WO1996009379A1 - Dna purification and isolation using a solid phase - Google Patents
Dna purification and isolation using a solid phase Download PDFInfo
- Publication number
- WO1996009379A1 WO1996009379A1 PCT/US1995/011839 US9511839W WO9609379A1 WO 1996009379 A1 WO1996009379 A1 WO 1996009379A1 US 9511839 W US9511839 W US 9511839W WO 9609379 A1 WO9609379 A1 WO 9609379A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- dna
- magnetic microparticles
- concentration
- solution
- microparticles
- Prior art date
Links
Classifications
-
- 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
- C12N15/1013—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 using magnetic beads
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/975—Kit
Definitions
- the present invention is a method of binding polynucieotides non-specifically and reversibly to a solid phase which reversibly binds polynucieotides, such as magnetic microparticles whose surfaces are coated with a functional group, such as a carboxyl group.
- the polynucieotides can be DNA, RNA or polyamide nucleic acids (PNAs) .
- the method comprises combining the solid phase, such as magnetic microparticles and a solution containing polynucieotides.
- the salt concentration and polyalkylene glycol concentration of the resulting combination are adjusted to concentrations suitable for binding polynucieotides to the surface of the solid phase, such as to surfaces of the magnetic microparticles; as a result, polynucieotides are bound non-specifically to the magnetic microparticles.
- the present invention also relates to a method of separating polynucieotides, such as DNA, RNA and PNA, from a solution containing polynucieotides.
- the method comprises binding polynucieotides non-specifically to a solid surface, such as to magnetic microparticles, as described above, washing the resulting bound polynucieotides with a high ionic strength buffer, and eluting the polynucieotides with a low ionic strength elution buffer.
- the method described herein is useful to separate double stranded (ds) or single stranded (ss) polynucieotides (e.g., DNA, RNA, PNA) of virtually any size and from a wide variety of sources.
- the present method can be used to separate DNA present in a transfected host cell, DNA resulting from an amplification process (e.g., polymerase chain reaction, PCR) and DNA in gels, such as agarose gels.
- amplification process e.g., polymerase chain reaction, PCR
- DNA in gels such as agarose gels.
- biochemical reactions and sequencing can be performed on DNA bound to the magnetic microparticles.
- the present invention also relates to a kit comprising magnetic microparticles and a binding buffer which contains a suitable salt and polyalkylene glycol at concentrations suitable for reversibly binding polynucleotide onto solid surfaces, such as to the surfaces of magnetic microparticles.
- the kit may additionally comprise a suitable wash buffer, elution buffer, reagents for preparing such buffers or reagents for preparing a cleared lysate.
- the method of the present invention is useful with both single and double stranded polynucieotides, as well as a wide range of polynucleotide fragment sizes, it has applicability in essentially any context in which polynucleotide separation is desired. In addition, this permits the standardization of manipulations and isolation carried out with polynucieotides.
- the present method simplifies the isolation of cloned DNA from lysate by obviating the need for centrifugation and produces a plasmid ready for sequencing and further characterization and processing.
- the present method also has the advantage that is fast, thus allowing for the rapid throughput in isolating polynucleotides, low cost and simple to perform and produces high yields of polynucieotides.
- polynucieotides bind reversibly and non-specifically to solid surfaces, such as certain magnetic microparticles, at certain concentrations of salt and polyalkylene glycol.
- a method for convenient and rapid separation of polynucieotides, such as DNA, RNA and PNA, from other biomolecules, such as proteins, monosaccharides, polysaccharides, lipids and RNA and from cellular components, such as cell membranes is available.
- a method for separation of polynucieotides on the basis of size. The following is a description of the present invention with reference to polynucieotides as exemplified by DNA.
- One embodiment of the present invention is a method of separating DNA from a solution containing DNA. The method comprises a first step of reversibly binding DNA non-specifically to a solid surface, such as magnetic microparticles whose surfaces are coated with functional groups.
- the magnetic microparticles are combined with a solution of DNA, after which the salt concentration and the polyethylene glycol concentration of the resulting combination are adjusted to a concentration suitable for binding DNA onto the surface of the magnetic particles.
- sufficient salt and polyethylene glycol are added to the solution containing magnetic microparticle-bound DNA to result in a final concentration of from about .5 M to about 5.0 M salt and from about 7% to about 13% polyethylene glycol.
- DNA is bound non-specifically to the surfaces of the magnetic microparticles.
- the magnetic microparticles in the resulting combination are separated from the supernatant.
- the magnetic microparticles having DNA bound thereto can, optionally, be washed with a suitable wash buffer before they are contacted with a suitable elution buffer, to elute and separate the DNA from the magnetic microparticles.
- the magnetic particles are separated from the elution buffer, which contains the polynucleotide, in solution.
- the magnetic microparticles are separated from the elution buffer by, for example, filtration or applying a magnetic field to draw down the microparticles.
- Magnetic microparticles are microparticles which are attracted by a magnetic field.
- the magnetic microparticles used in the method of the present invention comprise a magnetic metal oxide core, which is generally surrounded by an adsorptively or covalently bound silane coat to which a wide variety of bioaffinity adsorbents can be covalently bound through selected coupling chemistries, thereby coating the surface of the microparticles with functional groups.
- the magnetic metal oxide core is preferably iron oxide, wherein iron is a mixture of Fe 2* and Fe 3* .
- the preferred Fe 2* /Fe 3* ratio is preferably 2/1, but can vary from about 0.5/1 to about 4/1.
- Suitable amino silanes useful to coat the microparticle surfaces include p- aminopropyltrimethoxysilane, N-2-aminoethyl-3- aminopropyltrimethoxysilane, triaminofunctional silane (H 2 NCH 2 -NH-CH 2 CH 2 -NH-CH 2 -Si- (OCH 3 ) 3 , n-dodecyltriethoxysilane and n-hexyltrimethoxysilane.
- the term "functional group-coated surface” refers to a surface which is coated with moieties which each have a free functional group which is bound to the amino group of the amino silane on the microparticle; as a result, the surfaces of the microparticles are coated with the functional group containing moieties.
- the functional group acts as a bioaffinity absorbent for DNA in solution.
- the functional group is a carboxylic acid.
- a suitable moiety with a free carboxylic acid functional group is a succinic acid moiety in which one of the carboxylic acid groups is bonded to the amine of amino silanes through an amide bond and the second carboxylic acid is unbonded, resulting in a free carboxylic acid group attached or tethered to the surface of the magnetic microparticle.
- Carboxylic acid-coated magnetic microparticles are commercially available from PerSeptive Diagnostics (BioMag COOH, Catalog Number 8-4125) .
- Suitable functional groups which can used for coating the surface of the magnetic microparticles include, but are not limited to thiol groups (microparticles with thiol group coating are commercially available from PerSeptive Diagnostics, Division of PerSeptive Biosystems, Catalog Number 8-4135) and streptavidin (microparticles with a streptavidin coating are commercially available from PerSeptive Diagnostics, Division of PerSeptive Biosystems, Catalog Number 8-4135) and streptavidin (microparticles with a streptavidin coating are commercially available from
- Magnetic microparticles coated with thiol groups or streptavidin bind DNA less efficiently than carboxyl group-coated microparticles.
- the importance of having functional groups coat the surface of microparticles used is demonstrated by the observation that polymer encapsulated magnetic microparticles do not bind DNA in the method of the present invention.
- Polymer encapsulated microparticles are commercially available from Dynal, Incorporated, Dynabeads M-280, (Catalog Number 112.06) .
- Magnetic microparticles useful in the present method can be a variety of shapes, which can be regular or irregular; preferably the shape maximizes the surface areas of the microparticles.
- the magnetic microparticles should be of such a size that their separation from solution, for example by filtration or magnetic separation, is not difficult.
- the magnetic microparticles should not be so large that surface area is minimized or that they are not suitable for microscale operations. Suitable sizes range from about 0.1 ⁇ mean diameter to about 100 ⁇ mean diameter. A preferred size is about 1.0 ⁇ mean diameter. Suitable magnetic microparticles are commercially available from PerSeptive Diagnostics and are referred to as BioMag COOH (Catalog Number 8-4125) .
- Non-specific DNA binding refers to binding of different DNA molecules with approximately the same affinity to magnetic microparticles, despite differences in the DNA sequence or size of the different DNA molecules.
- a polynucleotide can be DNA, RNA or a synthetic DNA analog such as a PNA (Nielsen et al . , Science, 254:1497 (1991)) .
- Non-specific DNA binding refers to binding of different DNA molecules with approximately the same affinity to magnetic microparticles despite differences in the nucleic acid sequence or size of the different DNA molecules.
- a solution containing DNAs can be any aqueous solution, such as a solution containing DNA, RNA and/or PNAs.
- Such a solution can also contain other components, such as other biomolecules, inorganic compounds and organic compounds.
- the solution can contain DNA which is the reaction product of PCR amplification.
- the solution can also be a cleared lysate.
- a "lysate”, as used herein, is a solution containing cells which contain cloned DNA and genomic DNA and whose cell membranes have been disrupted, with the result that the contents of the cell, including the DNA contained therein, are in the solution.
- a “cleared lysate” is a lysate in which the chromosomal DNA, proteins and membranes of the host cells have been selectively removed, such as by chemical treatment or centrifugation of the lysate, thereby leaving a solution containing plasmid DNA.
- RNase can be added to create a "cleared lysate" free of RNA, thereby allowing DNA to bind to the magnetic microparticles free from RNA.
- Methods of creating a cleared lysate are well-known in the art. For example, a cleared lysate can be produced by treating the host cells with sodium hydroxide or its equivalent (0.2 N) and sodium dodecyl sulfate (SDS) (1%) . This method of creating a cleared lysate is described in detail in Birnboim and Doly, Nucl . Acids Res .
- a host cell is any cell, such as a bacterial cell such as E. coli , a mammalian cell or a yeast cell which contains exogenous or foreign D ⁇ A, in addition to genomic D ⁇ A.
- the foreign D ⁇ A may be introduced directly into the host cell by means known to one of ordinary skill in the art .
- Examples of foreign D ⁇ A introduced directly into a host cell include bacterial artificial chromosomes (BAC) , yeast artificial chromosomes (YAC) , plasmids, cosmids and PI.
- BACs are particularly difficult to separate and purify from cleared lysates due to their low concentrations in the lysates. (Shizuya et al . ) However, BACs are readily separated by the method of the present invention.
- the plasmid D ⁇ A may be introduced into the host cell by a phage into which the plasmid D ⁇ A has been packaged. Suitable plasmid D ⁇ As which can be packaged into a phage include a cosmid or PI . Host cells containing foreign D ⁇ A introduced by any method are referred to as transfected host cells.
- the solution with which the magnetic microparticles is combined may also contain single stranded polynucieotides.
- the present invention is useful to separate polynucieotides from a solution which is the supernatant from a recombinant D ⁇ A-containing M13 bacteriophage isolate which had been used to infect bacterial host cells.
- the host cells are removed from the supernatant by filtration (Kristensen, et al . , Nucl . Acids Res . , 15:550-16 (1987)) or by binding the host cells to amine coated surfaces (Hou and
- Single stranded D ⁇ A is released from the M13 bacteriophage into the solution by adding SDS to a final concentration of about 0.3% to about 3%, preferably about 1% and at a temperature from about 60°C to about 100°C, preferably 80°C.
- the DNA-containing solution may also be an agarose solution.
- a mixture of DNA is separated, according to methods known to one skilled in the art, such as by electrophoresis on an agarose gel.
- a plug of agarose containing DNA of interest can be excised from the gel and added to 1-10 volumes of 0.5 x SSC ( .75 M NaCI, .0075 M Sodium Citrate, pH 7.0) preferably 4 volumes.
- the mixture is then melted at a temperature of from about 60°C to about 100°C, preferably at about 80°C for about one to about twenty minutes, preferably ten minutes, to create an agarose solution containing DNA.
- the second step of the present method of binding DNA non-specifically to magnetic microparticles having a functional group-coated surface comprises adjusting the salt concentration and the polyalkylene glycol concentration of the combination to a concentration of each suitable for binding DNA reversibly onto the surface of the magnetic particles.
- Suitable polyalkylene glycols include polyethylene glycol (PEG) and polypropylene glycol.
- PEG poly(ethylene glycol)
- a sufficient quantity of a salt and a sufficient quantity of PEG are combined with the combination of magnetic microparticles and DNA-containing solution to produce a final salt concentration of from about 0.5 M to about 5.0 M and a final PEG concentration of from about 7% to about 13%.
- DNA binds non-specifically to the surface of the magnetic microparticles. The binding of the DNA to the magnetic microparticles is rapid; it is generally complete within thirty seconds.
- Salts which have been found to be suitable for binding DNA to the microparticles include sodium chloride (NaCI) , lithium chloride (LiCl) , barium chloride (BaCl 2 ) , potassium (KC1) , calcium chloride (CaCl 2 ) , magnesium chloride (MgCl 2 ) and cesium chloride (CeCl) .
- sodium chloride is used.
- the wide range of salts suitable for use in the method indicates that many other salts can also be used and can be readily determined by one of ordinary skill in the art. Yields of bound DNA decrease if the salt concentration is adjusted to less than about 0.5 M or greater than about 5.0 M. The salt concentration is preferably adjusted to about 1.25 M.
- the molecular weight of the polyethylene glycol (PEG) can range from about 6000 to about 10,000, with a molecular weight of about 8000 being preferred.
- the concentration of PEG is preferably adjusted to about 10%. Although concentrations of PEG as low as 7% and as high as 13% can be used, yields of bound DNA drop as the concentration of PEG deviates from 10%.
- the method of the present invention is useful to separate DNA from a solution containing polynucleotide. As discussed above, the method comprises binding DNA nonspecifically and reversibly to magnetic microparticles having a functional group coated (e.g., carboxyl-coated) surface.
- the microparticles are then separated from the supernatant, for example by applying a magnetic field to draw down the magnetic microparticles.
- the remaining solution i.e. supernatant
- the DNA can be removed from the magnetic microparticles particles by washing with a suitable elution buffer.
- An elution buffer is any aqueous solution in which the salt concentration and polyalkylene concentration are below the ranges required for binding of DNA onto magnetic microparticles, as discussed above.
- sucrose (20%) and formamide (100%) solutions can be used to elute the DNA.
- a preferred eluent is water. Elution of the DNA from the microparticles occurs in thirty seconds or less when an elution buffer of low ionic strength, for example water, is used. Once the bound DNA has been eluted, the magnetic microparticles are separated from the elution buffer that contains the eluted DNA. Preferably, the magnetic microparticles are separated from the elution buffer by magnetic means, as described above. Other methods known to those skilled in the art can be used to separate the magnetic microparticles from the supernatant; for example, filtration can be used. Yields of DNA following elution typically approach 100% when the magnetic microparticles are used in excess.
- the magnetic microparticles with bound DNA are washed with a suitable wash buffer solution before separating the DNA from the microparticles by washing with an elution buffer.
- a suitable wash buffer solution has several characteristics. First, the wash buffer solution must have a sufficiently high salt concentration (i.e., has a sufficiently high ionic strength) that the DNA bound to the magnetic microparticles does not elute off of the microparticles, but remains bound to the microparticles. Suitable salt concentrations are greater than about 1.0 M and is preferably about 5.0 M. Second, the buffer solution is chosen so that impurities that are bound to the DNA or microparticles are dissolved.
- the pH and solute composition and concentration of the buffer solution can be varied according to the type of impurities which are expected to be present.
- Suitable wash solutions include the following: 0.5 x 5 SSC ; 100 mM ammonium sulfate, 400 mM Tris pH 9, 25 mM MgCl 2 and 1% bovine serum albumine (BSA) ; and 5 M NaCI .
- a preferred wash buffer solution comprises 25 mM Tris acetate (pH 7.8), 100 mM potassium acetate (KOAc) , 10 mM magnesium acetate (Mg 2 0Ac) , and 1 mM dithiothreital (DTT) .
- the magnetic microparticles with bound DNA can also be washed with more than one wash buffer solution.
- the magnetic microparticles can be washed as often as required to remove the desired impurities.
- the number of washings is preferably limited to two or three in order to minimize loss of yield of the bound DNA. Yields of DNA when the microparticles are used in excess are typically about 80% after washing with a wash buffer and eluting with an elution buffer.
- the polynucleotide in the solution with which the magnetic microparticles are combined can be single stranded or double stranded.
- the polynucleotide can be homogeneous (i.e. polynucieotides which have the same nucleotide sequence) .
- the polynucleotide can be heterogeneous, (i.e., polynucieotides of differing nucleotide sequences) .
- the polynucleotide can also comprise a DNA library or partial library.
- the DNA can also comprise molecules of various lengths.
- DNA fragments of 23Kb, 9Kb, 64Kb, 4Kb, 2Kb and 12bp derived from the electrophoretic separation of a HINDIII cut lambda marker were cut out of the agarose gel and purified by the method of the present invention (see Example 7) .
- Temperature does not appear to be critical in the method of separating DNA of the present invention. Ambient temperature is preferred, but any temperature above the freezing point of water and below the boiling point of water can be used.
- DNA fragments of all sizes bind non-specifically to magnetic microparticles at high ionic strength. High ionic strength refers to salt concentrations greater than 0.5 M. However, smaller fragments of DNA bind with lower affinity than large DNA fragments at lower ionic strengths, for example, about 0.5 M salt concentration and lower.
- Another embodiment of the present invention refers to a method of separating a mixture of polynucleotide fragments, such as DNA fragments, based on size. For example, a solution of DNA fragments of different sizes is first combined with magnetic microparticles having a carboxyl group-coated surface under conditions appropriate for non ⁇ specific binding of DNA to the magnetic microparticles. The magnetic microparticles are then separated from the supernatant.
- the polynucleotide bound to the magnetic microparticles can be washed with a suitable wash buffer which dissolves bound impurities, but is of high enough ionic strength that the polynucleotide remains attached to the magnetic microparticles.
- the magnetic microparticles are then washed with an elution buffer of appropriate ionic strength to elute the smaller size polynucleotide fragments, but leave the larger size polynucleotide fragments bound to the magnetic microparticles.
- the smaller polynucleotide fragments, such as DNA, in the elution buffer can then be isolated in the usual manner or processed further, e.g., subjected to further biochemical reactions. This method has been used to separate PCR primers from the reaction product of a PCR amplification (see Example 6) .
