WO2000040697A1 - Procedes et compositions permettant d'isoler des molecules d'acide nucleique - Google Patents

Procedes et compositions permettant d'isoler des molecules d'acide nucleique Download PDF

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Publication number
WO2000040697A1
WO2000040697A1 PCT/US2000/000170 US0000170W WO0040697A1 WO 2000040697 A1 WO2000040697 A1 WO 2000040697A1 US 0000170 W US0000170 W US 0000170W WO 0040697 A1 WO0040697 A1 WO 0040697A1
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WIPO (PCT)
Prior art keywords
nucleic acid
acid molecules
matrix
molecular weight
cells
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PCT/US2000/000170
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English (en)
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WO2000040697A9 (fr
Inventor
Robert W. Blakesley
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Invitrogen Corporation
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Application filed by Invitrogen Corporation filed Critical Invitrogen Corporation
Priority to EP00904210A priority Critical patent/EP1141234A4/fr
Priority to JP2000592395A priority patent/JP2002534080A/ja
Priority to AU25998/00A priority patent/AU2599800A/en
Publication of WO2000040697A1 publication Critical patent/WO2000040697A1/fr
Publication of WO2000040697A9 publication Critical patent/WO2000040697A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification
    • 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
    • C12N15/101—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 by chromatography, e.g. electrophoresis, ion-exchange, reverse phase

Definitions

  • the present invention is in the fields of molecular biology and genetics.
  • the invention relates generally to compositions, methods and kits for use in isolating nucleic acid molecules. More specifically, the invention relates to such compositions, methods and kits that are useful in the isolation of small molecular weight nucleic acid molecules (e.g.. vectors, plasmids. and the like) from cells via lysis and one or more additional isolation steps, such as one or more chromatography steps.
  • the compositions, methods and kits of the invention are suitable for isolating a variety of forms of nucleic acid molecules from cells.
  • plasmid DNA isolation from bacterial cells is based on alkaline lysis, followed by batch chromatography.
  • lysis procedure Bornboim. H. and Doly, J. ( 1979) Nucleic Acids Res. 7. 1513
  • cells are completely ruptured, released proteins and nucleic acids are denatured, then plasmid DNA is preferentially renatured.
  • Precipitated denatured proteins and chromosomal DNA are then separated from soluble plasmid DNA.
  • plasmid DNA is further purified from residual contaminating proteins, lipids and nucleic acids by selective binding and release from a chromatography matrix.
  • the chromatography matrix in one instance, is an anion-exchange resin, e.g.. Qiagen- tip 20 (Qiagen. and U.S. Patent No. 4.997.932) or CONCERT High Purity Plasmid Miniprep System (Life Technologies. Inc. and U.S. Patent No. 5.843.312).
  • the chromatography matrix is a silica adsorption resin, such as Wizard Minipreps DNA Purification Resin (Promega. and U.S. Patent No.
  • compositions, methods and kits useful in the isolation of small molecular weight nucleic acid molecules, such as plasmids. vectors, and the like.
  • the present invention provides such compositions, methods and kits.
  • the present invention relates generally to compositions, methods and kits for use in isolating nucleic acid molecules. More specifically, the invention relates to such compositions, methods and kits that are useful in the isolation of nucleic acid molecules from cells (e.g., bacterial cells, animal cells, fungal cells, yeast cells or plant cells) via lysis and one or more additional isolation steps, such as one or more chromatography steps. In particular, the invention relates to compositions, methods and kits wherein desired nucleic acid molecules are isolated using an integrated lysis/chromatography matrix.
  • cells e.g., bacterial cells, animal cells, fungal cells, yeast cells or plant cells
  • additional isolation steps such as one or more chromatography steps.
  • the invention relates to compositions, methods and kits wherein desired nucleic acid molecules are isolated using an integrated lysis/chromatography matrix.
  • the invention relates to methods for isolating nucleic acid molecules, particularly low molecular weight nucleic acid molecules, comprising:
  • the cells may be lysed or disrupted before contacting the cells with the matrix, although cell lysis or disruption preferably takes place after the cells are contacted with the matrix and more preferably at the same time or approximately the same time (e.g.. simultaneously or substantially simultaneous! ⁇ ) the cells are contacted with the matrix.
  • the cells are preferably trapped within or on the matrix prior to or during cell lysis or disruption.
  • the cells are lysed/disrupted by contacting them with a composition or compound which causes or aids in cell lysis or disruption, although mechanical or physical forces (e.g. pressure, sonication. temperature (heating, freezing), and/or freeze-thawing etc.) may be used in accordance with the invention.
  • any combination of mechanical forces, physical forces or lysis compositions/compounds may be used to disrupt/iyse the cells.
  • the low molecular weight nucleic acid molecules are substantially separated from the high molecular weight nucleic acid molecules.
  • Such separation is preferably accomplished by the matrix binding/trapping the high molecular weight molecules and not substantially trapping/binding the low molecular weight molecules. Such action allows physical separation of such molecules where the smaller molecules of interest are allowed to substantially pass through the matrix while the larger molecules are trapped or bind to the matrix.
  • the matrix may be any porous matrix that traps or binds high molecular weight and/or does not substantially bind or trap low molecular weight nucleic acid molecules.
  • Such matrix may include but is not limited to a polyester matrix, a polyolefin matrix, a scintered polyethylene matrix, a nitrocellulose matrix, a cellulose acetate matrix, a cellulose matrix, a porous ceramic matrix, a silica matrix, a polysaccharide matrix (Sepharose. agarose. Sephadex. etc. ). a polymer matrix (Sephacryl. Trisacryl, Toyopearl. Bio-Gel. etc.) and the like.
  • the matrix is a solid matrix, although the matrix may be a semi-solid matrix.
  • Suitable matrix materials may be obtained commercially, for example from Filtrona Richmond. Inc. (Richmond. Virginia). Bio-Rad (Richmond. California). Gentra Systems (Minneapolis. MN). Tosohaas ( Montgomery ville. PA). BioSepra. Inc.. (Marlborough. MA), and Porex Technologies Corp. (Fairburn. GA).
  • the matrix may be prepared in various sizes, shapes, and forms including flat, wafer, cylindrical. rectangular. beads, gels, square, cartridge, swab tip. plug.
  • the invention involves the use of size separation chromatography and/or filtration to separate or substantially separate low molecular weight nucleic acid molecules (e.g.. vectors) from high molecular weight nucleic acid molecules.
  • size separation chromatography and/or filtration to separate or substantially separate low molecular weight nucleic acid molecules (e.g.. vectors) from high molecular weight nucleic acid molecules.
  • any matrix which provides desired size separation e.g., filters, chromatography supports, etc. may be used in the invention.
  • the invention combines such size separation chromatography/filtration with cell lysis/disruption (preferably such lysis/disruption is done when or approximately when the cellular source comes in contact with or after the cellular source is in contact with the chromatography/filtration matrix).
  • the pores or passage ways in the matrix are typically small enough to prevent passage of large nucleic acid molecules but large enough to permit passage of low molecular weight nucleic acid molecules, and may range from about 0.1 to about 10.000 micrometers in diameter, about 0.1 to about 5,000 micrometers in diameter, about 0.1 to about 1 ,000 micrometers in diameter, about 1 to about 500 micrometer in diameter, about 10 to about 500 micrometer in diameter, or about 25 to about 400 micrometers in diameter.
  • the composition or compound that disrupts the cellular membrane or cell wall integrity may comprise one or more detergents.
