WO2001045522A1

WO2001045522A1 - The removal of extraneous substances from biological fluids containing nucleic acids and the recovery of nucleic acids

Info

Publication number: WO2001045522A1
Application number: PCT/US2000/034514
Authority: WO
Inventors: John Krupey
Original assignee: Ligochem, Inc.
Priority date: 1999-12-20
Filing date: 2000-12-20
Publication date: 2001-06-28
Also published as: AU2440101A; EP1239739A1

Abstract

A method for removing proteins and unwanted aggregated DNA from biological media containing nucleic acids by subjecting the starting material to a water insoluble complex consisting of ProCipitateTM and protein interspersed with ferric oxide particles to a magnetic force.

Description

THE REMOVAL OF EXTRANEOUS SUBSTANCES FROM

BIOLOGICAL FLUIDS CONTAINING NUCLEIC ACIDS

AND THE RECOVERY OF NUCLEIC ACIDS

The use of fumed metallic oxide particles in nucleic acid purification was previously described in Provisional Patent Application entitled Method for Isolating DNA from Proteinaceous

Medium and Kit for Performing Method, filed November 10, 1999, the disclosure of which is incorporated herein by reference in its entirety

TECHNICAL FIELD

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 mateπal to a water insoluble complex consisting of ProCipitate™ -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 ProCιpιtate™-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 undeπvatized magnetic particles and is not constrained by solvent composition and ionic strength

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 monomeπc molecules In general the binding of a polyelectrolyte (e g DNA) to a surlace containing multiple permanent charges oi opposite sign is energetically more fa\ orable than the binding of a single isolated monomeπc unit (e g a deoxy πbonucleoside tπphosphate) to the same surface The simultaneous presence of multiple interactions w hen the polyelectrolyte and surface are brought together may produce cooperatn lty between them, and together they might be much stronger than might be expected from the sum of their individual bond strengths

In the case of single interactions involving the monomenc molecule and an oppositely charged surface, the single interactions are mutually exclusπ e or non-cooperative and hence the resulting bonds are relatively weak as compared to those between the polymer and the surface. Boom et al. 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 There are fundamental differences in the chemistry and physical properties of silica and the fumed metallic oxides of the present invention 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. 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

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

In order to release significant amounts of DNA from the nucleolnstone complex of mammalian cells, the cells are treated with a solution containing a chaotrope The accepted definition of 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) Examples of chaotropic anions are gua dine thiocyanate and potassium iodide

At the opposite extremes are the kosmotropic ions These substances are most effective as protein precipitants and lead to folding, coiling, and association The helical content of the protein is thereby increased as a result of this treatment Examples of kosmotropes are sodium chloride and sodium sulfate

The process of protein destabihzation is carried out in the presence of large amounts of chaotropes (3 molar to 10 molar for guamdine thiocyanate) At these concentrations, the extremely chaotic solution conditions overcome the molecular forces and cause destabihzation of proteins

Boom et al employed a 10 molar solution of guamdine thiocyanate to displace the DNA from the starting mateπal while a 3 molar solution of the same reagent was employed for dissociation purposes in the fumed metallic oxide procedure

There is, howev er a clear difference between the two methods with regard to the concentration of chaotrope that is employed dunng the adsorption process The chaotrope requirements for the adsorption process of Boom, et al, U S Patent No 5,234,809, are very stnngent in that high concentrations of this reagent must be maintained to permit the adsorption of DNA to the silica particles

In contrast, the chaotrope requirements for adsorption to fumed metallic oxide surfaces are far less stnngent, since the binding of DNA to this surface can occur at either high concentrations of chaotrope (5M) or at much lower concentrations of this reagent (0 01M) w ith equal efficiency 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 matπx is a mateπal tor chromatography hav ing been modified with respect to its surface, and the mateπal is based on a member selected from the group consisting of silica gel diatomite, aluminum oxide titanium oxide hydrox> lapatιte, dextran, agarose acrylamide polystyrene, polyv myl alcohol or other organic polymers and deriv ativ es or copolymers thereof

