WO1998023630A1 - Matrice pour la purification rapide d'acides nucleiques - Google Patents

Matrice pour la purification rapide d'acides nucleiques Download PDF

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Publication number
WO1998023630A1
WO1998023630A1 PCT/US1997/021907 US9721907W WO9823630A1 WO 1998023630 A1 WO1998023630 A1 WO 1998023630A1 US 9721907 W US9721907 W US 9721907W WO 9823630 A1 WO9823630 A1 WO 9823630A1
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WO
WIPO (PCT)
Prior art keywords
matrix
molecular weight
materials
proteins
migration
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Application number
PCT/US1997/021907
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English (en)
Inventor
Brian Seed
Wen Shao
John Seed
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Edge Biosystems, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Edge Biosystems, Inc. filed Critical Edge Biosystems, Inc.
Priority to AU55125/98A priority Critical patent/AU5512598A/en
Publication of WO1998023630A1 publication Critical patent/WO1998023630A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C07H1/06—Separation; Purification

Definitions

  • This invention is related to the area of purification of nucleic acids. BACKGROUND OF THE INVENTION
  • 4,923,978 teaches the use of a solid phase material with a large surface area and a high concentration of mildly acidic hydroxyls, e.g. silica, for the removal of proteins from nucleic acids.
  • Seed & Seed, EPO 0524800 teach the extraction of proteins from nucleic acids with aromatic polymers . While the purification of nucleic acids from enzymes and other reaction components used to modify
  • DNA is relatively straightforward, DNA purification from crude biological samples is considerably more complex.
  • the latter material contains large quantities of protein, lipid, carbohydrates and glycosylated macromolecules in addition to low molecular weight contaminants and in some cases detergents.
  • Ion exchange techniques efficiently separate many common contaminants of these samples from DNA and may also separate different forms of DNA such as supercoiled plasmid from linear DNA e.g., genomic DNA.
  • the principle disadvantage of this technique is that the procedure is slow, typically involving gravity flow processing, and generates large volumes of fluid from which DNA must be concentrated.
  • the adsorption techniques glass, silica, and diatomaceous earth), do not provide the same levels of purity, but are significantly faster than most ion exchange techniques and deliver the DNA in a small volume of low ionic strength buffer.
  • Resin contamination of the sample and limitations in either binding capacity or recovery of DNA from the resin are typical disadvantages to the adsorption methods. These techniques also require significant inputs of time and effort due to the requirement of binding the DNA to the matrix, washing out contaminants, and elution.
  • the composition of the wash buffer also can significantly impact the purity of the sample by contaminating the preparation with solvents and/or salts.
  • the technique of extracting contaminants from the DNA requires fewer manipulations than the techniques described above but typically leaves many low molecular weight residues in the sample and consequently requires an alcohol precipitation step. The latter is undesirable for safety reasons, substantially increases processing time, and carries a risk of catastrophic sample loss.
  • Kung, et al., EPO 0310251 teach the use of proteolytic enzymes in combination with gel filtration to purify
  • a matrix for purifying a desired nucleic acid having a defined minimum molecular weight has the ability to bind to proteins and in some cases detergents but not nucleic acids.
  • the matrix also retards the migration through the matrix of components having a an effective hydrodynamic radius smaller than that of nucleic acids of a minimum molecular weight.
  • the matrix also is hydrated with water or a buffer of known composition.
  • a method for purifying a desired nucleic acid having a defined minimum molecular weight comprises the step of applying a preparation comprising the desired nucleic acid to a column containing a matrix having the ability to bind to proteins and in some cases to detergents but not to nucleic acids.
  • the matrix retards the migration through the matrix of components having an effective hydrodynamic radius smaller than that of nucleic acids of a minimum molecular weight.
  • the matrix is hydrated with water or a buffer of known composition in which the purified nucleic acid is delivered.
  • the inventors have discovered that by formulating a matrix which has properties associated with a number of distinct chromatography matrices, that a single step chromatographic separation can be used to provide very clean preparations of nucleic acids from biological and experimental samples. Using such a separation, one can obtain preparations which are suitable for multiple analytic and therapeutic uses.
  • the nucleic acid can be eluted from the matrix in a minimum volume in water or a buffer of desired composition, thus obviating the need to precipitate for purposes of concentration or buffer exchange in most circustances.
  • the matrix of the present invention is typically provided in the form of a column from which the purified sample can be collected.
  • the matrix of the present invention has several important characteristics: it binds proteins; it may bind detergents; it retards the migration of molecules having a smaller hydrodynamic radius than the DNA molecule which is being purified; it does not bind to nucleic acids; it does not retard the migration of molecules having a hydrodynamic radius greater than of equal to the DNA molecule which is being purified; and it is hydrated with water or a buffer of known composition. All of these characteristics can be provided by a single physical component, or they may be provided by two or more components which are mixed together, or which are layered in a column.