- the polynucleotide (e.g., DNA) which remains bound to the magnetic particles can then be eluted with a suitable elution buffer.
- the DNA can then be isolated in the usual manner, or processed further, e.g. subjected to further biochemical reactions.
- the DNA which remains bound to the magnetic microparticles can be subjected to further size selection by washing with an elution buffer of sufficiently low ionic strength to elute the smaller remaining DNA fragment, but of sufficiently high enough ionic strength to allow the larger remaining polynucleotide fragments to remain bound to the magnetic microparticles.
- the separation of polynucleotide fragments (e.g., DNA fragments) based on size can also be accomplished by the method of the present invention by adjusting the PEG concentration, the molecular weight of the PEG used or both.
- One embodiment of the present invention is based on the discovery that the magnetic microparticles do not bind enzymes. The magnetic microparticles also do not inhibit the function of enzymes. It is therefore possible to carry out biochemical reactions on DNA bound to the magnetic microparticles, e.g., by exposing the bound DNA to enzymes capable of biochemically modifying the bound DNA under conditions which cause the biochemical modification to take place.
- the biochemical reactions are carried out on purified bound DNA (e.g., DNA bound to microparticles which have been separated from a cleared lysate or from a solution in which a biochemical reaction such as PCR was carried out) .
- the purified bound DNA can also be washed with a suitable wash buffer. Because residual salt can inhibit the activity of certain enzymes, it is preferable that washings with high ionic strength salt solutions be followed with a washing with a lower ionic strength solution. The ionic strength of this solution should be low enough that enough residual salt is removed to prevent enzyme inhibition, but not so low that substantial losses in bound DNA result.
- the DNA bound to the magnetic microparticles is digested with a restriction enzyme.
- the restriction enzyme-digested DNA can then be end-repaired, if necessary, for later ligation to a vector by suitable end- repair enzymes.
- the end-repaired DNA is typically eluted by the solvent in which the biochemical reaction takes place.
- the magnetic microparticles are washed with a suitable elution buffer to ensure complete separation of the end-repaired DNA from the microparticles.
- the magnetic microparticles are then separated from the reaction mixture or elution buffer, preferably by magnetic separation.
- the solution containing the end-repaired DNA can then be combined with a solution containing a pre-cut vector suitable for ligation to the eluted DNA.
- the end-repaired DNA can then be ligated to the pre-cut vector by methods known to those skilled in the art. After ligation, the DNA can be transformed into a host cell in the usual way.
- a DNA library is bound to the magnetic microparticles.
- the DNA bound to the magnetic microparticles will consist of molecules of various sizes and nucleotide sequences (heterogeneous DNA) . Specific size fragments can be eluted from the magnetic microparticles by varying the ionic strength of the elution buffer, as described earlier. Alternatively, the concentration, molecular weight or both of the PEG in the elution buffer can be varied, as described earlier, to selectively elute smaller DNA fragments.
- the DNA fragments which remain bound to the magnetic microparticles can be digested with one or more restriction enzymes and then ligated into a pre-cut vector, as described above. A vector is thereby created in which the DNA insert has a certain size. The vector can then be transformed into a host cell in the usual way.
- the nucleotide sequence of the DNA bound to the magnetic microparticles is determined directly without an elution step which releases the DNA from the magnetic microparticles.
- DNA is bound to the microparticles as described above.
- the microparticles with bound DNA are then separated from the supernatant and combined with the reagents used for determining nucleotide sequences under conditions suitable for sequence determination. Suitable reagents and conditions are known to those skilled in the art (See Sanger et al . , Proc . Na t . Acad. Sci . , 74:5463 (1977) and the ABI 373 Sequencer Manual) .
- a kit is also provided herein which contains the reagents necessary for separating polynucieotides, such as DNA, RNA and PNAs, from a solution containing polynucieotides by binding the polynucieotides to a solid surface, such as magnetic microparticles having a carboxyl group-coated surface.
- the kit comprises magnetic microparticles with a carboxyl group-coated surface and a binding buffer.
- the binding buffer comprises a suitable salt and a suitable polyalkylene glycol which are both present at a concentration suitable for binding DNA to the surface of the magnetic microparticles.
- the kit further comprises an elution buffer which is capable of dissolving the polynucleotide, such as DNA, bound to the magnetic microparticles.
- the kit can comprise the reagents for making the binding and/or elution buffer, to which a known amount of water can be added to create a binding and/or elution buffer of desired concentration.
- the kit further comprises a wash buffer which dissolves impurities bound to the magnetic microparticles, but does not result in elution of the polynucleotide bound to the magnetic microparticles.
- the kit can comprise the reagents for making the wash buffer, to which a known amount of water can be added to create a wash buffer of desired concentration.
- the kit comprises the reagents necessary for clearing a cell lysate.
- the reagents are present in solutions at a concentration suitable for direct use in preparing a cleared lysate without the need for further diluting the solutions.
- the magnetic particles used in the following examples were the carboxyl coated magnetic microparticles from PerSeptive Diagnostics Massachusetts, (Biomag COOH, Catalog Number 8-4125) particles which were 1 ⁇ m in diameter.
- the particles were stored in phosphate buffered saline (PBS) at a concentration of 20 mg/ml. All agarose gels were run using 1% final agarose (U.S. Biochemical #32827) with lx TBE buffers. The field strength was lOV/cm with run times from 40-60 minutes. The gels were post-stained with ethidium bromide and visualized under UV.
- PBS phosphate buffered saline
- a pUC plasmid (pUC 18, obtained from U.S. Biochemicals, Catalog Number 70070) was purified from its host cell by creating a cleared lysate, reversibly binding the plasmid to the magnetic microparticles, separating the magnetic microparticles and then eluting the DNA. The following procedure was used:
- a pUC plasmid (pUC 18, obtained from U.S. Biochemicals Catalog Number 70070) grown in microtitre plates was purified from its host cell by creating a cleared lysate, reversibly binding the plasmid to the magnetic microparticles, separating the magnetic microparticles and then eluting the DNA. The following procedure was used:
- This example yields 500-800ng plasmid DNA which is sufficient for thermal cycle DNA sequencing.
- the advantage of using a microtitre plate is that many samples can be isolated in parallel.
- electrophoretic analysis of the cleared lysate after binding to the magnetic microparticles showed no DNA, while electrophoretic analysis of the eluent from microparticles showed purified pUC plasmid.
- Example 3 Isolating Large DNA Vectors From 500 ml Cultures A cosmid (pWE15, obtained from Stratagene, Catalog Number 251201) containing a 35Kb insert and a Bacterial Artificial Chromosome were purified from their host cells by creating a cleared lysate, binding the DNA to the magnetic microparticles, separating the magnetic microparticles from the cleared lysate, and then eluting the DNA. The following procedure was used:
- Electrophoretic analysis of the solutions obtained from washing the magnetic microparticles with water showed purified cosmid cloned containing the 35Kb insert and the 150Kb BAC (Bacterial Artificial Chromosome) clone which had been cut with Notl to excise the 7Kb vector.
- Example 4 Single Stranded DNA Isolation from Bacteriophages Using the PEG Induced Precipitate Separation Single stranded DNA from M13 clones using E. coli (DH5 ⁇ F' culture, obtained from U.S. Biochemicals Catalog Number 75112) as a host was isolated by lysing the phage, binding the single stranded DNA to the magnetic microparticles, from separating the magnetic microparticles the supernatant and then eluting the DNA. The following procedure was used:
- Electrophoretic analysis of the solution obtained from washing the magnetic microparticles with water showed purified M13 single stranded DNA.
- Example 5 DNA Sequence from a Plasmid Clone Isolated Using the PEG Induced Precipitate Separation. DNA which had been isolated using the procedure described in example 1 was then sequenced using Taq poly erase and fluorescently labelled primers. The DNA sequence was then electrophoresed on an ABI 373A DNA sequence. The clarity of the data, the read length and the lack of ambiguous bases indicated DNA of high purity.
- DNA isolations may also be performed from amplified DNA such as PCR products.
- PCR product purification is the removal of residual nucleotides, oil and excess primers (18-25bp DNA fragments) , which may interfere with subsequent reactions.
- This example demonstrates the isolation of PCR products as well as the selective removal of the primers during the wash stages.
- Bind the DNA products Take 10 ⁇ l (@20 mg/ml) carboxyl coated magnetic particles, wash three times in 0.5 M EDTA pH 7.2 and resuspend in 10 ⁇ l 0.5 M EDTA. Add to the PCR amplifiers.
- Electrophoretic analysis of the PCR reaction mixture before binding to the magnetic microparticles showed the presence of primers as well as the final product. Electrophoretic analysis after elution from the magnetic microparticles show that the primers were removed from the amplification product.
- DNA fragments were isolated directly from molten agarose plugs.
- a Lambda marker was cut with HINDIII and electrophoresed. Bands corresponding to 23Kb, 9Kb, 6Kb, 4Kb, and 2Kb and 125bp bands were excised and isolated from the molten agarose according to the following procedure.
Abstract
A method of separating polynucleotides, such as DNA, RNA and PNA, from a solution containing polynucleotides by reversibly and non-specifically binding the polynucleotides to a solid surface, such as a magnetic microparticle, having a functional group-coated surface is disclosed. The salt and polyalkylene glycol concentration of the solution is adjusted to levels which result in polynucleotide binding to the magnetic microparticles. The magnetic microparticles with bound polynucleotides are separated from the solution and the polynucleotides are eluted from the magnetic microparticles.
Description
DNA PURIFICATION AND ISOLATION USING A SOLID PHASE
Background of the Invention
Preparation and manipulation of high quality DNA is a vital step in molecular biology. Although there are many methods reported for single and double stranded DNA isolations (Bankier, A. et al . , Meth . Enz . , 155:52-93 (1988); Birnboim, H. et al . , Nucl . Acids Res . , 7:1513 (1979) ; Ish-Horowicz, D. et al . , Nucl . Acids Res . , 9 : 2989 (1981) ; Kristensen, T. et al . , Nucl . Acids Res . , 25:5507-5516 (1987) ; Smith, V. et al . , DNA Seq. and Mapping , 1:73-78 (1990)) , there are few procedures that are rapid, low cost and procedurally identical for all DNA types, from PCR product to single copy BAC clone.
Summary of the Invention The present invention is a method of binding polynucieotides non-specifically and reversibly to a solid phase which reversibly binds polynucieotides, such as magnetic microparticles whose surfaces are coated with a functional group, such as a carboxyl group. The polynucieotides can be DNA, RNA or polyamide nucleic acids (PNAs) . The method comprises combining the solid phase, such as magnetic microparticles and a solution containing polynucieotides. The salt concentration and polyalkylene glycol concentration of the resulting combination are adjusted to concentrations suitable for binding polynucieotides to the surface of the solid phase, such as to surfaces of the magnetic microparticles; as a result, polynucieotides are bound non-specifically to the magnetic microparticles. The present invention also relates to a method of separating polynucieotides, such as DNA, RNA and PNA, from a
solution containing polynucieotides. The method comprises binding polynucieotides non-specifically to a solid surface, such as to magnetic microparticles, as described above, washing the resulting bound polynucieotides with a high ionic strength buffer, and eluting the polynucieotides with a low ionic strength elution buffer.
The method described herein is useful to separate double stranded (ds) or single stranded (ss) polynucieotides (e.g., DNA, RNA, PNA) of virtually any size and from a wide variety of sources. For example, the present method can be used to separate DNA present in a transfected host cell, DNA resulting from an amplification process (e.g., polymerase chain reaction, PCR) and DNA in gels, such as agarose gels. In addition, biochemical reactions and sequencing can be performed on DNA bound to the magnetic microparticles.
The present invention also relates to a kit comprising magnetic microparticles and a binding buffer which contains a suitable salt and polyalkylene glycol at concentrations suitable for reversibly binding polynucleotide onto solid surfaces, such as to the surfaces of magnetic microparticles. The kit may additionally comprise a suitable wash buffer, elution buffer, reagents for preparing such buffers or reagents for preparing a cleared lysate.
Because the method of the present invention is useful with both single and double stranded polynucieotides, as well as a wide range of polynucleotide fragment sizes, it has applicability in essentially any context in which polynucleotide separation is desired. In addition, this permits the standardization of manipulations and isolation carried out with polynucieotides. The present method simplifies the isolation of cloned DNA from lysate by obviating the need for centrifugation and produces a plasmid ready for sequencing and further characterization and processing. The present method also has the advantage that is fast, thus allowing for the rapid throughput in isolating
polynucleotides, low cost and simple to perform and produces high yields of polynucieotides. These properties, coupled with its applicability to many procedures useful in molecular biology, make the method amenable to automation.
Detailed Description of the Invention
As described herein, Applicant has shown that polynucieotides bind reversibly and non-specifically to solid surfaces, such as certain magnetic microparticles, at certain concentrations of salt and polyalkylene glycol. As a result, a method for convenient and rapid separation of polynucieotides, such as DNA, RNA and PNA, from other biomolecules, such as proteins, monosaccharides, polysaccharides, lipids and RNA and from cellular components, such as cell membranes, is available. Also available is a method for separation of polynucieotides on the basis of size. The following is a description of the present invention with reference to polynucieotides as exemplified by DNA. It is to be understood that the present invention is also useful for separation of RNA and PNAs in a similar manner. Because small polynucieotides require higher salt concentrations for strong binding to the microparticles, salt concentrations can be selectively manipulated to release polynucieotides bound to magnetic microparticles on the basis of size. One embodiment of the present invention is a method of separating DNA from a solution containing DNA. The method comprises a first step of reversibly binding DNA non-specifically to a solid surface, such as magnetic microparticles whose surfaces are coated with functional groups. In the method, the magnetic microparticles are combined with a solution of DNA, after which the salt concentration and the polyethylene glycol concentration of the resulting combination are adjusted to a concentration suitable for binding DNA onto the surface of the magnetic
particles. In one embodiment, sufficient salt and polyethylene glycol are added to the solution containing magnetic microparticle-bound DNA to result in a final concentration of from about .5 M to about 5.0 M salt and from about 7% to about 13% polyethylene glycol. As a result, DNA is bound non-specifically to the surfaces of the magnetic microparticles. Subsequently, the magnetic microparticles in the resulting combination are separated from the supernatant. The magnetic microparticles having DNA bound thereto can, optionally, be washed with a suitable wash buffer before they are contacted with a suitable elution buffer, to elute and separate the DNA from the magnetic microparticles. In a final step, the magnetic particles are separated from the elution buffer, which contains the polynucleotide, in solution. The magnetic microparticles are separated from the elution buffer by, for example, filtration or applying a magnetic field to draw down the microparticles.
Solid surfaces which bind DNA and have sufficient surface area to permit efficient binding can be used in the present invention. Microparticles, fibers, beads and supports contain suitable surfaces. Generally, magnetic microparticles are used in the present invention. As used herein, "magnetic microparticles" are microparticles which are attracted by a magnetic field. The magnetic microparticles used in the method of the present invention comprise a magnetic metal oxide core, which is generally surrounded by an adsorptively or covalently bound silane coat to which a wide variety of bioaffinity adsorbents can be covalently bound through selected coupling chemistries, thereby coating the surface of the microparticles with functional groups. The magnetic metal oxide core is preferably iron oxide, wherein iron is a mixture of Fe2* and Fe3* . The preferred Fe2*/Fe3* ratio is preferably 2/1, but can vary from about 0.5/1 to about 4/1. Suitable amino
silanes useful to coat the microparticle surfaces include p- aminopropyltrimethoxysilane, N-2-aminoethyl-3- aminopropyltrimethoxysilane, triaminofunctional silane (H2NCH2-NH-CH2CH2-NH-CH2-Si- (OCH3)3, n-dodecyltriethoxysilane and n-hexyltrimethoxysilane. Methods of preparing these microparticles are described in U.S. Patent Nos. 4,628,037, 4,554,088, 4,672,040, 4,695,393 and 4,698,302, the teachings of which are hereby incorporated by reference into this application in their entirety. These patents disclose other amino silanes which are suitable to coat the iron oxide core and which are encompassed by this invention. Magnetic microparticles comprising an iron oxide core, as described above, without a silane coat (BioMag Iron Oxide particles available from PerSeptive Diagnostics, Division of PerSeptive Biosystems, Catalog Number 8-4200) can also be used in the method of the present invention.
As used herein, the term "functional group-coated surface" refers to a surface which is coated with moieties which each have a free functional group which is bound to the amino group of the amino silane on the microparticle; as a result, the surfaces of the microparticles are coated with the functional group containing moieties. The functional group acts as a bioaffinity absorbent for DNA in solution. In one embodiment, the functional group is a carboxylic acid. A suitable moiety with a free carboxylic acid functional group is a succinic acid moiety in which one of the carboxylic acid groups is bonded to the amine of amino silanes through an amide bond and the second carboxylic acid is unbonded, resulting in a free carboxylic acid group attached or tethered to the surface of the magnetic microparticle. Carboxylic acid-coated magnetic microparticles are commercially available from PerSeptive Diagnostics (BioMag COOH, Catalog Number 8-4125) . Other suitable functional groups which can used for coating the surface of the magnetic microparticles include, but are not
limited to thiol groups (microparticles with thiol group coating are commercially available from PerSeptive Diagnostics, Division of PerSeptive Biosystems, Catalog Number 8-4135) and streptavidin (microparticles with a streptavidin coating are commercially available from
PerSeptive Diagnostics, BioMag Steptavidin, Catalog Number 8-MB4804) . As used herein, magnetic microparticles coated with thiol groups or streptavidin bind DNA less efficiently than carboxyl group-coated microparticles. The importance of having functional groups coat the surface of microparticles used is demonstrated by the observation that polymer encapsulated magnetic microparticles do not bind DNA in the method of the present invention. Polymer encapsulated microparticles are commercially available from Dynal, Incorporated, Dynabeads M-280, (Catalog Number 112.06) . The advantage of having a metal oxide core is illustrated by the observation that washing the magnetic microparticles with EDTA, which removes some of the iron, reduces the ability of the magnetic microparticles to bind DNA. Microparticles with a cellulose/iron oxide core, which are commercially available from Amersham International (Catalog Number NIF 876) did not bind DNA in the method of the present invention as it is described herein. Magnetic microparticles useful in the present method can be a variety of shapes, which can be regular or irregular; preferably the shape maximizes the surface areas of the microparticles. The magnetic microparticles should be of such a size that their separation from solution, for example by filtration or magnetic separation, is not difficult. In addition, the magnetic microparticles should not be so large that surface area is minimized or that they are not suitable for microscale operations. Suitable sizes range from about 0.1 μ mean diameter to about 100 μ mean diameter. A preferred size is about 1.0 μ mean diameter.