United States Patent No 5.470,463 relates to modified porous solid supports and processes for the preparation and use of same In particular 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 puπfication 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 Also descnbed is the use of the supports as a means to separate polynucleotides and hybrids thereof with a nucleotide probe from unhybπdized 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, compπsing 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 United States Patent No 5,705,628 discloses 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

There is a need in the art for improved methods for isolating DNA The present invention o ercomes pπor art deficiencies in methods of isolating DNA SUMMARY OF THE INVENTION

The present inv ention provides a means for remov ing 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 ProCipitate™ 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

In another embodiment the invention provides a means for removing proteins and aggregated DNA from biological media containing nucleic acids by reacting the ProCιpιtate™-proteιn 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

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-

Fe₃0₄ complex under mild alkali conditions in a magnetic field

In an alternative embodiment the invention provides a method for remov ing 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 ProCipitate™ and protein interspersed with feme oxide particles to form a mixture, or contacting a specimen including nucleic acids to a water insoluble complex comprising ProCipitate™ 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 In still another embodiment 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 ProCipitate™ and protein interspersed with a heavy metal oxide such as bismuth oxychlonde to form a mixture, or contacting a specimen including nucleic acids to a water insoluble complex compπsing ProCipitate™ aggregated DNA and protein interspersed with bismuth oxychlonde instead of feme oxide, and allowing the complex to settle under unit gravity

The above and other objects of the invention will become readily apparent to those of skill in the relevant art from the following detailed descnption and figures wherein only the prefened embodiments of the invention are shown and described, simply by way of illustration of the best mode of carrying out the invention As is readily recognized the in ention is capable of modifications w ithin the skill of the relevant art without departing from the spirit and scope of the invention

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 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, and

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, and 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

Figure 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

BEST MODE FOR CARRYING OUT THE INVENTION

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 ProCipitate™ 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.

Example 1

Isolation of DNA from whole blood usin ferric oxide particles Fifty microliters of whole mouse blood in a suitable container are treated with a 3M solution of guanidine thiocyanate containing O. IM ethylene diamine-tetraacetate (EDTA).

Two hundred and fifty microliters of the water insoluble, protein-aggregating agent, ProCipitate™ are then added.

One hundred microliters of a 10% aqueous suspension of finely divided ferric oxide (F^O,,) particles are then added and the tubes are mixed. At this stage of the procedure, the ferric oxide particles become interspersed within the protein ProCipitate™ 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

Application entitled Method for Isolating DNA from Proteinaceous Medium and Kit for Performing Method, filed November 10, 1999, incorporated herein by reference. An example of this procedure follows and is present in Example 5 below . 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, for example about 100 mM sodium acetate pH 7.0. After standing at room temperature for about 15 minutes a suspension of the protein precipitator ProCipitate™ (manufactured by LigoChem Inc., Fairfield NJ) is then added to precipitate the protein. 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) and 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 about 1000 x g for 30 seconds. The supernatant is discarded and 0.02M sodium hydroxide is added to the tube The tubes are then ortexed, followed by centπfugation at about 10 000 x g for 5 minutes T he supernatants are then removed neutralized with a 0 I M Tπs LICl solution and analyzed for DNA by spectrophotometπc absorption at 260 and 280 nm One ml of whole blood contains approximately 40 to 50 micrograms of DNA This 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

The electrophoretic profile of the genomic DNA isolated is shown in the enclosed Figure I Example 2 The isolation of plasmid DNA from bactenal lysates using ferric oxide particles

Two hundred fifty microliters (LB) of an overnight culture containing 10⁹ E coli plasmid containing cells per ml were prepared

The cells were centrifuged and the supernatants were discarded

The cells were then dispersed in 20ul of Tπs buffer pH 8 0 containing RNAse Twenty microliters of 1 0% sodium dodecyl sulphate (SDS) were added

One hundred microliters of a 10% suspension of feme oxide was then added and the tubes were mixed This was followed by the addition of 20 microliters of a 5M potassium acetate solution Ferric oxide becomes ter-spersed within the mucinous framework of the aggregated DNA ProCipitate™ can also be employed in this process to further remove extraneous substances along with the feme oxide particles

