CA2264688A1 - Apparatus and methods for active biological sample preparation - Google Patents

Abstract

Systems and methods for the electronic sample preparation of biological materials utilize the differential charge-to-mass ratio and/or the differential affinity of sample constituents to separation materials for sample preparation. An integrated system is provided for performing some or all of the processes of: receipt of biological materials, cell selection, sample purification, sample concentration, buffer exchange, complexity reduction and/or diagnosis and analysis. In one embodiment, one or more sample chambers adapted to receive a buffer solution are formed adjacent to a spacer region which may include a trap or other affinity material, electrophoretic motion of the materials to be prepared being effected through operation of electrodes. In another aspect of this invention, a transporter or dipstick serves to collect and permit transport of materials, such as nucleic acids, most preferably DNA and/or RNA. In one embodiment, a membrane or trap is held in a frame which is adapted to mate with a channel formed in the spacer region. In another aspect of this invention, an electrophoretic system for biological sample preparation is operated in a manner so as to utilize the differential charge-to-mass ratio so as to control the migration of materials within the solution. In one aspect, bunching of selected materials is achieved by operation of two electrodes in a manner so as to reduce the spatial dispersion of those materials. In another aspect of this invention, a vertically disposed sample preparation unit includes an upper reservoir and a collection chamber. A sample is preferably pre-prepared and densified, applied to the conductive polymer, electrophoresed so as to move nucleic acids into the conductive polymer and move undesired material away from the conductive polymer. Integrated systems are described in which cell separation, purification, complexity reduction and diagnosis may be performed together. In the preferred embodiment, cell separation and sample purification are performed in a first region, the steps of denaturation, complexity reduction and diagnosis being performed in a second region.

Description

WO 98/1027710152025CA 02264688 1999-03-05PCT/US97/ 135251DESCRIPTIONAPPARATUS AND METHODS FOR ACTIVEBIOLOGICAL SAMPLE PREPARATIONField of the InventionThis invention relates to devices and methods for performing active, multi-step molecular and biological sample preparation and diagnostic analyses. Moreparticularly, the invention relates to sample preparation, cell selection, biologicalsample purification, complexity reduction, biological diagnostics and general samplepreparation and handling.Background of the InventionMolecular biology comprises a wide variety of techniques for the analysis ofnucleic acid and protein. Many of these techniques and procedures form the basisof clinical diagnostic assays and tests. These techniques include nucleic acid hybrid-ization analysis, restriction enzyme analysis, genetic sequence analysis, and theseparation and purification of nucleic acids and proteins (See, e.g., J. Sambrook,E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed.,Cold spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989).Most of these techniques involve carrying out numerous operations (e.g.,pipetting, centrifugations, electrophoresis) on a large number of samples. They areoften complex and time consuming, and generally require a high degree of accuracy.Many a technique is limited in its application by a lack of sensitivity, specificity orreproducibility. For example, these problems have limited many diagnostic applica-tions of nucleic acid hybridization analysis.The complete process for carrying out a DNA hybridization analysis for agenetic or infectious disease is very involved. Broadly speaking, the completeprocess may be divided into a number of steps and substeps. In the case of geneticdisease diagnosis, the first step involves obtaining the sample (e.g. , blood or tissue).Depending on the type of sample, various pre-treatrnents would be carried out. TheW0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97/135252second step involves disrupting or lysing the cells, which then releases the crudeDNA and RNA (for simplicity, a reference to DNA in the following text also refersto RNA, where appropriate) material along with other cellular constituents. General-ly, several sub-steps are necessary to remove cell debris and to purify further thecrude lysate. At this point several options exist for further processing and analysis.One option involves denaturing the purified sample DNA and carrying out a directhybridization analysis in one of many formats (dot blot, microbead, microtiter plate,etc.). A second option, called Southern blot hybridization, involves cleaving DNAwith restriction enzymes, separating the DNA fragments on an electrophoretic gel,blotting to a membrane filter, and then hybridizing the blot with specific DNA probesequences. This procedure effectively reduces the complexity of the genomic DNAsample. and thereby helps to improve the hybridization specificity and sensitivity.Unfortunately, this procedure is long and arduous. A third option is to carry outthe polymerase chain reaction (PCR) or other amplification procedure. The PCRprocedure amplifies (increases) the number of target DNA sequences. Amplificationof target DNA helps to overcome problems related to complexity and sensitivity inanalysis of genomic DNA or RNA. All these procedures are time consuming,relatively complicated, and add significantly to the cost of a diagnostic test. Afterthese sample preparation and DNA processing steps, the actual hybridizationreaction is performed. Finally, detection and data analysis convert the hybridizationevent into an analytical result.The steps of sample preparation and processing have typically beenperformed separate and apart from the other main steps of hybridization anddetection and analysis. Indeed, the various substeps comprising sample preparationand DNA processing have often been performed as a discrete operation separate andapart from the other substeps. Considering these substeps in more detail, sampleshave been obtained through any number of means, such as obtaining of wholeblood, tissue, or other biological fluid samples. In the case of blood, the sample isoften processed to remove red blood cells and retain the desired nucleated (white)cells. This process is usually carried out by density gradient centrifugation. Celldisruption or lysis is then carried out, preferably by the technique of sonication,freeze/thawing, or by addition of lysing reagents.WO 98/102771015202530CA 02264688 1999-03-05PCT/US97/135253In certain cases, the blood is extensively processed to remove contaminants.One such system known to the prior art is the Qiagen system. This system involvesprior lysis followed by digestion with proteinase K, after which the sample is loadedonto a column and then eluted with a high salt buffer (e.g., 1.25 M NaCl). Thesample is concentrated by precipitation with isopropanol and then centrifuged toform a pellet. The pellet is then washed with ethanol and centrifuged, after whichit is placed in a desired buffer. The total purification time is greater thanapproximately two hours and the manufacturer claims an optical density ratio (260nm/280 nm) of 1.7 to 1.9 (OD 260-280). The high salt concentration can precludeperformance of certain enzymatic reactions on the prepared materials. Further,DNA prepared by the Qiagen method has relatively poor transport on an electropho-retic diagnostic system using free field electrophoresis.Returning now to the general discussion of sample preparation, crude DNAis often separated from the cellular debris by a centrifugation step. Prior tohybridization, double-stranded DNA is denatured into single—stranded form. Dena-turation of the double-stranded DNA has generally been performed by the techniquesinvolving heating (>Tm), changing salt concentration, addition of base (e.g.,NaOH), or denaturing reagents (e.g., urea, formamide). Workers have suggesteddenaturing DNA into its single-stranded form in an electrochemical cell. The theoryis stated to be that there is electron transfer to the DNA at the interface of an elec-trode, which effectively weakens the double—stranded structure and results in separa-tion of the strands. See, e.g., Stanley, "DNA Denaturation by an ElectricPotential", U.K. patent application 2,247,889 published March 18, 1992.Nucleic acid hybridization analysis generally involves the detection of a verysmall number of specific target nucleic acids (DNA or RNA) with an excess ofprobe DNA, among a relatively large amount of complex non-target nucleic acids.DNA complexity is sometimes overcome to some degree by amplification of targetnucleic acid sequences using polymerase chain reaction (PCR). (See, M.A. Inniset al, PCR Protocols: A Guide to Methods and Applications, Academic Press,1990). While amplification results in an enormous number of target nucleic acidsequences that improves the subsequent direct probe hybridization step, amplificationinvolves lengthy and cumbersome procedures that typically must be performed onWO 98/102771015202530CA 02264688 1999-03-05PCT/US97/135254a stand alone basis relative to the other substeps. Complicated and relatively largeequipment is required to perform the amplification step.The actual hybridization reaction represents an important step and occursnear the end of the process. The hybridization step involves exposing the preparedDNA sample to a specific reporter probe, at a set of optimal conditions forhybridization to occur to the target DNA sequence. Hybridization may beperformed in any one of a number of formats. For example, multiple samplenucleic acid hybridization analysis can be conducted on a variety of filter and solidsupport formats (See, G. A. Beltz et al., in Methods in Enzymology, Vol. 100, PartB, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press, New York, Chapter19, pp. 266-308, 1985).involves the non—covalent attachment