A 2.5 ⁇ l volume from each of these seven samples was added to a 22.5 PCR amplification mix for a total amplification volume of 25 ⁇ l.
  • Each amplification reaction contained IX amplification buffer (Promega, Madison, Wl), 1.5 mM Mg Cl 2 , 200 ⁇ M each deoxynucleotide, 1.25 units Taq DNA Polymerase (Promega, Madison, Wl), and 1 ⁇ M each primer.
  • Primers were sequences specific to a region of the ⁇ LA- ⁇ gene used for hereditary hemochromatosis genetic screening, 5'-TGG-CAA-GGG-TAA-ACA- GAT-CC-3' (SEQ ID NO:5) and 5'-CTC-AGG-CAC-TCC-TCT-CAA-CC-3' (SEQ ID 46).
  • a 10 ⁇ l volume of the amplification reaction was mixed with 5 ⁇ l of the Rsa I restriction enzyme mix containing 3.3 mg bovine serum albumin, 3.3 units Rsa I, and 1.5 ⁇ l 10X restriction enzyme buffer (all components from New England BioLabs, Beverley, MA). The samples were incubated at 37 °C for 30 minutes to allow the restriction enzyme to digest the amplified DNA.
  • a 15 ⁇ l reaction volume of each of the seven samples was electrophoresed in a 2% agarose gel run at 85 volts for 60 minutes. Ethidium bromide was present at 0.125 ⁇ g/ml in the gel and running buffer to allow visualization of the bands on a transilluminator.
  • Example 22 Restriction Enzyme Digestion and Southern blotting Analysis of DNA purified using DNA Purifying Reagents.
  • DNA from seven 5 mm diameter dried blood spots was purified, digested and electrophoresed as described in Example 6. Following electrophoreses, the restriction fragments were transferred over a 7 hour period to a nylon membrane (Biotrans+TM, ICN Biomedicals, Inc., Irvine, CA) by Southern blotting using a transfer solution containing 0.4 N 47
  • the nylon blot was hybridized for 14 hours at 65 °C in HYB-9 ® Hybridization Solution (Gentra Systems, Inc., Minneapolis, MN) and then washed according to the manufacturer's instructions.
  • the probe was prepared from an amplified 300 bp region of the glyceraldehyde 3-phosphate dehydrogenase labeled with 32 P-labeled dCTP using a random priming kit (Amersham Life Science, Inc., Arlington Heights, IL).
  • the membrane was placed against X-ray film (XAR5, Eastman Kodak Company, Rochester, NY) between two intensifying screens at -80°C for 14 hours.
  • the resulting autoradiogram showed bands in each of the lanes corresponding to digested DNA complementary to GAPDH sequences in the genome.
  • Test materials Whole blood (Memorial Blood Center of Minneapolis), 8E5 cultured cells (Folk, et al, 1986, Guenthner et al., 1998), or phosphate- buffered saline (PBS) were loaded on lysing matrix contained in two different vessel formats (Capture PlateTM, Gentra Systems Inc., Minneapolis, MN and Capture ColumnTM, Gentra Systems Inc., Minneapolis, MN).
  • the Capture ColumnTM consists of a lysing matrix enclosed in an insert which is placed in a centrifuge tube.
  • the Capture PlateTM consists of 96 flow-through wells each enclosing a lysing matrix. The bottom of each well has a tapered exit port.
  • Sample elution was performed by adding 200 ⁇ l DNA Elution SolutionTM to each well. Capture ColumnsTM were heated at 99 °C for 10 minutes in a block heater and Capture PlatesTM were heated for 25 minutes at 30% power in a 1100W Sharp microwave oven. Following centrifugation the eluates were ready for amplification.
  • Amplification A 2 ⁇ l sample of each eluate was amplified to detect contaminating DNA in the samples without DNA (PBS).
  • HLA-H primers (Feder et al, 1996) were used for experiments using blood and HIV-1 (gag) primers (Guenthner et al., 1998) were used for 8E5 cultured cells.
  • the cycling conditions used were: 40 cycles of: 94 °C for 30 seconds, 58 °C for 30 seconds, and 72 °C for 30 seconds in a PE2400 or 9700 thermal cycler.