Suitable magnetic microparticles are commercially available from PerSeptive Diagnostics and are referred to as BioMag COOH (Catalog Number 8-4125) .
"Non-specific DNA binding" refers to binding of different DNA molecules with approximately the same affinity to magnetic microparticles, despite differences in the DNA sequence or size of the different DNA molecules. A polynucleotide can be DNA, RNA or a synthetic DNA analog such as a PNA (Nielsen et al . , Science, 254:1497 (1991)) . "Non-specific DNA binding" refers to binding of different DNA molecules with approximately the same affinity to magnetic microparticles despite differences in the nucleic acid sequence or size of the different DNA molecules. "A solution containing DNAs" can be any aqueous solution, such as a solution containing DNA, RNA and/or PNAs. Such a solution can also contain other components, such as other biomolecules, inorganic compounds and organic compounds. The solution can contain DNA which is the reaction product of PCR amplification. The solution can also be a cleared lysate. A "lysate", as used herein, is a solution containing cells which contain cloned DNA and genomic DNA and whose cell membranes have been disrupted, with the result that the contents of the cell, including the DNA contained therein, are in the solution. A "cleared lysate" is a lysate in which the chromosomal DNA, proteins and membranes of the host cells have been selectively removed, such as by chemical treatment or centrifugation of the lysate, thereby leaving a solution containing plasmid DNA. RNase can be added to create a "cleared lysate" free of RNA, thereby allowing DNA to bind to the magnetic microparticles free from RNA. Methods of creating a cleared lysate are well-known in the art. For example, a cleared lysate can be produced by treating the host cells with sodium hydroxide or its equivalent (0.2 N) and sodium dodecyl sulfate (SDS) (1%) . This method of creating a cleared lysate is described
in detail in Birnboim and Doly, Nucl . Acids Res . , 7:1513 (1979) Horowicz and Burke, Nucleic Acids Research 9:2989 (1981) , the teachings of which are hereby incorporated in their entirety into this reference. A host cell is any cell, such as a bacterial cell such as E. coli , a mammalian cell or a yeast cell which contains exogenous or foreign DΝA, in addition to genomic DΝA. The foreign DΝA may be introduced directly into the host cell by means known to one of ordinary skill in the art . Examples of foreign DΝA introduced directly into a host cell include bacterial artificial chromosomes (BAC) , yeast artificial chromosomes (YAC) , plasmids, cosmids and PI. BACs are particularly difficult to separate and purify from cleared lysates due to their low concentrations in the lysates. (Shizuya et al . ) However, BACs are readily separated by the method of the present invention. Alternatively, the plasmid DΝA may be introduced into the host cell by a phage into which the plasmid DΝA has been packaged. Suitable plasmid DΝAs which can be packaged into a phage include a cosmid or PI . Host cells containing foreign DΝA introduced by any method are referred to as transfected host cells.
The solution with which the magnetic microparticles is combined may also contain single stranded polynucieotides. For example, the present invention is useful to separate polynucieotides from a solution which is the supernatant from a recombinant DΝA-containing M13 bacteriophage isolate which had been used to infect bacterial host cells. The host cells are removed from the supernatant by filtration (Kristensen, et al . , Nucl . Acids Res . , 15:550-16 (1987)) or by binding the host cells to amine coated surfaces (Hou and
Zaniewski, Bioche , 12:315 (1990)) . Single stranded DΝA is released from the M13 bacteriophage into the solution by adding SDS to a final concentration of about 0.3% to about 3%, preferably about 1% and at a temperature from about 60°C to about 100°C, preferably 80°C.
The DNA-containing solution may also be an agarose solution. For example, a mixture of DNA is separated, according to methods known to one skilled in the art, such as by electrophoresis on an agarose gel. A plug of agarose containing DNA of interest can be excised from the gel and added to 1-10 volumes of 0.5 x SSC ( .75 M NaCI, .0075 M Sodium Citrate, pH 7.0) preferably 4 volumes. The mixture is then melted at a temperature of from about 60°C to about 100°C, preferably at about 80°C for about one to about twenty minutes, preferably ten minutes, to create an agarose solution containing DNA.
As described above, the second step of the present method of binding DNA non-specifically to magnetic microparticles having a functional group-coated surface (e.g., a carboxyl-group-coated surface) comprises adjusting the salt concentration and the polyalkylene glycol concentration of the combination to a concentration of each suitable for binding DNA reversibly onto the surface of the magnetic particles. Suitable polyalkylene glycols include polyethylene glycol (PEG) and polypropylene glycol.
Generally, PEG is used. A sufficient quantity of a salt and a sufficient quantity of PEG are combined with the combination of magnetic microparticles and DNA-containing solution to produce a final salt concentration of from about 0.5 M to about 5.0 M and a final PEG concentration of from about 7% to about 13%. At appropriate concentrations of the two, DNA binds non-specifically to the surface of the magnetic microparticles. The binding of the DNA to the magnetic microparticles is rapid; it is generally complete within thirty seconds.
Salts which have been found to be suitable for binding DNA to the microparticles include sodium chloride (NaCI) , lithium chloride (LiCl) , barium chloride (BaCl2) , potassium (KC1) , calcium chloride (CaCl2) , magnesium chloride (MgCl2) and cesium chloride (CeCl) . In one embodiment sodium
chloride is used. The wide range of salts suitable for use in the method indicates that many other salts can also be used and can be readily determined by one of ordinary skill in the art. Yields of bound DNA decrease if the salt concentration is adjusted to less than about 0.5 M or greater than about 5.0 M. The salt concentration is preferably adjusted to about 1.25 M.
The molecular weight of the polyethylene glycol (PEG) can range from about 6000 to about 10,000, with a molecular weight of about 8000 being preferred. The concentration of PEG is preferably adjusted to about 10%. Although concentrations of PEG as low as 7% and as high as 13% can be used, yields of bound DNA drop as the concentration of PEG deviates from 10%. The method of the present invention is useful to separate DNA from a solution containing polynucleotide. As discussed above, the method comprises binding DNA nonspecifically and reversibly to magnetic microparticles having a functional group coated (e.g., carboxyl-coated) surface. The microparticles are then separated from the supernatant, for example by applying a magnetic field to draw down the magnetic microparticles. The remaining solution, i.e. supernatant, can then be removed, leaving the microparticles with the bound DNA. Once separated from the supernatant, the DNA can be removed from the magnetic microparticles particles by washing with a suitable elution buffer. As a result, an elution buffer containing unbound DNA and magnetic microparticles is produced. An elution buffer is any aqueous solution in which the salt concentration and polyalkylene concentration are below the ranges required for binding of DNA onto magnetic microparticles, as discussed above. In addition, sucrose (20%) and formamide (100%) solutions can be used to elute the DNA. A preferred eluent is water. Elution of the DNA from the microparticles occurs in thirty seconds or less
when an elution buffer of low ionic strength, for example water, is used. Once the bound DNA has been eluted, the magnetic microparticles are separated from the elution buffer that contains the eluted DNA. Preferably, the magnetic microparticles are separated from the elution buffer by magnetic means, as described above. Other methods known to those skilled in the art can be used to separate the magnetic microparticles from the supernatant; for example, filtration can be used. Yields of DNA following elution typically approach 100% when the magnetic microparticles are used in excess. In one embodiment, the magnetic microparticles with bound DNA are washed with a suitable wash buffer solution before separating the DNA from the microparticles by washing with an elution buffer. A suitable wash buffer solution has several characteristics. First, the wash buffer solution must have a sufficiently high salt concentration (i.e., has a sufficiently high ionic strength) that the DNA bound to the magnetic microparticles does not elute off of the microparticles, but remains bound to the microparticles. Suitable salt concentrations are greater than about 1.0 M and is preferably about 5.0 M. Second, the buffer solution is chosen so that impurities that are bound to the DNA or microparticles are dissolved. The pH and solute composition and concentration of the buffer solution can be varied according to the type of impurities which are expected to be present. Suitable wash solutions include the following: 0.5 x 5 SSC ; 100 mM ammonium sulfate, 400 mM Tris pH 9, 25 mM MgCl2 and 1% bovine serum albumine (BSA) ; and 5 M NaCI . A preferred wash buffer solution comprises 25 mM Tris acetate (pH 7.8), 100 mM potassium acetate (KOAc) , 10 mM magnesium acetate (Mg20Ac) , and 1 mM dithiothreital (DTT) . The magnetic microparticles with bound DNA can also be washed with more than one wash buffer solution. The magnetic microparticles can be washed as often as required to remove
the desired impurities. However, the number of washings is preferably limited to two or three in order to minimize loss of yield of the bound DNA. Yields of DNA when the microparticles are used in excess are typically about 80% after washing with a wash buffer and eluting with an elution buffer.
The polynucleotide in the solution with which the magnetic microparticles are combined can be single stranded or double stranded. In addition, the polynucleotide can be homogeneous (i.e. polynucieotides which have the same nucleotide sequence) . Alternatively, the polynucleotide can be heterogeneous, (i.e., polynucieotides of differing nucleotide sequences) . The polynucleotide can also comprise a DNA library or partial library. The DNA can also comprise molecules of various lengths. For example, DNA fragments of 23Kb, 9Kb, 64Kb, 4Kb, 2Kb and 12bp derived from the electrophoretic separation of a HINDIII cut lambda marker were cut out of the agarose gel and purified by the method of the present invention (see Example 7) . Temperature does not appear to be critical in the method of separating DNA of the present invention. Ambient temperature is preferred, but any temperature above the freezing point of water and below the boiling point of water can be used. DNA fragments of all sizes bind non-specifically to magnetic microparticles at high ionic strength. High ionic strength refers to salt concentrations greater than 0.5 M. However, smaller fragments of DNA bind with lower affinity than large DNA fragments at lower ionic strengths, for example, about 0.5 M salt concentration and lower.
Another embodiment of the present invention refers to a method of separating a mixture of polynucleotide fragments, such as DNA fragments, based on size. For example, a solution of DNA fragments of different sizes is first combined with magnetic microparticles having a carboxyl
group-coated surface under conditions appropriate for non¬ specific binding of DNA to the magnetic microparticles. The magnetic microparticles are then separated from the supernatant. Optionally, the polynucleotide bound to the magnetic microparticles can be washed with a suitable wash buffer which dissolves bound impurities, but is of high enough ionic strength that the polynucleotide remains attached to the magnetic microparticles. The magnetic microparticles are then washed with an elution buffer of appropriate ionic strength to elute the smaller size polynucleotide fragments, but leave the larger size polynucleotide fragments bound to the magnetic microparticles. The smaller polynucleotide fragments, such as DNA, in the elution buffer can then be isolated in the usual manner or processed further, e.g., subjected to further biochemical reactions. This method has been used to separate PCR primers from the reaction product of a PCR amplification (see Example 6) .
The polynucleotide (e.g., DNA) which remains bound to the magnetic particles can then be eluted with a suitable elution buffer. The DNA can then be isolated in the usual manner, or processed further, e.g. subjected to further biochemical reactions. Alternatively, the DNA which remains bound to the magnetic microparticles can be subjected to further size selection by washing with an elution buffer of sufficiently low ionic strength to elute the smaller remaining DNA fragment, but of sufficiently high enough ionic strength to allow the larger remaining polynucleotide fragments to remain bound to the magnetic microparticles. The separation of polynucleotide fragments (e.g., DNA fragments) based on size can also be accomplished by the method of the present invention by adjusting the PEG concentration, the molecular weight of the PEG used or both. One embodiment of the present invention is based on the discovery that the magnetic microparticles do not bind
enzymes. The magnetic microparticles also do not inhibit the function of enzymes. It is therefore possible to carry out biochemical reactions on DNA bound to the magnetic microparticles, e.g., by exposing the bound DNA to enzymes capable of biochemically modifying the bound DNA under conditions which cause the biochemical modification to take place. Preferably the biochemical reactions are carried out on purified bound DNA (e.g., DNA bound to microparticles which have been separated from a cleared lysate or from a solution in which a biochemical reaction such as PCR was carried out) . The purified bound DNA can also be washed with a suitable wash buffer. Because residual salt can inhibit the activity of certain enzymes, it is preferable that washings with high ionic strength salt solutions be followed with a washing with a lower ionic strength solution. The ionic strength of this solution should be low enough that enough residual salt is removed to prevent enzyme inhibition, but not so low that substantial losses in bound DNA result. In one embodiment, the DNA bound to the magnetic microparticles is digested with a restriction enzyme. The restriction enzyme-digested DNA can then be end-repaired, if necessary, for later ligation to a vector by suitable end- repair enzymes. The end-repaired DNA is typically eluted by the solvent in which the biochemical reaction takes place. Alternatively, the magnetic microparticles are washed with a suitable elution buffer to ensure complete separation of the end-repaired DNA from the microparticles. The magnetic microparticles are then separated from the reaction mixture or elution buffer, preferably by magnetic separation. The solution containing the end-repaired DNA can then be combined with a solution containing a pre-cut vector suitable for ligation to the eluted DNA. The end-repaired DNA can then be ligated to the pre-cut vector by methods
known to those skilled in the art. After ligation, the DNA can be transformed into a host cell in the usual way.
In another embodiment, a DNA library is bound to the magnetic microparticles. The DNA bound to the magnetic microparticles will consist of molecules of various sizes and nucleotide sequences (heterogeneous DNA) . Specific size fragments can be eluted from the magnetic microparticles by varying the ionic strength of the elution buffer, as described earlier. Alternatively, the concentration, molecular weight or both of the PEG in the elution buffer can be varied, as described earlier, to selectively elute smaller DNA fragments. The DNA fragments which remain bound to the magnetic microparticles can be digested with one or more restriction enzymes and then ligated into a pre-cut vector, as described above. A vector is thereby created in which the DNA insert has a certain size. The vector can then be transformed into a host cell in the usual way.
In another embodiment, the nucleotide sequence of the DNA bound to the magnetic microparticles is determined directly without an elution step which releases the DNA from the magnetic microparticles. DNA is bound to the microparticles as described above. The microparticles with bound DNA are then separated from the supernatant and combined with the reagents used for determining nucleotide sequences under conditions suitable for sequence determination. Suitable reagents and conditions are known to those skilled in the art (See Sanger et al . , Proc . Na t . Acad. Sci . , 74:5463 (1977) and the ABI 373 Sequencer Manual) . A kit is also provided herein which contains the reagents necessary for separating polynucieotides, such as DNA, RNA and PNAs, from a solution containing polynucieotides by binding the polynucieotides to a solid surface, such as magnetic microparticles having a carboxyl group-coated surface. The kit comprises magnetic
microparticles with a carboxyl group-coated surface and a binding buffer. The binding buffer comprises a suitable salt and a suitable polyalkylene glycol which are both present at a concentration suitable for binding DNA to the surface of the magnetic microparticles. In one embodiment, the kit further comprises an elution buffer which is capable of dissolving the polynucleotide, such as DNA, bound to the magnetic microparticles. Alternatively, instead of a binding buffer and/or elution buffer, the kit can comprise the reagents for making the binding and/or elution buffer, to which a known amount of water can be added to create a binding and/or elution buffer of desired concentration.
In another embodiment, the kit further comprises a wash buffer which dissolves impurities bound to the magnetic microparticles, but does not result in elution of the polynucleotide bound to the magnetic microparticles. Alternatively, instead of a wash buffer, the kit can comprise the reagents for making the wash buffer, to which a known amount of water can be added to create a wash buffer of desired concentration.
In yet another embodiment, the kit comprises the reagents necessary for clearing a cell lysate. In a preferred embodiment, the reagents are present in solutions at a concentration suitable for direct use in preparing a cleared lysate without the need for further diluting the solutions.
The present invention will now be illustrated by the following examples, which are not limiting in any way.
General Methodolocrv The magnetic particles used in the following examples were the carboxyl coated magnetic microparticles from PerSeptive Diagnostics Massachusetts, (Biomag COOH, Catalog Number 8-4125) particles which were 1 μm in diameter. The particles were stored in phosphate buffered saline (PBS) at
a concentration of 20 mg/ml. All agarose gels were run using 1% final agarose (U.S. Biochemical #32827) with lx TBE buffers. The field strength was lOV/cm with run times from 40-60 minutes. The gels were post-stained with ethidium bromide and visualized under UV.
Example 1 - Double Stranded DNA Isolation using the PEG Induced Precipitate Separation
A pUC plasmid (pUC 18, obtained from U.S. Biochemicals, Catalog Number 70070) was purified from its host cell by creating a cleared lysate, reversibly binding the plasmid to the magnetic microparticles, separating the magnetic microparticles and then eluting the DNA. The following procedure was used:
1. Take 1 ml of overnight culture containing the plasmid clone in an Eppeπdorf™ tube.
2. Create a cleared lysate.
Centrifuge for 2 minutes to pellet the cells. Pour off the supernatant and resuspend the pellet in 30 μl Solution 1 (50 mM Glucose, 25 mM Tris.Cl pH 8, 10 mM EDTA pH 8, 100 μg/ml RNase) .
Add 60 μl Solution 2 (0.2N NaOH, 1% SDS) and mix by shaking. Leave at room temperature for 5 minutes. Add 45 μl Solution 3 (3 M KOAc) , mix by shaking and leave on ice for 10 minutes. Centrifuge for 10 minutes and remove 100 μl of the supernatant to a new Eppendorf tube.
3. Take 10 μl (@20 mg/ml) carboxyl coated magnetic particles, wash three times in 0.5 M EDTA pH 7.2 and resuspend in 10 μl 0.5 M EDTA. Add to the cleared lysate.
4. Add 100 μl of the binding buffer (20% PEG 8000, 2.5 M NaCI) and mix.
5. Allow to incubate at room temperature for 5 minutes.
6. Wash the magnetic particles twice with 5 M NaCI and once with wash buffer (25 mM TrisAcetate pH 7.8, 100 mM KOAc, 10 mM Mg2OAc, 1 mM DTT) . There is no need to resuspend the particles during each wash. 7. Resuspend the particles in 50 μl water and incubate at room temperature for 1 minute.