  • one or more detergents such as sodium dodecylsulfate (SDS) or Sarkosyl. Triton X-100. NP-40. deoxycholate or Brij 35; one or more chaeotropic agents such as sodium iodide, sodium perchlorate or guanidine or a salt thereof; one or more enzymes such as zymolyase. lyticase. lysozyme or lysostaphin; one or more inorganic salts such as sodium chloride, potassium chloride, or lithium chloride: one or more acids and/or bases or buffering agents (e.g..).
  • the composition may comprise one or more compounds or enzymes to degrade, destroy or remove unwanted components or contaminants (e.g..
  • compositions may also include any combination of the above described components.
  • the composition may be adsorbed onto or complexed with or associated with the matrix prior to applying the one or more cells or cellular source to the matrix.
  • the composition is dried in or on the matrix.
  • the matrix comprises a cell lysis/disruption compound or composition.
  • the cell disruption/lysis may occur when or about the same time the cells come into contact with the composition containing matrix.
  • the composition is added after the cells are added to (e.g.. bound to or associated with) the matrix. In yet another aspect, the composition is added to the cells prior to adding the cells to the matrix. In this aspect, the composition may be formulated to weaken the cell membrane/cell wall such that the cells will substantially disrupt/lyse when contacted with the matrix.
  • composition will substantially lyse/disrupt the cells before addition to the matrix.
  • the nucleic acid molecules of interest may be removed from the matrix by elution with an aqueous solution, such as a buffered salt solution or elution buffer.
  • an aqueous solution such as a buffered salt solution or elution buffer.
  • the unwanted molecules e.g.. chromosomal or genomic DNA
  • Such elution or removal of the desired nucleic acid molecules may be facilitated by centrifugation. gravity, vacuum. pressure, etc.. which provides flow of the desired nucleic acid sample from the matrix.
  • the isolated nucleic acid molecules of interest mav then be further purified by standard nucleic acid purification techniques and/or further manipulated by standard molecular biology techniques such as sequencing, amplification, endonuclease digestion (e.g.. restriction enzyme digestion), nucleic acid synthesis, transformation, transfection. and the like.
  • the methods according to the invention are suitable for isolation of low molecular weight nucleic acid molecules from any cell or cellular source, including bacterial cells (particularly Escherichia coli cells), yeast cells, fungal cells, animal cells (particularly insect cells, and mammalian cells including human cells, CHO cells. VERO cells. Bowes melanoma cells.
  • the methods of the invention are particularly well-suited for isolation of extrachromosomal nucleic acid molecules, including but not limited to plasmids. vectors, phagemids. cosmids. B ACs, PACs. YACs. cDNA molecules or cDNA libraries, mitochondrial nucleic acid molecules, and chloroplast nucleic acid molecules, any of which may be single-stranded or double-stranded, linear or circular, supercoiled. and which may be DNA or RNA molecules.
  • the invention also relates to isolated nucleic acid molecules produced by the methods of the invention, which are preferably low molecular weight nucleic acid molecules, such as the extrachromosomal nucleic acid molecules described herein and particularly plasmids and vectors.
  • isolated nucleic acid molecules produced by the methods of the invention, which are preferably low molecular weight nucleic acid molecules, such as the extrachromosomal nucleic acid molecules described herein and particularly plasmids and vectors.
  • the invention also relates to vectors
  • the invention also relates to further manipulation of the isolated nucleic acid molecules of the invention by standard molecular biology techniques such as sequencing, nucleic acid synthesis, cloning, amplification, endonuclease digestion (restriction endonuclease digestion), transformation, transfection and the like.
  • compositions for use in isolating low molecular weight nucleic acid molecules preferably comprise one or more components, such as: (a) a cellular source of the low molecular weight nucleic acid molecules of interest: (b) a nucleic acid-binding portion comprising at least one pore- containing matrix which binds or traps high molecular weight nucleic acid molecules but not substantially trap or bind low molecular weight nucleic acid molecules; and (c) a cell disrupting or cell lysis portion comprising at least one compound that disrupts the integrity of the cellular membrane or cell wall when the cellular source comes into contact with said compound.
  • a cellular source of the low molecular weight nucleic acid molecules of interest a nucleic acid-binding portion comprising at least one pore- containing matrix which binds or traps high molecular weight nucleic acid molecules but not substantially trap or bind low molecular weight nucleic acid molecules
  • a cell disrupting or cell lysis portion comprising at least one compound that disrupts
  • Preferred cellular sources, solid matrices, and lysis/disrupting compounds for use in the compositions of the invention include those described and used in the methods of the present invention.
  • the compound that disrupts the integrity of the cellular membrane and/or cell wall is adsorbed onto or complexed with or associated with the matrix, for example by ionic, hydrophobic, or covalent or non-covalent attachment of the cell membrane/cell wall disrupting compound to the matrix material.
  • such compound is dried in or on the matrix.
  • the compositions of the invention are useful in isolating a variety of low molecular weight nucleic acid molecules, particularly those described herein and most particularly plasmids and vectors.
  • kits for use in isolating low molecular weight nucleic acid molecules comprising one or more of the components for carrying out the methods of the invention or one or more of the compositions of the invention.
  • kits of the invention may comprise one or more components. which may be contained in one or more containers such as boxes, cartons, tubes. vials, ampules, bags, and the like.
  • the kits of the invention may comprise:
  • a cell disrupting/lysing composition comprising at least one compound that disrupts the integrity of the cellular membrane or cell wall when the cellular source comes into contact with the compound or composition, such that the high and low molecular weight nucleic acid molecules (or portion thereof) are released from the cellular source.
  • the matrix comprises the cell disrupting/lysing composition or compound.
  • Such cell disrupting/lysing composition or compound may be adsorbed onto or complexed with or associated with the matrix, for example by ionic, hydrophobic. non-covalent or covalent attachment to the matrix material.
  • Such cell disrupting/lysing composition is preferably dried in or on the matrix.
  • kits of the invention further comprise one or more additional reagents, such as one or more restriction enzymes, one or more polypeptides having nucleic acid polymerase activity (e.g.. one or more DNA polymerases which may be thermostable DNA polymerases and/or one or more reverse transcriptases which may be substantially reduced in RNase H activity).
  • additional reagents such as one or more restriction enzymes, one or more polypeptides having nucleic acid polymerase activity (e.g.. one or more DNA polymerases which may be thermostable DNA polymerases and/or one or more reverse transcriptases which may be substantially reduced in RNase H activity).
  • kits of the invention may also comprise protocols or instructions for carrying out the methods of the invention.
  • Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of what is known in the art, the following drawings and description of the invention, and the claims.
  • Figure 1 is a diagram of one aspect of the invention, depicting a thin- walled tube (preferably a micro fuge tube of any size) 1 containing a porous, matrix material in the form of a frit or plug or cartridge or swab tip 2 which divides the airspace within the tube into an upper sample application section 3 and a lower sample collection or sample elution section 4.
  • the matrix material 2 may comprise one or more cell disrupting/lysing compounds or compositions.
  • the matrix material may be in the form of beads or a gel or other semi-solid matrix in which case the matrix is preferably encased or associated with a solid support material to maintain the upper sample-application section 3 and the lower sample collection section 4.
  • the matrix material (solid or semi-solid) is in the form of a cartridge or plug or swab tip which can be easily removed from the tube 1 to facilitate sample collection.
  • one or more additional matrices or resins may be included in the upper sample application section 3 and/or in the sample-collection section 4, to further facilitate isolation or purification of the desired nucleic acid molecules.
  • nucleic acid binding matrices such as silica adsorption resins, anion-exchange resins, reverse phase resins, and/or affinity resins
  • a size separation matrix of the invention may be included below a size separation matrix of the invention to further remove or substantially remove undesired components including proteins, lipids, lysis/disruption compositions used to lyse/disrupt the cellular source, solvents, detergents, etc.