Plasmid DNA is recovered in the clear supernatant after subjecting the aggregate to a magnetic field

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

The electrophoretic profile of the plasmid DNA isolated is shown in the enclosed Figure 1 Example 3

Isolation of Plasmids from Bacteria Recovery In small-scale production process, growth is earned out in multi-well micro-titer plates to a high cell density (OD₆₀₀=30- 100) The cells are then recovered by centπfugation Cells are then re- suspended and concentrated in a buffer appropπate for the following step and designed to disrupt cells and release the plasmid This buffer usually contains agents that disrupt the non-covalent bonds between hpids/and or proteins, for instance, ethylene diamine tetra-acetic acid (EDTA) is often used as a chelat g agent The remov al of divalent cations (e g , Ca⁷ and Mg^2* ) from the cell w all, outer membrane (in Gram-negativ e bacteria) and plasma membrane destabilizing their structure, facilitating

Ivsis

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) At this stage of processing, aggregated chromosomal DNA, protein containing bound SDS molecules (SDS-protein) and 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

The aggregates of chromosomal DNA and SDS-protein are highly gelatinous and in most cases will occlude the membrane filter when one attempts to recover the desired plasmid DNA using a preliminary filtration step This situation presents an intractable problem particularly in robotic systems where the isolation of plasmid DNA in a multi-microtiter well format must be rapid and efficient

We have found that this problem may be circumvented by first binding ferric oxide (FejO,,) to the aggregate driving the Fe_jO^chromosomal DNA- SDS protein complex to the bottom of the container by applying a magnetic force and recovering the plasmid DNA in the clear supernatant

Alternatively, a heavy metal oxide such as bismuth oxy-chloπde (BiOCl) can be employed in place of Fe₃0₄ as an aggregate binding agent The BiOCl-chromosomal DNA protein-SDS complex that is formed settles rapidly under unit gravity and thus permits the recovery of the desired plasmid DNA in the clear supernatant

The plasmids, that are recovered, using either protocol may be puπfied further by using the traditional alcohol precipitation method or the LigoChem fumed metallic oxide (DNAble) method

ProCipitate™, 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 Fe30₄ or BiOCl However, 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 ProCipitate™ 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.

Example 4 Isolation of BAC DNA from bacterial lysates using ferric oxide particles

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.

The electrophoretic profiles of the BAC DNA, isolated is shown in the enclosed Figure 1.

Example 5

Isolation of DNA from whole blood and bacterial lysate using heavy metal oxides

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

No. 456808

The electrophoretic profiles of the DNA, isolated are shown in Figure 2.

Example 6

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 ProCipitate™ (manufactured by LigoChem Inc., Fairfield NJ) is then added to precipitate the protein. 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 centπfugation at, say, 10 000 x g for 5 minutes The supernatants are then removed neutralized ith a 0 I M Tπs HCI solution and analyzed for DNA by spectrophotometπc 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

In another version of this procedure one volume of blood is treated with two v olumes of 3M guanidine thiocyanate in 100 mM sodium acetate (ED LA is not present) The mixture is then heated at 65 degrees Celsius for 10 minutes After standing at room temperature for 5 minutes, a suspension of ProCipitate™ is added to precipitate the protein The supernatant is recovered by centπfugation and this DNA containing solution is processed and analyzed for DNA as described abov e

Alternatively, 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 Tπs buffer and l OOmM EDTA pH

8 0 After remaining at room temperature for 15 minutes, 3 v olumes of a 3M solution of potassium acetate is added to neutralize the SDS and to precipitate the hemoglobin that is present The tubes are then centrifuged and the supernatant is recovered 1 5ml of Titanium Oxide P-25 suspension is then added The aggregate is allowed to settle under unit gravity After settling the supernatant is discarded and the DNA-metalhc oxide complex is washed with three washings of deionized water

The tubes were then centπfuged at 1000 x g for 30 seconds and the supernatant discarded Dissociation of the complex was accomplished by the same method that was used in the ProCipitate™ guanidine thiocyanate procedure