8. Magnetically separate the particles and remove the DNA to a new tube.
An electrophoretic analysis was done on the cleared lysate after binding to the magnetic microparticles. A complete absence of DNA was observed. An electrophoretic analysis was also done on the elution solution, which showed the purified pUC plasmid.
Example 2 - Double Stranded DNA Isolation using the PEG Induced Precipitate Separation Using a Microtitre Plate
A pUC plasmid (pUC 18, obtained from U.S. Biochemicals Catalog Number 70070) grown in microtitre plates was purified from its host cell by creating a cleared lysate, reversibly binding the plasmid to the magnetic microparticles, separating the magnetic microparticles and then eluting the DNA. The following procedure was used:
1. Grow a single plasmid clone in the well of a microtitre plate containing 300 μl of growth media. 96 different clones may be grown in each plate. Grow the plasmids at 37°C overnight (12-15 hours) shaking at 300rpm.
2. Create a cleared lysate.
Centrifuge for 10 minutes at 3000rpm to pellet the cells.
Aspirate off the supernatant and resuspend the pellet in 30 μl Solution 1 (50 mM Glucose, 25 mM Tris Cl pH 8, 10 mM EDTA pH 8, 100 μg/ml RNase) .
Add 60 μl Solution 2 (0.2N NaOH, 1% SDS) and mix by shaking. Leave at room temperature for 5 minutes. Add 45 μl Solution 3 (3 M KOAc) , mix by shaking and leave on ice for 10 minutes. Centrifuge for 15 minutes at 3000rpm and remove
100 μl of the supernatant to a new microtitre plate. 3. Take 10 μl (@ 20 mg/ml) carboxyl coated magnetic particles, wash three times in 0.5 M EDTA pH 7.2 and resuspend in 10 μl 0.5 M EDTA. Add to each well. 4. Add 100 μl of the binding buffer to each well (20% PEG 8000, 2.5 M NaCI) and mix.
5. Allow to incubate at room temperature for 5 minutes.
6. Wash the magnetic particles twice with 5 M NaCI and once with wash buffer (25 mM Tris.Acetate pH 7.8, 100 mM KOAc, 10 mM Mg2OAc, 1 mM DTT) . There is no need to resuspend the particles during each wash.
7. Resuspend the particles in 50 μl water and incubate at room temperature for 1 minute.
8. Magnetically separate the particles and remove the DNA to a new microtitre plate.
This example yields 500-800ng plasmid DNA which is sufficient for thermal cycle DNA sequencing. The advantage of using a microtitre plate is that many samples can be isolated in parallel. As in Example 1, electrophoretic analysis of the cleared lysate after binding to the magnetic microparticles showed no DNA, while electrophoretic analysis of the eluent from microparticles showed purified pUC plasmid.
Example 3 - Isolating Large DNA Vectors From 500 ml Cultures A cosmid (pWE15, obtained from Stratagene, Catalog Number 251201) containing a 35Kb insert and a Bacterial Artificial Chromosome were purified from their host cells by creating a cleared lysate, binding the DNA to the magnetic
microparticles, separating the magnetic microparticles from the cleared lysate, and then eluting the DNA. The following procedure was used:
1. Grow a single plasmid clone in 500 ml of growth media. Grow the plasmids at 37°C overnight (12-15 hours) shaking at 300rpm.
2. Create a cleared lysate.
Centrifuge for 10 minutes at 4000rpm to pellet the cells. Pour off the supernatant and aspirate any further liquid. Resuspend the pellet in 3 ml Solution 1 (50 mM Glucose, 25 mM Tris.Cl pH 8, 10 mM EDTA pH 8, 100 μg/ml RNase) .
Add 6 ml Solution 2 (0.2N NaOH, 1% SDS) and mix by shaking. Leave at room temperature for 5 minutes.
Add 4.5 ml Solution 3 (3 M KOAc) , mix by shaking and leave on ice for 15 minutes.
Centrifuge for 15 minutes at 5000rpm and remove 10 ml of the supernatant to a new Falcon tube. (Filter through a 0.45m filter if there are any visible signs of precipitate) .
3. Take 1 ml (@20 mg/ml) carboxyl coated magnetic particles, wash three times in 0.5 M EDTA pH 7.2 and resuspend in 1 ml 0.5 M EDTA. Add to the supernatant. 4. Add 11 ml of the binding buffer to each well (20% PEG 8000, 2.5 M NaCI) and mix.
5. Allow to incubate at room temperature for 15 minutes.
6. Wash the magnetic particles twice with 5 M NaCI and once with wash buffer (25 mM Tris Acetate pH 7.8, 100 mM KOAc, 10 mM Mg2OAc, 1 mM DTT) . There is no need to resuspend the particles during each wash.
7. Resuspend the particles in 1 ml water and incubate at room temperature for 1 minute.
8. Magnetically separate the particles and remove the DNA to a new tube.
Electrophoretic analysis of the solutions obtained from washing the magnetic microparticles with water showed purified cosmid cloned containing the 35Kb insert and the 150Kb BAC (Bacterial Artificial Chromosome) clone which had been cut with Notl to excise the 7Kb vector.
Example 4 - Single Stranded DNA Isolation from Bacteriophages Using the PEG Induced Precipitate Separation Single stranded DNA from M13 clones using E. coli (DH5αF' culture, obtained from U.S. Biochemicals Catalog Number 75112) as a host was isolated by lysing the phage, binding the single stranded DNA to the magnetic microparticles, from separating the magnetic microparticles the supernatant and then eluting the DNA. The following procedure was used:
1. Grov: the M13 clones for 6 hours in 500 μl of lxLB media with a 1/100 dilution an overnight E. coli F' culture. Use of the Beckman deepwell plates will facilitate 96 clones being grown at one time.
2. Centrifuge the growth plate at 3000rpm for 10 minutes to pellet the cells.
3. Add 20 μl of 10% SDS to each well of a Falcon 9311 using a multidispensing pipette. 4. Using a 12 channel pipette, add 100 μl of the phage supernatant to each well, mixing the lysis solution with the supernatant. 5. Incubate the Falcon plate at room temperature for 5 minutes. 6. For a whole plate of 96 clones, remove 1 ml of magnetic particles from the stock and wash three times in 0.5 M
EDTA pH 7.2. Take up the washed particles in 1 ml 0.5 M EDTA pH 7.2 and add 10 μl to each well. 7. Add 100 μl of the binding buffer to each well (20% PEG 8000, 2.5 M NaCI) and mix. 8. Incubate the Falcon plate at room temperature for 5 minutes.
9. Wash the magnetic particles twice with 5 M NaCI and once with wash buffer (25 mM Tris Acetate pH 7.8, 100 mM KOAc, 10 mM Mg2OAc, 1 mM DTT) . There is no need to resuspend the particles during each wash.
10. Resuspend the particles in 50 μl water and incubate at room temperature for 1 minute.
11. Magnetically separate the particles and remove the DNA to a new microtitre plate.
Electrophoretic analysis of the solution obtained from washing the magnetic microparticles with water showed purified M13 single stranded DNA.
Example 5 - DNA Sequence from a Plasmid Clone Isolated Using the PEG Induced Precipitate Separation. DNA which had been isolated using the procedure described in example 1 was then sequenced using Taq poly erase and fluorescently labelled primers. The DNA sequence was then electrophoresed on an ABI 373A DNA sequence. The clarity of the data, the read length and the lack of ambiguous bases indicated DNA of high purity.
Example 6 - Selective Removal of DNA From the Solid Phase Based on the Size of the DNA Fragments
DNA isolations may also be performed from amplified DNA such as PCR products. One desirable feature of PCR product purification is the removal of residual nucleotides, oil and excess primers (18-25bp DNA fragments) , which may interfere with subsequent reactions. This example demonstrates the
isolation of PCR products as well as the selective removal of the primers during the wash stages.
1. PCR amplify the DNA.
2. Bind the DNA products : Take 10 μl (@20 mg/ml) carboxyl coated magnetic particles, wash three times in 0.5 M EDTA pH 7.2 and resuspend in 10 μl 0.5 M EDTA. Add to the PCR amplifiers.
Add an equal volume of the binding buffer to each reaction (20% PEG 8000, 2.5 M NaCI) and mix.
Allow to incubate at room temperature for 5 minutes.
3. Wash the magnetic particles twice with 5 M NaCI and once with wash buffer (25 mM Tris.Acetate pH 7.8, 100 mM LOAC, 10 mM Mg2OAc, 1 mM DTT) . There is no need to resuspend the particles during each wash.
4. Resuspend the particles in 50 μl water and incubate at room temperature for 1 minute.
5. Magnetically separate the particles and remove the DNA to a new tube.
Electrophoretic analysis of the PCR reaction mixture before binding to the magnetic microparticles showed the presence of primers as well as the final product. Electrophoretic analysis after elution from the magnetic microparticles show that the primers were removed from the amplification product.
Example 7 - DNA Isolation from Agarose Gels Using the PEG Induced Precipitate Separation
DNA fragments were isolated directly from molten agarose plugs. A Lambda marker was cut with HINDIII and electrophoresed. Bands corresponding to 23Kb, 9Kb, 6Kb,
4Kb, and 2Kb and 125bp bands were excised and isolated from the molten agarose according to the following procedure.
1. Electrophorese the DNA samples on a 0.5% agarose gel.
2. Cut out the band of interest. 3. Add 4 volumes of 0.5 x SSC to the agarose plug and melt for 10 minutes at 80°C. 4. Take 10 μl (@ 20 mg/ml) carboxyl coated magnetic particles, wash three times in 0.5 M EDTA pH 7.2 and resuspend in 10 μl 0.5 M EDTA. Add to the supernatant. 5. Add an equal volume of the binding buffer to each well (20% PEG 8000, 2.5 M NaCI) and mix.
6. Allow to incubate at room temperature for 15 minutes.
7. Wash the magnetic particles twice with 5 M NaCI and once with wash buffer (25 mM Tris Acetate pH 7.8, 100 mM KOAc, 10 mM Mg20Ac, 1 mM DTT) . There is no need to resuspend the particles during each wash.
8. Resuspend the particles in 50 μl water and incubate at room temperature for 1 minute.
9. Magnetically separate the particles and remove the DNA to new tube.
Electrophoretic analysis after elution from the magnetic microparticles showed DNA fragments of the sizes excised from the original gel. This approach is rapid and gives the same high yield as the more standard gel extraction applications such as 3-agarose (Eppicenter technologies, WI or Geneclean II (Bio 101 Inc. CA) .
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.
Claims
1. A method of reversibly binding polynucieotides non- specifically to magnetic microparticles, comprising the steps of : a) combining magnetic microparticles whose surfaces have bound thereto a functional group which reversibly binds polynucleotide and a solution containing polynucieotides, thereby producing a combination; and b) adjusting the salt and polyalkylene glycol concentration of the combination to a concentration suitable for binding the polynucleotide onto the surfaces of the magnetic microparticles, whereby polynucleotide in the solution binds non-specifically to the magnetic microparticles.
2. The method of Claim 1, wherein the polynucleotide is DNA and the polyalkylene glycol is polyethylene glycol .
3. The method of Claim 2, wherein the polyethylene glycol has a molecular weight of between about 6000 and about 10,000, and wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride.
4. The method of Claim 3, wherein the concentration of the polyethylene glycol is adjusted to between about 5% and about 15% and wherein the concentration of sodium chloride is adjusted to between about 0.5 M and about 5.0 M.
5. The method of Claim 4, wherein the concentration of the polyethylene glycol is about 10% and the concentration of sodium chloride is about 1.25 M.
6. A method of binding DNA non-specifically to magnetic microparticles, comprising the steps of: a) combining magnetic microparticles whose surfaces have bound thereto a functional group which reversibly binds DNA and a solution containing DNA, thereby producing a combination; and b) adjusting the salt and polyethylene glycol concentration of the combination to a concentration suitable for binding the DNA onto the surfaces of the magnetic microparticles, whereby DNA in the solution binds non-specifically to the magnetic microparticles.
7. The method of Claim 6, wherein the polyethylene glycol has a molecular weight of between 6000 and 10,000, and wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride.
8. The method of Claim 7, wherein the polyethylene glycol has a molecular weight of about 8000 and the salt is sodium chloride.
9. The method of Claim 8, wherein the concentration of the polyethylene glycol is adjusted to between about 5% and about 15% and wherein the concentration of sodium chloride is adjusted to between about 0.5 M and about 5.0 M.
10. The method of Claim 9, wherein the concentration of the polyethylene glycol is about 10% and the concentration of sodium chloride is about 1.25 M.
11. The method of Claim 10, wherein the DNA solution is a cleared lysate.
12. The method of Claim 10, wherein the DNA solution comprises the reaction product of a PCR amplification.
13. The method of Claim 10, wherein the DNA solution comprises M13 phage DNA, wherein the host cells have been removed.
14. A method of reversibly binding DNA non-specifically to magnetic microparticles having a carboxyl group-coated surface, comprising the steps of: a) combining magnetic microparticles having a carboxyl group-coated surface to a solution containing DNA; and b) adjusting the salt and polyethylene glycol concentration of the solution to a concentration suitable for binding the DNA onto the surfaces of the magnetic microparticles, thereby binding the DNA non-specifically to the magnetic microparticles.
15. The method of Claim 14, wherein the polyethylene glycol has a molecular weight between about 6000 and about 10,000, and wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride.
16. The method of Claim 15, wherein the polyethylene glycol has a molecular weight of about 8000 and the salt is sodium chloride.
17. The method of Claim 16, wherein the concentration of the polyethylene glycol is adjusted to between about 5% and about 15% and wherein the concentration of sodium chloride is adjusted to between about 0.5 M and about 5.0 M.
18. The method of Claim 17, wherein the concentration of the polyethylene glycol is about 10% and the concentration of sodium chloride is about 1.25 M.
19. The method of Claim 17, wherein the DNA solution is a cleared lysate.
20. The method of Claim 17, wherein the DNA solution comprises the reaction product of a PCR amplification.
21. The method of Claim 17, wherein the DNA solution comprises M13 phage DNA, wherein the host cells have been removed.
22. A method of separating DNA from a solution containing DNA, comprising the steps of: a) combining magnetic microparticles having carboxyl group-coated surfaces and a solution containing DNA, thereby producing a first combination; b) adjusting the salt concentration and the polyethylene glycol concentration of the first combination to concentrations suitable for binding DNA to surfaces of the magnetic microparticles, thereby producing a second combination comprising DNA bound non-specifically to magnetic microparticles; c) separating the magnetic microparticles from the second combination; 5 d) contacting the magnetic microparticles separated in c) with the bound DNA in an elution buffer, whereby DNA is dissolved in the elution buffer and DNA bound from the magnetic microparticles is separated from the magnetic microparticles; and 10 e) separating the magnetic microparticles from the elution buffer.
23. The method of Claim 22, wherein the separation of the magnetic microparticles in steps b) and d) is done magnetically.
15 24. The method of Claim 23, wherein the DNA bound to the magnetic microparticles is washed with a buffer solution, wherein the buffer solution dissolves impurities bound to the magnetic microparticles while leaving the DNA bound to the magnetic microparticles.
20 25. The method of Claim 23, wherein the DNA bound to the magnetic microparticles is washed with a selective elution solution, wherein the selective elution solution dissolves small DNA fragments bound to the microparticles while leaving large DNA fragments bound
25 to the magnetic microparticles.
26. The method of Claim 25, wherein the DNA dissolved by the elution solution is isolated.
27. The method of Claim 24, wherein the DNA is digested by one or more enzymes while bound to the magnetic
30 microparticles.
28. The method of Claim 24, wherein the DNA solution of step a) is a cleared lysate.
29. The method of Claim 24, wherein the DNA of the DNA solution of step a) is the reaction product of a PCR
5 amplification procedure.
30. The method of Claim 24, wherein the DNA solution of step a) comprises M13 phage DNA, wherein the host cells have been removed.
31. The method of Claim 24, wherein the DNA solution of 10 step a) comprises DNA of interest, wherein the DNA of interest has been separated from undesired DNA by electrophoresis in an agarose gel, and a melted agarose plug excised from the electrophoresis gel, wherein the agarose plug contains the DNA of interest.
15 32. A method of separating DNA from a solution containing DNA, comprising the steps of: a) combining a solution containing DNA and carboxyl group-coated magnetic micro-particles, thereby producing a first combination;
20 b) combining the first combination with sufficient quantities of polyethylene glycol and sodium chloride to produce a second combination having a final polyethylene glycol concentration of from about 5% to about 15% and a final sodium chloride 5 concentration of from about 0.5 M to about 5.0 M, whereby DNA in the solution binds nonspecifically to the magnetic microparticles, producing magnetic microparticles having DNA bound thereto; c) separating magnetic microparticles from the second 0 combination, thereby producing separated magnetic microparticles having DNA bound thereto; d) contacting the separated magnetic microparticles having DNA bound thereto with an elution buffer, whereby the DNA is released from the magnetic microparticles and an elution buffer containing
5 DNA and magnetic microparticles is produced; and e) separating DNA released from the magnetic microparticles from the elution buffer.
33. The method of Claim 32, wherein the polyethylene glycol has a molecular weight of between about 6000 and about
10 10,000 and wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, calcium chloride, potassium chloride, lithium chloride, barium chloride and cesium chloride.
34. The method of Claim 33, wherein the polyethylene glycol 15 has a molecular weight of about 8000 and the salt is sodium chloride.
35. The method of Claim 34, wherein the concentration of the polyethylene glycol is adjusted to between about 5% and about 15% and wherein the concentration of sodium
20 chloride is adjusted to between about 0.5 M and about 5.0 M.
36. The method of Claim 35, wherein the concentration of the polyethylene glycol is about 10% and the concentration of sodium chloride is about 1.25 M.
25 37. A method of determining the nucleotide sequence of a
DNA molecule, comprising binding the DNA molecule non- specifically to magnetic microparticles having a carboxyl group-coated surface by the method of Claim 17 and sequencing the DNA while the DNA is bound to the
30 magnetic microparticles.
38. A kit comprising a binding buffer and magnetic microparticles with carboxyl group-coated surfaces, wherein the binding buffer comprises a suitable salt and a suitable polyethylene glycol at a concentration suitable for binding DNA to the surface of the magnetic microparticles .
39. The kit of Claim 38, wherein the kit further comprises a suitable elution buffer, wherein the elution buffer is capable of dissolving DNA bound to the magnetic microparticles.