  • matrices such as cation-exchange resins, reverse phase resins, and/or hydrophobic interaction resins which bind undesired such components but which do not substantially bind the desired nucleic acids may be used.
  • nucleic acid binding matrices and contaminant binding matrices may be used.
  • the optional nucleic acid binding resin and/or contaminant binding resin 5 is shown.
  • additional matrices are preferably in cartridge or plug or swab tip form.
  • the sample- collection section 4 may contain an opening or access port (which may be closed if desired) to collect samples without the need to remove the matrix or matrices.
  • the desired nucleic acid molecules pass through the size separation matrix and bind to the binding matrix. Then be applied to remove unwanted materials through the access port or opening within the sample-collection section 4.
  • the seze separation matrix may be removed from the tube 1.
  • the desired isolated nucleic acid molecules may then be removed from the access port/opening when an elution buffer is applied.
  • the removal of desired nucleic acid molecules is accomplished by removal of the matrix or matrices to access the sample-collection section 4.
  • Figure 2 is a photograph of an ethidium bromide-stained 1 % agarose gel, analyzing samples of nucleic acid molecules at various stages of isolation by the methods of the invention.
  • Lane 1 l O ⁇ l of flow through from swab tip (Sample 1);
  • lane 2 l O ⁇ l of trapped DNA released from swab tip (Sample 2);
  • lane 3 l O ⁇ l of eluant from Concert Rapid spin cartridge (Sample 3):
  • lane 4 Purified plasmid pRPA-1 ;
  • lane 5 l O ⁇ l of Sample 1 digested with 10 units of Hind III;
  • lane 6 lO ⁇ l of Sample 3 digested with 10 units of Hind III;
  • lane 7 :
  • Figure 3 is a diagram of one aspect of the invention, depicting a thin- walled tube or column (preferably microspin or spin cartridges of any size) 1 containing a size separation matrix (for separating large nucleic acid molecules from small nuclei acid molecules) 2 and a second matrix material 5 for further purifying small molecular weight nucleic acid molecules.
  • the matrix material 5 is a nucleic acid binding matrix or a contaminant binding matrix, or combinations thereof.
  • the size separation matrix 2 and the preferred nucleic acid binding matrix 5 may be separated by a space within the tube or column 1, although such matrices are preferably in close proximity and preferably separated by a frit or other material.
  • the tube of column 1 contains a sample application section 3 and an opening or access port 6 (which may be closed if desired) to collect the sample.
  • An optional collection tube, well or container 7 is provided for collecting samples passing through the opening or access port 6.
  • the size separation matrix 2 comprises a cell lysis/disruption compound or composition.
  • a sample containing a cellular source of large and small molecular weight nucleic acid molecules are applied to the sample application section 3 preferably to the upper surface of the matrix 2.
  • the cell lysis/disruption composition or compound causes release of the low and/or high molecular weight nucleic acid molecules which separate according to size in the size separation matrix 2.
  • the size separation matrix 2 may optionally be removed from the column or tube 1 (before or after washing) to minimize large molecular weight nucleic acid molecules from passing through the size separation matrix 2 during subsequent washing and elution steps. Washing buffers or solutions may then be applied to remove unwanted materials. An elution buffer or solution may then be applied to elute the desired low molecular weight nucleic acid molecules from the nucleic acid binding matrix and through the opening or access port 6. During washing, the collection tube 7 (containing the unwanted materials) can be replaced with a second or new collection tube 7 to collect the desired nucleic acid molecules upon elution.
  • the present invention provides compositions, methods, and kits that may be used in isolating nucleic acid molecules from a nucleic acid-containing sample.
  • an ⁇ cell, tissues, organs, populations ot cells, etc can be used as a nucleic acid source Preferabh .
  • nucleic acid sources are used to isolate an ⁇ ector. or other extragenomic or extrachromosomal structure
  • a population of different ⁇ ectors for example, a cDNA libran or genomic library contained b ⁇ the cell population or culture
  • mvnetion allows isolation of such population of vectors/cDNA hbrary/genomic hbrar ⁇
  • Low molecular weight nucleic acid molecule refers to any nucleic acid molecule or population of nucleic acid molecules which is smaller in size or molecular mass than a genomic nucleic acid molecule such as a chromosome from a given cell or cellular source
  • Low molecular weight nucleic acid molecules according to the invention typically are no larger than about 500 kilobases in size, and are preferabh about 1 to about 300. more preierabl ⁇ about 1 to about 200. more preferabh about 1 to about 100, still more preferabh about 1 to about 50.
  • high molecular weight nucleic acid molecule is used to mean any nucleic acid molecule or population of nucleic acid molecules that is larger than about 500 kilobases in size, particularly genomic nucleic acid molecules or chromosomes which ma ⁇ range in size from about 1 to about 15.000 megabases
  • Examples of low molecular weight nucleic acid molecules include, but are not limited to. plasmids. large molecular weight plasmids (BAC ' s. PAC ' s. YAC ' s). ⁇ ectors. phagemids. cosmids.
  • cDNA molecules or cDNA libraries amplification fragments (e g . PCR generated nucleic acid molecules) and fragments of nucleic acid molecules regardless of the means b% which such fragments are generated.
  • Such fragments may be generated by enzymatic digestion (e.g.. endonuclease digestion for example with type I and/or II restriction enzymes), mechanical forces (shearing) and the like.
  • fragments of genomic or chromosomal nucleic acid molecules which are smaller in size than the complete genomic or chromosomal nucleic acid molecule from which they are derived are included in this definition.
  • Amplification refers to any in vitro method for increasing the number of copies of a nucleotide sequence with the use of a polymerase. Nucleic acid amplification results in the inco ⁇ oration of nucleotides into a nucleic acid (e.g.. DNA) molecule or primer thereby forming a new nucleic acid molecule complementary to the nucleic acid template. The formed nucleic acid molecule and its template can be used as templates to synthesize additional nucleic acid molecules. As used herein, one amplification reaction may consist of many rounds of nucleic acid synthesis. Amplification reactions include, for example, polymerase chain reactions (PCR). One PCR reaction may consist of 5 to 100 "cycles" of denaturation and synthesis of a nucleic acid molecule.
  • PCR polymerase chain reactions
  • prokaryotic or eukaryotic cell that is the recipient of a replicable expression vector or cloning vector.
  • the terms "host” or “host cell” may be used interchangeably herein.
  • Preferred prokaryotic hosts include, but are not limited to. bacteria of the genus Escherichia (e.g., E. coli). Bacillus. Staphylococcus, Agrobacter (e.g..A. tumefaciens), Streptomyces, Pseudomonas, Salmonella. Serratia. Caryophanon. etc.
  • the most preferred prokaryotic host is
  • E. coli Bacterial hosts of particular interest in the present invention include E. coli strains K12. DH 10B. DH5 ⁇ and HB 101.
  • Preferred eukaryotic hosts include, but are not limited to. fungi, fish cells, yeast cells, plant cells and animal cells. Particularly preferred animal cells are insect cells such as Drosophila cells. Spodoplera Sf9. SO 1 cells and Trichoplusa High-Five cells: nematode cells such as C. elega ⁇ s cells: and mammalian cells such as COS cells. CHO cells. VERO cells. 293 cells. PERC6 cells. BHK cells and human cells.
  • a host or host cell may serve as the cellular source for the desired nucleic acid molecule to be isolated.
  • a vector is a nucleic acid molecule (preferably DNA) capable of replicating autonomously in a host cell. Such vectors may also be characterized by having a small number of endonuclease restriction sites at which such sequences may be cut without loss of an essential biological function and into which nucleic acid molecules may be spliced to bring about its replication and cloning. Examples include plasmids. autonomously replicating sequences (ARS), centromeres, cosmids and phagemids. Vectors can further provide primer sites, e.g.. for PCR.
