EP1239739A1 - Suppression de substances etrangeres de liquides biologiques contenant des acides nucleiques et recuperation de ces acides nucleiques - Google Patents

Suppression de substances etrangeres de liquides biologiques contenant des acides nucleiques et recuperation de ces acides nucleiques

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Publication number
EP1239739A1
EP1239739A1 EP00988165A EP00988165A EP1239739A1 EP 1239739 A1 EP1239739 A1 EP 1239739A1 EP 00988165 A EP00988165 A EP 00988165A EP 00988165 A EP00988165 A EP 00988165A EP 1239739 A1 EP1239739 A1 EP 1239739A1
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EP
European Patent Office
Prior art keywords
dna
nucleic acids
specimen
protein
procipitate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP00988165A
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German (de)
English (en)
Inventor
John Krupey
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LigoChem Inc
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LigoChem Inc
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Publication date
Application filed by LigoChem Inc filed Critical LigoChem Inc
Publication of EP1239739A1 publication Critical patent/EP1239739A1/fr
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    • 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

Definitions

  • the present invention relates to a means for removing proteins and unwanted aggregated DNA from biological media containing the desired nucleic acids by subjecting the starting mate ⁇ al to a water insoluble complex consisting of ProCipitateTM -protein and aggregated DNA interspersed with feme oxide particles to a magnetic force or by interspersing heavy metal oxides such as bismuth ox> chloride into the ProC ⁇ p ⁇ tateTM-protem-aggregated DNA complex and allowing the resulting aggregate to settle under unit gravity Hawkins
  • U S Patent No 5,705,628 descnbes a method for separating nucleic acids using magnetic micro-particles The method as descnbed involves many steps and is expensive First the magnetic particles must be chemically denvatized to permit nucleic acid attachment Secondly, the binding conditions are very stnngent since they require different iterations of salt and polyethylene ghcol In contradistinction to this procedure, the method employed in the present invention uses unde ⁇ vatized magnetic particles and is not constrained by
  • Nucleic acids are polymenc acids In addition to having large numbers of nucleotides and nbose moieties, they possess a plurality of negatively charged phosphate groups Because of their strong negative charge they should bind tightly to a positively charged fumed metallic oxide surface metallic oxide surface It has been demonstrated (Kummert R , and Strum W , International Journal of Colloid and Interface Science, 75(2) 373, 1980) that organic molecules with molecular masses smaller than 200 daltons and with the functional groups carboxylic, phenolic -OH or an amino group hich can form covalent bonds with the structural metal, bind to the fumed aluminum oxide surface
  • the compounds that were employed in these studies were phthahc acid, benzoic acid, salicylic acid and catechol Since the pnmary focus and objective is the binding of polymenc acids to the oxide surface, very little is to be gamed from the studies which employ monome ⁇ c molecules In general the binding of a polyelectrolyte (e g DNA) to a sur
  • U S Patent No 5,234,809 discloses a method for adsorbing nucleic acids onto silica particles in the presence of chaotropic agents
  • the silica-nucleic acid complex is then washed with organic solvents to prevent desorption of the nucleic acid from the solid phase
  • the nucleic acid is then eluted from silica using a mild buffer
  • Silica is an oxide of the element Silicon Silicon has properties between metals and non- metals and is called a metalloid
  • Metallic oxides, such as titanium oxide are an oxide of metals such as titanium A metal is a substance having a charactenstic luster, malleability and high electncal conductivity, that is, metals readily loose electrons to form positive ions.
  • a metal can be thought of as an anay of nuclei immersed in a sea of electrons, some of the electrons present roam through the array of nuclei and acid and act as an all prevailing electrostatic glue This is not the case with metalloids (silicon) where the electrons are less promiscuous and have a lesser tendency to wander about All the atoms of metalloids are held together by a network of electron pair bonds Substances with this type of structure are referred to as "network covalent solids" The entire crystal, in effect consists of one huge molecule.
  • fumed titanium oxide of the present invention When fumed titanium oxide of the present invention is placed in contact with water, its surface acquires a permanent positive charge When this positively charged matnx is placed into contact with an aqueous solution of nucleic acid in either pure water, chaotropic salts or non- chaotropic salts (kos otropes), a strong ionic bond is formed between the positively charged metallic surface and the negatively charged phosphate groups of the nucleic acid The resulting nucleic acid- fumed titanium oxide complex is stable and cannot be dissociated by treatment with either pure water, alcohol, chaotropic ions or kosmotropic ions under neutral conditions Dissociation is promoted by treatment with mild alkali