  • Binding of proteins to the matrix can occur by any intermolecular interaction, such as affinity binding, ionic interactions, hydrophobic interactions, hydrogen binding, and Van der Waals interactions.
  • the matrix bind to other substances which typically contaminate nucleic acid preparations, detergents in particular. These contaminants are frequently found in cell lysates produced by the alkaline detergent method (Birnboim and Doly, Nucl. Acids Res. 7, p.1513, 1979) and the contaminants may form aggregates which are difficult to separate from nucleic acids on the basis of size.
  • a preferred material for protein binding, according to the invention is poly(4-hydroxystyrene), although any material known in the art to bind protein with high affinity for protein can be used.
  • This protein binding material may not carry a surface positive charge which is accessible for binding by nucleic acids.
  • suitable protein binding materials include silica, glass beads, diatomaceous earth, various metal and semiconductor oxides, beads or similar packing material prepared from protein-binding polymers such as polyvinylidenefluoride, nitrocellulose and polymers bearing uncharged or negatively charged protein binding dyes, and resins with affinity for particular proteins or which are substituted with molecules, e.g. antibodies, which bind certain proteins with high affinity.
  • Positively charged protein binding matrices can be used if the positive charges are sequestered within the matrix so that proteins having an effective hydrodynamic radius less than that of the minimum molecular weight nucleic acid are admitted, but all species having a larger radius are excluded.
  • Retardation of lower molecular weight substances is accomplished by using a "molecular sieve", i.e., a substance which has pores of a defined size range such that only molecules smaller than a certain hydrodynamic radius will enter the pores and be retarded by such entry, whereas molecules with a hydrodynamic radius larger than the pore size will not enter and therefore their migration will not be retarded by the porous substance.
  • the pore size which is useful for isolating DNA from crude biological samples is the same as is found in porous chromatographic media useful for fractionating globular proteins of 1 x 10 3 to 8 x 10 6 .
  • the matrix should be resistant to deformation and have a particle size in the range of 2.5 - 1000 microns, with a preferred particle size of 30-120 microns.
  • the sieving matrices of the invention are cross-linked hydrophilic polymers or copolymers. These include by way of example but without limitation, dextran, agarose, surface modified silica, porous glass and substituted acrylamides. The precise degree of cross linking required to achieve the retention characteristics described above are well known to those of skill in the art.
  • a singel component matrix for purification of nucleic acids comprises beads of a cross-linked hydroxylated aromatic polymer having a suitable pore size as defined above.
  • a variety of components, each having a desirable characteristic as identified above may be mixed and added to a column or layered onto a column. See Examples below. It is also possible to have separate components which may not have useful 5 sieving properties, and which do not bind DNA, but are useful for binding detergents and lipids, such as polystyrene.
  • Crude biological samples comprise cells or cell lysates or combinations thereof.
  • Such combinations include by way of example but without limitation, blood, tissues, soil samples, body fluids, scrapings and plant materials.
  • cells can be lysed by physical, chemical or enzymatic means, or combinations thereof.
  • RNase may be added to the lysis solution to facilitate the degradation of RNA and its subsequent separation from DNA on
  • Lysis in the presence of RNase occurs most favorably with a salt concentration of 0.1 M or less and optimally with a salt concentration of 0.01 M or less.
  • the lysate may be chemically processed to facilitate removal of detergents, proteins and lipids by the addition of salt.
  • the precipitates formed by the addition of salt may be removed by centrifugation or filtration
  • Preferred salts for the formation of precipitates are those which form insoluble complexes with both the detergents and proteins present in the sample.
  • potassium acetate is used in plasmid DNA preparations of the alkaline lysis
  • transition metal ion salts 25 type to form insoluble dodecylsulfate/protein complexes. Addition of transition metal ion salts in low concentration to the lysate in alkaline lysis purifications facilitates the formation of condensed precipitates, thus allowing the samples to be processed in a minimum volume.
  • concentration of transition metal ion salt is 0.01 - 0.1 M with a preferred concentration of 0.02 - 0.06 M.
  • the preferred transition metal ion salt is zinc chloride, although other salts of zinc and other transition metal ions may be useful in the method of the invention.
  • the sample can be processed through the
  • the sample may be eluted from the matrix in a minimum volume using either water or a suitable low salt buffer.
  • the buffer composition of the eluate may be changed by pre-equilibrating the matrix with the buffer which is to be used in subsequent processing of the sample.
  • the eluate typically contains a sample which is ready to use for further applications.
  • the DNA may be concentrated by precipitation from alcohol, ultrafiltration, or other methods known in the art.
  • EXAMPLE 1 A column was prepared in an 800 ⁇ l well, in a 96 well filter plate. Well dimensions were 1.087 inches long, tapered from a diameter of 0.274 inches at top to 0.204 inches at the base. The well was filled sequentially with 400 ⁇ l of an 80% suspension of Sephacryl® S-400 HR (Pharmacia) in water and 300 ⁇ l of an 80% suspension of Sephadex® G100 fine (Pharmacia) in water. Excess fluid was removed between additions by vacuum filtration and 200 ⁇ l of a 1 : 1 mixture of 80% Sephadex G100 and 3.3% poly(4-hydroxystyrene) beads (AdvamaxTM beads, Edge BioSystems) was added to the column.