of target DNAs to a filter, which are"Dot blot"hybridization has gained wide~spread use, and many versions have been developed(See, M. L. M. Anderson and B. D. Young, in Nucleic Acid Hybridization - APractical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press, Washington,D.C. Chapter 4, pp. 73-111, 1985). "Dot blot" assays have been developed forOne format, the so-called "dot blot" hybridization,subsequently hybridized with a radioisotope labelled probe(s).the multiple analysis of genomic mutations (D. Nanibhushan and D. Rabin, in EPA0228075, July 8, 1987) and for the detection of overlapping clones and the con-struction of genomic maps (G. A. Evans, in US Patent Number 5,219,726, June 15,1993).New techniques are being developed for carrying out multiple sample nucleicacid hybridization analysis on micro—formatted multiplex or matrix devices (e.g.,DNA chips) (See, M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10Bio/Technology, pp. 757-758, 1992). These methods usually attach specific DNAsequences to very small specific areas of a solid support, such as micro-wells of aDNA chip. These hybridization fortnats are micro-scale versions of the convention-al "dot blot" and "sandwich" hybridization systems.The micro—formatted hybridization can be used to carry out "sequencing byhybridization" (SBH) (See, M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains,10 Bio/Technology, pp. 757-758, 1992). SBH makes use of all possible n-nucleotide oligomers (n—mers) to identify n-mers in an unknown DNA sample,WO 98/102771015202530CA 02264688 1999-03-05PCT/US97/135255which are subsequently aligned by algorithm analysis to produce the DNA sequence(R. Drmanac and R. Crkvenjakov, Yugoslav Patent Application #570/87, 1987; R.Drmanac et al., 4 Genomics, 114, 1989; Strezoska et al., 88 Proc. Natl. Acad. Sci.USA 10089, 1992; and R. Dramanac and R. B. Crkvenjakov, U.S. Patent#5,202,231, April 13, 1993).There are two formats for carrying out SBH. The first format involvescreating an array of all possible n—mers on a support, which is then hybridized withthe target sequence. The second format involves attaching the target sequence toa support, which is sequentially probed with all possible n—mers. Both formats havethe fundamental problems of direct probe hybridizations and additional difficultiesrelated to multiplex hybridizations.Southern, United Kingdom Patent Application GB 8810400, 1988; E. M.Southern et al. , 13 Genomics 1008, 1992, proposed using the first format to analyzeor sequence DNA. Southern identified a known single point mutation using PCRamplified genomic DNA. Southern also described a method for synthesizing anarray of oligonucleotides on a solid support for SBH. However, Southern did notaddress how to achieve an optimal stringency condition for each oligonucleotide onan array.Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, used thesecond format to sequence several short (116 bp) DNA sequences. Target DNAswere attached to membrane supports ("dot blot" format). Each filter wassequentially hybridized with 272 labelled 10-mer and 11-mer oligonucleotides. Awide range of stringency conditions was used to achieve specific hybridization foreach n-mer probe; washing times varied from 5 minutes to overnight, andtemperatures from 0°C to 16°C. Most probes required 3 hours of washing at 16°C.The filters had to be exposed for 2 to 18 hours in order to detect hybridizationsignals. The overall false positive hybridization rate was 5 % in spite of the simpletarget sequences, the reduced set of oligomer probes, and the use of the moststringent conditions available.A variety of methods exist for detection and analysis of hybridization events.Depending on the reporter group (fluorophore, enzyme, radioisotope, etc.) used tolabel the DNA probe, detection and analysis are carried out fluorometrically,W0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97/135256colorimetrically, or by autoradiography. By observing and measuring emittedradiation, such as fluorescent radiation or particle emission, information may beobtained about the hybridization events. Even when detection methods have veryhigh intrinsic sensitivity, detection of hybridization events is difficult because of thebackground presence of non—specifically bound materials. A number of other factorsalso reduce the sensitivity and selectivity of DNA hybridization assays.Attempts have been made to combine certain processing steps or substepstogether. For example, various microrobotic systems have been proposed forpreparing arrays of DNA probes on a support material. For example, Beattie et al.,in The 1992 San Diego Conference: Genetic Recognition, November, 1992, useda microrobotic system to deposit micro-droplets containing specific DNA sequencesinto individual microfabricated sample wells on a glass substrate. Various attemptshave been made to describe integrated systems formed on a single chip or substrate,wherein multiple steps of an overall sample preparation and diagnostic system wouldbe included. For example, A. Manz gt a_l_., in "Miniaturized Total ChemicalAnalysis System: A Novel Concept For Chemical Sensing", Sensors AndActuators, B1(1990), pp. 244-248, describe a ’total chemical analysis system’ (TAS)which comprises a modular construction of a miniaturized total chemical analysissystem. Sampling, sample transport, any necessary chemical reactions, chromato-graphic separations as well as detection were to be automatically carried out. Yetanother proposed integrated system is Stapleton, U.S. Patent No. 5,451,500, whichdescribes a system for the automated detection of target nucleic acid sequences inwhich multiple biological samples are individually incorporated into matricescontaining carriers in a 2—dimensional format. Different types of carriers aredescribed for different kinds of diagnostic tests or test panels.Various multiple electrode systems are disclosed which purport to performmultiple aspects of biological sample preparation or analysis. Pace, U.S. Patent4,908,112, entitled "Silicon Semiconductor Wafer for Analyzing MicronicBiological Samples" describes an analytical separation device in which a capillary-sized conduit is formed by a channel in a semiconductor device, wherein electrodesare positioned in the channel to activate motion of liquids through the conduit. Pacestates that the dimension transverse to the conduit is less than 100 pm. Pace statesW0 98/102771015202530CA 02264688 1999-03-05PCT/US97/135257that all functions of an analytical instrument may be integrated within a singlesilicon wafer: sample injection, reagent introduction, purification, detection, signalconditioning circuitry, logic and on-board intelligence. Soane _e_t a_1., in U.S. Patent5,126,022, entitled "Method and Device for Moving Molecules by the Applicationof a Plurality of Electrical Fields", describes a system by which materials are movedthrough trenches by application of electric potentials to electrodes in which selectedcomponents may be guided to various trenches filled with antigen-antibodies reactivewith given charged particles being moved in the medium or moved into contact withcomplementary components, dyes, fluorescent tags, radiolabels, enzyme—specifictags or other types of chemicals for any number of purposes such as varioustransformations which are either physical or chemical in nature. It is said thatbacterial or mammalian cells, or viruses may be sorted by complicated trenchnetworks by application of potentials to electrodes where movement through thetrench network of the cells or viruses by application of the fields is based upon thesize, charge or shape of the particular material being moved. Clark, U.S. Patent5,194,133, entitled "Sensor Devices", discloses a sensor device for the analysis ofa sample fluid which includes a substrate in a surface of which is formed anelongate micro-machined channel containing a material, such as starch, agarose,alginate, carrageenan or polyacrylamide polymer gel, for causing separation of thesample fluid as the fluid passes along the channel. The biological material maycomprise, for example, a binding protein, an antibody, a lectin, an enzyme, asequence of enzymes or a lipid.Various devices for eluting DNA from various surfaces are known. ShuklaU.S. Patent 5,340,449, entitled "Apparatus for Electroelution" describes a systemand method for the elution of macromolecules such as proteins, DNA and RNAfrom solid phase matrix materials such as polyacrylamide, agarose and membranessuch as PVDF in an electric field. Materials are eluted from the solid phase intoa volume defined in part by molecular weight cut-off membranes. Okano, U.S.Patent 5,434,049, entitled "Separation of Polynucleotides Using Supports Havinga Plurality of Electrode—Containing Cells" discloses a method for detecting aplurality of target polynucleotides in a sample, the method including the step ofapplying a potential to individual chambers so as to serve as electrodes to eluteCA 02264688 1999-03-05W0 98/ 10277 PCT/US97/135258captured target polynucleotides, the eluted material then available for collection.Generally, the prior art processes have been extremely labor and timeintensive. For example, the PCR amplification process is time consuming and addscost to the diagnostic assay. Multiple steps requiring human intervention either5 during the process or between processes is suboptimal in that there is a possibilityof contamination and operator error. Further, the use of multiple machines orcomplicated robotic systems for performing the individual processes is often prohibi-tive except for the largest laboratories. both in terms of the expense and physicalspace requirements.10 As is apparent from the preceding discussion, numerous attempts have beenmade to provide effective techniques to conduct sample preparation reactions.However, for the reasons stated above, these techniques are limited and lacking.These various approaches are not easily combined to form a system which can carryout a complete DNA diagnostic assay. Despite the long-recognized need for such15 a system, no satisfactory solution has been proposed