  • DNA is purified from whole blood, bone marrow, buffy coat, body fluids, cultured cells and solid tissue on a lysing matrix (Capture ColumnTM, Gentra Systems Inc., Minneapolis, MN). Prior to purification on the Capture ColumnTM, samples were processed as follows: i. Whole Blood and Bone Marrow
  • the samples are thawed quickly in a 37° C water bath and kept on ice until use. If the blood or bone marrow is not frozen, it is recommended that it be stored at 4°C. A 200 ⁇ l volume of blood or bone marrow may be used for DNA purification. If it is necessary to purify DNA from a larger volume, a buffy coat sample may be prepared, i. Buffy Coat
  • DNA may be purified from up to 200 ⁇ l buffy coat preparation containing a maximum of 10 million white blood cells, iii. Body Fluids
  • Examples include saliva, synovial fluid, cerebrospinal fluid, urine, amniotic fluid, plasma and serum.
  • Body fluid samples are collected and stored at 4°C and used as quickly as possible. Alternatively, they may be stored frozen at -80 °C. For body fluids with low cell numbers, it is preferable to concentrate samples by centrifugation. Cells from a 3-40 ml volume of body fluid were pelleted by centrifuging at 2,000 x g for 10 minutes. The 50
  • Fresh samples and those stored frozen at -80 °C were used. The samples were kept on ice at all times to reduce DNase activity. 20 mg tissue was added to a 1.5 ml microfuge tube containing 30 ⁇ l cold PBS 1 ' (preferably containing 1 mM EDTA to reduce Dnase activity) and was quickly homogenized with a microfuge tube pestle. The sample was placed on ice to allow cell clumps to settle for 2 to 10 minutes. The upper 200 ⁇ l cell suspension was then removed excluding any cell clumps, vi. Gram-negative Bacteria Fresh samples and those stored frozen at -80° C were used. The samples were kept on ice. Typically, an overnight culture contains 1-3 billion cells per ml. However, due to the smaller genome size of Gram-negative bacteria, up to 3 billion cells were applied to the column for DNA purification. The culture was centrifuged, washed, resuspended and applied to the column.
  • PBS with EDTA 8 gm NaCI, 0.2 gm KCl, 2.72 gm Na 2 HPO 4 «7H 2 O, 0.24 gm KH 2 PO , 0.372 gm EDTA disodium salt dissolved in ultrapure water, brought up to a volume of 1,000 ml and autoclaved. 51
  • Example 25 DNA Purification on a Lysing Matrix.
  • Example 24 The samples from Example 24 were purified on a lysing matrix (Capture ColumnTM Gentra Systems, Minneapolis, MN) as follows: i. Sample Purification 1. A volume of 200 ⁇ l of a well-mixed sample was added to the lysing matrix (Capture ColumnTM Gentra Systems, Minneapolis, MN) as follows: i. Sample Purification 1. A volume of 200 ⁇ l of a well-mixed sample was added to the lysing matrix (Capture ColumnTM Gentra Systems, Minneapolis, MN) as follows: i. Sample Purification 1. A volume of 200 ⁇ l of a well-mixed sample was added to the
  • Capture ColumnTM and allowed to absorb at room temperature for at least 1 minute or up to 1 hour.
  • DNA Purification SolutionTM DNA Purification SolutionTM, Gentra Systems, Minneapolis, MN
  • the Capture ColumnTM was centrifuged for 10 seconds at 2,000-12,000 x g. A waste volume of 600 ⁇ l was collected in the waste collection tube. 4. A volume of 400 ⁇ l DNA Purification SolutionTM was added again to the Capture ColumnTM and allowed to incubate for 1 minute at room temperature. 5. The Capture ColumnTM was then centrifuged for 10 seconds at
  • the Capture ColumnTM was centrifuged as described above.
  • the Capture ColumnTM was transferred to a DNA collection tube and the waste was discarded.
  • a volume of 200 ⁇ l DNA Elution SolutionTM was added and allowed to incubate for 10 minutes in a dry block heater preheated to 99 °C.