In view of the above, 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

(b) Forensic medicine, molecular bioinformatics

(c) In an automated system for the isolation of bacterial and viral constructs for genomic sequencing (d) Non invasiv e diagnostics-capture and quantification of DNA in saliva-capture and quantification of small quantities of DNA present in large volumes of uπne

(e) Removal of contaminating nucleic acids in the do nstream processing of recombinant proteins

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

Example 7

Isolation of DNA from Whole Blood

In order to obtain a DNA specimen which is suitable for amplification and sequencing from a highly proteinaceous medium such as whole blood, the proteins must first be removed Most available methods are either arduous or painstaking and may require the use of organic solvents to effect protein removal

A reagent, ProCipitate™ 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

ProCipitate™ 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,

Krupey J U S Patent No 5,453,493 September 26, 1995, Krupey, J , U S Patent No 5,534,597 July 9, 1996, Krupey, J , Smith, AD , Arnold, E, and Donnell. U S Patent No 5,658,779 August 19, 1997, Krupey, J U S Patent No 5,976,382, November 2, 1999) These reagents have been collectivelv named protein bπdgmg network polyelectrolytes (PBNP) The skeletal framework of these polymers is generated by reacting a polymeric maleic anhydride co-polymer with an aliphatic diam e to yield a network of polycarboxyhc acid chains covalentlv cross-linked by diamide bonds (Figure 7 ) By controlling the chemistry, architecture and charge properties of these structures, it was possible to produce a polymenc configuration that can specificallv bind and aggregate the molecules of interest These network polyelectrolytes have been engineered to have a slight imbalance between the

Coulombic attractive forces that cause the network to shnnk or to collapse and the repulsiv e forces that cause the network to expand The polymer is routinely employed with only a fraction of its total number of carboxylic acid groups in the ionized fonn Therefore, the repulsive interaction predominates, but only marginally In addition to slightly expanding the network, the repulsive force increases the chemical potential of the polyelectrolyte, a condition that fav ors its binding to oppositely charged groups present on proteins

In order for the network polyelectrolytes to bind and aggregate molecules, they must be sufficiently flexible Since the polymers are chemically cross-lmked, parts of the polymer chain could be entwined This would result in decreased flexibility, since rotations about single bonds w ould be restricted The only remaining way in which the chains can flex is by deformation of the bond angles by periodic lengthening and shortening of covalent bonds Consequently, all the deformations add up along the chain and result in some degree of flexibility The number and distribution of charged and polar to apolar residues at the surface of protein molecules is the primary aspect that deteπnines their solubility in a given solvent Although apolar or hydrophobic groups tend to be concentrated around the interior of protein molecules, some hydrophobic side chains remain exposed to water at the molecular surface or in crevices These hydrophobic clusters in contact with an aqueous environment cause an ordering of water molecules into extensive hydrogen bonded configurations effectively "freezing" them about the side chains

One important aspect of this phenomenon is a reduction in the number of permitted configurations, equivalent to a decrease in entropy

Assuming there are no other considerations, the presence of clusters of hydrophobic residues on the surface will favor protein-protein interaction and the formation of multi-unit complexes Thus in those proteins possessing quaternary structure, the sub units appear to be held together by interactions between hydrophobic clusters on their surfaces

It is postulated that ordered water structures can be disrupted by treatment with the cross- linked polyelectro-lytes (Figure 8) These reagents are flexible and can effectiv ely bπdge two or more protein molecules by salt bπdges formed by the carboxylate ions of the polymer and the ionized amino groups of the protein As a consequence of this interaction, the apolar moieties of the individual molecules are brought into close proximity; water is passively excluded and the proteins aggregate while being electrostatically bound to the polymer The end result is a net increase in the entropy of the system

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 When these groups interact with the oppositely charged ammo groups on the protein, 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 bπdge 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 polycarboxyhc acids with different chemistries, architectures, electπcal properties and functional binding properties have been prepared

ProCipitate™ which was prepared from a linear high molecular mass (:_ 20 kD) aliphatic polyanhydπde 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