40. The kit of Claim 39, wherein the kit additionally comprises a wash buffer, wherein the wash buffer is capable of dissolving impurities bound to magnetic microparticles but is incapable of dissolving DNA bound to magnetic microparticles.
41. The kit of Claim 39, wherein the kit additionally comprises reagents necessary for clearing a cell lysate .
42. A method of isolating polynucieotides from a solution containing a mixture of biomolecules, comprising the steps of : a) combining a solution containing a mixture of biomolecules with carboxyl-coated magnetic microparticles; b) adjusting the polarity and the hydrophobicity of the resulting solution so as to adsorb polynucieotides onto the surface of the microparticles; c) separating the microparticles from the solution containing the biomolecules; d) combining the separated magnetic microparticles with a second solution whose polarity and hydrophobicity results in the polynucieotides being desorbed from the surface of the magnetic microparticle, and dissolved in the second solution; and e) separating the magnetic microparticles from the second solution.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/309,267 US5705628A (en) | 1994-09-20 | 1994-09-20 | DNA purification and isolation using magnetic particles |
US08/309,267 | 1994-09-20 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996009379A1 true WO1996009379A1 (en) | 1996-03-28 |
Family
ID=23197456
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1995/011839 WO1996009379A1 (en) | 1994-09-20 | 1995-09-19 | Dna purification and isolation using a solid phase |
Country Status (3)
Country | Link |
---|---|
US (2) | US5705628A (en) |
IL (1) | IL115352A (en) |
WO (1) | WO1996009379A1 (en) |
Cited By (30)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0885958A1 (en) * | 1996-02-25 | 1998-12-23 | Precision System Science Co., Ltd. | Method for treating biopolymers, microorganisms or materials by using more than one type of magnetic particles |
WO1999058664A1 (en) * | 1998-05-14 | 1999-11-18 | Whitehead Institute For Biomedical Research | Solid phase technique for selectively isolating nucleic acids |
EP1002860A1 (en) * | 1998-10-30 | 2000-05-24 | Becton Dickinson and Company | Method for purification and manipulation of nucleic acids using paramagnetic particles |
WO2001040459A2 (en) * | 1999-12-03 | 2001-06-07 | InViTek Gesellschaft für Biotechnik & Biodesign mbH | Surface modified supporting materials for binding biological materials, method for the production and use thereof |
WO2001046404A1 (en) * | 1999-12-22 | 2001-06-28 | Abbott Laboratories | Nucleic acid isolation method and kit |
WO2001059098A2 (en) * | 2000-02-11 | 2001-08-16 | Eppendorf Ag | Method for purifying nucleic acids |
WO2003040687A2 (en) | 2001-11-06 | 2003-05-15 | Cortex Biochem, Inc. | Isolation and purification of nucleic acids |
EP1368629A1 (en) * | 2001-02-16 | 2003-12-10 | Cortex Biochem Inc. | Magnetic isolation and purification of nucleic acids |
WO2005089929A2 (en) | 2004-03-18 | 2005-09-29 | Ambion , Inc. | Modified surfaces as solid supports for nucleic acid purification |
US7081192B1 (en) * | 2000-08-08 | 2006-07-25 | Aviva Biosciences Corporation | Methods for manipulating moieties in microfluidic systems |
US7119194B2 (en) | 1995-07-07 | 2006-10-10 | Toyo Boseki Kabushiki Kaisha | Nucleic acid-bondable magnetic carrier and method for isolating nucleic acid using the same |
EP1799847A2 (en) * | 2004-09-16 | 2007-06-27 | Lumigen, Inc. | Simplified methods for isolating nucleic acids from cellular materials |
EP1799846A2 (en) * | 2004-09-16 | 2007-06-27 | Lumigen, Inc. | Methods for isolating nucleic acids from biological and cellular materials |
DE102007009347A1 (en) | 2007-02-27 | 2008-08-28 | Agowa Gmbh | Isolating nucleic acids comprises binding the nucleic acids adsorbtive to polar surfaces, washing the surfaces with substances solution after removing the binding mixture, which have an affinity to polar surfaces and to the nuclei acids |
US7527929B2 (en) | 2004-07-30 | 2009-05-05 | Agencourt Bioscience Corporation | Methods of isolating nucleic acids using multifunctional group-coated solid phase carriers |
WO2009070465A1 (en) * | 2007-11-29 | 2009-06-04 | New England Biolabs, Inc. | Selective purification of small rnas from mixtures |
GB2455780A (en) * | 2007-12-21 | 2009-06-24 | Zainulabedin Mohamedali Saiyed | Nucleic acid separation |
EP2157181A1 (en) | 2008-08-13 | 2010-02-24 | AGOWA Gesellschaft für molekularbiologische Technologie mbH | Method for isolating nucleic acids and test kit |
US8026068B2 (en) | 2002-01-08 | 2011-09-27 | Roche Molecular Systems, Inc. | Use of silica material in an amplification reaction |
WO2012069660A1 (en) * | 2010-11-26 | 2012-05-31 | Invitrogen Dynal As | Use of polyols in nucleic acid processing |
EP2969140A1 (en) * | 2013-03-15 | 2016-01-20 | Abbott Molecular Inc. | One-step procedure for the purification of nucleic acids |
WO2016077294A1 (en) * | 2014-11-14 | 2016-05-19 | Corning Incorporated | Methods and kits for post-ivt rna purification |
WO2016079509A1 (en) * | 2014-11-18 | 2016-05-26 | Cambridge Epigenetix Limited | Methods for nucleic acid isolation |
EP3061823A1 (en) * | 2015-02-25 | 2016-08-31 | QIAGEN GmbH | Method for extracting nucleic acids from an agarose matrix |
EP3141298A1 (en) | 2015-09-09 | 2017-03-15 | National Center For Scientific Research "Demokritos" | Polymeric microfluidic device for nucleic acid purification fabricated via plasma micro-nanotexturing |
WO2018109075A1 (en) * | 2016-12-15 | 2018-06-21 | Qiagen Gmbh | Method for isolating highly pure nucleic acid with magnetic particles |
US10344274B2 (en) | 2016-02-16 | 2019-07-09 | Life Magnetics, Inc. | Methods for separating nucleic acids with graphene coated magnetic beads |
WO2020260620A1 (en) * | 2019-06-28 | 2020-12-30 | Qiagen Gmbh | Method for enriching nucleic acids by size |
WO2020260618A1 (en) * | 2019-06-28 | 2020-12-30 | Qiagen Gmbh | Method for separating nucleic acid molecules by size |
WO2021122846A1 (en) * | 2019-12-16 | 2021-06-24 | Qiagen Gmbh | Enrichment method |
Families Citing this family (346)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
GB9425138D0 (en) * | 1994-12-12 | 1995-02-08 | Dynal As | Isolation of nucleic acid |
KR100463475B1 (en) | 1995-06-08 | 2005-06-22 | 로셰 디아그노스틱스 게엠베하 | Magnetic Pigment |
DE19520398B4 (en) * | 1995-06-08 | 2009-04-16 | Roche Diagnostics Gmbh | Magnetic pigment |
US20020031771A1 (en) * | 1995-12-07 | 2002-03-14 | Short Jay M. | Sequence based screening |
EP0929694A4 (en) * | 1996-03-15 | 2002-05-02 | Penn State Res Found | Detection of extracellular tumor-associated nucleic acid in blood plasma or serum using nucleic acid amplification assays |
US6251691B1 (en) | 1996-04-25 | 2001-06-26 | Bioarray Solutions, Llc | Light-controlled electrokinetic assembly of particles near surfaces |
JP2001502179A (en) * | 1996-10-15 | 2001-02-20 | ユニバーシティ・オブ・ユタ・リサーチ・ファウンデイション | Compositions and methods for rapid isolation of plasmid DNA |
US6017457A (en) * | 1996-11-13 | 2000-01-25 | Transgenomic, Inc. | Method for performing polynucleotide separations using liquid chromatography |
US5997742A (en) * | 1996-11-13 | 1999-12-07 | Transgenomic, Inc. | Method for performing polynucleotide separations using liquid chromatography |
US6030527A (en) * | 1996-11-13 | 2000-02-29 | Transgenomic, Inc. | Apparatus for performing polynucleotide separations using liquid chromatography |
US6174441B1 (en) | 1996-11-13 | 2001-01-16 | Transgenomic, Inc. | Method for performing polynucleotide separations using liquid chromatography |
US5986085A (en) * | 1997-04-25 | 1999-11-16 | Transgenomic, Inc. | Matched ion polynucleotide chromatography (MIPC) process for separation of polynucleotide fragments |
US6177559B1 (en) * | 1998-04-24 | 2001-01-23 | Transgenomic, Inc. | Process for separation of polynucleotide fragments |
JP2000516638A (en) | 1997-06-10 | 2000-12-12 | トランスジエノミツク・インコーポレーテツド | Systems and methods for performing polynucleotide separation using liquid chromatography |
DE19743518A1 (en) * | 1997-10-01 | 1999-04-15 | Roche Diagnostics Gmbh | Automated, universally applicable sample preparation method |
US6914137B2 (en) * | 1997-12-06 | 2005-07-05 | Dna Research Innovations Limited | Isolation of nucleic acids |
ATE218140T1 (en) * | 1997-12-06 | 2002-06-15 | Dna Res Innovations Ltd | ISOLATION OF NUCLEIC ACIDS |
US6265168B1 (en) | 1998-10-06 | 2001-07-24 | Transgenomic, Inc. | Apparatus and method for separating and purifying polynucleotides |
US7078224B1 (en) | 1999-05-14 | 2006-07-18 | Promega Corporation | Cell concentration and lysate clearance using paramagnetic particles |
FI990082A0 (en) * | 1999-01-18 | 1999-01-18 | Labsystems Oy | Purification process using magnetic particles |
EP1147226B1 (en) * | 1999-01-27 | 2013-01-23 | Folim G. Halaka | Materials and methods for the purification of polyelectrolytes |
CA2270106C (en) | 1999-04-23 | 2006-03-14 | Yousef Haj-Ahmad | Nucleic acid purification and process |
US6310199B1 (en) * | 1999-05-14 | 2001-10-30 | Promega Corporation | pH dependent ion exchange matrix and method of use in the isolation of nucleic acids |
EP1621618B1 (en) * | 1999-05-14 | 2011-10-26 | Promega Corporation | Cell concentration and lysate clearance using paramagnetic particles |
JP4551568B2 (en) * | 1999-05-14 | 2010-09-29 | プロメガ コーポレイション | Cell collection and lysate clarification using paramagnetic particles |
AU6281200A (en) * | 1999-08-09 | 2001-03-05 | Bilatec Ag | Laboratory robot and method and reagent kit for isolating nucleic acids |
DE19943374A1 (en) * | 1999-09-10 | 2001-03-29 | Max Planck Gesellschaft | Method for binding nucleic acids to a solid phase |
ES2256068T3 (en) * | 1999-11-17 | 2006-07-16 | Roche Diagnostics Gmbh | MAGNETIC GLASS PARTICLES, METHOD FOR THEIR PREPARATION AND USES OF THE SAME. |
AU2440101A (en) * | 1999-12-20 | 2001-07-03 | Ligochem, Inc. | The removal of extraneous substances from biological fluids containing nucleic acids and the recovery of nucleic acids |
US9709559B2 (en) | 2000-06-21 | 2017-07-18 | Bioarray Solutions, Ltd. | Multianalyte molecular analysis using application-specific random particle arrays |
EP1311839B1 (en) | 2000-06-21 | 2006-03-01 | Bioarray Solutions Ltd | Multianalyte molecular analysis using application-specific random particle arrays |
DE10033583A1 (en) * | 2000-07-11 | 2002-01-24 | Bayer Ag | Superparamagnetic polymer beads |
US20030228600A1 (en) * | 2000-07-14 | 2003-12-11 | Eppendorf 5 Prime, Inc. | DNA isolation method and kit |
US6548256B2 (en) | 2000-07-14 | 2003-04-15 | Eppendorf 5 Prime, Inc. | DNA isolation method and kit |
US20030082605A1 (en) * | 2000-09-06 | 2003-05-01 | Hodge Timothy A. | Genomic DNA detection method and system thereof |
US20050272085A1 (en) * | 2000-09-06 | 2005-12-08 | Hodge Timothy A | Methods for forensic and congenic screening |
US7494817B2 (en) * | 2000-09-06 | 2009-02-24 | Transnet Yx, Inc. | Methods for genotype screening using magnetic particles |
US20050266494A1 (en) * | 2000-09-06 | 2005-12-01 | Hodge Timothy A | System and method for computer network ordering of biological testing |
EP1978110B1 (en) * | 2000-09-06 | 2010-05-26 | Transnetyx, Inc. | Computer-based method and system for screening genomic DNA |
US20030087286A1 (en) * | 2000-09-06 | 2003-05-08 | Hodge Timothy A. | Isolation of eukaryotic genomic DNA using magnetically responsive solid functionalized particles |
US20050239125A1 (en) | 2000-09-06 | 2005-10-27 | Hodge Timothy A | Methods for genotype screening |
US20020106686A1 (en) * | 2001-01-09 | 2002-08-08 | Mckernan Kevin J. | Methods and reagents for the isolation of nucleic acids |
DE10104025B4 (en) * | 2001-01-31 | 2008-07-10 | Qiagen North American Holdings, Inc. | Process for the purification and subsequent amplification of double-stranded DNA |
US6855499B1 (en) | 2001-02-16 | 2005-02-15 | Cortex Biochem, Inc. | Magnetic isolation and purification of nucleic acids |
CN1152055C (en) * | 2001-03-20 | 2004-06-02 | 清华大学 | Surface cladding and radical functino modification method of magnetic microsphere, thus obtained microsphere and its application |
GB0108287D0 (en) * | 2001-04-03 | 2001-05-23 | Imp College Innovations Ltd | Methods of crystal optimisation |
WO2002099824A2 (en) * | 2001-04-16 | 2002-12-12 | Zornes David A | Nanotube deposition on adsorbents in water maker heat pump |
AU2002320058A1 (en) * | 2001-06-06 | 2002-12-16 | The General Hspital Corporation | Magnetic-nanoparticle conjugates and methods of use |
US7262063B2 (en) | 2001-06-21 | 2007-08-28 | Bio Array Solutions, Ltd. | Directed assembly of functional heterostructures |
US20060014186A1 (en) * | 2001-09-04 | 2006-01-19 | Hodge Timothy A | Methods for genotype screening of a strain disposed on an adsorbent carrier |
US20040137449A1 (en) * | 2001-10-05 | 2004-07-15 | Nargessi R D | Magnetic isolation and purification of nucleic acids |
US7253609B2 (en) * | 2001-10-08 | 2007-08-07 | Dr. Johannes Heidenhain Gmbh | Encapsulated measuring apparatus for detecting a velocity and/or acceleration of a rotationally or linearly moved component |
US20040002073A1 (en) | 2001-10-15 | 2004-01-01 | Li Alice Xiang | Multiplexed analysis of polymorphic loci by concurrent interrogation and enzyme-mediated detection |
US6667165B2 (en) | 2001-11-13 | 2003-12-23 | Eppendorf Ag | Method and compositions for reversible inhibition of thermostable polymerases |
JP4106026B2 (en) * | 2001-11-28 | 2008-06-25 | アプレラ コーポレイション | Selective nucleic acid isolation methods and compositions |
AU2003202026A1 (en) | 2002-01-16 | 2003-09-02 | Dynal Biotech Asa | Method for isolating nucleic acids and protein from a single sample |
US7052840B2 (en) * | 2002-04-03 | 2006-05-30 | Capitol Genomix, Inc. | Reversible association of nucleic acid with a carboxylated substrate |
US7202907B2 (en) * | 2002-04-09 | 2007-04-10 | Zoran Corporation | 2:2 and 3:2 pull-down detection techniques |
GB0210766D0 (en) * | 2002-05-10 | 2002-06-19 | Genovision As | Isolating nucleic acid |
GB0215185D0 (en) | 2002-07-01 | 2002-08-07 | Genovision As | Binding a target substance |
AU2003291931A1 (en) † | 2002-11-08 | 2004-06-07 | Invitek Gesellschaft Fur Biotechnik And Biodesign Mbh | Novel buffer formulations for isolating, purifying and recovering long-chain and short-chain nucleic acids |
US7526114B2 (en) | 2002-11-15 | 2009-04-28 | Bioarray Solutions Ltd. | Analysis, secure access to, and transmission of array images |
GB0229287D0 (en) * | 2002-12-16 | 2003-01-22 | Dna Res Innovations Ltd | Polyfunctional reagents |
US7893222B2 (en) * | 2002-12-20 | 2011-02-22 | University Of Houston | Introduction of structural affinity handles as a tool in selective nucleic acid separations |
CA2515075C (en) * | 2003-02-05 | 2012-10-02 | Iquum, Inc. | Sample processing |
US20040157219A1 (en) * | 2003-02-06 | 2004-08-12 | Jianrong Lou | Chemical treatment of biological samples for nucleic acid extraction and kits therefor |
US7601491B2 (en) * | 2003-02-06 | 2009-10-13 | Becton, Dickinson And Company | Pretreatment method for extraction of nucleic acid from biological samples and kits therefor |
US20040197780A1 (en) * | 2003-04-02 | 2004-10-07 | Agencourt Bioscience Corporation | Method for isolating nucleic acids |
US20140011201A1 (en) * | 2003-05-13 | 2014-01-09 | Ibis Biosciences, Inc. | Method for the purification of targeted nucleic acids from background nucleic acids |
TWI303275B (en) * | 2003-05-30 | 2008-11-21 | Advisys Inc | Devices and methods for biomaterial production |
US20050277204A1 (en) * | 2003-08-12 | 2005-12-15 | Massachusetts Institute Of Technology | Sample preparation methods and devices |
US20050118570A1 (en) * | 2003-08-12 | 2005-06-02 | Massachusetts Institute Of Technology | Sample preparation methods and devices |
EP1510577A1 (en) | 2003-08-29 | 2005-03-02 | Qiagen GmbH | Method for magnetic bead isolation of nucleic acids |
US7927796B2 (en) | 2003-09-18 | 2011-04-19 | Bioarray Solutions, Ltd. | Number coding for identification of subtypes of coded types of solid phase carriers |