  • the Capture ColumnTM was then centrifuged as previously described to release DNA from the lysing matrix.
  • the purified DNA is stable for at least 3 months at 4°C. For long term storage, it can be stored at -20°C.
  • Example 26 DNA Quantification Using UV Spectrophotometric Analysis. Water is often used for diluting DNA for UV spectrophotometric analysis. However, there can be significant variability in both the A 26O A 2g0 ratio and the yield determined when water is used as a diluent. Commercial buffers such as Tris-based or phosphate based buffers may be used to overcome these problems. Consistent results are obtained by diluting DNA samples in TE BufferTM (10 mM Tris, 11 mM EDTA pH 8.0) (Gentra Systems, Minneapolis, MN) as described below. Additional consistency is obtained by using a masked quartz cuvette (e.g., Beckman Instruments, Inc. Semi- Microcell Masked Cuvette Cat. No. 533041). ii. Sample Preparation and UV Spectrophotometric Analysis
  • a blank solution was prepared by diluting a volume of 10 ⁇ l DNA Eluting reagent (DNA Elution SolutionTM, Gentra Systems, Minneapolis, MN) with 190 ⁇ l TE BufferTM in a 0.6 ml microfuge tube.
  • the diluted samples were then vortexed at high speed for 5 seconds.
  • a volume of 200 ⁇ l diluted sample was used to determine yield and purity using a UV spectrophotometer by determining absorbances at 26 nm, 280 nm and 320 nm. 53
  • Fresh or frozen biological samples were collected and processed as described in Example 24. Large sample volumes (e.g. 300 ⁇ l) were pipetted onto a flat solid support ( Collection Cards TM, Gentra Systems, Minneapolis, MN), dried and then sampled by punching out a disk prior to DNA purification. Samples may be stored on the Collection Cards at room temperature for at least 9 months or at -20 °C for long term storage.
  • the sample was removed from the Collection Card by punching a 3 mm disk with a clean hole punch, and then purified in a 96-well plate, a 0.2 ml or 0.6 ml microfuge tube.
  • the 3 mm disk was placed in a well of a 96-well plate and the plate was positioned in a robotic workstation.
  • the samples may be processed manually in a 96-well plate using a multichannel pipet or in a 0.2 or 0.6 ml tube using a micropipet).
  • a volume of 200 ⁇ l of DNA purifying reagent (DNA Purification Solution, Gentra Systems, Minneapolis, MN) was added and allowed to incubate for 15 minutes at room temperature causing the DNA to remain bound to the disk while the contaminants were released.
  • the solution was mixed by pipetting and then removed. This process was repeated twice. A volume of 200 ⁇ l 100% isopropanol or 100% ethanol was then added and allowed to incubate for 1 minute at room temperature. The alcohol was removed, and the alcohol wash repeated. The disk was then dried at room temperature for at least 1-16 hours to evaporate the alcohol. After drying, the sample disks were light orange to white in color. The purified disks are stable for at least 9 months at room temperature or at -20 °C for long term storage.
  • the disk was purified in a 0.2 or 0.6 ml amplification tube, at least 50 ⁇ l amplification solution was added directly to the tube. If the disk was purified in a 96-well flat bottom plate, the purified DNA sample disk was transferred to an amplification tube at least 50 ⁇ l amplification solution was then added. The disk was completely submerged in the amplification solution. The sample was then amplified using standard conditions. 2. The disk may be stored in amplification solution for at least 4 months at room temperature. Re-Use for DNA Sample Disks The purified DNA disks may be washed and re-used at least 5 times. 55