Two network polyelectrolytes with similar chemistries, but with different geometπc profiles, were also evaluated These configurations were prepared from styrene maleic anhydride copolymers, which differed in their respective molecular masses Viraffimty™, a virus capture reagent. was denved from a styrene maleic anhydnde co-polymer with an average molecular mass of 350 kD

HemogloBind™. 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

The purpose of the above descnption and examples is to illustrate some embodiments of the present invention without implying any limitation It will be apparent to those of skill in the art that vaπous modifications and vanations may be made to the composition and method of the present invention without departing from the spiπt or scope of the invention All patents and publications cited herein are incorporated by reference in their entireties

Claims

1 A method for removing proteins and unw anted aggregated DNA from biological specimen containing nucleic acids, compnsmg (A) contacting a specimen including nucleic acids to a water insoluble complex comprising ProCipitate™ and protein interspersed with ferric oxide particles to form a mixture, and (B) applying a magnetic force to said mixture 2 The method of claim 1 , wherein said nucleic acids are selected from the group consisting of DNA and R A 3 The method of claim 1 further comprising isolating the nucleic acid from the specimen by (A) treating the specimen with a chaotropic agent containing a metal chelator, or alternatively heating the specimen in the presence of the chaotropic agent without the chelator being present, (B) adding a water insoluble protein aggregating agent, ProCipitate™ and isolating a liquid phase, (C) treating the liquid phase with an adsorbent consisting of alumina, titania or zirconia generated by flame hydrolysis, (D) separating the supernatant, (E) washing the residue with deionized water, (F) removing deionized water wash and then dissociating the DNA from the fumed alumina, titania, or zirconia by treatment with aqueous alkali borate or phosphate or a metal hydroxide, and (G) recovering and neutralizing the liquid phase containing DNA 4 A method for removing proteins and aggregated DNA from biological specimens and removing the desired nucleic acids compπsing, contacting a specimen including nucleic acids to a water insoluble complex consisting of ProCipitate™ and protein interspersed with feme oxide particles to form a mixture, or contacting a specimen including nucleic acids to a water insoluble complex compπsing ProCipitate™ 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 feme oxide, and applying a magnetic force to said mixture 5 A method for removing proteins and aggregated DNA from biological specimens and recovenng the desired nucleic acids compnsmg, contacting a specimen including nucleic acids to a water insoluble complex consisting of ProCipitate™ and protein interspersed with a heavy metal oxide such as bismuth oxychlonde to foπn a mixture, or contacting a specimen including nucleic acids to a water insoluble complex compnsmg ProCipitate™ aggregated DNA and protein interspersed with bismuth oxychlonde instead of feme oxide, and allowing the complex to settle under unit gravity 6 The method of any one of claims 4 or 5, wherein said nucleic acids are selected from the group consisting of DNA and RNA 7 The method of any one of claims 4 or 5 further comprising isolating the desired nucleic acid from the specimen by (A) treating the liquid phase with an adsorbent consisting of alumina, titania, or zirconia generated bv flame hydrolysis, (B) separating the supernatant (C) washing the residue with deionized water (D) removing the deionized water wash and then dissociating the DNA from the fumed alumina, titania, or zirconia by treatment with aqueous alkali borate or phosphate or a metal hydroxide and (E) recovering and neutralizing the liquid phase containing DNA 8 The method of claims 1 and 2 further compπsing isolating the desired nucleic acid from the specimen by precipitating the desired nucleic acid by adding the specimen to an alcohol contained in a vessel equipped with a filtration membrane by removing the alcohol by vacuum or pressure filtration, drying the membrane containing the nucleic acid by vacuum suction or by applying pressure, and adding a small volume of water or buffer to the membrane to solubihze the nucleic acid and permit its recovery 9 A method for removing proteins and unwanted aggregated DNA from a biological specimen containing nucleic acids, compnsmg (A) contacting a specimen including nucleic acids with a water insoluble complex compnsmg aggregated DNA and protein interspersed with feme oxide particles or bismuth oxychloride. to form a mixture and (B ) applying a magnetic force to said mixture