WO2005031305A2 (en) | 2003-09-22 | 2005-04-07 | Bioarray Solutions, Ltd. | Surface immobilized polyelectrolyte with multiple functional groups capable of covalently bonding to biomolecules |
US20050072674A1 (en) * | 2003-10-03 | 2005-04-07 | Agencourt Bioscience Corporation | Method and device for introducing a sample into an electrophoretic apparatus |
US7563569B2 (en) | 2003-10-28 | 2009-07-21 | Michael Seul | Optimization of gene expression analysis using immobilized capture probes |
JP2007509629A (en) | 2003-10-29 | 2007-04-19 | バイオアレイ ソリューションズ リミテッド | Complex nucleic acid analysis by cleavage of double-stranded DNA |
WO2005061724A1 (en) * | 2003-12-10 | 2005-07-07 | The General Hospital Corporation | Self-assembling nanoparticle conjugates |
US20050266394A1 (en) * | 2003-12-24 | 2005-12-01 | Massachusette Institute Of Technology | Magnetophoretic cell clarification |
KR20050071751A (en) * | 2004-01-02 | 2005-07-08 | 삼성전자주식회사 | A method for isolating a nucleic acid by using a carbon nanotube |
KR101077603B1 (en) * | 2004-01-28 | 2011-10-27 | 삼성전자주식회사 | A method for amplifying a nucleic acid using a solid phase material coated with a carboxyl group or amino group |
US20050181378A1 (en) * | 2004-02-18 | 2005-08-18 | Applera Corporation | Polyelectrolyte-coated size-exclusion ion-exchange particles |
US20060160122A1 (en) * | 2004-02-18 | 2006-07-20 | Applera Corporation | Polyelectrolyte-coated size-exclusion ion-exchange particles |
US20050196856A1 (en) * | 2004-02-18 | 2005-09-08 | Applera Corporation | Polyelectrolyte-coated size-exclusion ion-exchange particles |
JP2005292007A (en) * | 2004-04-01 | 2005-10-20 | Seiko Epson Corp | Nucleic acid immobilizing method, and manufacturing method for biosensor using same |
US20050239091A1 (en) * | 2004-04-23 | 2005-10-27 | Collis Matthew P | Extraction of nucleic acids using small diameter magnetically-responsive particles |
JP4476050B2 (en) * | 2004-06-30 | 2010-06-09 | 株式会社ニデック | Perimeter |
US7848889B2 (en) | 2004-08-02 | 2010-12-07 | Bioarray Solutions, Ltd. | Automated analysis of multiplexed probe-target interaction patterns: pattern matching and allele identification |
JP2008511816A (en) * | 2004-08-03 | 2008-04-17 | ベクトン・ディキンソン・アンド・カンパニー | Use of magnetic materials to separate samples |
AU2005271688B2 (en) * | 2004-08-03 | 2011-10-06 | Becton, Dickinson And Company | Use of magnetic material to direct isolation of compounds and fractionation of multipart samples |
JP4810164B2 (en) * | 2004-09-03 | 2011-11-09 | 富士フイルム株式会社 | Nucleic acid separation and purification method |
CN102759466A (en) * | 2004-09-15 | 2012-10-31 | 英特基因有限公司 | Microfluidic devices |
US7166680B2 (en) * | 2004-10-06 | 2007-01-23 | Advanced Cardiovascular Systems, Inc. | Blends of poly(ester amide) polymers |
EP1650297B1 (en) * | 2004-10-19 | 2011-04-13 | Samsung Electronics Co., Ltd. | Method and apparatus for the rapid disruption of cells or viruses using micro magnetic beads and laser |
KR100601972B1 (en) | 2004-11-03 | 2006-07-18 | 삼성전자주식회사 | Apparatus and method for the purification of nucleic acids by phase separation using Laser and beads |
KR100601974B1 (en) * | 2004-11-25 | 2006-07-18 | 삼성전자주식회사 | Apparatus and method for the purification of nucleic acids by different laser absorption of beads |
KR100624450B1 (en) | 2004-12-10 | 2006-09-18 | 삼성전자주식회사 | Isolation and purification method of biomolecules using hydrogel |
KR100624452B1 (en) * | 2004-12-21 | 2006-09-18 | 삼성전자주식회사 | Method for isolating and purifying nucleic acids using immobilized hydrogel or PEG-hydrogel co-polymer |
US8288169B2 (en) * | 2005-01-21 | 2012-10-16 | Argylla Technologies | Surface mediated self-assembly of nanoparticles |
JP2008528040A (en) | 2005-02-01 | 2008-07-31 | アジェンコート バイオサイエンス コーポレイション | Reagents, methods and libraries for bead-based sequencing |
EP2233582A1 (en) | 2005-02-01 | 2010-09-29 | AB Advanced Genetic Analysis Corporation | Nucleic acid sequencing by performing successive cycles of duplex extension |
US8569477B2 (en) | 2005-02-11 | 2013-10-29 | Life Technologies As | Method for isolating nucleic acids comprising the use of ethylene glycol multimers |
US20060186110A1 (en) * | 2005-02-22 | 2006-08-24 | Mark Campello | Electric heater with resistive carbon heating elements |
US20060234251A1 (en) * | 2005-04-19 | 2006-10-19 | Lumigen, Inc. | Methods of enhancing isolation of RNA from biological samples |
US20070105181A1 (en) * | 2005-05-04 | 2007-05-10 | Invitrogen Corporation | Identification of cancer biomarkers and phosphorylated pdroteins |
EP2471805A3 (en) | 2005-05-06 | 2013-01-16 | Gen-Probe Incorporated | Compositions and assays to specifically detect nucleic acid of influenza virus A or B |
US8486629B2 (en) | 2005-06-01 | 2013-07-16 | Bioarray Solutions, Ltd. | Creation of functionalized microparticle libraries by oligonucleotide ligation or elongation |
DE102005025640A1 (en) * | 2005-06-03 | 2006-12-07 | Scienion Ag | Microdispenser and associated operating method |
JP2008546424A (en) * | 2005-06-28 | 2008-12-25 | アジェンコート パーソナル ジェノミクス コーポレイション | Methods for making and sequencing modified polynucleotides |
JP2009500019A (en) * | 2005-07-01 | 2009-01-08 | プロメガ・コーポレーション | Suspended particle network for the purification of biomolecules, and the use of buoyant particles or buoyant particle networks for the purification of biomolecules |
US20070026435A1 (en) * | 2005-07-28 | 2007-02-01 | Polysciences, Inc. | Hydroxysilane functionalized magnetic particles and nucleic acid separation method |
AU2006284832B2 (en) * | 2005-08-31 | 2011-06-02 | T2 Biosystems Inc. | NMR device for detection of analytes involving magnetic particles |
US7727473B2 (en) | 2005-10-19 | 2010-06-01 | Progentech Limited | Cassette for sample preparation |
US7754148B2 (en) | 2006-12-27 | 2010-07-13 | Progentech Limited | Instrument for cassette for sample preparation |
KR101157174B1 (en) * | 2005-11-24 | 2012-06-20 | 삼성전자주식회사 | Method and apparatus for rapidly lysing cells or virus |
US8030034B2 (en) * | 2005-12-09 | 2011-10-04 | Promega Corporation | Nucleic acid purification with a binding matrix |
EP1979079A4 (en) * | 2006-02-03 | 2012-11-28 | Integenx Inc | Microfluidic devices |
US20080003564A1 (en) * | 2006-02-14 | 2008-01-03 | Iquum, Inc. | Sample processing |
WO2007111937A1 (en) | 2006-03-23 | 2007-10-04 | Applera Corporation | Directed enrichment of genomic dna for high-throughput sequencing |
US20090062129A1 (en) * | 2006-04-19 | 2009-03-05 | Agencourt Personal Genomics, Inc. | Reagents, methods, and libraries for gel-free bead-based sequencing |
EP3617321A3 (en) | 2006-05-31 | 2020-04-29 | Sequenom, Inc. | Methods and compositions for the extraction and amplification of nucleic acid from a sample |
US20080026374A1 (en) * | 2006-07-31 | 2008-01-31 | Sigma Aldrich Co. | Compositions and Methods for Isolation of Biological Molecules |
US20080026375A1 (en) * | 2006-07-31 | 2008-01-31 | Sigma Aldrich Co. | Compositions and Methods for Isolation of Biological Molecules |
US20080023395A1 (en) * | 2006-07-31 | 2008-01-31 | Sigma Aldrich Co. | Compositions and Methods for Isolation of Biological Molecules |
US20080044884A1 (en) * | 2006-08-21 | 2008-02-21 | Samsung Electronics Co., Ltd. | Method and device for separating cells from a sample using a nonplanar solid substrate |
US8158411B2 (en) * | 2006-08-21 | 2012-04-17 | Samsung Electronics Co., Ltd. | Method of separating microorganism using nonplanar solid substrate and device for separating microorganism using the same |
JP2011503244A (en) * | 2006-12-21 | 2011-01-27 | インヴィトロジェン ダイナル エーエス | Particles and their use in nucleic acid isolation methods or phosphorylated protein isolation methods |
JP2008167722A (en) * | 2007-01-15 | 2008-07-24 | Konica Minolta Medical & Graphic Inc | Nucleic acid isolation method by heating on magnetic support |
CN101715483A (en) * | 2007-02-05 | 2010-05-26 | 微芯片生物工艺学股份有限公司 | microfluidic and nanofluidic devices, systems, and applications |
WO2008101192A1 (en) * | 2007-02-16 | 2008-08-21 | Applied Biosystems, Llc | Method for recovering nucleic acid from a mixed cell suspension, without centrifugation |
US20100190240A1 (en) * | 2007-03-21 | 2010-07-29 | Ibis Biosciences, Inc. | Reagents for nucleic acid purification |
US9458451B2 (en) | 2007-06-21 | 2016-10-04 | Gen-Probe Incorporated | Multi-channel optical measurement instrument |
JP5232858B2 (en) * | 2007-06-29 | 2013-07-10 | ベクトン・ディキンソン・アンド・カンパニー | Method for extracting and purifying components of biological samples |
CN103443275B (en) * | 2007-07-23 | 2016-03-16 | 应用生物系统有限责任公司 | The method of spermanucleic acid is reclaimed by forensic samples |
US9097644B2 (en) * | 2007-08-17 | 2015-08-04 | Massachusetts Institute Of Technology | Magnetic resonance-based viscometers and methods |
EP2191012A1 (en) * | 2007-09-21 | 2010-06-02 | Streck, Inc. | Nucleic acid isolation in preserved whole blood |
WO2009046149A1 (en) * | 2007-10-01 | 2009-04-09 | Applied Biosystems Inc. | Chase ligation sequencing |
US8685322B2 (en) * | 2007-11-13 | 2014-04-01 | Stratec Biomedical Ag | Apparatus and method for the purification of biomolecules |
US8815576B2 (en) * | 2007-12-27 | 2014-08-26 | Lawrence Livermore National Security, Llc. | Chip-based sequencing nucleic acids |
US20090253181A1 (en) * | 2008-01-22 | 2009-10-08 | Microchip Biotechnologies, Inc. | Universal sample preparation system and use in an integrated analysis system |
JP5693449B2 (en) | 2008-04-30 | 2015-04-01 | グラダリス インク.Gradalis, Inc. | High-purity plasmid DNA preparation and preparation method thereof |
EP2163621A1 (en) * | 2008-09-03 | 2010-03-17 | Qiagen GmbH | Method for isolating and cleaning nucleic acids |
DE102008047790A1 (en) * | 2008-09-17 | 2010-04-15 | Qiagen Gmbh | Method for normalizing the content of biomolecules in a sample |
US8672532B2 (en) * | 2008-12-31 | 2014-03-18 | Integenx Inc. | Microfluidic methods |
US8368882B2 (en) | 2009-01-30 | 2013-02-05 | Gen-Probe Incorporated | Systems and methods for detecting a signal and applying thermal energy to a signal transmission element |
EP2394175B1 (en) * | 2009-02-09 | 2016-02-03 | caprotec bioanalytics GmbH | Devices, systems and methods for separating magnetic particles |
EP3211095B1 (en) | 2009-04-03 | 2019-01-02 | Sequenom, Inc. | Nucleic acid preparation compositions and methods |
CN113145298B (en) | 2009-05-15 | 2023-09-22 | 简·探针公司 | Method and apparatus for automatic movement of magnet in instrument for performing magnetic separation process |
US8388908B2 (en) * | 2009-06-02 | 2013-03-05 | Integenx Inc. | Fluidic devices with diaphragm valves |
WO2010141921A1 (en) | 2009-06-05 | 2010-12-09 | Integenx Inc. | Universal sample preparation system and use in an integrated analysis system |
US8222397B2 (en) | 2009-08-28 | 2012-07-17 | Promega Corporation | Methods of optimal purification of nucleic acids and kit for use in performing such methods |
US8039613B2 (en) * | 2009-08-28 | 2011-10-18 | Promega Corporation | Methods of purifying a nucleic acid and formulation and kit for use in performing such methods |
WO2011026030A1 (en) * | 2009-08-31 | 2011-03-03 | Mbio Diagnostics Corporation | Integrated sample preparation and analyte detection |
US10174368B2 (en) | 2009-09-10 | 2019-01-08 | Centrillion Technology Holdings Corporation | Methods and systems for sequencing long nucleic acids |
US10072287B2 (en) | 2009-09-10 | 2018-09-11 | Centrillion Technology Holdings Corporation | Methods of targeted sequencing |
US8536322B2 (en) * | 2009-10-19 | 2013-09-17 | Zhiqiang Han | Method for nucleic acid isolation by solid phase reversible binding of nucleic acids |
TWI407994B (en) | 2009-10-22 | 2013-09-11 | Ind Tech Res Inst | Method, agent, and kit for isolating nucleic acids |
US8584703B2 (en) | 2009-12-01 | 2013-11-19 | Integenx Inc. | Device with diaphragm valve |
EP2513335A4 (en) * | 2009-12-14 | 2013-09-11 | Betty Wu | Method and materials for separating nucleic acid materials |
CN105004596B (en) | 2010-02-23 | 2018-12-21 | 卢米尼克斯公司 | Instrument and method for the sample preparation of integration, reaction and detection |
US20110223588A1 (en) * | 2010-03-09 | 2011-09-15 | Biosample Llc | Solid Phase Nucleic Acid Extraction From Small Sample Volumes, and Release of Controlled Quantities |
US8512538B2 (en) | 2010-05-28 | 2013-08-20 | Integenx Inc. | Capillary electrophoresis device |
US9186668B1 (en) | 2010-06-04 | 2015-11-17 | Sandia Corporation | Microfluidic devices, systems, and methods for quantifying particles using centrifugal force |
US8945914B1 (en) | 2010-07-08 | 2015-02-03 | Sandia Corporation | Devices, systems, and methods for conducting sandwich assays using sedimentation |
US9795961B1 (en) * | 2010-07-08 | 2017-10-24 | National Technology & Engineering Solutions Of Sandia, Llc | Devices, systems, and methods for detecting nucleic acids using sedimentation |
US8962346B2 (en) | 2010-07-08 | 2015-02-24 | Sandia Corporation | Devices, systems, and methods for conducting assays with improved sensitivity using sedimentation |
EP2593569B1 (en) | 2010-07-12 | 2018-01-03 | Gen-Probe Incorporated | Compositions and assays to detect seasonal h1 influenza a virus nucleic acids |
US9046507B2 (en) | 2010-07-29 | 2015-06-02 | Gen-Probe Incorporated | Method, system and apparatus for incorporating capacitive proximity sensing in an automated fluid transfer procedure |
US20130040375A1 (en) | 2011-08-08 | 2013-02-14 | Tandem Diagnotics, Inc. | Assay systems for genetic analysis |
US20120034603A1 (en) | 2010-08-06 | 2012-02-09 | Tandem Diagnostics, Inc. | Ligation-based detection of genetic variants |
US11031095B2 (en) | 2010-08-06 | 2021-06-08 | Ariosa Diagnostics, Inc. | Assay systems for determination of fetal copy number variation |
US11203786B2 (en) | 2010-08-06 | 2021-12-21 | Ariosa Diagnostics, Inc. | Detection of target nucleic acids using hybridization |
US8700338B2 (en) | 2011-01-25 | 2014-04-15 | Ariosa Diagnosis, Inc. | Risk calculation for evaluation of fetal aneuploidy |
US20130261003A1 (en) | 2010-08-06 | 2013-10-03 | Ariosa Diagnostics, In. | Ligation-based detection of genetic variants |
US20140342940A1 (en) | 2011-01-25 | 2014-11-20 | Ariosa Diagnostics, Inc. | Detection of Target Nucleic Acids using Hybridization |
US10533223B2 (en) | 2010-08-06 | 2020-01-14 | Ariosa Diagnostics, Inc. | Detection of target nucleic acids using hybridization |
US10167508B2 (en) | 2010-08-06 | 2019-01-01 | Ariosa Diagnostics, Inc. | Detection of genetic abnormalities |
EP2606242A4 (en) | 2010-08-20 | 2016-07-20 | Integenx Inc | Microfluidic devices with mechanically-sealed diaphragm valves |
WO2012024658A2 (en) | 2010-08-20 | 2012-02-23 | IntegenX, Inc. | Integrated analysis system |
US8563298B2 (en) | 2010-10-22 | 2013-10-22 | T2 Biosystems, Inc. | NMR systems and methods for the rapid detection of analytes |
US8409807B2 (en) | 2010-10-22 | 2013-04-02 | T2 Biosystems, Inc. | NMR systems and methods for the rapid detection of analytes |
ES2576927T3 (en) | 2010-10-22 | 2016-07-12 | T2 Biosystems, Inc. | NMR systems and methods for rapid analyte detection |
EP2646551B1 (en) | 2010-11-30 | 2017-06-07 | Life Technologies Corporation | Alkylene glycols and polymers and copolymers thereof for direct isolation of nucleic acid from embedded samples |
US10131947B2 (en) | 2011-01-25 | 2018-11-20 | Ariosa Diagnostics, Inc. | Noninvasive detection of fetal aneuploidy in egg donor pregnancies |
US9994897B2 (en) | 2013-03-08 | 2018-06-12 | Ariosa Diagnostics, Inc. | Non-invasive fetal sex determination |
US8756020B2 (en) | 2011-01-25 | 2014-06-17 | Ariosa Diagnostics, Inc. | Enhanced risk probabilities using biomolecule estimations |
US11270781B2 (en) | 2011-01-25 | 2022-03-08 | Ariosa Diagnostics, Inc. | Statistical analysis for non-invasive sex chromosome aneuploidy determination |
US20120252682A1 (en) | 2011-04-01 | 2012-10-04 | Maples Corporate Services Limited | Methods and systems for sequencing nucleic acids |