  • the disk is removed from the amplification tube and subsequently transferred to a filter insert contained within a 2 ml receiver centrifuge tube.
  • DNA purifying reagent (DNA Purification Solution, Gentra Systems, Minneapolis, MN) is pipetted into the filter insert and centrifuged at 13,000-16,000 x g for 5 seconds to wash the disk. This wash is repeated once more for a total of 2 washes.
  • the wash solution in the receiver tube is discarded and the washed disk is transferred to a new amplification tube.
  • the amplification solution is added and amplified as described above.
  • Example 28 Purification of DNA from Biological Samples on a Lysing Matrix Disk and Subsequent Amplification
  • DNA was purified from five biological samples on lysing matrix disks (Capture Disk, Gentra Systems, Minneapolis, MN). The quality of the DNA was ascertained using PCR amplification.
  • Biological samples were collected and prepared as follows: human whole blood and bone marrow were collected in tubes containing EDTA (Becton Dickinson No. 6457); a buffy coat was isolated from 0.2 ml whole blood collected in EDTA; a 0.5 ml urine sediment was prepared by centrifuging a 40 ml urine sample at 800 x g for 10 minutes; and 1 million K562 cells were suspended in 0.3 ml culture medium.
  • DNA was purified from 3 ⁇ l of each prepared sample as described in the following sections. Each 3 mm disk containing purified DNA was amplified in a 50 ⁇ l reaction using primers specific of the HLA-H locus (used for hereditary hemochromatosis screening). A 388 base pair amplification product was expected.
  • White blood cells were isolated from the red blood cells in the sample by using -in RBC lysis reagent (PUREGENE ® RBC Lysis Solution, Gentra Systems, Minneapolis, MN). Alternatively, the buffy coat may be prepared by centrifuging the sample at 800 x g for 10 minutes at room temperature. A thin layer of white blood cells (buffy coat) should be visible between the upper plasma layer and the lower red blood cell layer. The upper plasma layer was removed and the buffy coat collected with a pipet and kept on ice.
  • ACD citrate
  • heparin heparin
  • a 3 ⁇ l suspension of buffy coat containing at least 2, 100 white blood cells was then added to the Capture Disk, ii.
  • body Fluids examples include saliva, synovial fluid, cerebrospinal fluid, urine, amniotic fluid, plasma and serum.
  • samples are concentrated by centrifugation.
  • Cells from a 3-40 ml volume of body fluid are pelleted by centrifuging at 800 x g for 10 minutes. The supernatant is removed and the pellet is suspended in the residual fluid and kept on ice.
  • the number of cells was determined using a hemacytometer or other cell counter. A 3 ⁇ l suspension containing at least 2,100 cultured cells was added to the lysing matrix disk. iv. Gram-negative Bacteria
  • the above samples are purified as follows: 1. A volume of 3 ⁇ l well-mixed sample was pipetted onto a 3 mm lysing matrix disk in the insert of a 2 ml spin tube. The sample was allowed to absorb at room temperature for at least 1 minute or up to 2 hours.
  • DNA purifying reagent DNA Purification Solution, Gentra Systems, Minneapolis, MN
  • the spin tube was centrifuged at 2,000-16,000 x g for 10 seconds to collect the wash solution in the receiver tube.
  • the disk was placed directly into an amplification tube. A volume of 25-50 ⁇ l amplification master mix was added to the tube, and the DNA was amplified using standard conditions. The disk was stored in the amplification solution for at least 4 months at room temperature.
  • the disk may be re-used for further amplification.
  • the protocol is as follows: The disk is washed twice with 200 ⁇ l of DNA Purification Solution and centrifuged at 2,000-16,000 x g for 5 seconds before amplification.