  • the vector can further contain one or more selectable markers suitable for use in the identification of cells transformed or transfected with the vector, such as kanamycin, tetracycline. amplicillin, etc.
  • any vector may be used.
  • vectors known in the art and those commercially available (and variants or derivatives thereof) may be used in accordance with the invention.
  • Such vectors may be obtained from, for example. Vector Laboratories Inc.. InVitrogen. Promega, Novagen. NEB, Clontech. Boehringer Mannheim, Pharmacia. EpiCenter, OriGenes Technologies Inc.. Stratagene. Perkin Elmer. Pharmingen. Life Technologies. Inc.. and Research Genetics.
  • Such vectors may be used for cloning or subcloning nucleic acid molecules of interest and therefore recombinant vectors containing inserts, nucleic acid fragments or genes may also be isolated in accordance with the invention.
  • vectors of particular interest include prokaryotic and/or eukaryotic cloning vectors, expression vectors, fusion vectors, two-hybrid or reverse two-hybrid vectors, shuttle vectors for use in different hosts, mutagenesis vectors, transcription vectors, vectors for receiving large inserts (yeast artifical chromosomes (YAC ' s). bacterial artificial chromosomes (BAC ' s) and PI artifical chromosomes (PAC ' s)) and the like.
  • Other vectors of interest include viral origin vectors (M 13 vectors, bacterial phage ⁇ vectors, baculovirus vectors, adenovirus vectors, and retrovirus vectors), high.
  • vectors which have compatible replicons for use in combination in a single host e.g.. pACYC184 and pBR322
  • eukaryotic episomal replication vectors e.g.. pCDM8
  • the vectors contemplated by the invention include vectors containing inserted or additional nucleic acid fragments or sequences (e.g.. recombinant vectors) as well as derivatives or variants of any of the vectors described herein.
  • Expression vectors useful in accordance with the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids or bacteriophages. and vectors derived from combinations thereof, such as cosmids and phagemids. and will preferably include at least one selectable marker (such as a tetracycline or ampicillin resistance genes) and one or more promoters such as the phage lambda P L promoter, and/or the E. coli lac, trp and tac promoters. Other suitable promoters will be known to the skilled artisan.
  • vectors preferred for use in the present invention include pQE70. pQE60 and pQE-9. available from Qiagen; pBS vectors. Phagescript vectors,
  • Bluescript vectors pNH8A. pNH16a. pNH18A. pNH46A. available from Stratagene; pcDNA3 available from Invitrogen: pGEX. pTrxfus. pTrc99a. pET-5. pET-9. pKK223-3. pKK233-3, pDR540, pRIT5 available from Pharmacia: and pSPORTl. pSPORT2 and pSV SPORTl . available from Life Technologies. Inc. Other suitable vectors will be readily apparent to the skilled artisan.
  • Plasmid means an extrachromosomal genetic element, typically less than about 25 kilobases (kb) in size and more typically about 15 kb to about 2 kb in size.
  • isolated means that the isolated material, component, or composition has been at least partially purified away from other materials, contaminants, and the like which are not part of the material, component, or composition that has been isolated.
  • an "isolated low molecular weight nucleic acid molecule” is a nucleic acid molecule that has been treated in such a way as to remove at least some of the other nucleic acid molecules (e.g.. large nucleic acid molecules) with which it may be associated in the cell, tissue, organ or organism.
  • isolated low molecular weight nucleic acid molecule or "isolated vector” refers to a low molecular weight nucleic acid molecule preparation or vector preparation which contains no more than about 50%, 45%. 40%, 35%. 30%. 25%. 20%. 15%. 10%. and 7%. preferably no more than 5%. 2.5%. and 2%. and most preferably less than 1%. 0.5%. and 0.1 % (percentages by weight) of high molecular weight nucleic acid molecules (e.g.. chromosomal/genomic DNA).
  • a solution comprising an isolated nucleic acid molecule may comprise one or more buffer salts and/or a solvents, e.g.. water or an organic solvent such as acetone, ethanol. methanol. and the like, and yet the nucleic acid molecule may still be considered an " isolated" nucleic acid molecule with respect to its starting materials.
  • Cell disrupting or cell lysing compound or composition refers to a composition or a component of a composition that effects lysis, rupture, or poration of the cells, tissues, or organisms used as the source of the nucleic acid molecules to be isolated, such that the nucleic acid molecules (or portion thereof) that are contained in the cell, tissue, or organism source are released from the cell, tissue, or organism.
  • the cells, tissues, or organisms need not be completely lysed. ruptured or porated. and all of the nucleic acid molecules contained in the source cells, tissues or organisms need not be released therefrom.
  • a cell disrupting or cell lysis compound or composition at least 25%. 50%.
  • nucleic acid molecules 75%. 80%. 85%. 90%. 95%. 97%. 99%. or more of the total nucleic acid molecules, particularly the total low molecular weight nucleic acid molecules (such as vectors, plasmids. and the like) that are contained in the cell, tissue, or organism.
  • the total low molecular weight nucleic acid molecules such as vectors, plasmids. and the like
  • Other terms used in the fields of recombinant DNA technology and molecular and cell biology as used herein will be generally understood by one of ordinary skill in the applicable a ⁇ s.
  • the methods, compositions and kits of the invention are suitable for isolation of low molecular weight nucleic acid molecules from any cellular source, including a variety of cells, tissues, organs or organisms, which may be natural or which may be obtained through any number of commercial sources (including American Type Culture Collection (ATCC). Rockville. Maryland: Jackson
  • Cells that may be used as cellular nucleic acid sources may be prokaryotic (bacterial, including members of the genera Escherichia (particularly E. coli), Serratia, Salmonella. Staphylococcus, Streptococcus. Clostridium, Chlamydia, Neisseria. Treponema. Mycoplasma,
  • Any virus may also be used as a cellular source of nucleic acid molecules in accordance with the invention.
  • suitable for use as sources of low molecular weight nucleic acid molecules are mammalian tissues or organs such as those derived from brain, kidney, liver, pancreas, blood, bone marrow, muscle, nervous, skin, genitourinary, circulatory, lymphoid.
  • gastrointestinal and connective tissue sources as well as those derived from a mammalian (including human) embryo or fetus.
  • These cells, tissues and organs may be normal, transformed, or established cell lines, or they ma ⁇ ' be pathological such as those involved in infectious diseases (caused by bacteria, fungi or yeast, viruses (including AIDS) or parasites), in genetic or biochemical pathologies (e.g.. cystic fibrosis. hemophilia. Alzheimer's disease, schizophrenia, muscular dystrophy or multiple sclerosis), or in cancers and cancerous processes.
  • the methods, compositions and kits of the invention are particularly well-suited for isolation of extrachromosomal nucleic acid molecules, including but not limited to plasmids. vectors, phagemids.
  • cosmids any of which may be single- stranded or double-stranded, linear or circular, supercoiled. and which may be DNA or RNA molecules.
  • the methods of the invention are useful in the isolation of plasmid or vector DNA from bacterial cells.
  • Other cells, tissues, viruses, organs and organisms that will be familiar to one of ordinary skill in the art may also be used as sources of nucleic acid molecules for the preparation of isolated nucleic acid molecules according to the present invention.
  • the invention relates to methods for isolating nucleic acid molecules, particularly small molecular weight nucleic acid molecules such as plasmids. vectors, organellar nucleic acid molecules, and the like.
  • Methods according to this aspect of the invention may comprise one or more steps which result in the isolation of one or more nucleic acid molecules or populations of nucleic acid molecules (e.g., a cDNA library) from the natural environment in which the nucleic acid molecules are found.