AU2012250619B2 (en) | 2011-05-04 | 2015-11-26 | Luminex Corporation | Apparatus and methods for integrated sample preparation, reaction and detection |
US8712697B2 (en) | 2011-09-07 | 2014-04-29 | Ariosa Diagnostics, Inc. | Determination of copy number variations using binomial probability calculations |
US11021733B2 (en) | 2011-09-26 | 2021-06-01 | Qiagen Gmbh | Stabilization and isolation of extracellular nucleic acids |
CA2849354C (en) | 2011-09-26 | 2021-11-09 | Preanalytix Gmbh | Stabilisation and isolation of extracellular nucleic acids |
CN104080958A (en) | 2011-10-19 | 2014-10-01 | 纽亘技术公司 | Compositions and methods for directional nucleic acid amplification and sequencing |
US20150136604A1 (en) | 2011-10-21 | 2015-05-21 | Integenx Inc. | Sample preparation, processing and analysis systems |
US10865440B2 (en) | 2011-10-21 | 2020-12-15 | IntegenX, Inc. | Sample preparation, processing and analysis systems |
US9340828B2 (en) | 2011-10-27 | 2016-05-17 | Ge Healthcare Bio-Sciences Ab | Purification of nucleic acid |
US9441265B2 (en) | 2011-12-29 | 2016-09-13 | Ibis Biosciences, Inc. | Compositions and methods for sample preparation |
GB2513793B (en) | 2012-01-26 | 2016-11-02 | Nugen Tech Inc | Compositions and methods for targeted nucleic acid sequence enrichment and high efficiency library generation |
US9244065B1 (en) | 2012-03-16 | 2016-01-26 | Sandia Corporation | Systems, devices, and methods for agglutination assays using sedimentation |
US9562271B2 (en) | 2012-04-20 | 2017-02-07 | T2 Biosystems, Inc. | Compositions and methods for detection of Candida species |
US9803237B2 (en) | 2012-04-24 | 2017-10-31 | California Institute Of Technology | Slip-induced compartmentalization |
EP2664914A1 (en) | 2012-05-16 | 2013-11-20 | Koninklijke Philips N.V. | Magnetically assisted processing of a medium |
US10289800B2 (en) | 2012-05-21 | 2019-05-14 | Ariosa Diagnostics, Inc. | Processes for calculating phased fetal genomic sequences |
EP2861787B1 (en) | 2012-06-18 | 2017-09-20 | Nugen Technologies, Inc. | Compositions and methods for negative selection of non-desired nucleic acid sequences |
DE102012012523B4 (en) * | 2012-06-26 | 2015-02-12 | Magnamedics Gmbh | Purification of nucleic acids |
US20150011396A1 (en) | 2012-07-09 | 2015-01-08 | Benjamin G. Schroeder | Methods for creating directional bisulfite-converted nucleic acid libraries for next generation sequencing |
CN104812947B (en) | 2012-07-17 | 2018-04-27 | 考希尔股份有限公司 | The system and method for detecting hereditary variation |
US9092401B2 (en) | 2012-10-31 | 2015-07-28 | Counsyl, Inc. | System and methods for detecting genetic variation |
EP2875131B1 (en) | 2012-07-18 | 2018-03-14 | Siemens Healthcare Diagnostics Inc. | A method of normalizing biological samples |
EP2875156A4 (en) | 2012-07-19 | 2016-02-24 | Ariosa Diagnostics Inc | Multiplexed sequential ligation-based detection of genetic variants |
US9903001B1 (en) | 2012-07-19 | 2018-02-27 | National Technology & Engineering Solutions Of Sandia, Llc | Quantitative detection of pathogens in centrifugal microfluidic disks |
US9951386B2 (en) | 2014-06-26 | 2018-04-24 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US9701998B2 (en) | 2012-12-14 | 2017-07-11 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10273541B2 (en) | 2012-08-14 | 2019-04-30 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10752949B2 (en) | 2012-08-14 | 2020-08-25 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US11591637B2 (en) | 2012-08-14 | 2023-02-28 | 10X Genomics, Inc. | Compositions and methods for sample processing |
US10584381B2 (en) | 2012-08-14 | 2020-03-10 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10323279B2 (en) | 2012-08-14 | 2019-06-18 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10221442B2 (en) | 2012-08-14 | 2019-03-05 | 10X Genomics, Inc. | Compositions and methods for sample processing |
MX364957B (en) | 2012-08-14 | 2019-05-15 | 10X Genomics Inc | Microcapsule compositions and methods. |
US9422602B2 (en) | 2012-08-15 | 2016-08-23 | Bio-Rad Laboratories, Inc. | Methods and compositions for determining nucleic acid degradation |
JP6608280B2 (en) | 2012-09-25 | 2019-11-20 | キアゲン ゲーエムベーハー | Biological sample stabilization |
US10364455B2 (en) | 2012-09-27 | 2019-07-30 | Bioo Scientific Corporation | Methods and compositions for improving removal of ribosomal RNA from biological samples |
JP1628115S (en) | 2012-10-24 | 2019-04-01 | ||
US20140322706A1 (en) | 2012-10-24 | 2014-10-30 | Jon Faiz Kayyem | Integrated multipelx target analysis |
AR093878A1 (en) * | 2012-12-10 | 2015-06-24 | Agrigenetics Inc | RECOVERY OF GENOMIC DNA FROM SAMPLES OF REMOVED REMOVED SEEDS |
EP2931661B1 (en) | 2012-12-11 | 2017-11-08 | Qiagen GmbH | Preparation of silica particles |
CA2894694C (en) | 2012-12-14 | 2023-04-25 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10533221B2 (en) | 2012-12-14 | 2020-01-14 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
WO2014118166A1 (en) * | 2013-01-31 | 2014-08-07 | Ge Healthcare Uk Limited | Improvements in and relating to the transfer and storage of biological material |
US9411930B2 (en) | 2013-02-01 | 2016-08-09 | The Regents Of The University Of California | Methods for genome assembly and haplotype phasing |
GB2519255B (en) | 2013-02-01 | 2016-01-06 | Univ California | Methods for genome assembly and haplotype phasing |
US9304128B1 (en) | 2013-02-01 | 2016-04-05 | Sandia Corporation | Toxin activity assays, devices, methods and systems therefor |
US9493766B2 (en) | 2013-02-04 | 2016-11-15 | Corning Incorporated | PCR reaction cleanup buffers |
US10745686B2 (en) | 2013-02-08 | 2020-08-18 | Qiagen Gmbh | Method for separating DNA by size |
BR112015019159A2 (en) | 2013-02-08 | 2017-07-18 | 10X Genomics Inc | polynucleotide barcode generation |
WO2014160233A1 (en) * | 2013-03-13 | 2014-10-02 | Abbott Molecular Inc. | Systems and methods for isolating nucleic acids |
AU2013202805B2 (en) | 2013-03-14 | 2015-07-16 | Gen-Probe Incorporated | System and method for extending the capabilities of a diagnostic analyzer |
WO2014144092A1 (en) | 2013-03-15 | 2014-09-18 | Nugen Technologies, Inc. | Sequential sequencing |
EP3520895A1 (en) | 2013-03-15 | 2019-08-07 | Genmark Diagnostics Inc. | Fluid container with cantilevered lance |
CN105283550A (en) | 2013-03-18 | 2016-01-27 | 凯杰有限公司 | Stabilisation of biological samples |
CN105164258B (en) | 2013-03-18 | 2021-05-18 | 凯杰有限公司 | Stabilization and isolation of extracellular nucleic acids |
SG11201600550WA (en) | 2013-07-25 | 2016-02-26 | Dch Molecular Diagnostics Inc | Methods and compositions for detecting bacterial contamination |
USD881409S1 (en) | 2013-10-24 | 2020-04-14 | Genmark Diagnostics, Inc. | Biochip cartridge |
US9498778B2 (en) | 2014-11-11 | 2016-11-22 | Genmark Diagnostics, Inc. | Instrument for processing cartridge for performing assays in a closed sample preparation and reaction system |
US10415083B2 (en) * | 2013-10-28 | 2019-09-17 | The Translational Genomics Research Institute | Long insert-based whole genome sequencing |
US9486756B2 (en) | 2013-11-08 | 2016-11-08 | Covaris, Inc. | Method and apparatus for shearing of genomic material using acoustic processing |
CA2929596C (en) | 2013-11-13 | 2022-07-05 | Nugen Technologies, Inc. | Compositions and methods for identification of a duplicate sequencing read |
EP3071333A4 (en) | 2013-11-18 | 2017-11-15 | IntegenX Inc. | Cartridges and instruments for sample analysis |
US9803238B1 (en) | 2013-11-26 | 2017-10-31 | National Technology & Engineering Solutions Of Sandia, Llc | Method and apparatus for purifying nucleic acids and performing polymerase chain reaction assays using an immiscible fluid |
EP3077072B1 (en) | 2013-12-02 | 2019-03-06 | Biocartis N.V. | Extraction of circulating nucleic acids |
US11859246B2 (en) | 2013-12-11 | 2024-01-02 | Accuragen Holdings Limited | Methods and compositions for enrichment of amplification products |
US11286519B2 (en) | 2013-12-11 | 2022-03-29 | Accuragen Holdings Limited | Methods and compositions for enrichment of amplification products |
WO2015089243A1 (en) | 2013-12-11 | 2015-06-18 | The Regents For Of The University Of California | Methods for labeling dna fragments to recontruct physical linkage and phase |
AU2014362227B2 (en) | 2013-12-11 | 2021-05-13 | Accuragen Holdings Limited | Compositions and methods for detecting rare sequence variants |
US20150167065A1 (en) * | 2013-12-13 | 2015-06-18 | General Electric Company | Isothermal amplification of nucleic acids within a porous matrix |
WO2015131107A1 (en) | 2014-02-28 | 2015-09-03 | Nugen Technologies, Inc. | Reduced representation bisulfite sequencing with diversity adaptors |
EP3119197A1 (en) | 2014-03-18 | 2017-01-25 | Qiagen GmbH | Stabilization and isolation of extracellular nucleic acids |
EP2921556A1 (en) | 2014-03-21 | 2015-09-23 | Lexogen GmbH | Copy number preserving RNA analysis method |
CN114534806B (en) | 2014-04-10 | 2024-03-29 | 10X基因组学有限公司 | Fluidic devices, systems and methods for packaging and partitioning reagents and uses thereof |
WO2015179098A1 (en) | 2014-05-21 | 2015-11-26 | Integenx Inc. | Fluidic cartridge with valve mechanism |
WO2015200893A2 (en) | 2014-06-26 | 2015-12-30 | 10X Genomics, Inc. | Methods of analyzing nucleic acids from individual cells or cell populations |
WO2016019360A1 (en) | 2014-08-01 | 2016-02-04 | Dovetail Genomics Llc | Tagging nucleic acids for sequence assembly |
EP3552690A1 (en) | 2014-10-22 | 2019-10-16 | IntegenX Inc. | Systems and methods for sample preparation, processing and analysis |
WO2016065218A1 (en) | 2014-10-23 | 2016-04-28 | Corning Incorporated | Polymer-encapsulated magnetic nanoparticles |
CN107002128A (en) | 2014-10-29 | 2017-08-01 | 10X 基因组学有限公司 | The method and composition being sequenced for target nucleic acid |
US9975122B2 (en) | 2014-11-05 | 2018-05-22 | 10X Genomics, Inc. | Instrument systems for integrated sample processing |
AU2015342907A1 (en) * | 2014-11-07 | 2017-05-25 | The Johns Hopkins University | Chaotrope- and volatile-free method for purifying nucleic acids from plasma |
US9598722B2 (en) | 2014-11-11 | 2017-03-21 | Genmark Diagnostics, Inc. | Cartridge for performing assays in a closed sample preparation and reaction system |
WO2016075701A2 (en) * | 2014-11-11 | 2016-05-19 | Scigenom Labs Pvt. Ltd. | A method for extraction of dna using naked magnetic nanoparticles |
US10005080B2 (en) | 2014-11-11 | 2018-06-26 | Genmark Diagnostics, Inc. | Instrument and cartridge for performing assays in a closed sample preparation and reaction system employing electrowetting fluid manipulation |
US9702871B1 (en) | 2014-11-18 | 2017-07-11 | National Technology & Engineering Solutions Of Sandia, Llc | System and method for detecting components of a mixture including a valving scheme for competition assays |
SG11201705615UA (en) | 2015-01-12 | 2017-08-30 | 10X Genomics Inc | Processes and systems for preparing nucleic acid sequencing libraries and libraries prepared using same |
EP3259696A1 (en) | 2015-02-17 | 2017-12-27 | Dovetail Genomics LLC | Nucleic acid sequence assembly |
US10697000B2 (en) * | 2015-02-24 | 2020-06-30 | 10X Genomics, Inc. | Partition processing methods and systems |
CA2975958A1 (en) | 2015-02-24 | 2016-09-01 | 10X Genomics, Inc. | Methods for targeted nucleic acid sequence coverage |
US10254298B1 (en) | 2015-03-25 | 2019-04-09 | National Technology & Engineering Solutions Of Sandia, Llc | Detection of metabolites for controlled substances |
US11807896B2 (en) | 2015-03-26 | 2023-11-07 | Dovetail Genomics, Llc | Physical linkage preservation in DNA storage |
EP3303630B1 (en) | 2015-06-05 | 2022-01-05 | Qiagen GmbH | Method for separating dna by size |
WO2017041013A1 (en) | 2015-09-04 | 2017-03-09 | Mo Bio Laboratories, Inc. | Small-molecule mediated size selection of nucleic acids |
EP3344643A4 (en) | 2015-09-04 | 2019-05-01 | Qiagen Sciences LLC | Methods for co-isolation of nucelic acids and proteins |
WO2017042819A1 (en) | 2015-09-11 | 2017-03-16 | Molecular Detection Israel Ltd. | Methods for isolating microbial cells from a blood sample |
AU2016334233B2 (en) | 2015-10-09 | 2023-01-05 | Accuragen Holdings Limited | Methods and compositions for enrichment of amplification products |
CN108368542B (en) | 2015-10-19 | 2022-04-08 | 多弗泰尔基因组学有限责任公司 | Methods for genome assembly, haplotype phasing, and target-independent nucleic acid detection |
EP3368668B1 (en) | 2015-10-28 | 2023-11-29 | Silicon Valley Scientific, Inc. | Method and apparatus for encoding cellular spatial position information |
CN108291250B (en) | 2015-11-20 | 2022-05-27 | 凯杰有限公司 | Method for preparing sterilized composition for stabilizing extracellular nucleic acid |
CN115927547A (en) | 2015-12-03 | 2023-04-07 | 安可济控股有限公司 | Methods and compositions for forming ligation products |
EP4144861A1 (en) | 2015-12-04 | 2023-03-08 | 10X Genomics, Inc. | Methods and compositions for nucleic acid analysis |
CA3011901A1 (en) | 2016-01-21 | 2017-07-27 | T2 Biosystems, Inc. | Nmr methods and systems for the rapid detection of bacteria |
JP7441003B2 (en) | 2016-02-23 | 2024-02-29 | ダブテイル ゲノミクス エルエルシー | Generation of phased read sets and haplotype phasing for genome assembly |
EP3954771A1 (en) | 2016-05-13 | 2022-02-16 | Dovetail Genomics, LLC | Recovering long-range linkage information from preserved samples |
WO2017197338A1 (en) | 2016-05-13 | 2017-11-16 | 10X Genomics, Inc. | Microfluidic systems and methods of use |
US11427866B2 (en) | 2016-05-16 | 2022-08-30 | Accuragen Holdings Limited | Method of improved sequencing by strand identification |
US10774393B2 (en) | 2016-05-20 | 2020-09-15 | Roche Molecular Systems, Inc. | Cell surface marker depletion in a sample processing device |
WO2017218777A1 (en) | 2016-06-17 | 2017-12-21 | California Institute Of Technology | Nucleic acid reactions and related methods and compositions |
WO2018005811A1 (en) | 2016-06-30 | 2018-01-04 | Grail, Inc. | Differential tagging of rna for preparation of a cell-free dna/rna sequencing library |
GB2551801A (en) * | 2016-06-30 | 2018-01-03 | Lgc Genomics Ltd | Methods |
WO2018023033A1 (en) | 2016-07-29 | 2018-02-01 | Western Michigan University Research Foundation | Magnetic nanoparticle-based gyroscopic sensor |
US10981174B1 (en) | 2016-08-04 | 2021-04-20 | National Technology & Engineering Solutions Of Sandia, Llc | Protein and nucleic acid detection for microfluidic devices |
US10406528B1 (en) | 2016-08-04 | 2019-09-10 | National Technology & Engineering Solutions Of Sandia, Llc | Non-contact temperature control system for microfluidic devices |
JP6966052B2 (en) | 2016-08-15 | 2021-11-10 | アキュラーゲン ホールディングス リミテッド | Compositions and Methods for Detecting Rare Sequence Variants |
US10786811B1 (en) | 2016-10-24 | 2020-09-29 | National Technology & Engineering Solutions Of Sandia, Llc | Detection of active and latent infections with microfluidic devices and systems thereof |
DE102016121483B4 (en) * | 2016-11-09 | 2020-06-18 | Axagarius Gmbh & Co. Kg | Particulate solid composite material for nucleic acid purification, containing magnetic nanoparticles, process for its production and its use |
US11231347B2 (en) | 2016-11-29 | 2022-01-25 | S2 Genomics, Inc. | Method and apparatus for processing tissue samples |
US10815525B2 (en) | 2016-12-22 | 2020-10-27 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10550429B2 (en) | 2016-12-22 | 2020-02-04 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
US10011872B1 (en) | 2016-12-22 | 2018-07-03 | 10X Genomics, Inc. | Methods and systems for processing polynucleotides |
WO2018140966A1 (en) | 2017-01-30 | 2018-08-02 | 10X Genomics, Inc. | Methods and systems for droplet-based single cell barcoding |
WO2018140452A1 (en) | 2017-01-30 | 2018-08-02 | Counsyl, Inc. | Enrichment of cell-free dna from a biological sample |
EP3612646A1 (en) | 2017-04-18 | 2020-02-26 | Dovetail Genomics, LLC | Nucleic acid characteristics as guides for sequence assembly |