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Abstract

La présente invention concerne des réactifs d'élution et des procédés permettant d'isoler l'ADN de matières biologiques.
PCT/US1999/002190 1998-02-02 1999-02-02 Reactifs d'elution, procedes et trousses permettant d'isoler un adn WO1999039010A1 (fr)

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JP2000529467A JP2004500002A (ja) 1998-02-02 1999-02-02 Ｄｎａを単離するための溶出試薬、方法およびキット
CA2319691A CA2319691C (fr) 1998-02-02 1999-02-02 Reactifs d'elution, procedes et trousses permettant d'isoler un adn
AU25742/99A AU2574299A (en) 1998-02-02 1999-02-02 Eluting reagents, methods and kits for isolating dna
EP99905618A EP1053353A1 (fr) 1998-02-02 1999-02-02 Reactifs d'elution, procedes et trousses permettant d'isoler un adn

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1141234A1 (fr) * 1999-01-06 2001-10-10 Invitrogen Corporation Procedes et compositions permettant d'isoler des molecules d'acide nucleique
WO2002005930A1 (fr) * 2000-07-18 2002-01-24 Invitrogen Corporation Dispositif et procedes de subdivision et de filtration de matiere de gel et d'extraction de molecules de celle-ci
WO2003057910A2 (fr) 2002-01-08 2003-07-17 Roche Diagnostics Gmbh Utilisation d'un materiau de silice dans une reaction d'amplification
US6750059B1 (en) 1998-07-16 2004-06-15 Whatman, Inc. Archiving of vectors
US6803200B2 (en) 2000-12-12 2004-10-12 Invitrogen Corporation Compositions and methods for the release of nucleic acid molecules from solid matrices
JP2006501850A (ja) * 2002-10-04 2006-01-19 ワットマン インコーポレイテッド 核酸精製システムの媒体上に核酸を保存するツールとして、化学物質を用いる方法および材料
JP2008220380A (ja) * 2003-10-31 2008-09-25 Fujifilm Corp 核酸の分離精製方法
US7776616B2 (en) 1997-09-17 2010-08-17 Qiagen North American Holdings, Inc. Apparatuses and methods for isolating nucleic acid
WO2018170186A1 (fr) 2017-03-15 2018-09-20 Ancestry.Com Dna, Llc Dispositif et procédé de prélèvement d'échantillon

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050032105A1 (en) * 2001-10-12 2005-02-10 Bair Robert Jackson Compositions and methods for using a solid support to purify DNA
KR20070097430A (ko) * 2004-11-05 2007-10-04 퀴아젠 노쓰 아메리칸 홀딩즈, 인크. 안정화 시약으로부터 핵산을 정제하기 위한 조성물 및 방법
US9243289B2 (en) * 2013-12-23 2016-01-26 Roche Molecular Systems, Inc. Method for screening reagents used in PCR assays
CA2967008A1 (fr) * 2014-11-07 2016-05-12 The Johns Hopkins University Methode sans agents chaotropiques ni composes volatils pour la purification d'acides nucleiques issus de plasma
CA2983415A1 (fr) * 2015-05-12 2016-11-17 Gen-Probe Incorporated Procede de regroupement de prelevements sanguins

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007863A1 (fr) * 1990-10-26 1992-05-14 Diagen Institut Für Molekularbiologische Diagnostik Gmbh Procede et dispositif de separation d'acides nucleiques contenus dans des suspensions de cellules
EP0580305A2 (fr) * 1992-07-02 1994-01-26 Advanced Genetic Technologies Corporation Procédé et matériau pour la purification d'acides nucléiques
WO1995002049A1 (fr) * 1993-07-09 1995-01-19 Cambridge Molecular Technologies Limited Appareil et procede de purification
WO1998003674A1 (fr) * 1996-07-23 1998-01-29 University Of North Dakota Medical Education Research Foundation Procedure de purification d'adn

Family Cites Families (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL8900725A (nl) * 1989-03-23 1990-10-16 Az Univ Amsterdam Werkwijze en combinatie van middelen voor het isoleren van nucleinezuur.
JPH04158784A (ja) * 1990-10-24 1992-06-01 Tax Adm Agency 酵母細胞壁溶解酵素の製造法及びその利用
US5654179A (en) * 1990-11-14 1997-08-05 Hri Research, Inc. Nucleic acid preparation methods
JPH06277040A (ja) * 1991-02-13 1994-10-04 Tax Adm Agency 酵母細胞壁溶解酵素の製造法及び溶解法
JP3294288B2 (ja) * 1991-02-22 2002-06-24 明治乳業株式会社 乳酸桿菌由来の新規なプラスミドｐＢＵＬ１ 及びその誘導体
MX9202037A (es) * 1991-05-03 1992-11-01 Becton Dickinson Co Purificacion por filtracion de adn.
DE4143639C2 (de) * 1991-12-02 2002-10-24 Qiagen Gmbh Verfahren zur Isolierung und Reinigung von Nukleinsäuren
DE4321904B4 (de) * 1993-07-01 2013-05-16 Qiagen Gmbh Verfahren zur chromatographischen Reinigung und Trennung von Nucleinsäuregemischen
US5438127A (en) * 1993-09-27 1995-08-01 Becton Dickinson And Company DNA purification by solid phase extraction using a PCl3 modified glass fiber membrane
ATE302208T1 (de) * 1994-02-11 2005-09-15 Qiagen Gmbh Verfahren zur trennung von doppelstrang/einzelstrangnukleinsäurestrukturen
WO1995034569A1 (fr) * 1994-06-14 1995-12-21 Invitek Gmbh Procede universel d'isolement et de purification d'acides nucleiques a partir de quantites extremement reduites de differents materiaux de depart fortement contamines
DE69633239T2 (de) * 1995-06-07 2005-09-08 Whatman Plc, Maidstone Trockenes festes medium zur lagerung und analyse von genetischem material
US5702884A (en) * 1996-03-12 1997-12-30 Johnson & Johnson Clinical Diagnostics, Inc. Whole blood sample preparation for polymerase chain reaction using ammonium chloride and a carboxylic acid or metal carboxylate for selective red blood cell lysis
JP4025399B2 (ja) * 1997-10-28 2007-12-19 株式会社日立製作所 核酸の回収方法及び装置
WO1999039009A1 (fr) * 1998-02-02 1999-08-05 Gentra Systems, Inc. Procedes permettant d'isoler, d'amplifier et de caracteriser un adn