previously.Summary of the InventionThis invention relates broadly to the methods and apparatus for electronicsample preparation of biological materials for their ultimate use in diagnosis oranalysis. An integrated system is provided for performing some or all of the20 processes of: receipt of biological materials, cell selection, sample purification,complexity reduction and/or diagnosis and analysis. Separation of desiredcomponents, such as DNA, RNA or proteins from crude mixtures such as biologicalmaterials or cell lysates. Electronic sample preparation utilizes the differentialmobility and/or differential affinity for various materials in the sample for purposes25 of preparation and separation. These methods and apparatus are especially usefulfor the free field electrophoretic purification of DNA from a crude mixture orlysate. In one aspect of this invention, a device comprises at least a first central orsample chamber adapted to receive a buffer solution and a second central or samplechamber adapted to receive a same or different buffer solution, where the first30 sample chamberand the second sample chamber are separated by a spacer regionwhich preferably includes a trap, membrane or other affinity material. A firstW0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97/135259electrode in electrical Contact with the conductive solution of the first samplechamber and a second electrode in electrical contact with the conductive solution ofthe second sample chamber are provided. Preferably, each electrode is containedwithin its own electrode chamber, the electrode chamber being separated from thecorresponding sample chamber via a protective layer or separation medium, e.g.,a membrane, such as an ultrafiltration membrane, a polymer or a gel. In operation,the sample mixture is placed within the first central chamber. The central chamberscontain a solution, preferably a low conductivity buffer solution, such as 50 mMhistidine, 250 mM HEPES, or 0.5 X TBE. A mixture of biological substances, forexample a crude lysate, is added to the first chamber and then the electrodes areactivated. Substances with mobility in an electric field will move towards oneelectrode or the other depending on the charge of the substance. In one embodi-ment, the desired and undesired substances with similar charges will be attracted toan electrode biased with the opposite charge to the desired substance, located in thesecond chamber. Therefore, the desired substance and the undesired substances ofsimilar charge will move towards the affinity material which is between the first andsecond chambers.In one embodiment, the sample mixture is composed of a desired substancewhich has charge mixed with undesired substances some of which have charges.After the electrodes are activated, the desired substance travels toward the secondchamber where the electrode, biased with opposite charge, is located and binds tothe affinity material. In contrast, the similarly charged, undesired substances movetoward the electrode biased with the opposite charge and pass through the affinitymaterial into the second chamber. Other undesired substances with the oppositeAfter theundesired substances have passed through the affinity material, the electrolytecharge will be attracted toward the electrode in the first chamber.solution may be changed in both chambers to remove the undesired substances.Then the desired substance is eluted into the fresh electrolyte solution. Elution canbe accomplished by continued electrophoresis at the same or increased current orby the addition of a chemical, such as a detergent, salt, a base or an acid, that willcause elution from the affinity material. In addition, a change in temperature couldbe used to elute the desired substance.W0 98/102771015202530CA 02264688 1999-03-05PCT/U S97/ 1352510In one particular embodiment, the affinity material is composed of a gel withsufficient volume to hold a substantial fraction, e.g., preferably 50%, and morepreferably, 80%, of the desired substance in the sample. The gel composition andconcentration is chosen such that the mobility of the desired substance which hashigh molecular weight (30,000 to 3,000,000 daltons) is retarded by the gel but themobility of the undesired substances which have low molecular weight (100 to10,000 daltons) is relatively unaffected. Consequently, the gel will release thedesired substance only after a longer period of electrophoresis or a higher currentthan is necessary for the passage of the undesired substances. In effect the gel isa trap for the desired substance but does not provide a relatively significant barrierto undesired substances. The desired and undesired substances preferably have avery large difference in electrophoretic mobility while traveling through the gel forthe gel to serve as a trap. Preferably, the trap does not effectively resolve mobilitydifferences among fragments which have similar compositions and molecularweights which are within a factor of substantially 10 of each other.In accordance with this invention, the resolution of different molecularweights is preferentially sacrificed in favor of rapid mobility. Preferably, the gelin the device is relatively compact (e.g., 0.5 to 10 mm) in the direction of migrationto permit rapid electrophoretic transport. Thus, the speed of this technique is notcompatible with conventional resolution of substances by size except betweensubstances with very gross differences in size. Consequently, it is inherent in thistechnique that desired substances of very different molecular weights will becopurified. The preparation of substances of similar composition but different sizesmay be advantageous to the user as for example in the purification of DNA ofdifferent sizes for the purpose of cloning of a whole or representative portion of agenome of an organism.In accordance with one aspect of this invention, the desired substance ispropelled into and out of the gel in the same device. That is, in the preferredembodiment, different regions within the whole system serve as traps and therefore,help to separate analytes or materials to be eluted. The integration of electropho-resis and elution steps also provides significant advantages for the user in savingtime, reducing the number of steps required and decreasing the amount of spaceWO 98/102771015202530CA 02264688 1999-03-05PCT/US97ll352511required for the apparatus.In one aspect of the invention, a device containing multiple electrodechambers in electrical communication with a first and second end sample chamberand one or more intermediate sample chambers is advantageously utilized. In thepreferred embodiment, each of the end sample chambers and the intermediatesample chamber or chambers is in electrical communication with an electrodechamber, preferably having the sample chamber separated from the electrodechamber by a membrane. Preferably, the electrode chamber has a buffer volumewhich is larger than and preferably much larger than (e.g., at least 10 to 1) thevolume of the sample which is loaded into the sample chamber.In another aspect of this invention, the traps, membranes or other affinitymaterials serve as a transporter or ’dipstick’ to collect and permit transport ofmaterials. In one embodiment of this device, the membrane or other affinitymaterial disposed between adjacent sample chambers is provided with structuralintegrity to permit the removal of the trap or affinity material from the chamberstructures. In one preferred embodiment, a membrane or trap is held in a frameThetransporter or dipstick may be utilized to transport the collected material for furtherwhich is adapted to mate with a channel formed at the spacer region.processing, such as further purification, complexity reduction or assaying ordiagnosis. Optionally, the transporter may be disposed adjacent to or formed inelectrical communication with a power source.In yet another aspect of this invention, first and second electrodes aredisposed within an intervening sample solution, the system further including a thirdelectrode between the first and second electrodes. The third electrode may serveas a control electrode to modulate the flow of charged materials within the samplesolution. The third electrode is preferably formed as a grid, and may be advanta-geously formed by sputtering a metal coating on a membrane. The third electrode,or grid, is preferably disposed within the sample solution closer to the first electrodethan the second electrode. In one mode of operation, a sample is placed within thesample solution between the first and third electrodes, and the first and secondelectrodes are biased for net migration of charged materials of a first charge towardthe second electrode. The third electrode or grid is preferably biased slightlyW0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97/1352512negative or neutral. Once the desired DNA or other charged materials passesthrough or by the third electrode or grid, the third electrode or grid is preferablymade relatively negative. This increased negativity serves to move the negativelycharged DNA towards the second electrode, and to repel other, more slowly movingnegatively charged materials which still remain between the first electrode and thethird or grid electrode.In yet another aspect of this invention, a pair of electrodes are located withina sample solution and are operated so as to bunch or concentrate a subset of thecharged macromolecules within the sample solution. Biasing one electrode so as toaccelerate motion of charged macromolecules towards the other electrode, andbiasing the other electrode so as to retard motion of the charged macromoleculeswhich are closer to that electrode in the region between the electrodes. Suchbunching serves to physically concentrate the charged macromolecules within theregion.Thisstructure serves to bunch materials contained within the region bounded by the C-In a preferred embodiment, a "C" shaped electrode is utilized.shaped electrode, as well as to repel like charged materials which are external to theregion bounded by the C-shaped region. Further, the materials contained within theC-shaped region are subject to a force in a sideways (i.e., a transverse or obliquedirection to the net flow direction apart from the C-shaped electrode). The C-shaped electrode may be an integrated, continuous electrode or may be segmented.Other shapes are advantageously utilized, such as parabolic structures. The C-shaped region is sized to include within the region the desired or target material.A complexity reduction device includes one or more probe areas whichcomprise a support material, preferably a polymer gel, such as an agarose,acrylamide or other conductive polymer, to which capture probes are attached. Thesupport material is formed in contact with an electrode to permit the electrophoreticattraction and hybridization of the capture probes with the target materials.Electrical elution or electronic stringency control of the captured materials may beused. In one embodiment, the complexity reduction device is formed from thecombination of a chamber mated to a printed circuit board. The chamber includesvias in which the support material is located. The printed circuit board preferablyWO 98/102771015202530CA 02264688 1999-03-05PCT/U S97/ 1352513including concentric vias to provide a continuous space for the inclusion of thesupport material.’ In this embodiment, gases or other reaction products may bevented through the via, in part because the gas generally does not rise into the viasince it is filled with polymer. Therefore, these gases or other reaction products donot come in contact with the analytes, such as DNA. The complexity reductionsystem optionally may further include disposal regions for the attraction and/ordisposal of undesired materials. The disposal regions include an electrode inelectrical communication through the conductive polymer. In operation, thecomplexity reduction device performs free field electrophoretic transport.Alternatively, an uncovered electrode in contact with the solution may be utilizedfor disposal of undesired materials.In yet another aspect of this invention, a DNA or other nucleic acidpurification device is provided. An upper reservoir containing an electrode, whichmay be identified as a cathode, is adapted to receive a buffer solution and a samplesolution. Preferably, the upstream reservoir includes a tube in fluid communicationwith the upstream reservoir, the tube having an internal diameter less than thediameter of the upstream reservoir. The tube includes at least a first differentialmobility section, preferably a gel, which provides a plug or trap region within thetube. Optionally, the gel may be cast on top of a support membrane. A collectionchamber is adjacent to the differential mobility region. In the preferred embodi-ment, the collection region has a smaller volume than the differential mobilityregion and is smaller than the sample volume. The reduction in volume fromsample to collection permits an increase in volume concentration of DNA andAnanode is provided in a lower reservoir. In yet another aspect of this invention, theexchanges the DNA into the desired buffer formulation of known volume.format and transport of DNA is in the horizontal plane instead of vertical.In operation, a sample is subject to a cell lysing and shearing pre—preparationstep. Preferably, the proteins are then reduced in size, such as through applicationof a proteinase, such as proteinase K. Preferably, when the unit is operated in avertical format, sucrose or other densifier is added to the sample. The densifierserves to collect and concentrate the sample in a region immediately above the firstdifferential mobility section. The prepared sample including densifier is thenWO 98/102771015202530CA 02264688 1999-03-05PCT/US97/1352514injected onto the separation unit in the tube, in a region immediately upstream ofthe differential mobility region. The cathode and anode are then energized toprovide electrophoretic transport of the charged materials within the system, causingthe reduced size protein materials to pass through the differential separation mediumfirst, while retaining the relatively slower moving DNA, as well as resulting in theproteinase K or other positively charged materials being removed to the cathode.After a time sufficient to permit desired amounts of protein to pass through thedifferential mobility region and support membrane, the DNA is eluted from thedifferential movement region into the sample chamber. Optionally, the buffersolution in the lower reservoir which received the proteinaceous material havingpassed through the support membrane may be removed and replaced with newbuffer solution, and optionally provided with a membrane which serves to retaineluted DNA within the sample chamber.In yet another aspect of this invention, the cathode and anode electrodes arein communication with the sample through conductive fluids or gel or polymers, butare resident in the power supply or other controlling instrumentation. Conductionis made through fluidic ports and the electrode (noble metal) preferably is not a partof the consumable device.Integrated systems may be advantageously formed which include some or allof the functions of cell separation, purification, complexity reduction anddiagnostics. In one embodiment, a purification chamber includes an input andmultiple electrodes for providing an electrophoretic driving force to the chargedmaterials. A protein trap disposed between the sample input port and an outlet tapserves to trap undesired proteins or other charged macromolecules. In an alternativeembodiment, the undesired materials such as proteins are reduced in size ormodified in charge so as to increase their degree of mobility relative to the desiredmaterials, such as DNA. The materials for further processing are then removedfrom the trap or other transportation device for further processing, such ascomplexity reduction and/or diagnostic procedures. This separation of targetmaterials from the undesired material is accomplished by modifying the electric fieldto steer the target materials into a region of higher purity (e.g., pure buffer).Electric field modification can be implemented through C-shape electrodes orW0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97/ 1352515activation of a new configuration of electrodes which favorably influence thedirection of mobility of the target.In yet another aspect of this invention, an integrated sample preparation,complexity reduction and diagnostic system is provided. An input region receivescrude lysate including proteins which have been reduced in size, such as through useof a proteinase. The materials pass through a DNA trap, in which the DNA movesrelatively slower than the proteins. The proteins arrive at a protein trapelectrophoretically prior to the arrival of the DNA. The DNA then exit the DNAtrap towards a collection region. Preferably, the collection region has a volumewhich is less than, preferably substantially less than the volume of the DNA trap,such as 50 microliters. This volume is in communication with the complexityreduction and diagnostic regions. In one aspect of this invention, fluidic transportwithin the device is accomplished by input ports located at each end of the collectionregion. These dual inputs are adapted to receive any fluid, such as a buffer, an airslug or a reagent. By selective operation of supplies or pumps, the materialscontained within the collection region, e.g., substantially purified DNA, may beforced from the region to the complexity reduction device. By utilization of airslugs, various liquids may be separated from one another. By operation of the inputliquids or gas, a hydraulic or pneumatic ram serves to move materials within thefluid section of the device. While materials may be pushed forward throughout thesystem, such as from the collection volume to the complexity reduction to thediagnostic region, the materials may be moved in the opposite direction, e.g. , fromthe complexity reduction region to the concentration region.Accordingly, it is an object of this invention to provide for a samplepreparation system useful for biological sample preparation.It is yet a further object of this invention to provide a system for purificationof DNA from biological materials or other crude samples.It is yet a further object of this invention to provide methods for theseparation and purification of desired biological charged macromolecules throughdifferential solution phase mobility and/or differential affinity.Brief Description of the DrawingsW0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97/1352516Fig. 1 is a top, plan view of a multiple sample chamber, multiple electrodechamber device.Fig. 2 is a top, plan view of a device including multiple electrode chambers,end sample chambers and multiple intermediate sample chambers.Fig. 3 is a perspective, exploded view of one embodiment of the invention.Fig. 4 is a cross-sectional view of a multiple electrode embodiment.Fig. 5 is a cross-sectional view of a multiple electrode embodiment includinga trap electrode.Fig. 6 is a perspective view of a multiple electrode structure including a gridor control electrode.Fig. 7 is a perspective view of a multiple electrode system further includingbunching electrodes.Fig. 8 is a plan view of one implementation of an integrated samplepreparation an diagnostic device.Fig. 9 is a plan view of another integrated sample preparation and diagnosticdevice.Fig. 10 is a perspective, cross-sectional view of the complexity reductiondevice.Fig. 11 is a perspective, cross-sectional close-up view of the complexityreduction device.Fig. 12 is a perspective view of the complexity reduction device with theprinted circuit board and complexity reduction chamber exploded from each other.Fig. 13 is a cutaway perspective drawing of a vertically disposed samplepreparation device.Fig. 14 shows a plan view of a horizontal integrated sample preparation,complexity reduction, diagnostic and disposal device.Fig. 14A shows a cross-sectional view along the line A—A’ of Fig. 14.Fig. 15 is a graph of target DNA transport in the presence of S. aureusGenomic DNA purified by the device of Fig. 13 compared to the prior art Qiagenmethod, as a function of time.Fig. 16 is a graph comparing the operation of the complexity reductiondevice in different buffer solutions.W0 98/102771015202530CA 02264688 1999-03-05PCT/US97/1352517Fig. 17 is a graph of the signal accumulation for a microelectronic device incomparison to a printed circuit board based device for various buffers.Fig. 18 is a graph of relative target signal levels determined for specific andnonspecific probes following transport and electronic wash, and afterdehybridization using a Texas Red Bodipyf“ labelled Streptococcal target sequencein the presence of irrelevant human DNA per 40 pL.Fig. 19 is a graph of hybridization of labeled Streptococcal target DNA inthe presence of an equimolar concentration of nonlabeled complementary DNA andirrelevant human DNA.Detailed Description of the InventionFig. 