  • Sephacryl® S-400 HR Pharmacia
  • Sephadex® G100 fine Pharmacia
  • the column was then centrifuged at 1400 x g for 3 minutes to remove excess fluid volume. Lysed bacterial sample containing plasmid DNA was applied to the column and centrifuged at 1400 x g for 3 minutes to elute the sample.
  • the plasmid was comprised of an unknown clone of human cDNA inserted in a pBLUESCRIPT vector (Stratagene).
  • the host was a commercial strain of E. coli known as SOLRTM cells (Stratagene).
  • Bacteria containing the plasmid were cultured overnight for 22 hours in 1 ml of terrific broth (Life Technologies) at 36° in the presence of 100 ⁇ g ml ampicillin in a 96 well plate (2 ml plate with square well configuration) shaking at 325 rpm. The cell suspension was pelleted by centrifugation for 5 minutes at 1400 xg. The bacteria were resuspended in 40 ⁇ l of water containing 300 ⁇ g/ml RNase A and 200 ⁇ g/ml lysozyme. After complete resuspension, the cells were incubated at room temperature for 5 minutes, lysed with 0.5 M NaOH containing 1.3% SDS, mixed by vortexing and allowed to sit for 8 minutes at room temperature.
  • EXAMPLE 2 Porous beads used for binding proteins in the matrix of the invention in Example 1 were prepared as follows. Poly(4-hydroxystyrene)[MW 104,000] was dissolved in 50% aqueous ethanol at 8 mg/ml. The solution was pumped into flowing water at a rate of 80 ml minute through an 18 gauge orifice mounted perpendicular to the flow of water in a closed system. Polymer was added until the concentration of alcohol reached 10-12%. The precipitate that formed was then concentrated by tangential flow filtration over a 3 OK membrane filter, and washed until the concentration of alcohol was less than
  • EXAMPLE 3 A column was prepared in a 760 ⁇ l microcentrifuge tube bucket 1.00 inches long, tapered from a diameter of 0.290 inches at top to 0.280 inches at the base. The bucket is supported at the base with an open plastic grid. Column components are retained by a porous polyethylene frit (15 - 30 micron nominal pore size) press fit into the bucket on top of the open plastic grid. The bucket was filled with 400 ⁇ l of an 80% suspension of Sephacryl® S-400 HR (Pharmacia) in water and 400 ⁇ l of an 80% suspension of Sephadex® Gl 00 fine (Pharmacia) in water.
  • Bacteria containing the plasmid were cultured, lysed and precipitated as in Example 1..
  • the precipitated lysate was transferred to the column and processed as described above.
  • the eluate was free of detectable protein and detergent and contained approximately 26 ⁇ g of purified plasmid.
  • EXAMPLE 4 A column was prepared in a 760 ⁇ l microcentrifuge tube bucket 1.00 inches long, tapered from a diameter of 0.290 inches at top to 0.280 inches at the base. The bucket is supported at the base with an open plastic grid. Column components are retained by a porous polyethylene frit (15 - 30 micron nominal pore size) press fit into the bucket on top of the open plastic grid. The bucket was filled with 400 ⁇ l of an 80% suspension of Sephacryl® S-400 HR
  • coli known as SOLRTM cells (Stratagene). Bacteria containing the plasmid were cultured, lysed and precipitated as described in Example 1. The precipitated lysate was transferred to the column and processed as described above. The eluate was free of detectable protein and detergent and contained approximately 30 ⁇ g of purified plasmid.
  • EXAMPLE 5 A column was prepared in a 760 ⁇ l microcentrifuge tube bucket 1.00 inches long, tapered from a diameter of 0.290 inches at top to 0.280 inches at the base. The bucket is supported at the base with an open plastic grid. Column components are retained by a porous polyethylene frit (15 - 30 micron nominal pore size) press fit into the bucket on top of the open plastic grid. The bucket was filled with 400 ⁇ l of an 80% suspension of Sephacryl® S-400 HR
  • coli known as SOLRTM cells (Stratagene). Bacteria containing the plasmid were cultured, lysed and precipitated as described in Example 1. The precipitated lysate was transferred to the column and processed as described above. The eluate was free of detectable protein and detergent and contained approximately 30 ⁇ g of purified plasmid.
  • a column was prepared in a 760 ⁇ l microcentrifuge tube bucket 1.00 inches long, tapered from a diameter of 0.290 inches at top to 0.280 inches at the base.
  • the bucket is supported at the base with an open plastic grid.
  • Column components are retained by a porous polyethylene frit (15 - 30 micron nominal pore size) press fit into the bucket on top of the open plastic grid.
  • the bucket was filled with 1500 ⁇ l of a 1:2 mix of an 80% suspension of crosslinked poly-
  • Example 7 4-hydroxystyrene beads prepared according to details provided in Example 7 and an 80% suspension of Sephadex® GlOO fine (Pharmacia) in water.
  • the gel was added in 2 steps to allow for limitiations of column volume. Excess fluid was removed between additions by vacuum filtration. The column was then centrifuged at 1400 x g for 3 minutes to remove excess fluid volume. Lysed bacterial sample containing plasmid DNA was applied to the column and centrifuged at 1400 x g for 3 minutes to elute the sample.
  • the plasmid was comprised of an unknown clone of human cDNA inserted in a pBLUESCRTPT vector (Stratagene). The host was a commercial strain of E.
  • coli known as SOLRTM cells (Stratagene). Bacteria containing the plasmid were cultured, lysed and precipitated as described in Example 1. The precipitated lysate was transferred to the column and processed as described above. The eluate was free of detectable protein and detergent and contained approximately 50 ⁇ g of purified plasmid.
  • EXAMPLE 7 Porous beads used for binding proteins in the matrix of the invention in Example 6 were prepared as follows. Poly(4-hydroxystyrene)[MW 104,000] was dissolved in 2 M NaOH at a concentration of 300 mg/ml. Butane-diol- diglycidyl ether (15 mole percent of PHS) was added and the solution incubated