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992007863A1 (fr) * 1990-10-26 1992-05-14 Diagen Institut Für Molekularbiologische Diagnostik Gmbh Procede et dispositif de separation d'acides nucleiques contenus dans des suspensions de cellules
EP0580305A2 (fr) * 1992-07-02 1994-01-26 Advanced Genetic Technologies Corporation Procédé et matériau pour la purification d'acides nucléiques
WO1995002049A1 (fr) * 1993-07-09 1995-01-19 Cambridge Molecular Technologies Limited Appareil et procede de purification
WO1998003674A1 (fr) * 1996-07-23 1998-01-29 University Of North Dakota Medical Education Research Foundation Procedure de purification d'adn

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
REED K C ET AL: "RAPID PREPARATION OF DNA DOT BLOTS FROM TISSUE SAMPLES,USING HOT ALKALINE LYSIS AND FILTRATION ONTO CHARGE-MODIFIED NYLON MEMBRANE", NUCLEIC ACIDS RESEARCH, vol. 18, no. 10, 25 May 1990 (1990-05-25), pages 3093, XP000132494 *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7776616B2 (en) 1997-09-17 2010-08-17 Qiagen North American Holdings, Inc. Apparatuses and methods for isolating nucleic acid
US7833705B2 (en) 1998-07-16 2010-11-16 Whatman, Inc. Archiving of vectors
US6750059B1 (en) 1998-07-16 2004-06-15 Whatman, Inc. Archiving of vectors
EP1141234A1 (fr) * 1999-01-06 2001-10-10 Invitrogen Corporation Procedes et compositions permettant d'isoler des molecules d'acide nucleique
EP1141234A4 (fr) * 1999-01-06 2004-05-19 Invitrogen Corp Procedes et compositions permettant d'isoler des molecules d'acide nucleique
WO2002005930A1 (fr) * 2000-07-18 2002-01-24 Invitrogen Corporation Dispositif et procedes de subdivision et de filtration de matiere de gel et d'extraction de molecules de celle-ci
US6916423B2 (en) 2000-07-18 2005-07-12 Invitrogen Corporation Device and methods for subdividing and filtering gel material and extracting molecules therefrom
US6803200B2 (en) 2000-12-12 2004-10-12 Invitrogen Corporation Compositions and methods for the release of nucleic acid molecules from solid matrices
WO2003057910A3 (fr) * 2002-01-08 2004-03-25 Roche Diagnostics Gmbh Utilisation d'un materiau de silice dans une reaction d'amplification
WO2003057910A2 (fr) 2002-01-08 2003-07-17 Roche Diagnostics Gmbh Utilisation d'un materiau de silice dans une reaction d'amplification
US8026068B2 (en) 2002-01-08 2011-09-27 Roche Molecular Systems, Inc. Use of silica material in an amplification reaction
JP2006501850A (ja) * 2002-10-04 2006-01-19 ワットマン インコーポレイテッド 核酸精製システムの媒体上に核酸を保存するツールとして、化学物質を用いる方法および材料
JP2008220380A (ja) * 2003-10-31 2008-09-25 Fujifilm Corp 核酸の分離精製方法
WO2018170186A1 (fr) 2017-03-15 2018-09-20 Ancestry.Com Dna, Llc Dispositif et procédé de prélèvement d'échantillon
US11826027B2 (en) 2017-03-15 2023-11-28 Ancestry.Com Dna, Llc Sample collection device and method

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CA2319691C (fr) 2012-12-18

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