1 shows a top down, plan view of one embodiment of this invention.A frame 10 supports a first central or sample chamber 12 and a second central orsample chamber 14. The designations first and second are arbitrary, and may bereversed, or the chambers may be referred to as the left central chamber 12 andright central chamber 14. Disposed between the left central chamber 12 and rightcentral chamber 14 is a spacer region 16. The spacer region is adapted to receivea trap, membrane or other affinity material or material for relative differentiationof biological materials within the spacer region 16. In one aspect, the spacer region16 may be filled with a gel cast in the region of the spacer 16 or a membrane maybe disposed covering the spacer region 16. Adjacent to the left central chamber 12is a first electrode chamber 18. The electrode chamber 18 is separated from thecentral chamber 12 by a membrane 22, preferably an ultrafiltration membrane, mostpreferably a cellulose acetate membrane. Similarly, a right or second electrodechamber 20 is disposed adjacent to the right central chamber 14, separated by amembrane 24. A function of the membranes 22, 24 is to reduce or prevent contact' of the sample materials or selected components thereof from directly contacting theelectrodes 26, 28. A first electrode 26 and a second electrode 28 are disposed inContact with the first electrode chamber 18 and second electrode chamber 20,respectively. The electrodes are preferably formed of a noble metal, most especiallyplatinum.In the preferred embodiment, the frame 10 is formed of a relatively rigidW0 98/102771015202530CA 02264688 1999-03-05PCT/US97l1352518support material which is non—reactive with the materials placed in contact with it.Desired materials include: polymethacrylate, plastics, polyproplene, polycarbonate,PTFE, TEFLON" or other non—reactive materials. Generally, the frame materialsare non-reactive, non-interactive or non-binding with the sample materials. Theframe 10 may have formed within it regions comprising the electrode chambers 18,20, central chambers 12, 14 and the spacer region 16. In one embodiment, theelectrode chambers 18, 20, are 0.4 cm wide, 0.6 cm deep and 5 cm long (thedirection parallel to a line between the first electrode 26 and second electrode 28).The central chambers 12, 14 have the same width and depth as the electrodechambers 18, 20 and are 1.5 cm long. The spacer region 16 has the same widthand depth as the chamber components 12, 14, 18 and 20, and is 0.4 cm long.Generally, the volume of the spacer region 16 is small relative to the volume of thecentral chamber 12, 14. Preferably, the volume of the spacer region 16 is less than50%, and more preferably less than 30% of the volume of the central chamber 12,14. The embodiment described above as a spacer region 16 with a volume ofapproximately 25% of that of the central chamber 12, 14. As an alternativemeasure, the spacer region 16 may be characterized by its distance along thedirection the direction from the first electrode 26 to the second electrode 28.Preferably, the linear distance is less than 5 mm, more preferably 4 mm or less, andoften in the range of 1-2 mm. In yet another characterization, the thickness of thespacer region 16 in a direction between the first electrode 26 and the secondelectrode 28 is short relative to the distance between the first electrode 26 andsecond electrode 28. Preferably, this length is less than 20%, more preferably lessthan 10% and most preferably less than approximately 5%. Preferably, the size ofthe spacer region 16 is such, relative to the other structures of the device, that thedesired materials may be separated from the undesired materials, so as to permitisolation of the desired materials.The spacer region 16 includes a material which serves to discriminateamongst macromolecules within the device. One class of materials would compriseprotein traps, materials such as PVDF, nitrocellulose and hydrophobic materials.Yet another class of materials are negatively charged materials. Yet a third classof materials are positively charged materials. A modified class of materials utilizesW0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97/1352519a detergent in combination with the trap which permits the DNA to pass through thetrap. Various other surfactants, detergents and materials which achieve thefunctions of the trap to differentiate amongst materials may be utilized as known tothose skilled in the art. DNA/RNA traps would especially include low densitypolymers (e.g., 0.5-3% agarose, or 5%—15% acrylamide) and PVDF. The lattermaterial is a material which traps DNA to some extent, but from which DNA orRNA may be selectively eluted by adding a detergent.The electrode chambers 18, 20 and central chambers 12, 14 are filled witha buffer solution. Preferably, the buffer solution is a relatively low conductivitybuffer solution. Other functional characteristics of the buffer may include:chemically low reactivity, Zwitterionic or ampholytes with no net charge. Theapplication entitled "Methods and Materials for Optimization of ElectronicHybridization Reactions", filed on the same date as the instant application, andincorporated herein by reference more fully describes such techniques. Examplesof buffers which are suitably acceptable for separation of DNA include: histidine,especially about 50 millimolar histidine, HEPES and 0.5 X TBE. HEPES is 4(—2-Hydroxyethyl)-1—Piperazine Ethanesulfonic acid. TAE is prepared by diluting 10x TAE (0.4 M Tris Acetate, 0.1 M EDTA, 0.2 M glacial acetic acid pH 8.4) 1 to10 in deionized water. 0.5 X TBE is prepared by diluting TBE (0.89M Tris-Borate,0.89 M Boric Acid, 0.02 M EDTA, pH 8.0) 1 to 20 in deionized water.In operation, a sample is placed in a central chamber 12, 14, for purposesof discussion assumed to be the left central chamber 12. A potential is appliedacross the first electrode 26 and second electrode 28, permitting a current to flowthrough the electrode chambers 18, 20 and central chambers 12, 14. The chargedmacromolecules in the sample are electrophoretically moved through the left centralchamber 12, towards the spacer region 16. If the spacer region 16 includes aprotein trap, the DNA is moved through the spacer region 16 into the right centralchamber 14. Alternatively, if the spacer region 16 includes a material designed tohold the DNA, but pass the proteins and other undesired materials, the DNAremains on the left portion of the spacer region 16, from which it may be eluted.In the later mode of operation, the buffer in the left central chamber 12 may bereplaced prior to eluting the DNA back into the left central chamber 12.W0 98/102771015202530CA 02264688 1999-03-05PCTIU S97/ 1352520Broadly, the method of this invention provides for active biological samplepreparation of a sample comprising a collection of materials including desiredmaterials and undesired materials having differential charge-to—mass ratios, theseparation being achieved in an electrophoretic system including solution phaseregions and at least one trap region having differential effect on desired materialsas compared to undesired materials. The differential effect of the trap region is afunction of‘the physical size, composition and structure of the trap, which areselected so as to selectively retain or pass either the desired or undesired material.In the preferred embodiment, the method includes the step of providing the samplematerials to a first solution phase region of the device. Subsequently, the sampleis electrophoresed within the system to affect net differential migration between thedesired material and the undesired material whereby one of the desired or undesiredmaterials is located within the trap and the other material is in a solution phaseregion, that is, either the desired materials are substantially retained (e.g.,preferably _2_ 50%, and more preferably, _>_ 80%) in the trap, and the undesiredmaterials are substantially not retained in the trap, or vice versa. Subsequently, thedesired material are removed from the system, whereby relatively purified desiredmaterials are prepared. If the desired materials are the bound or trapped materials,they may be removed in any number of ways, including but not limited to physicalremoval from the system, such as by a dipstick, subsequent removal into a fluid orother medium, such as by moving through the trap or moving back into the chamberfrom which they came, especially if the buffer solution has been changed within thatchamber.Fig. 2 shows a plan view of a multichamber arrangement. A frame 30includes multiple electrode chambers 32. Each electrode chamber 32 includes anelectrode 34. Preferably, the electrode is formed of a noble metal, such asplatinum, and may be placed in electrical contact with a buffer solution placedwithin the electrode chamber 32. End sample chambers 36 sandwich one or moreintermediate sample chambers 38. The end sample chamber 36 is separated fromthe adjacent intennediate sample chamber 38 by a separator 40. The separator 40may comprise an affinity media, a membrane, or other material which selectivelydifferentiates between passage or affinity for various biological materials. TheW0 98/102771015202530CA 02264688 1999-03-05PCT/US97/1352521sample