  • the methods of the invention may comprise:
  • the invention relates to a method for obtaining one or more vectors comprising:
  • the desired low molecular weight nucleic acid molecules or vectors are preferably separated by the matrix based on size separation or size exclusion and thus the matrix can be designed (e.g.. varying pore size, matrix material, size and dimension of the matrix, etc.) to separate the desired nucleic acid molecules (e.g.. vectors) from unwanted nucleic acid molecules (e.g.. chromosomal or genomic nucleic acid molecules) contained by the cellular source.
  • This separation may be facilitated by any means for moving the desired nucleic acid molecules through the matrix including centrifugation, vacuum, gravity, pressure and the like.
  • the matrix may be any porous matrix that substantially traps or binds (reversibly or irreversibly) high molecular weight nucleic acid molecules but not substantially bind or trap low molecular weight nucleic acid molecules.
  • Suitable materials for preparing the solid matrices used in this aspect of the invention include, but are not limited to. polyester, scintered polyethylene, nitrocellulose, polyolefin. cellulose acetate, cellulose, silica, and the like.
  • This solid matrix may be provided in any convenient format for use in isolation of nucleic acid molecules, for example, as an insert (e.g...
  • the matrix may be provided as a frit or cartridge or as a membrane suitable for insertion into a tube or column, providing a partitioning of upper and lower chambers of the tube or column by the matrix: such an aspect of the invention is diagramed in Figure 1.
  • the matrix may also be provided in other convenient forms, such as sheets, frits, plugs, cartridges or inserts suitable to fit multi-well plates typically used in filtration of multiple samples, including, for example.6-well plates. 12-well plates. 24-well plates. 48-well plates. 96-well plates.
  • the solid matrix is provided as a frit or insert or cartridge or swab suitable to fit into a microcentrifuge tube, microspin tube or spin cartridges.
  • the frit/insert/cartridge/swab has a size of 8 mm diameter x 1 cm length.
  • Such tubes are available for example from N /Lida Manufacturing. Naperville. IL.
  • the pores in the separation matrix are typically small enough to prevent passage of large molecular weight (e.g.. genomic or chromosomal) nucleic acid molecules, but large enough to permit passage of small molecular weight nucleic acid molecules, and may range from about 0.1 to about 10,000 micrometers in diameter, about 0.1 to about 5.000 micrometers in diameter, about 0.1 to about
  • the cellular source is applied onto the matrix, preferably in an aqueous solution, and then is introduced into or on the matrix either by unit gravity incubation or preferably by centrifugation or vacuum.
  • the cellular source will optionally be trapped within or on the matrix in preparation for release of the nucleic acid molecules. Lysis/disruption compositions, physical forces and/or mechanical forces (or combinations thereof) may be used for disrupting the integrity of the cell membrane/cell wall of the cellular source of the nucleic acid molecules. In accordance w ith the invention, any physical or mechanical forces (freezing. heating, freeze-thawing.
  • the matrix comprises such lysis/disruption compounds or compositions
  • the lysis/disruption composition or compound may be either applied to the matrix containing the cellular source or preferably may be adsorbed, complexed or associated with (e.g.. by ionic, hydrophobic. covalent or non- covalent binding) to the matrix prior to applying the cellular source to the matrix, for example by soaking or saturating the matrix in the disrupting/lysing composition and then allowing the matrix to dry under air.
  • the composition may be applied to the matrix material just prior to its use or prior to the preparation of the matrix plug. frit, insert, membrane, etc. from the matrix material. Any method of pre-treating the matrix results in the formation of a matrix that has been impregnated with a disrupting/lysing composition.
  • the matrix compises the lysis/disruption compositions or compounds. In this preferred aspect of the invention, contacting of the cellular source and the lysis/disrupting steps of the present methods are thus accomplished concurrently or nearly concurrently, thereby reducing the amount of time and manipulation required for the isolation of the small molecular weight nucleic acid molecules.
  • the composition that disrupts the cellular membrane/cell wall integrity that is applied to the matrix, or that is pre-adsorbed onto the matrix may comprise one or more detergents, such as sodium dodecylsulfate (SDS) or Sarkosyl. Triton X-100. Tween 20. NP-40. N- alkylglucosides. N-alkylmaltosides. glucamides. digitonin. deoxycholate, 3-[(3- cholamidopropyl)-dimethylammonio]-l -propane-sulfonate (CHAPS) or cetyltrimethvl-ammoniumbromide (CTAB).
  • SDS sodium dodecylsulfate
  • Sarkosyl Sarkosyl
  • Triton X-100 Tween 20. NP-40. N- alkylglucosides. N-alkylmaltosides. glucamides. digitonin. deoxycholate, 3-[(3
  • Brij 35 at a concentration of about 0.01 %- 10% (w/v). more preferably about 0.1 %-5%. and most preferably about 0.5%; one or more chaeotropic agents such as sodium iodide, sodium perchlorate. guanidine or a salt thereof or urea at a concentration of about 300-1000 mM. more preferably about 500-2000 mM. and most preferably about 1500 mM; one or more enzymes such as lysozyme. lyticase. zymolyase. neuraminidase. Novozym 234. streptolysin. cellulysin.
  • mutanolysin or lysostaphin at a concentration of about 0.1 to 5 mg/ml one or more inorganic salts such as sodium chloride, potassium chloride, magnesium chloride. lithium chloride, or praseodymium chloride at a concentration of about 1 mM to 5M: one or more organic solvents such as toluene, phenol, butanol. isopropyl alcohol, isoamyl alcohol, ethanol. an ether (e.g., diethyl ether, dimethyl ether, or ethylmethyl ether), or chloroform at a concentration of 25 to 60% (v/v); or any other compound which disrupts the integrity of (i.e..
  • compositions may also comprise other components, such as chelating agents (e.g., disodium ethylenediaminetetraacetic acid (Na 2 EDTA), EGTA, CDTA. most preferably at a concentration of about 10 mM) and/or one or more ribonucleases (RNase A, TI, T2. and the like) at concentrations ranging from I to 400 ⁇ g/ml, proteases (Protinase K. Pronase, pepsin, trypsin. papain.
  • chelating agents e.g., disodium ethylenediaminetetraacetic acid (Na 2 EDTA), EGTA, CDTA. most preferably at a concentration of about 10 mM
  • RNase A, TI, T2. and the like ribonucleases
  • proteases proteases
  • subtilisin at concentrations ranging from 50 to 1000 ⁇ g/ml. or any combination of the foregoing.
  • the composition comprises 0.5% SDS or a combination of triton X- 100 and lysozyme. Desired concentrations and combinations of the active ingredients of the lysis/disruption compositions may be readily determined by those skilled in the art.
  • the high and low molecular weight nucleic acid molecules contained within the cellular source are released from the cell and the high molecular weight (e.g.. genomic or chromosomal) nucleic acid molecules are bound to or trapped within or on the matrix material, while the low- molecular weight nucleic acid molecules, such as plasmids. vectors, phagemids. cosmids. and the like, substantially pass through the matrix material without being bound thereby or trapped therein.
  • the high molecular weight (e.g. genomic or chromosomal) nucleic acid molecules are bound to or trapped within or on the matrix material, while the low- molecular weight nucleic acid molecules, such as plasmids. vectors, phagemids. cosmids. and the like, substantially pass through the matrix material without being bound thereby or trapped therein.
  • These low molecular weight nucleic acid molecules may be collected with the flow-through, for example by washing the atrix with an aqueous solution sufficient to wash or elute the low molecular weight nucleic acid molecules through the matrix, but insufficient to remove the large (genomic or chromosomal) nucleic acid molecules from the matrix to which they are bound or in which they are trapped; such an approach is described in detail in the Example below.