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Abstract

L'invention concerne une matrice conçue pour la purification en une étape d'acides nucléiques d'un lysat de cellules, cette matrice liant certains contaminants et en retardant d'autres, et les acides nucléiques traversant l'éluat.
PCT/US1997/021907 1996-11-27 1997-11-26 Matrice pour la purification rapide d'acides nucleiques WO1998023630A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU55125/98A AU5512598A (en) 1996-11-27 1997-11-26 Matrix for rapid purification of nucleic acids

Applications Claiming Priority (2)

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US3195296P 1996-11-27 1996-11-27
US60/031,952 1996-11-27

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020029477A (ko) * 2000-10-13 2002-04-19 박제철 한 단계에 의한 유전자 추출방법
KR20020029476A (ko) * 2000-10-13 2002-04-19 박제철 한 단계에 의해 유전자를 추출하기 위한 라이시스버퍼시약
WO2012004619A1 (fr) 2010-07-07 2012-01-12 Diagon Kft. Processus d'isolement spécifique d'une teneur en adn total de germes bactériens, et trousse associée

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4923978A (en) * 1987-12-28 1990-05-08 E. I. Du Pont De Nemours & Company Process for purifying nucleic acids
EP0580305A2 (fr) * 1992-07-02 1994-01-26 Advanced Genetic Technologies Corporation Procédé et matériau pour la purification d'acides nucléiques

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4923978A (en) * 1987-12-28 1990-05-08 E. I. Du Pont De Nemours & Company Process for purifying nucleic acids
EP0580305A2 (fr) * 1992-07-02 1994-01-26 Advanced Genetic Technologies Corporation Procédé et matériau pour la purification d'acides nucléiques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
J. CHROMATOG., 1988, Vol. 456, BOOS et al., "On-Line Sample Processing and Analysis of Diol Compounds in Biological Fluids", pages 93-104. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020029477A (ko) * 2000-10-13 2002-04-19 박제철 한 단계에 의한 유전자 추출방법
KR20020029476A (ko) * 2000-10-13 2002-04-19 박제철 한 단계에 의해 유전자를 추출하기 위한 라이시스버퍼시약
WO2012004619A1 (fr) 2010-07-07 2012-01-12 Diagon Kft. Processus d'isolement spécifique d'une teneur en adn total de germes bactériens, et trousse associée

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Publication number Publication date
AU5512598A (en) 1998-06-22

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