chambers 36, 38 are separated from the electrode chamber 32 by amembrane 42. Preferably, the volume of the electrode chamber 32 is larger,preferably a factor of 10 times, and most preferably a factor of 20 times, relativeto the size of the sample chamber 36, 38. This is so since electrosmosis throughthe membrane 42 may result in liquid-level differences. Further, this relativelylarge volume ratio minimizes the ion concentration gradient between the adjacentelectrode chamber 32 and sample chamber 36, 38 and provides a larger bufferingcapacity around the electrode 34 which increases pH stability which, in turn,optimizes the working time and power (V x 1) input to the sample before thedetrimental effects of the electrophoretically driven osmosis begin to dominate andnegatively impact the process. Yet another object of the relatively large electrodechamber 32 is that adequate spatial separation between the chamber membrane 42and the electrode 34 is provided so as to prevent bubble attachment to the membrane42, after the bubbles are formed at the electrode 34. It is desirable to avoid bubblecontact with the membrane 42 as they obstruct the membrane and preventconduction through them, and the bubbles may have large pHs. Further, it isdesirable that the electrodes 34 have relatively large surface area, e.g., 5-20 mm?"which minimize bubble formation by reducing local current density near theelectrode surfaces and the inherent surface nucleation sites.The frame 30 may be formed of any material which is non-reactive with thematerials to be placed in contact with it. Preferably, the materials are selected tohave low autofluorescence at 380 nmUV, and between 480 nm and 630 nm, in orderto minimize background signal during quantitation. The sample chambers 36, 38may be of different sizes. Preferably, the sample chamber 36, 38 is in the rangefrom 300 pl to 3 ml, most preferably 1 ml. After purification steps, the relativelypurified material may be reduced in sample volume, and the volume may be on theorder of tens of microliters or less.Fig. 3 shows a perspective, exploded view of a multichamber device. Aframe 50 has formed, such as by milling or molding, one or more end samplechambers 56, sample chambers 58 and electrode chambers 52 having the functionsand sizes described in connection with Fig. 2. The electrode 54 preferably exits theelectrode chamber 52 and is connected via a connector 86, such as a threadedW0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97l1352522connector as is known to those skilled in the art. Adjacent sample chambers 56, 58are separated by a membrane holder 60. The membrane holder 60 optionally isformed of membrane holder halves 62 connected via connector 66. An opening 64in the membrane holder 66. An opening 64 in the membrane holder 60 is adaptedto receive a material which differentiates or discriminates the passage of biologicalmaterials, such as a membrane or affinity material. The membrane holder 60 isadapted to matingly engage with holder 66. The sample chamber 56, 58 is incommunication with the electrode chamber 52 via passage 68. In this embodiment,insert 70 threadingly engages with the frame 50 by threading 72 in receptivethreading 74. A barrel 76 includes a counterbore 78 and includes holes 80 to permitpassage from the electrode chamber 52 through the holes 80, through thecounterbore 78, to the sample chamber 56, 58. The insert 70 preferably terminatesat a ring 82, opposite the threaded end of the insert 70, where the ring 82 is adaptedto sandwich a filter or membrane 84 between the ring 82 and the bore 68 of theelectrode chamber 52. The size of the sample chambers 56, 58 may vary from oneto another. Further, the various membrane holders 60 may be utilized, or not,providing yet an additional degree of flexibility in determining the size of the samplechamber 56, 58.The membrane holder 60 is removable from the frame 50. The membraneholder 60 may include membrane, mesh or beads with functional groups covalentlylinked to oligonucleotides. After material is captured within the opening 64 of themembrane holder 60, the membrane holder 60 may be removed from the frame 50and the materials transported to another site.Fig. 4 shows a cross—sectional view of an embodiment of this invention. Afirst or left electrode 90 and a second or right electrode 92 are adapted to providean electrophoretic force on charged macromolecules disposed within the solutionphase region 94. The solution phase region 94 is shown with a dashed boundary,the physical boundary of which may be formed through any desired support mediumnot inconsistent with the materials or methods to be achieved with this invention.A trap 96 is disposed at, near or substantially surrounding the second electrode 92.The trap 96 may be formed of the materials, and have the attributes as described forthe trap or spacer materials, above. Optionally, a protective or permeable layer 98W0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97/1352523is disposed between at least a portion of the solution phase region 94 and the firstor right electrode 90. As shown in Fig. 4, the permeable layer 98 serves to blockthe solution phase region 94 from direct contact with the left electrode 90. Inoperation, a sample is placed in an input region 100, such as through a port or otheropening in the device. The solution phase region 94 contains a buffer or othersuitable transport medium, comprised of or having the functions described for thesolution phase, above. A sample initially placed in the input region 100 iselectrophoretically moved towards the trap 96 by application of potential to theelectrodes 90, 92. When the desired material, e.g., DNA, contacts the trap 96, thesystem is then operated so as to remove the now trapped materials. This could beby removal of the trap 96 from the system, or by eluting the trapped material fromthe trap 96 back into the solution phase region 94. Preferably, the elution of thetrapped material into the solution phase region 94 is preceded by replacing thesolution in the solution phase region 94 with a new solution. The solution may bethe same or different from that previously existing.Fig. 5 shows a cross—sectional view of an alternate embodiment of theinvention. A first electrode 110 and a second electrode 112 provide overallelectrophoretic movement of charged materials within the first solution phase region114 and second solution phase region 115. The first electrode 110 optionally hasa first permeable layer 118 disposed at, near or substantially around the firstelectrode 110, so as to minimize contact of the charged macromolecules with thefirst electrode 110. A second layer 109 is formed at, near or substantiallysurrounding the second electrode 112. In one version, the second layer 109comprises a trap, the materials and functionality being those described above.Alternatively, the second layer 109 may comprise a second permeable layer, adaptedprincipally for the protection of charge macromolecules from directly contacting thesecond electrode 112. Optionally, a trap electrode 122 is located between the firstelectrode 110 and the second electrode 112, dividing the solution phase region intoa first solution phase region 114 and a second solution phase region 115. Further,optionally, the trap electrode 122 may include a trap 116 formed at or integral withthe trap electrode 122. In operation, a sample is provided to the input region 120,which, under operation of the electric fields created via the first electrode 110,W0 98/102771015202530CA 02264688 1999-03-05PCT/U S97/ 1352524second electrode 112 and trap electrode 122 cause the electrophoretic movement ofthe charged macromolecules. Optionally, an additional electrode maybe locatedwithin the solution phase regions 114, 115.Fig. 6 shows a perspective view of a first electrode 130, a second electrode132 and a control electrode 134. Generally, the first electrode 130 may beidentified as the cathode and the second electrode 132 the anode, though those termsmay be interchanged depending on the polarity of the connections. The controlelectrode 134 may be spaced equidistant between the first electrode 130 and secondelectrode 132, though optionally it may be placed closer to the electrodes 130, 132,most preferably to the cathode 130. Variation of the potential applied to the controlelectrode 134 may be used to modulate the flow of charged macromolecules withinthe region between the first electrode 130 and second electrode 132. In one modeof operation, the sample is placed between the first electrode 130, the cathode, andthe control electrode 134. The net flow of negatively charged materials is from thecathode to the anode (second electrode 132). If the control electrode 134 is madeneutral, or even slightly negative, negatively charged materials, such as DNA,would flow in a direction from the cathode to the anode. Once a desired fractionof the DNA passes through or by the control electrode 134, the control electrode134 may be made more negative, thereby aiding the motion of the DNA towards thesecond electrode 132 and repelling undesired material which remains between thecontrol electrode 134 and the first electrode 130 (cathode).Fig. 7 shows a perspective view of electrodes advantageously used in abuncher structure. A first electrode 140 and a second electrode 142 are arrangedas cathode and anode, respectively (though the terminology may be reversed). Afirst control electrode 144 and a second control electrode 146 are disposed betweenthe cathode and anode. Bunching of charged macromolecules between the firstcontrol electrode 144 and the second control electrode 146 may be achieved byapplying a potential to the first control electrode 144 so as to accelerate the speedof transit of charged materials relatively closer to the first control electrode 144 thanto the second control electrode 146. The second control electrode 146 is biased soas to retard the speed of charged materials which are relatively closer to the secondcontrol electrode 146 than to the first control electrode 144. Since the rate ofW0 98/102771015202530CA 02264688 1999-03-05PCT/US97l 