  • silica particles When silica particles are placed in contact with w ater they do not acquire a permanent positive charge Silica particles are mildly acid Based on the experiments of Boom et al U S Patent No 5 234,809 it appears that the interactiv e forces between the silica particles are weak in companson to the strong electrostatic force that exists between the fumed metallic oxide and the nucleic acid since washing of the complex w ith pure w ater or neutral salt solutions tend to release significant amounts of nucleic acid from the surface As a result of this property.
  • Boom uses organic solvents to wash off extraneous proteins that are co-adsorbed onto the particles Treating the nucleic acid-silica complex with an aqueous organic soh ent to remov e contaminating protein might be counterproductive, particularly if the protein is insoluble in that solvent composition
  • a chaoptrope or chaotropic ion is a substance or a on which is least effective as a protein precipitant, and promotes unfolding, extension, and dissociation (Dandhker, W B and de Saussure, V A in The Chemistry of Biosurfaces, Ed M L Hair Marcel Dekker, New York, 1971 , pi 8)
  • chaotropic anions are gua dine thiocyanate and potassium iodide
  • kosmotropic ions are sodium chloride and sodium sulfate
  • Boom et al employed a 10 molar solution of guamdine thiocyanate to displace the DNA from the starting mate ⁇ al while a 3 molar solution of the same reagent was employed for dissociation purposes in the fumed metallic oxide procedure
  • U S Patent No 5 057,426 discloses a method for separating long chain nucleic acids compnsmg fixing the nucleic acids onto a porous matrix, washing the porous matnx to separate the other substances from the long chain nucleic acids, and removing the fixed long chain nucleic acids from the porous matrix 1 he porous mat ⁇ x is a mate ⁇ al tor chromatography hav ing been modified with respect to its surface, and the mate ⁇ al is based on a member selected from the group consisting of silica gel diatomite, aluminum oxide titanium oxide hydrox> lapat ⁇ te, dextran, agarose acrylamide polystyrene, polyv myl alcohol or other organic polymers
  • United States Patent No 5.470,463 relates to modified porous solid supports and processes for the preparation and use of same
  • passiv ated porous mineral oxide supports are disclosed which are characterized by a reversible high sorptiv e capacity substantially unaccompanied by non-specific adsorption of or interaction with biomolecules
  • Passivation is achieved by use of a passivation mixture compnsmg a mam monomer, a passivat g monomer and a crosslinking agent, which mixture upon polymerization results in the substantial elimination of the undesirable non-specific interaction with biomolecules
  • United States Patent No 5,599,667 discloses the use of polycatio c solid supports in the pu ⁇ fication of nucleic acids from solutions containing contaminants
  • the nucleic acids non-covalently bind to the support without significant binding of contaminants permitting their separation from the contaminants
  • the bound nucleic acids can be recovered from the support
  • the supports descnbed is the use of the supports as a means to separate polynucleotides and hybrids thereof with a nucleotide probe from unhyb ⁇ dized probe
  • Assays for target nucleotide sequences are described which employ this separation procedure
  • United States Patent No 5,635,405 discloses an aqueous colloidal dispersion for diagnostic or immunodiagnostic tests, comp ⁇ sing non-polymer nuclei surrounded by a hydrophihc copolymer that contains functional groups, a method for the detection of a specifically binding substance or immunochemically active component in a test fluid, and test kit containing the aqueous colloidal dispersion
  • 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
  • the present inv ention provides a means for removing proteins and unw anted aggregated DNA from biological media containing nucleic acids by subjecting the starting material of specimen to a water insoluble complex consisting of ProCipitateTM and protein interspersed with ferric oxide particles to a magnetic force Alternatively, the aggregated DNA interspersed w ith feme oxide particles are subjected to a magnetic force The clear supernatants are recovered and analyzed for nucleic acid
  • the invention provides a means for removing proteins and aggregated DNA from biological media containing nucleic acids by reacting the ProC ⁇ p ⁇ tateTM-prote ⁇ n complex with heavy metal oxides (e g bismuth oxychloride) or by interacting the aggregated DNA with heavy metal oxides, and allowing the respective complexes to settle under unit gravity The clear supernatants are recovered and analyzed for nucleic acids
  • heavy metal oxides e g bismuth oxychloride
  • the invention also provides a means for binding fumed metallic oxides to fe e oxide particles
  • the object is to enable the dissociation of the DNA or RNA from the fumed metallic oxide-
  • the invention provides a method for removing proteins and aggregated DNA from biological specimens and removing the desired nucleic acids compnsmg, contacting a specimen including nucleic acids to a water insoluble complex consisting of ProCipitateTM and protein interspersed with feme oxide particles to form a mixture, or contacting a specimen including nucleic acids to a water insoluble complex comprising ProCipitateTM aggregated DNA and protein interspersed with feme oxide particles, or contacting a specimen including nucleic acids to a water insoluble complex compnsmg aggregated DNA and protein interspersed with fe e oxide, and applying a magnetic force to said mixture
  • the invention provides a method for removing proteins and aggregated DNA from biological specimens and recovenng the desired nucleic acids comprising, contacting a specimen including nucleic acids to a water insoluble complex consisting of ProCipitateTM and protein interspersed with a heavy metal oxide such as bismuth oxy