  • these large molecular weight molecules may be removed from the matrix to which they are bound once being released from the cellular source, by elution with an aqueous solution such as a buffered salt solution, according to methods of nucleic acid chromatography that are well- known in the art.
  • the desired nucleic acid molecules obtained may be further purified by well known nucleic acid purification or chromatography techniques.
  • further purification steps may involve adso ⁇ tion chromatography. reverse phase ion pair chromatography. ionic exchange chromatography. extraction (e.g.. with organic solvents such as phenyl/chloroform), ethanol precipitation, density /gradient centrifugation (CsCl). and the like.
  • the invention further comprises purifying the desired nucleic acid molecules by any known techniques available in the art. Such additional purification may facilitate removal of unwanted contaminants such as proteins, lipids. nucleotides. oligonucleotides.
  • nucleic acid molecules obtained by the method of the invention may be manipulated directly by standard molecular biology techniques.
  • one or more addition further purification resins e.g. ion exchange resins, and/or abso ⁇ tion resins
  • additional purification may be accomplished in separate steps, although in a preferred aspect, the additional purification is accomplished simultaneously or in conjunction with the separation method of the invention.
  • the one or more separation matrices and the one or more nucleic acid binding resins are associated in series in a fluid channel such that a sample containing the desired nucleic acid molecules may pass from one matrix to another.
  • the separation matrix and binding resin combination may be provided in any format to provide a fluid channel to associate the various matrices in fluid connection such as a column format, a tube format, a well format. a multi-well plate format, etc.
  • the desired nucleic acid molecules passing through the separation matrix would bind or absorb onto the nucleic acid binding resin. Removal of unwanted materials (such as lipids.
  • nucleic acid molecules proteins, lysis/disruption compositions, and components which may inhibit further manipulation of nucleic acid molecules are removed with a wash buffer or solution which allows the desired nucleic acid molecule to be retained on the binding resin.
  • An elution buffer or solution for removing the desired nucleic acid molecule from the binding resin may then be used to isolate the purified nucleic acid molecule.
  • compositions for use in isolating low molecular weight nucleic acid molecules may comprise one or more components or portions. such as:
  • the matrix comprises the compound that disrupts the integrity of the cellular membrane or cell wall.
  • Such compound is preferably adsorbed onto or complexed with or associated with the matrix, for example by ionic, hydrophobic. non-covalent or covalent attachment of the lysis/dis ⁇ uting compound or composition to the matrix material.
  • the compositions of the invention are useful in isolating a variety of low molecular weight nucleic acid molecules, particularly those described herein and most particularly plasmids or vectors from bacterial cells.
  • kits for use in isolating low molecular weight nucleic acid molecules may comprise one or more components, which may be contained in or include one or more containers such as boxes, cartons, tubes, microspin tubes, micro fuge tubes, spin cartridges, multi-well plates, vials, ampules, bags, and the like.
  • the kits of the invention may comprise one or more of the compositions of the invention described in detail herein.
  • the kits of the invention may comprise:
  • the matrix comprises a cell disrupting/lysing composition or compound which may be may be adsorbed onto or complexed with or associate with the matrix, for example by ionic, hydrophobic. non-covalent or covalent attachment of the composition or compound to the matrix material.
  • the kits comprise additional nucleic acid purification resins (e.g.. nucleic acid binding resins), wash buffers, elution buffers etc.
  • Preferred matrix materials, cell lysis/disrupting compositions and compounds, and elution and wash compositions for use in the kits of the invention include those described herein for use in the methods and compositions of the present invention.
  • kits of the invention may further comprise one or more additional components or reagents that may be useful in further processing, analysis, or use of the nucleic acid molecules isolated or purified according to the invention, for example components or reagents useful in nucleic acid amplification, sequencing, cloning, transfection. transcription, translation, and the like.
  • additional components or reagents that may be useful in further processing, analysis, or use of the nucleic acid molecules isolated or purified according to the invention, for example components or reagents useful in nucleic acid amplification, sequencing, cloning, transfection. transcription, translation, and the like.
  • Such reagents or components may, for example, include one or more restriction enzymes, one or more polypeptides having nucleic acid polymerase activity, one or more polypeptides having reverse transcriptase activity, one or more cells competent for transformation, one or more transfection reagents (e.g., lipids) and other reagents that will be familiar to one of ordinary skill in the art.
  • Polypeptides having nucleic acid polymerase activity for use in the kits of the invention may be any polypeptide that can synthesize a nucleic acid molecule from a nucleic acid template, typically in the 5 ' to 3" direction.
  • the nucleic acid polymerases used in the kits of the present invention may be mesophilic or thermophilic. and are preferably thermophilic.
  • Preferred mesophilic DNA polymerases include T7 DNA polymerase. T5 DNA polymerase. Klenow fragment DNA polymerase. DNA polymerase III and the like.
  • Preferred thermostable DNA polymerases that may be used in the kits of the invention include Taq, Tne. Tma, Pf . Tfl. Tih. Stoffel fragment. VENTTM and DEEPVENTTM DNA polymerases. and mutants, variants and derivatives thereof (U.S. Patent No. 5.436.149; U.S.
  • DNA polymerases For amplification oflong nucleic acid molecules (e.g.. nucleic acid molecules longer than about 3-5 Kb in length), at least two DNA polymerases (one substantially lacking 3 ' exonuclease activity and the other having 3 ' exonuclease activity) are typically used. See U.S. Patent No. 5.436.149: and U.S. Patent No. 5.512.462: Barnes. W.M.. Gene 172:29-35 ( 1992), the disclosures of which are inco ⁇ orated herein in their entireties. Examples of DNA polymerases substantially lacking in 3 * exonuclease activity include, but are not limited to. Taq. Tne(exo ' ). Tma(exo ' ).
  • Polypeptides having reverse transcriptase activity for use in the kits of the invention may include any polypeptide having the ability to synthesize an RNA molecule from a DNA template molecule. In one embodiment, the polypeptides having reverse transcriptase activity may be substantially reduced in RNase H activity. Suitable polypeptides having reverse transcriptase activity for use in the kits of the invention include, but are not limited to. M-ML V reverse transcriptase.
  • RSV reverse transcriptase AM V reverse transcriptase. RA V reverse transcriptase. MAV reverse transcriptase or HIV reverse transcriptase.
  • These polypeptides having reverse transcriptase activity may be substantially reduced in RNase H activity; preferred such polypeptides include M-MLV H " reverse transcriptase. RSV H " reverse transcriptase. AMV H ' reverse transcriptase. RAV H " reverse transcriptase. MAV H “ reverse transcriptase and HIV H “ reverse transcriptase.
  • Methods for the production and use of such polypeptides having reverse transcriptase activity, including those which are substantially reduced in RNase H activity, are described in detail in commonly owned, co-pending U.S. Application No. 09/064.057. filed April 22. 1998. the disclosure of which is inco ⁇ orated herein in its entirety.
  • the invention also relates to isolated nucleic acid molecules that are prepared according to the methods of the invention.
  • the isolated nucleic acid molecules of the invention preferably are low molecular weight nucleic acid molecules.
  • Preferred such low molecular eight nucleic acid molecules that may be isolated according to the present invention include, but are not limited to. plasmids. large molecular weight plasmids (BAC * s. PAC ' s and YAC ' s). vectors. cDNA molecules or libraries, cosmids. phagemids. organellar nucleic acid molecules (e.g.. those isolated from organelles such as mitochondria or chloroplasts). RNA transcripts, and the like.
  • the nucleic acid molecules may be single stranded or double stranded, circular or linear, supercoiled. and may be comprised of DNA. RNA. or a combination of DNA and RNA.