1352525diffusion of charged macromolecules in a solution phase environment is significantcompared to the transit time through the chamber (e.g. , the region defined betweenthe first electrode 140 and second electrode 142), the bunching process serves tolocalize the desired charged materials within a smaller region, counteracting theeffects of diffusion. In one mode of operation, once the desired amount of DNApasses the first control electrode 144, that control electrode may be placed at anegative potential which serves to further cause the negatively charged materialstowards the second electrode 142 (anode). While Fig. 7 shows a 4-electrodearrangement, a buncher may be formed from the structure of Fig. 6, by operationof the electrodes in a manner described above.Broadly stated, this aspect of the invention involves a method for selectiveisolation of desired charged biological materials from undesired charged biologicalmaterials in a electrophoretic system having a solution phase region, the methodincluding at least the step of applying a repulsive potential to a first electrode so asto accelerate motion of the desired charged materials which are relatively closer tothe first electrode than to the second electrode, and applying a repulsive potentialto a second electrode so as to decelerate motion of the desired charged materialswhich are relatively closer to the second electrode than to the first electrode. Suchoperation results in a spatial distribution of the desired charged materials betweenthe first and second electrodes is reduced.Figs. 8 and 9 show two implementations in plan view of an integrated samplepreparation system of this invention. For convenience, commonly identifiedstructures in Figs. 8 and 9 will be labeled with the same reference numerals. Fig.8 shows a system in which the tap 166 is disposed downstream from a protein trapregion 162. Fig. 9 shows a system in which the protein trap region 162 isdownstream of the tap region 166.A purification chamber 150 has disposed at the end thereof a first electrode152 and a second electrode 154. A sample addition port 156 may comprise an inputregion of the purification chamber 150. Preferably, the sample addition port 156comprises a liquid interconnect or cover seal (e.g., luer lock, face seal, slide seal).Optionally, the sample addition port 156 includes a filter, such as a 0.2 micronfilter. The filter optionally serves the function of debris removal and may alsoWO 98/102771015202530CA 02264688 1999-03-05PCT /U S97/ 1352526provide some shearing of DNA which will reduce the viscosity of the DNA.Optionally, membranes 158 may be utilized at one or both ends of the purificationchamber 150, the principal function of the membranes 158 being to isolate thesample material from the electrodes 152, 154. Optionally, a cell separation region160 (shown in Fig. 8) is formed in the purification chamber 150 downstream of thesample addition port 156. A protein trap region 162 is disposed within thepurification chamber 150. In one embodiment, as shown in Fig. 8, a protein trapregion 162 is disposed between the sample addition port 156 (and the cell separationregion 160 if optionally included) and the tap 166. This option is selectedprincipally if the desired materials for diagnosis have higher electrophoretic mobilitythan the protein materials to be trapped. An alternative mode of operation involvesthe step of causing the proteins or other undesired materials to have higher degreeof mobility than the desired materials, e.g., DNA. In this mode of operation, adevice such as shown in Fig. 9 may be used and the undesired materials are movedthrough the purification chamber 150 past the tap 166 prior to arrival at the tap 166of the desired material, e.g., DNA. Optionally, a protein trap region 162 may bethen included between the tap 166 and the second electrode 154.An electrode 164 is preferably disposed within the purification chamber 150at a point adjacent the tap 166 which intersects the purification chamber 150. Fig. 9shows a "C-shaped" electrode 165 generally disposed adjacent to and symmetricalwith respect to the tap 166. When the DNA band is passing through the regiondefined by the "C" and the bias of the C-electrode is then changed to negative (-),the C-shaped electrode serves to concentrate the DNA or other charged materialscontained within the space defined by the "C" and to provide a focusing of the‘charged materials within that region, while further repelling undesired moleculesoutside the C-region. Additionally, when a positive (+) potential is switched to aelectrode located in line with the open portion of the "C", the entire band of DNAwith the "C" is focused and propelled in the new direction toward the positiveelectrode location. The C-shaped electrode may be considered as various subparts,which may be formed as a continuous C-shaped structure or as discreet components.First and second electrode portions 165a are disposed generally perpendicular to theline connecting the first electrode 152 and the second electrode 154. TheseW0 98/102771015202530CA 02264688 1999-03-05PCT/US97/1352527perpendicular electrodes 165a generally serve to provide a bunching function. Theside electrode portion 165b generally provides a sideways or transverse forcecausing the charged materials contained within the C—shaped region towards the trap166.The tap 166 comprises a channel or chamber leading from the purificationchamber 150 to the denaturation region 168. Optionally, the denaturation may beperformed by heating, such as through a resistive heater 170, or by other modes ormethods known to those skilled in the art, including, but not limited to: other formsof energy input sufficient to break the DNA strands or other chemical methodsknown in the art. In the preferred embodiment, the width of the tap 166 is greaterthan 100 microns, and most preferably on the order of 1 mm. Generally, it isdesired to have a medium to low surface-to—volume ratio, the preferred embodimentreducing the amount of surface area for non—specific binding of sample or othermaterials to the walls of the device. The tap 166 leads to the complexity reductionchamber 172. One example of a complexity reduction chamber is described belowin connection with Figs. 10-12. Optionally, a valve 174 is disposed between thecomplexity reduction chamber 172 and the diagnostic assay 176. In the preferredembodiment, the diagnostic assay 176 comprises an active programmable matrixelectronic device of the type described in the various application identified in therelated application information section, above. Optionally, a disposal path 178 isconnected to a waste chamber.Figs. 10, 11 and 12 show one embodiment of a complexity reduction device.The device 180 comprises a printed circuit board 182 and a chamber 200 mountedthereon. The printed circuit board 182 preferably includes an edge connector 184to permit the interfacing of the complexity reduction system 180 to controlelectronics. The edge connector 184 includes a plurality of conductive fingers 186which contact corresponding conductive portions in a mating edge connector (notshown). The printed circuit board 182 in conventional manner may include asubstrate 188. The printed circuit board 182 includes conductors 190 which arepatterned into conductive strips and disposed on the substrate 188. Via holes 192are optionally formed in the printed circuit board 182, and preferably, theconduction portions 194 extend into the via holes 192. A conductive gel, such asWO 98/102771015202530CA 02264688 1999-03-05PCT/US97/1352528a polymer gel, most preferably agarose, acrylamide or other conductive polymer,is placed within the via holes 192. Optionally, these materials may be cured in situ,the curing optionally enhanced or promoted by application of a potential to theconductor 194. In the preferred embodiment, a chamber 200 is attached to theprinted circuit board 182. A seal 198 serves to form a hermetic seal between thechamber 200 and the substrate 188. Within the chamber 200, a sample volume 201is formed to contain a sample for complexity reduction. Optionally, an input portand an output port may be included within the chamber 200 to provide access to thechamber volume 201. Alternatively, the sample may be supplied into the samplevolume 201 through an opening at the surface. Within the chamber 200, one ormore probe areas 202 form the upper portion of the via holes 192. The gel 196preferably fills this space and terminates at the bottom of the sample volume 201.Optionally, disposal or dump areas 204 may be included within the chamber 200.Preferably, an index detent 206 is provided within the substrate 188. A matchingkey 208 is preferably formed on the underside of the chamber 200 to aid in indexingof the chamber 200 relative to the printed circuit board 182. As shown in Fig. 12,the chamber 200 may then be matingly engaged with the printed circuit board 182,with the keys 208 joining with the index detents 206. In operation, the polymer gel196 includes capture probes. These capture probes then interact with the targetmaterials in the sample and hybridize thereto. Conductive polymer may be used tofill the vias of the complexity reduction device in order to provide a matrix forDNA probe attachment, and DNA target hybridization and separation. The polymercan be mixed with protein bound DNA capture probe prior to filing the vias of thedevice in order to introduce polymer functionality or covalently bound capture probemay be pre-mixed with the conductive polymer. Alternatively, the probe may beelectrophoretically transported into the polymer and linked by enzymatic or covalentmeans in order to provide additional means for attachment to the polymeric support.In operation, the target DNA may be placed directly in the sample well ofthe complexity reduction device of fluidically or electrophoretically introduced intothe sample chamber. The sample can be introduced in one of several differentbuffers, including 50 mM sodium borate pH 8, or 0.5 X TBE. These buffersprovide for free field electrophoretic transport of DNA at relatively low ionicW0 98/102771015202530CA 02264688 