  • FIG 1 shows electrophoretic patterns of DNA isolated using magnetized ferric oxide particles
  • Lane 1 Control genomic DNA
  • Lane 2 DNA isolated from whole blood
  • Lane 3 BAC DNA from a strain of E coli
  • Lane 4 Plasmid DNA from a strain of E coli
  • Figure 2 shows electrophoretic patterns of DNA isolated using bismuth oxychlonde
  • Lane 1 Control genomic DNA
  • Lane 2 DNA isolated from whole blood
  • Lane 3 BAC DNA from a strain of E coli
  • L ane 4 Plasmid DNA from a strain of E coli
  • Figure 3 shows a schematic of a method of isolation of DNA from whole blood using feme oxide
  • Figure 4 shows a schematic of a method of isolation of DNA from hole blood using bismuth oxychlonde
  • Figure 5 shows a schematic of a method of isolation of plasmid DNA using feme oxide
  • Figure 6 shows a schematic of a method of isolation of plasmid DNA using bismuth oxychlonde
  • FIG. 7 shows the preparation of protein bndging network polyelectrolytes (PBNP)
  • Figure 8 shows a proposed mechanism for the aggregation of proteins by protein bndging network polyelectrolytes (PBNP) and their desorbtion
  • the present invention relates to a means for removing proteins and unwanted aggregated DNA from biological media containing nucleic acids by subjecting the starting matenal to a water insoluble complex consisting of ProCipitateTM and protein interspersed with feme oxide particles to a magnetic force
  • the method of the invention can advantageously be used, for example in high throughput diagnostics, molecular bioinformatics, nucleic acid isolation and characterization.
  • Advantages of the present invention include: a) The methods described obviate the necessity for centrifugation or filtration process steps b) The methods described are user friendly, cost effective and amenable for process automation c) The methods described permit the rapid removal of contaminating nucleic acids in the downstream processing.
  • ProCipitateTM Two hundred and fifty microliters of the water insoluble, protein-aggregating agent, ProCipitateTM are then added.
  • ferric oxide particles become interspersed within the protein ProCipitateTM aggregate thus making this configuration amenable to the action of a magnetic force.
  • the aggregates are drawn to the inner wall of the tube using a magnet.
  • the clear supernatant containing the nucleic acids is withdrawn from the tube by pipette.
  • the nucleic acid is isolated according to the procedure described in Provisional Patent
  • Example 5 Application entitled Method for Isolating DNA from Proteinaceous Medium and Kit for Performing Method, filed November 10, 1999, incorporated herein by reference.
  • a chaotropic agent such as 3M guanidine thiocyanate in a buffer, for example about 100 mM sodium acetate pH 7.0.
  • a suspension of the protein precipitator ProCipitateTM manufactured by LigoChem Inc., Fairfield NJ
  • the composition of ProCipitateTM is disclosed in U.S. Patent Nos.
  • Example 2 The isolation of plasmid DNA from bactenal lysates using ferric oxide particles
  • the cells were centrifuged and the supernatants were discarded
  • the cells were then dispersed in 20ul of T ⁇ s buffer pH 8 0 containing RNAse Twenty microliters of 1 0% sodium dodecyl sulphate (SDS) were added
  • the plasmid DNA is further punfied by the procedure described in Provisional Patent Application entitled Method for Isolating DNA from Proteinaceous Medium and Kit for Performing Method, filed November 10, 1999
  • Lysis Bacterial cell lysis is traditionally carried out under alkaline conditions in the presence of the ionic surfactant sodium dodecyl sulfate (SDS) (Ref Birnboim H C and Doly J Nucleic Acids Research 7 15 13- 1523 1979)
  • SDS sodium dodecyl sulfate
  • SDS-protein protein containing bound SDS molecules
  • free plasmid DNA are released into the surrounding milieu which results m an increase the viscosity of the solution
  • the next step in the lysis procedure is the addition of a high-salt neutralization solution usually potassium acetate which neutralizes the negative charges on the SDS-protein as well as other components and promotes the formation of aggregates of chromosomal DNA and SDS protein complexes
  • the plasmid DNA remains in solution after this treatment
  • BiOCl bismuth oxy-chlo ⁇ de
  • the plasmids, that are recovered, using either protocol may be pu ⁇ fied further by using the traditional alcohol precipitation method or the LigoChem fumed metallic oxide (DNAble) method
  • ProCipitateTM a protein aggregating reagent may be added to the neutralized cell lysate to affect the removal of residual proteins, followed by the addition of either Fe30 4 or BiOCl
  • Fe30 4 or BiOCl it cannot be stated absolute certainty at this time as to whether this treatment is absolutely necessary in all cases to obtain amplifiable and sequenceable plasmid and BAC DNA Since bactenal cultures show a marked variation in protein content it can only be sunnised that high protein containing cultures require ProCipitateTM pretreatment while those containing lesser amounts of protein do not.
  • Figure 5 shows a schematic of a method of isolation of plasmid DNA using feme oxide.
  • Figure 6 shows a schematic of a method of isolation of plasmid DNA using bismuth oxychloride.
  • the method employed for the isolation of BAC DNA was essentially the same employed for the isolation of plasmid DNA except that 2.0ml of bacterial culture was employed instead of 250 microliters.