  • the isolated nucleic acid molecules of the invention are double- stranded DNA plasmids or vectors (which are optionally supercoiled). particularly those isolated, for example, from bacterial cells.
  • the invention provides the ability quickly to screen and evaluate recombinant vectors prepared by recombinant technologies (e.g., by cloning or subcloning).
  • the invention thus may be used to quickly isolate such recombinant vectors, providing a ready source of the recombinant vectors for such evaluation or screening (e.g.. by sequencing, restriction digestion, restriction mapping, etc.).
  • Vectors and recombinant vectors obtained by the invention may be introduced into host cells using any of the techniques for introducing nucleic acid molecules into host cells that are described herein.
  • the invention also provides recombinant host cells comprising the isolated nucleic acid molecules, vectors or recombinant vectors of the invention.
  • Representative host cells prokaryotic or eukaryotic
  • Representative host cells include, but are not limited to. bacterial cells, yeast cells, plant cells and animal cells.
  • Such suitable host cells are available commercially, for example from Life Technologies. Inc. (Rockville. Maryland), ATCC (Manassas. Virginia), and other commercial sources that will be familiar to one of ordinary skill in the art.
  • Host cells comprising the vectors, recombinant vectors or isolated nucleic acid molecules of the invention may be prepared by inserting the isolated nucleic acid molecules or vectors of the invention into the host cells, using well-known transformation, electroporation or transfection techniques that will be familiar to one of ordinary skill in the art.
  • introduction of the isolated nucleic acid molecules into a host cell to produce a host cell comprising the nucleic acid molecules can be effected by any known method of introducing nucleic acid molecules into host cells, including but not limited to calcium phosphate transfection. DEAE-dextran mediated transfection. cationic lipid-mediated transfection. electroporation. transduction. transformation (e.g.. of competent cells particularly E.
  • the invention provides methods for producing a recombinant polypeptide encoded by the isolated nucleic acid molecules of the invention, and polypeptides produced by these methods.
  • a recombinant polypeptide may be produced by culturing any of the above recombinant host cells comprising the isolated nucleic acid molecules, recombinant vectors or vectors of the invention, under conditions favoring production of a polypeptide therefrom, and isolation of the polypeptide. Methods for culturing recombinant host cells, and for production and isolation of polypeptides therefrom, are well-known to one of ordinary skill in the art.
  • nucleic acid molecules that are isolated by the compositions, methods and kits of the present invention may be further characterized or manipulated, for example by cloning, sequencing, amplification, nucleic acid synthesis, endonuclease digestion and the like.
  • the isolated nucleic acid molecules of the invention may be used in methods for amplifying and sequencing nucleic acid molecules.
  • Amplification methods which may be used in accordance with the present invention include PCR (U.S. Patent Nos. 4.683.195 and 4.683.202). Strand Displacement Amplification (SDA: U.S. Patent No. 5.455.166: ⁇ P 0 684 315). and Nucleic Acid Sequence- Based Amplification (NASBA: U.S. Patent No. 5.409.818: ⁇ P 0 329 822).
  • the isolated nucleic acid molecules may also be used in complex PCR-based nucleic acid finge ⁇ rinting techniques such as Random Amplified Polymo ⁇ hic DNA (RAPD) analysis (Williams.
  • RAPD Random Amplified Polymo ⁇ hic DNA
  • the invention may be used in methods of amplifying or sequencing a nucleic acid molecule comprising one or more polymerase chain reactions (PCRs), such as any of the PCR-based methods described above.
  • Nucleic acid sequencing methods according to this aspect of the invention may comprise both cycle sequencing (sequencing in combination with linear amplification) and standard sequencing reactions, according to methods that are well-known in the art and as described in commonly owned, co-pending U.S.
  • nucleic acid molecules isolated according to the present invention may be used for the manufacture of various materials in industrial processes by methods that are well-known in the art. Such materials include, but are not limited to. hybridization probes, therapeutic proteins (dependent upon transcription and translation of the isolated nucleic acid molecules, or the production of synthetic peptides or proteins with amino acid sequences deduced from the nucleotide sequences of the specific nucleic acid molecules), gene therapy vehicles and compositions, molecular weight markers, and the like.
  • the aim of this project was to improve the process of preparing plasmid DNA from bacterial cells. Specifically, the objectives were first, to develop a more rapid lysis procedure where there are fewer manipulations and the manipulations are more forgiving, and second, to eliminate a separate centrifugation or filtration step for the removal of precipitated denatured proteins and genomic DNA. According to the present invention, these objectives are accomplished by integration of the lysis and filtration processes into a single operation. The output from this operation is soluble plasmid DNA ready for further purification, if necessary, by matrix chromatography. By the present invention, it is further possible to combine the matrix chromatography step with the lysis and precipitate removal step, to make a single unit operation of the entire plasmid preparation method.
  • the spin cartridge was washed twice with 500 ⁇ l of wash buffer, then plasmid DNA was eluted with 75 ⁇ l of TE buffer pre-warmed to 75°C into a 1 5- ml microcent ⁇ fuge tube
  • the Micro Spin housing the swab tip was incubated in a 100°C water bath for 10 min Centrifugation of the Micro Spin at 14.000 ⁇ m for 1 mm remo ⁇ ed the eluant from the swab tip
  • the ⁇ olume was collected in a 1 5-ml microcent ⁇ fuge tube Restriction Digestion of Plasmid DNA. Aliquots ( 10 ⁇ l) of plasmid DNA were incubated for 30 min at 37 n C with 10 units of Hind III (Life Technologies) according to the manufacturer ' s instructions.
  • Adsorption to silica is preferred, since the process is quick and DNA elutes in a ready-to-use form.
  • the composition of the eluant solution from the Capture Column was found to be incompatible with direct plasmid DNA adsorption to a silica matrix.
  • Adsorption to silica requires that the DNA solution be in a specific chemical environment, which includes a high concentration of a chaeotropic agent, such as guanidine»HCl.
  • a chaeotropic agent such as guanidine»HCl.
  • the cell lysis reagent was simplified, creating a new lysis matrix with 0.5% SDS (described above).
  • plasmid DNA may bind directly from the eluant solution to an anion exchange chromatography matrix, e.g.. in the CONCERT High Purity Plasmid Miniprep System.
  • Bacterial cells containing plasmid pRPA- 1 were introduced into Suspension Buffer, then applied to an SDS-impregnated swab tip in a Micro Spin.
  • the cell lysis eluant (sample 1 ) was adjusted to silica adsorption conditions by adding two volumes of GE buffer, forming a precipitate removable by centrifugation.
  • the plasmid-containing supernate was applied to a silica membrane spin cup from the CONCERT Rapid Plasmid Miniprep System. Bound DNA was washed, then eluted in TE buffer (sample 2). To determine which nucleic acids were trapped in the lysis matrix, the Micro Spin was incubated in TE buffer at 100°C for lO min. then the eluant collected by centrifugation (sample 3).