WO2018199689A2 (en) * | 2017-04-28 | 2018-11-01 | 주식회사 이지다이아텍 | Automated immunoassay device and method using large magnetic particle complex |
WO2018213803A1 (en) | 2017-05-19 | 2018-11-22 | Neon Therapeutics, Inc. | Immunogenic neoantigen identification |
US20190093155A1 (en) | 2017-05-25 | 2019-03-28 | Roche Molecular Systems, Inc. | Multiplex Nucleic Acid Amplification Assay |
EP4230746A3 (en) | 2017-05-26 | 2023-11-01 | 10X Genomics, Inc. | Single cell analysis of transposase accessible chromatin |
US10844372B2 (en) | 2017-05-26 | 2020-11-24 | 10X Genomics, Inc. | Single cell analysis of transposase accessible chromatin |
US11384351B2 (en) | 2017-07-27 | 2022-07-12 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Methods and tools for purifying nucleic acids and using polymerized tubulin |
WO2019060722A2 (en) | 2017-09-22 | 2019-03-28 | X Gen Us Co. | Methods and compositions for preparing polynucleotides |
US11099202B2 (en) | 2017-10-20 | 2021-08-24 | Tecan Genomics, Inc. | Reagent delivery system |
SG11201913654QA (en) | 2017-11-15 | 2020-01-30 | 10X Genomics Inc | Functionalized gel beads |
US10829815B2 (en) | 2017-11-17 | 2020-11-10 | 10X Genomics, Inc. | Methods and systems for associating physical and genetic properties of biological particles |
WO2019106568A1 (en) | 2017-11-29 | 2019-06-06 | Basf Se | Plants having increased tolerance to herbicides |
CA3090102A1 (en) | 2018-01-31 | 2019-08-08 | Dovetail Genomics, Llc | Sample prep for dna linkage recovery |
US11203782B2 (en) | 2018-03-29 | 2021-12-21 | Accuragen Holdings Limited | Compositions and methods comprising asymmetric barcoding |
CN112262218A (en) | 2018-04-06 | 2021-01-22 | 10X基因组学有限公司 | System and method for quality control in single cell processing |
CN113383083A (en) | 2018-04-27 | 2021-09-10 | 埃克斯基因美国公司 | Methods and compositions for preparing polynucleotides |
WO2019232504A2 (en) | 2018-06-01 | 2019-12-05 | S2 Genomics, Inc. | Method and apparatus for processing tissue samples |
EP3824484A1 (en) | 2018-07-19 | 2021-05-26 | Beckman Coulter Inc. | Magnetic particles |
US11515012B1 (en) | 2018-09-22 | 2022-11-29 | Mark Gordon Arnold | Method and apparatus for a pipelined DNA memory hierarchy |
WO2020069385A1 (en) | 2018-09-28 | 2020-04-02 | Beckman Coulter, Inc. | Isolation of dna and rna from a single sample |
EP3905881A1 (en) | 2019-01-04 | 2021-11-10 | QIAGEN GmbH | Urine stabilization |
US11807909B1 (en) | 2019-09-12 | 2023-11-07 | Zymo Research Corporation | Methods for species-level resolution of microorganisms |
US11857981B2 (en) | 2019-12-23 | 2024-01-02 | 10X Genomics, Inc. | Magnetic separator for an automated single cell sequencing system |
CA3131632A1 (en) | 2019-12-04 | 2021-06-10 | Tong Liu | Preparation of dna sequencing libraries for detection of dna pathogens in plasma |
CN115104033A (en) | 2019-12-23 | 2022-09-23 | 贝克曼库尔特有限公司 | Method for transferring liquid from reagent reservoir using mechanical processor |
EP4126374A1 (en) | 2020-04-03 | 2023-02-08 | Beckman Coulter Inc. | Electromagnetic assemblies for processing fluids |
US11701668B1 (en) | 2020-05-08 | 2023-07-18 | 10X Genomics, Inc. | Methods and devices for magnetic separation |
US11946038B1 (en) | 2020-05-29 | 2024-04-02 | 10X Genomics, Inc. | Methods and systems including flow and magnetic modules |
CN116601308A (en) | 2020-12-22 | 2023-08-15 | 豪夫迈·罗氏有限公司 | Method for multiplex real-time PCR using large Stokes shift fluorescent dyes |
CN112501161B (en) * | 2020-12-23 | 2023-03-28 | 华南师范大学 | Double-magnetic-particle-intervention DNA extraction and purification method |
GB2603968A (en) | 2021-02-23 | 2022-08-24 | Imperial College Innovations Ltd | Lid assembly for a sample tube, method of using the same to collect magnetic beads, and sample processing kit |
WO2022183205A1 (en) | 2021-02-25 | 2022-09-01 | Beckman Coulter, Inc. | Rapidly-sedimenting magnetic particles and applications thereof |
WO2023031161A1 (en) | 2021-09-03 | 2023-03-09 | BASF Agricultural Solutions Seed US LLC | Plants having increased tolerance to herbicides |
CN113881644A (en) * | 2021-09-28 | 2022-01-04 | 江苏金迪克生物技术股份有限公司 | Method for removing host DNA in rabies vaccine |
WO2023131803A1 (en) | 2022-01-10 | 2023-07-13 | Imperial College Innovations Limited | Lyophilised nucleic acid amplification reaction composition |
GB2620799A (en) | 2022-07-22 | 2024-01-24 | Life Tech As | Particles |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993025709A1 (en) * | 1992-06-09 | 1993-12-23 | Medical Research Council | Preparation of nucleic acids |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4628037A (en) * | 1983-05-12 | 1986-12-09 | Advanced Magnetics, Inc. | Binding assays employing magnetic particles |
US4698302A (en) * | 1983-05-12 | 1987-10-06 | Advanced Magnetics, Inc. | Enzymatic reactions using magnetic particles |
US4695393A (en) * | 1983-05-12 | 1987-09-22 | Advanced Magnetics Inc. | Magnetic particles for use in separations |
US4672040A (en) * | 1983-05-12 | 1987-06-09 | Advanced Magnetics, Inc. | Magnetic particles for use in separations |
US4554088A (en) * | 1983-05-12 | 1985-11-19 | Advanced Magnetics Inc. | Magnetic particles for use in separations |
GB9003253D0 (en) * | 1990-02-13 | 1990-04-11 | Amersham Int Plc | Precipitating polymers |
-
1994
- 1994-09-20 US US08/309,267 patent/US5705628A/en not_active Expired - Lifetime
-
1995
- 1995-09-19 WO PCT/US1995/011839 patent/WO1996009379A1/en active Application Filing
- 1995-09-19 IL IL115352A patent/IL115352A/en not_active IP Right Cessation
-
1998
- 1998-01-02 US US09/002,412 patent/US5898071A/en not_active Expired - Lifetime
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1993025709A1 (en) * | 1992-06-09 | 1993-12-23 | Medical Research Council | Preparation of nucleic acids |
Non-Patent Citations (2)
Title |
---|
HAWKINS, TREVOR L. ET AL: "DNA purification and isolation using a solid-phase", NUCLEIC ACIDS RES. (1994), 22(21), 4543-4 CODEN: NARHAD;ISSN: 0305-1048 * |
HAWKINS, TREVOR: "M13 single-strand purification using a biotinylated probe and streptavidin coated magnetic beads", DNA SEQUENCE (1992), 3(2), 65-9 CODEN: DNSEES;ISSN: 1042-5179 * |
Cited By (59)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7119194B2 (en) | 1995-07-07 | 2006-10-10 | Toyo Boseki Kabushiki Kaisha | Nucleic acid-bondable magnetic carrier and method for isolating nucleic acid using the same |
EP0885958A4 (en) * | 1996-02-25 | 2002-05-22 | Prec System Science Co Ltd | Method for treating biopolymers, microorganisms or materials by using more than one type of magnetic particles |
EP0885958A1 (en) * | 1996-02-25 | 1998-12-23 | Precision System Science Co., Ltd. | Method for treating biopolymers, microorganisms or materials by using more than one type of magnetic particles |
WO1999058664A1 (en) * | 1998-05-14 | 1999-11-18 | Whitehead Institute For Biomedical Research | Solid phase technique for selectively isolating nucleic acids |
US6534262B1 (en) | 1998-05-14 | 2003-03-18 | Whitehead Institute For Biomedical Research | Solid phase technique for selectively isolating nucleic acids |
US6433160B1 (en) | 1998-10-30 | 2002-08-13 | Becton, Dickinson And Company | Method for purification and manipulation of nucleic acids using paramagnetic particles |
EP1674571A3 (en) * | 1998-10-30 | 2008-08-13 | Becton, Dickinson and Company | Use of paramagnetic material in the purification and manipulation of nucleic acids |
EP1002860A1 (en) * | 1998-10-30 | 2000-05-24 | Becton Dickinson and Company | Method for purification and manipulation of nucleic acids using paramagnetic particles |
WO2001040459A2 (en) * | 1999-12-03 | 2001-06-07 | InViTek Gesellschaft für Biotechnik & Biodesign mbH | Surface modified supporting materials for binding biological materials, method for the production and use thereof |
WO2001040459A3 (en) * | 1999-12-03 | 2001-12-27 | Invitek Biotechnik & Biodesign | Surface modified supporting materials for binding biological materials, method for the production and use thereof |
US6936414B2 (en) | 1999-12-22 | 2005-08-30 | Abbott Laboratories | Nucleic acid isolation method and kit |
WO2001046404A1 (en) * | 1999-12-22 | 2001-06-28 | Abbott Laboratories | Nucleic acid isolation method and kit |
WO2001059098A2 (en) * | 2000-02-11 | 2001-08-16 | Eppendorf Ag | Method for purifying nucleic acids |
WO2001059098A3 (en) * | 2000-02-11 | 2002-05-23 | Eppendorf Ag | Method for purifying nucleic acids |
US7081192B1 (en) * | 2000-08-08 | 2006-07-25 | Aviva Biosciences Corporation | Methods for manipulating moieties in microfluidic systems |
EP2363476A1 (en) * | 2001-02-16 | 2011-09-07 | Promega Corporation | Magnetic isolation and purification of nucleic acids |
EP1368629A4 (en) * | 2001-02-16 | 2005-07-06 | Cortex Biochem Inc | Magnetic isolation and purification of nucleic acids |
EP1368629A1 (en) * | 2001-02-16 | 2003-12-10 | Cortex Biochem Inc. | Magnetic isolation and purification of nucleic acids |
EP2090655A1 (en) * | 2001-02-16 | 2009-08-19 | Promega Corporation | Magnetic isolation and purification of nucleic acids |
US7264927B2 (en) | 2001-11-06 | 2007-09-04 | Cortex Biochem, Inc. | Isolation and purification of nucleic acids |
EP2000533A3 (en) * | 2001-11-06 | 2009-07-08 | Promega Corporation | Isolation and purification of nucleic acids |
EP2258845A3 (en) * | 2001-11-06 | 2012-03-21 | Promega Corporation | Isolation and purification of nucleic acids |
EP1442045A2 (en) * | 2001-11-06 | 2004-08-04 | Cortex Biochem Inc. | Isolation and purification of nucleic acids |
WO2003040687A2 (en) | 2001-11-06 | 2003-05-15 | Cortex Biochem, Inc. | Isolation and purification of nucleic acids |
EP1442045A4 (en) * | 2001-11-06 | 2006-09-27 | Cortex Biochem Inc | Isolation and purification of nucleic acids |
US8026068B2 (en) | 2002-01-08 | 2011-09-27 | Roche Molecular Systems, Inc. | Use of silica material in an amplification reaction |
WO2005089929A3 (en) * | 2004-03-18 | 2005-11-24 | Ambion Inc | Modified surfaces as solid supports for nucleic acid purification |
EP2292789A1 (en) * | 2004-03-18 | 2011-03-09 | Ambion, Inc. | Modified surfaces as solid supports for nucleic acid purification |
WO2005089929A2 (en) | 2004-03-18 | 2005-09-29 | Ambion , Inc. | Modified surfaces as solid supports for nucleic acid purification |
US9464315B2 (en) | 2004-03-18 | 2016-10-11 | Applied Biosystems, Llc | Modified surfaces as solid supports for nucleic acid purification |
US8426126B2 (en) | 2004-03-18 | 2013-04-23 | Applied Biosystems, Llc | Modified surfaces as solid supports for nucleic acid purification |
US7527929B2 (en) | 2004-07-30 | 2009-05-05 | Agencourt Bioscience Corporation | Methods of isolating nucleic acids using multifunctional group-coated solid phase carriers |
EP1799846A2 (en) * | 2004-09-16 | 2007-06-27 | Lumigen, Inc. | Methods for isolating nucleic acids from biological and cellular materials |
EP1799847A2 (en) * | 2004-09-16 | 2007-06-27 | Lumigen, Inc. | Simplified methods for isolating nucleic acids from cellular materials |
EP1799846A4 (en) * | 2004-09-16 | 2008-01-16 | Nexgen Diagnostics Llc | Methods for isolating nucleic acids from biological and cellular materials |
EP1799847A4 (en) * | 2004-09-16 | 2007-12-12 | Nexgen Diagnostics Llc | Simplified methods for isolating nucleic acids from cellular materials |
DE102007009347A1 (en) | 2007-02-27 | 2008-08-28 | Agowa Gmbh | Isolating nucleic acids comprises binding the nucleic acids adsorbtive to polar surfaces, washing the surfaces with substances solution after removing the binding mixture, which have an affinity to polar surfaces and to the nuclei acids |
WO2009070465A1 (en) * | 2007-11-29 | 2009-06-04 | New England Biolabs, Inc. | Selective purification of small rnas from mixtures |
GB2455780A (en) * | 2007-12-21 | 2009-06-24 | Zainulabedin Mohamedali Saiyed | Nucleic acid separation |
EP2157181A1 (en) | 2008-08-13 | 2010-02-24 | AGOWA Gesellschaft für molekularbiologische Technologie mbH | Method for isolating nucleic acids and test kit |
WO2012069660A1 (en) * | 2010-11-26 | 2012-05-31 | Invitrogen Dynal As | Use of polyols in nucleic acid processing |
US11261480B2 (en) | 2010-11-26 | 2022-03-01 | Life Technologies As | Nucleic acid preparation method |
EP2993232A1 (en) * | 2010-11-26 | 2016-03-09 | Life Technologies AS | Nucleic acid preparation method |
EP3409777A1 (en) * | 2010-11-26 | 2018-12-05 | Life Technologies AS | Nucleic acid preparation method |
US9909165B2 (en) | 2010-11-26 | 2018-03-06 | Life Technologies Corporation | Nucleic acid preparation method |
US9708645B2 (en) | 2010-11-26 | 2017-07-18 | Life Technologies Corporation | Nucleic acid preparation method |
EP2969140A4 (en) * | 2013-03-15 | 2017-05-03 | Abbott Molecular Inc. | One-step procedure for the purification of nucleic acids |
US9803230B2 (en) | 2013-03-15 | 2017-10-31 | Abbott Molecular Inc. | One-step procedure for the purification of nucleic acids |
EP3828284A1 (en) * | 2013-03-15 | 2021-06-02 | Abbott Molecular Inc. | One-step procedure for the purification of nucleic acids |
EP2969140A1 (en) * | 2013-03-15 | 2016-01-20 | Abbott Molecular Inc. | One-step procedure for the purification of nucleic acids |
WO2016077294A1 (en) * | 2014-11-14 | 2016-05-19 | Corning Incorporated | Methods and kits for post-ivt rna purification |
WO2016079509A1 (en) * | 2014-11-18 | 2016-05-26 | Cambridge Epigenetix Limited | Methods for nucleic acid isolation |
EP3061823A1 (en) * | 2015-02-25 | 2016-08-31 | QIAGEN GmbH | Method for extracting nucleic acids from an agarose matrix |
EP3141298A1 (en) | 2015-09-09 | 2017-03-15 | National Center For Scientific Research "Demokritos" | Polymeric microfluidic device for nucleic acid purification fabricated via plasma micro-nanotexturing |
US10344274B2 (en) | 2016-02-16 | 2019-07-09 | Life Magnetics, Inc. | Methods for separating nucleic acids with graphene coated magnetic beads |
WO2018109075A1 (en) * | 2016-12-15 | 2018-06-21 | Qiagen Gmbh | Method for isolating highly pure nucleic acid with magnetic particles |
WO2020260620A1 (en) * | 2019-06-28 | 2020-12-30 | Qiagen Gmbh | Method for enriching nucleic acids by size |
WO2020260618A1 (en) * | 2019-06-28 | 2020-12-30 | Qiagen Gmbh | Method for separating nucleic acid molecules by size |
WO2021122846A1 (en) * | 2019-12-16 | 2021-06-24 | Qiagen Gmbh | Enrichment method |
Also Published As
Publication number | Publication date |
---|---|
US5705628A (en) | 1998-01-06 |
US5898071A (en) | 1999-04-27 |
IL115352A0 (en) | 1995-12-31 |
IL115352A (en) | 2009-02-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5705628A (en) | DNA purification and isolation using magnetic particles | |
JP4369619B2 (en) | Rapid and simple isolation method for circular nucleic acids | |
US6534262B1 (en) | Solid phase technique for selectively isolating nucleic acids | |
US20060078923A1 (en) | Method for isolating nucleic acids | |
EP0508985B1 (en) | Method and kit for purifying nucleic acids | |
US7560228B2 (en) | Solid-phase nucleic acid isolation | |
US20020106686A1 (en) | Methods and reagents for the isolation of nucleic acids | |
US20070054285A1 (en) | Method for isolating nucleic acids | |
JP2002531126A (en) | Formulations and methods for isolation of nucleic acids from any complex starting material and subsequent complex gene analysis | |
EP0792355A1 (en) | Method for purifying nucleic acids from homogeneous mixtures | |
US20060160085A1 (en) | Novel buffer formulations for isolating purifying and recovering long-chain and short-chain nucleic acids | |
US20090306359A1 (en) | Non-alcoholic buffer formulations for isolating, purifying and recovering long-chain and short-chain nucleic acids | |
ENGELSTEIN et al. | An efficient, automatable template preparation for high throughput sequencing | |
CA2315257A1 (en) | Method for isolating short and long-chain nucleic acids | |
JP3811767B6 (en) | Method for purifying nucleic acids from a homogeneous mixture |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): CA JP |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
122 | Ep: pct application non-entry in european phase |