  • Genomic DNA, plasmid DNA, and BAC DNA were isolated from the respective sources by treating lysates with a 10.0% suspension of bismuth oxychloride (BiOCl) and allowing the resulting complexes consisting of extraneous substances and BiOCl to settle under unit gravity in the absence of a magnetic field.
  • the DNA was recovered and purified as described in the Disclosure Document
  • Nucleic Acid Isolation In this procedure, one volume of whole blood is treated with two volumes of a chaotropic agent such as 3M guanidine thiocyanate in a buffer, say, 100 mM sodium acetate pH 7.0. After standing at room temperature for 15 minutes a suspension of the protein precipitator ProCipitateTM (manufactured by LigoChem Inc., Fairfield NJ) is then added to precipitate the protein.
  • a chaotropic agent such as 3M guanidine thiocyanate
  • a buffer say, 100 mM sodium acetate pH 7.0.
  • a suspension of the protein precipitator ProCipitateTM manufactured by LigoChem Inc., Fairfield NJ
  • the composition of ProCipitate is disclosed in U.S. Patent Nos. 5,294,681 ; 5,453,493; and 5,534,597, and U.S. Application Serial No. 08/676,668 (now allowed) incorporated herein by reference in their entireties.
  • the tubes are then centrifuged at 10,000 x g for 15 minutes, and the supernatant recovered, 1.5 volumes of Titanium Oxide P-25 is then added.
  • the resulting aggregate consisting of DNA and metallic oxide is allowed to settle under unit gravity. After settling the supernatant is removed by aspiration and the settled complex is washed with three washings using deionized water.
  • the tubes are then centrifuged at 1000 x g for 30 seconds The supernatant is discarded and 0 02M sodium hvdroxide is added to the tube T he tubes are then v ortexed followed by cent ⁇ fugation at, say, 10 000 x g for 5 minutes The supernatants are then removed neutralized ith a 0 I M T ⁇ s HCI solution and analyzed for DNA by spectrophotomet ⁇ c absorption at 260 and 280 nm
  • One ml of whole blood contains approximately 40 to 50 micrograms of DNA 1 his quantity translates into about one absorbance unit (AU) at 260 nm and 0 8 AU at 280 nm
  • the DNA specimens are also subjected to agarose gel electrophoresis in which the DNA bands were identified by ethidium bromide staining
  • one volume of whole blood is treated w ith three volumes of a 1 0% w/v of sodium dodecyl sulfate (SDS) in a buffer, say, 10 mM solution of T ⁇ s buffer and l OOmM EDTA pH
  • the methods of the present inv ention can advantageously be used for (a) General screening of blood samples in a 96 well-automated microtiter plate format for genetic aberrations
  • the procedure is m the DNA recovery protocol
  • the DNA is routinely eluted from the fumed titanium oxide particles bv mild alkali treatment Under these conditions the metallic oxide particles ill not sediment so the suspension must be filtered or centri luged to iecover the DNA
  • An ideal configuration consists of an alkali stable complex of fumed metallic oxides and ferric oxide that binds DNA and is attracted by a magnet under mild alkali conditions Under such conditions the DNA appears in the clear supernatant after magnetization
  • a complex consisting of fumed titanium oxide and feme oxide has been prepared in the presence of polyethy lene glycol This complex binds DNA and is attracted by a magnet However, this complex is unstable under mild alkali conditions, dissociating into free fumed metallic oxide, free fe e oxide and free polyethylene glycol
  • a reagent, ProCipitateTM has been shown to be effective in aggregating large quantities of protein present m biological media while leav ing the DNA intact in the supernatant This reagent is currently employed in the isolation of DNA from whole blood
  • ProCipitateTM belongs to a class of water insoluble network polyelectrolytes that selectively bind and aggregate proteins and viruses (Krupey, J , U S Patent No 5,294,681 March 15 1994,
  • a fundamental aspect of protein bndging by the network polyelectrolyte is the energy change that occurs in the course of binding
  • the polyelectrolyte is initially in a high energy (unfavorable) state because of the strong electrostatic repulsions between the negatively charged monomenc units
  • energy is released and salt bridges may be formed between the carboxylate ions and positively charged ammo groups
  • the release of immobilized water surrounding the ionic groups provides an additional driv ing force for salt b ⁇ dge formation
  • the complex that results then collapses to a state of lower energv which is fav orable
  • the protein can be dissociated from the complex under mild alkaline conditions (pH 8 ⁇ - 9 5) Under these conditions, the undissociated carbox> hc acid groups on the polymer ionize and strongly repel each other As a result of this repulsive interaction, the polymeric network expands and the protein is released A number of cross-linked poly
  • ProCipitateTM which was prepared from a linear high molecular mass (:_ 20 kD) aliphatic polyanhyd ⁇ de and was found to be functional in the (3-6 2 pH range) This reagent has a high protein aggregating capacity and is capable of aggregating at least an equivalent weight of either serum albumin or immunoglobulin G originally present in a physiological medium
  • HemogloBindTM a reagent with a high affinity from hemoglobin was prepared from a styrene maleic anhydnde co-polymer with an average molecular mass of 1 0 kD
  • the functional pH range of both types of polyelectrolytes was found to be between 5 5 and 7 5
  • Figure 3 shows a schematic of a method of isolation of DNA from whole blood using feme oxide
  • Figure 4 shows a schematic of a method of isolation of DNA from whole blood using bismuth oxychlonde