  • Plasmid DNA was present in the eluant from the lysis matrix, free of most RNAs and contaminated with a relatively small amount of chromosomal

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Abstract

La présente invention concerne, de manière générale, des compositions, des procédés et des kits destinés à être utilisés pour isoler des molécules d'acide nucléique. De manière plus spécifique, cette invention concerne des compositions, des procédés et des kits qui sont utiles pour isoler des molécules d'acide nucléique à partir de cellules (telles que par exemple des cellules bactériennes, des cellules animales, des cellules de champignons, des virus, des cellules de levure ou des cellules de plantes) au moyen d'une étape de lyse et d'au moins une étape additionnelle d'isolement, telle qu'au moins une étape d'analyse chromatographique. De manière plus spécifique, cette invention concerne des compositions, des procédés et des kits dans lesquels les molécules d'acide nucléique sont isolées au moyen d'une matrice mixte de lyse/chromatographie pouvant elle-même comprendre un ou plusieurs supports (par exemple en polyoléfine, en polyéthylène fritté, en nitrocellulose, en polypropylène, en polycarbonate, en acétate de cellulose, en silice et autres) qui ont été traités avec un ou plusieurs réactifs de lyse chimique (par exemple un ou plusieurs détergents, un ou plusieurs chaéotropes, une ou plusieurs enzymes et autres) ou bien qui ont été associés à ces derniers. Les compositions, les procédés et les kits de l'invention sont adaptés pour isoler diverses formes de molécules d'acide nucléique à partir de cellules, comprenant sans limitation aucune, des plasmides, des vecteurs, de l'ADN, de l'ADNc, de l'ARN, de l'ADN mitochondrial, de l'ADN chloroplastique, et autre, ces derniers pouvant être de type monocaténaire ou bicaténaire, linéaire ou circulaire. Les compositions, les procédés et les kits selon l'invention sont particulièrement adaptés pour isoler rapidement un plasmide soluble ou un ADN vecteur présent dans des cellules bactériennes.
PCT/US2000/000170 1999-01-06 2000-01-06 Procedes et compositions permettant d'isoler des molecules d'acide nucleique WO2000040697A1 (fr)

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JP2000592395A JP2002534080A (ja) 1999-01-06 2000-01-06 核酸分子の単離のための方法および組成物
AU25998/00A AU2599800A (en) 1999-01-06 2000-01-06 Methods and compositions for isolation of nucleic acid molecules

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WO2002008368A1 (fr) * 2000-07-21 2002-01-31 Cargill, Incorporated Procede d'elimination de contaminants presents dans de l'huile vegetale et dans de la lecithine
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WO2002074954A1 (fr) * 2001-03-21 2002-09-26 Eppendorf Ag Procede d'isolation de plasmides a partir de cellules bacteriennes ou de levure en suspension
EP1301591A1 (fr) * 2000-07-13 2003-04-16 Invitrogen Corporation Methodes et compositions d'extraction et d'isolation rapides de proteines et de peptides au moyen d'une matrice de lyse
WO2003046178A1 (fr) * 2001-11-20 2003-06-05 Glaxo Group Limited Traitement d'acides nucleiques
EP1388588A1 (fr) * 2002-07-29 2004-02-11 JSR Corporation Méthode de séparation d'acide nucléiques et solutions pour les extraire
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EP1585825A2 (fr) * 2002-12-17 2005-10-19 Sigma Aldrich Company Compositions et procedes permettant d'extraire l'acide nucleique de prelevements biologiques
WO2009058414A1 (fr) 2007-10-31 2009-05-07 Akonni Biosystems Appareil, système et procédé de purification d'acides nucléiques
US7608399B2 (en) 2006-06-26 2009-10-27 Blood Cell Storage, Inc. Device and method for extraction and analysis of nucleic acids from biological samples
US8048681B2 (en) 2005-11-04 2011-11-01 Hitachi High-Technologies Corporation Methods for nucleic acid isolation and instruments for nucleic acid isolation
US8163535B2 (en) 2006-06-26 2012-04-24 Blood Cell Storage, Inc. Devices and processes for nucleic acid extraction
US8629264B2 (en) 2011-05-19 2014-01-14 Blood Cell Storage, Inc. Gravity flow fluidic device for nucleic acid extraction
WO2017196285A2 (fr) 2016-05-12 2017-11-16 Akdeniz Universitesi Procédé et kit d'isolement d'adn génomique
CN111867728A (zh) * 2018-01-08 2020-10-30 摇篮基因组公司 分离核酸的方法和试剂盒
EP3783113A1 (fr) * 2019-08-21 2021-02-24 New England Biolabs, Inc. Isolation d'adn à poids moléculaire élevé au moyen de billes

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KR20130143677A (ko) * 2004-11-05 2013-12-31 퀴아젠 노쓰 아메리칸 홀딩즈, 인크. 안정화 시약으로부터 핵산을 정제하기 위한 조성물 및 방법
JP5328547B2 (ja) * 2009-07-31 2013-10-30 一般財団法人電力中央研究所 有機物の抽出方法、有機物の製造方法、有機物抽出装置組立体、湿潤材料の処理方法
JP6161205B2 (ja) * 2010-07-14 2017-07-12 キアゲン ゲーエムベーハー 生体分子の単離および／または精製のためのデバイス
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WO2001042456A3 (fr) * 1999-12-10 2002-01-03 Genespan Corp Isolation et purification d'acides nucleiques
EP1301591A4 (fr) * 2000-07-13 2004-05-26 Invitrogen Corp Methodes et compositions d'extraction et d'isolation rapides de proteines et de peptides au moyen d'une matrice de lyse
EP1301591A1 (fr) * 2000-07-13 2003-04-16 Invitrogen Corporation Methodes et compositions d'extraction et d'isolation rapides de proteines et de peptides au moyen d'une matrice de lyse
WO2002008368A1 (fr) * 2000-07-21 2002-01-31 Cargill, Incorporated Procede d'elimination de contaminants presents dans de l'huile vegetale et dans de la lecithine
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WO2003046178A1 (fr) * 2001-11-20 2003-06-05 Glaxo Group Limited Traitement d'acides nucleiques
EP1388588A1 (fr) * 2002-07-29 2004-02-11 JSR Corporation Méthode de séparation d'acide nucléiques et solutions pour les extraire
US7214484B2 (en) 2002-12-17 2007-05-08 Sigma-Aldrich Co. Compositions and methods for nucleic acid extraction from biological samples
EP1585825A2 (fr) * 2002-12-17 2005-10-19 Sigma Aldrich Company Compositions et procedes permettant d'extraire l'acide nucleique de prelevements biologiques
EP1585825A4 (fr) * 2002-12-17 2007-01-03 Sigma Aldrich Co Compositions et procedes permettant d'extraire l'acide nucleique de prelevements biologiques
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US8148115B2 (en) 2006-06-26 2012-04-03 Blood Cell Storage, Inc. Device and method for extraction and analysis of nucleic acids from biological samples
US8771955B2 (en) 2006-06-26 2014-07-08 Blood Cell Storage, Inc. Device and method for extraction and analysis of nucleic acids from biological samples
WO2009058414A1 (fr) 2007-10-31 2009-05-07 Akonni Biosystems Appareil, système et procédé de purification d'acides nucléiques
US8629264B2 (en) 2011-05-19 2014-01-14 Blood Cell Storage, Inc. Gravity flow fluidic device for nucleic acid extraction
WO2017196285A2 (fr) 2016-05-12 2017-11-16 Akdeniz Universitesi Procédé et kit d'isolement d'adn génomique
CN111867728A (zh) * 2018-01-08 2020-10-30 摇篮基因组公司 分离核酸的方法和试剂盒
EP3783113A1 (fr) * 2019-08-21 2021-02-24 New England Biolabs, Inc. Isolation d'adn à poids moléculaire élevé au moyen de billes
US11591591B2 (en) 2019-08-21 2023-02-28 New England Biolabs, Inc. Isolation of high molecular weight DNA using beads
EP4219748A1 (fr) * 2019-08-21 2023-08-02 New England Biolabs, Inc. Isolement d'adn de poids moléculaire élevé à l'aide de billes

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EP1141234A4 (fr) 2004-05-19
WO2000040697A9 (fr) 2001-08-02
AU2599800A (en) 2000-07-24
EP1141234A1 (fr) 2001-10-10
JP2002534080A (ja) 2002-10-15

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