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Abstract

La présente invention concerne une technique de suppression de protéines et d'ADN agrégés indésirables de milieu biologique contenant des acides nucléiques par la soumission du matériau de départ à un complexe non soluble dans l'eau constitué de ProCipitateTM et de protéine intercalé avec des particules d'oxyde ferrique soumises à une force magnétique.
EP00988165A 1999-12-20 2000-12-20 Suppression de substances etrangeres de liquides biologiques contenant des acides nucleiques et recuperation de ces acides nucleiques Withdrawn EP1239739A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US17258599P 1999-12-20 1999-12-20
US172585P 1999-12-20
PCT/US2000/034514 WO2001045522A1 (fr) 1999-12-20 2000-12-20 Suppression de substances etrangeres de liquides biologiques contenant des acides nucleiques et recuperation de ces acides nucleiques

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EP1239739A1 true EP1239739A1 (fr) 2002-09-18

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EP00988165A Withdrawn EP1239739A1 (fr) 1999-12-20 2000-12-20 Suppression de substances etrangeres de liquides biologiques contenant des acides nucleiques et recuperation de ces acides nucleiques

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EP (1) EP1239739A1 (fr)
AU (1) AU2440101A (fr)
WO (1) WO2001045522A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004005553A1 (fr) 2002-07-10 2004-01-15 Massachusetts Institute Of Technology Appareil et procede permettant d'isoler un acide nucleique d'un echantillon
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
EP1992689B1 (fr) * 2006-02-15 2015-03-18 Tosoh Corporation Procédé d'extraction d'acide nucléique à partir d'un matériau biologique
GB2455780A (en) * 2007-12-21 2009-06-24 Zainulabedin Mohamedali Saiyed Nucleic acid separation
CN103063494A (zh) * 2012-12-13 2013-04-24 宁波大学 一种实验室用磁性复合微粒悬浊液离析装置

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Publication number Priority date Publication date Assignee Title
US5453493A (en) * 1992-03-20 1995-09-26 Affinity Technology, Inc. Method of precipitating proteins from aqueous media with a polyhydroxy polycarboxylic acid
US5705628A (en) * 1994-09-20 1998-01-06 Whitehead Institute For Biomedical Research DNA purification and isolation using magnetic particles

Non-Patent Citations (1)

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Title
See references of WO0145522A1 *

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WO2001045522A1 (fr) 2001-06-28

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