1999-03-05PCT/US97ll352529strengths. Test results are shown in Fig. 17. Also, enhanced hybridization isshown for 0.5 X TBE and histidine in Fig. 16. During transport, the electrodes arebiased with a positive current and the target DNA is transported electrophoreticallyinto the polymer filled vias of the device allowing the complementary target DNAto hybridize to the specific capture probe. Next, during the electronic washprocedure, the DNA which is not a specific match to the capture DNA is removedfrom the vias using a mild negative current. A fluidic wash removes any irrelevantDNA from the sample well and fresh buffer is then introduced. The hybridizedtarget DNA then may be dehybridized electrophoretically using a strong negativecurrent.In order to maximize DNA purification, electrophoretic transport,hybridization, electronic wash and dehybridization can be performed using a varietyof electronic settings. For transport and accumulation, the settings include apositive DC current of between 5 to 2,500 p.A/mmz per polymer filled via for 10s to 180 s (preferably: 200 to 500 uA/mmz for 15 to 60 s), a pulsed current ofbetween 5 to 2,500 p.A/mmz at a 25 to a 75% duty cycle for 15 to 180 s(preferably: 200 to 1,000 [LA/IIIIIIZ, 50% duty, 15 Hz for 20 to 180 s), and areverse linear stair starting at between 100 to 500 ;.tA/mmz and ending at 0 to 150,uA/mmz in 15 to 90 s (preferably: starting at 250 ,uA/mmz and ending at 25pA/mmz in 90 s). An electronic washes is conducted with a negative DC biasbetween 200 to 300 ;lA/mmz or a pulsed current of between 200 and 500 p.A/mmzfor 15 to 180 s. Dehybridization is performed at a negative DC current of 400 to750 ptA/mmz for 60 to 420 s.For purposes of fluorometric detection, target DNA can be labeled with afluorophore, "reverse dot blot" hybridization, Figures 16 and 19. In addition, thetarget DNA can be detected after hybridization to the capture probe by theintroduction of a fluorophore labeled strand of DNA complementary to anonhybridized region of the target DNA "sandwich" hybridization. Alternatively,the fluorophore label can be incorporated into the target DNA which has beenamplified by PCR.Fig. 13 shows a perspective, cutaway view of a vertically disposed DNA ornucleic acid purification device of this invention. An upper reservoir tube 210W0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97/1352530communicates with a lower reservoir 212 via tube 214 permitting fluidcommunication from the upper reservoir 210 to the lower reservoir 212. Thereservoirs 210, 212 are adapted to receive a buffer solution 216. Optionally, theupper reservoir 210 and lower reservoir 212 may be formed so as to permitformation of a closed system, such as by causing the bottom 218 of the upperreservoir 210 to sealingly contact the top 220 of the lower reservoir 212.Alternatively, the system may be an open system. The upper reservoir 210 containsa first electrode 222 and the lower reservoir contains a second electrode 224. Thefirst electrode 222 and the second electrode 224 may be referred to as the cathodeand anode, respectively, though those terms may be interchanged given the polarityof operation. The tube 214 preferably has an inner diameter which is smaller thanthe inner diameter of the reservoirs 210, 212. The tube 214 contains a conductivepolymer region 226. The conductive polymer region 226 is a molecular sieve whichcomprises a differential mobility region. Materials which provide differentialmobility for charged biological macromolecules include materials such as agaroseand polyacrylamide.In the preferred embodiment, the differential mobility material is a cast 1.5 %agarose gel in 50 mM histidine, formed on a supporting membrane 228 in tube 214.Optionally, the conductive polymer region 226 is disposed adjacent a membrane228. Preferably, the membrane 228 is porous, e.g., 5 micron pore size, and servesin part to provide a support for the formation of the conductive polymer 226. Achamber 230 is disposed adjacent the conductive polymer region 226. Preferably,the chamber 230 has a volume which is less than that of the conductive polymerregion 226, e.g., preferably being approximately 50% or less in volume, and mostparticularly approximately 1/3 or less in volume than the conductive polymer region226. If the chamber 230 has a reduced volume relative to the conductive polymerregion 226, a reduced inner diameter region 232 may be formed in the tube 214.This reduced inner diameter region 232 advantageously forms a ledge 234 providingan annular region on which the membrane 228 may disposed. Optionally, a secondmembrane 236 may define a portion of the boundary of chamber 230. The secondmembrane 236 preferably constitutes a molecular weight cut-off membrane, such asan ultrafiltration membrane, which serves to retain the DNA within the chamberWO 98/102771015202530CA 02264688 1999-03-05PCT/US97l1352531230, but passes smaller materials, such as proteins subjected to proteinase K. Suchultrafiltration membranes include those formed from cellulose acetate or cellulose.In operation, a sample is previously lysed such as by motion of glass beadsacting on cells of the sample. Additionally, the sample is preferably subject toshearing, such as by movement through a relatively narrow aperture, such as anaperture of diameter of 250 microns. The sample is preferably subject to atreatment step which reduces the size of the proteins or other undesired materialsso as to increase their differential mobility relative to the desired material, e.g.,DNA. For example, addition of proteinase K may reduce the size of proteins, suchas to 20,000 daltons or less. This application of proteinase K may be done at roomtemperature, though performing it at an elevated temperature, e.g., 37°C to 50°C,increases the rate of reaction. In the preferred embodiment, the lysate cells aredigested with 250 pg/ml proteinase K, 0.5 X TBE 50 mM EDTA buffer (37—50°C)with a total volume of 50 ptl. Next, a densification agent, such as sucrose, e.g.,preferably 5%—20%, and most preferably substantially 8-10% serves to densify thesample. Optionally, the densified material may be combined with a dye, such asbromphenol blue. The. densified, pre-prepared sample is then injected or placedabove the conductive polymer region 226, such as by use of a syringe. Use of thedensified sample serves to locate and concentrate the sample immediately above theconductive polymer region 226. The system is then operated to cause theelectrophoretic motion of charged materials. The system is activated for a time topermit the DNA to migrate into the conductive polymer region 226, and to permitthe reduced size proteins to substantially traverse the conductive polymer region 226into the chamber 230 and lower reservoir 212. In the preferred embodiment, thesample is run into the gel for six to eight minutes, at a current of 2.25 mA, with a1,000 V limit. The cathode additionally serves to permit attraction and destructionof the proteinase K and other positively charged materials. Optionally, fresh buffer216 is provided in the lower reservoir 212, and the second membrane 236 may beadded at this time (though it may be initially included within the device). In thepreferred embodiment, the membrane 236 is a 25 kD molecular weight cutoffmembrane. Next, the DNA is eluted from the conductive polymer region 226 intothe chamber 230. In the preferred embodiment, the sample is eluted out of the gelW0 98/ 102771015202530CA 02264688 1999-03-05PCT/U S97/ 1352532into the elution chamber 230 for approximately two minutes, with the 1,000 V limit.The DNA is then extracted from the chamber 230, such as by piercing or providinga port and valve arrangement.Though the system of Fig. 13 is shown in a vertical arrangement, it may beperformed in a horizontal arrangement. The vertical arrangement permits a constantcontact area between the sample solution 236 and the conductive polymer 226.Further, the use of the densification agent permits the localization of the samplesolution 236 immediately adjacent the conductive polymer region 226, reducing thetime necessary for the sample solution 236 to reach the conductive polymer region226. In a horizontal arrangement, as shown in Figure 17, a polymer (or gel ormembrane) dam can be used to maintain the separation of the sample from theelectrode buffer to prevent mixing.Fig. 14 shows a plan view of an integrated device including a samplepreparation, complexity reduction, diagnostic region and disposal region. Fig. 14Ashows a cross-sectional view of Fig. 14 along the line A-A’. A support member240, such as a printed circuit board, preferably serves to support the variouscomponents described below. Optionally, an edge connector 242 may provideelectrical connection to control electronics, as discussed previously in connection inFigs. 10-12. A sample preparation region 244 preferably includes a first buffercontaining region 246 which also is in electrical communication with an electrode.A material 248 such as a gel or other conductive material is disposed between thebuffer region 246 and the input port 250. In the preferred embodiment, the inputport 250 includes a cover, which may be optionally removed for sample input orwhich can be sealed and pierced once the sample is placed within the sample port250. A DNA trap 252 is disposed between the input port 250 and the protein trap260.. Preferably, the DNA trap 252 narrows or constricts as materials areelectrophoresed through the DNA trap 252. In one embodiment, a sloped upperportion 254 and inwardly sloped side walls 256 serve to form a constriction at thegel boundary 258. Such a structure provides a concentrating effect. Preferably, theprotein trap 260 and the buffer space 264 are formed in a y-shaped manner. Theprotein trap 260 then contacts the buffer space 262 which includes an electrode.The sample preparation structure 244 operates generally as follows. First,W0 98/ 102771015202530CA 02264688 1999-03-05PCT/U S97/ 1352533a sample is placed in the input port 250. Electrophoretic action of the electrodescauses conduction of the charged macromolecules from a direction connecting thebuffer region 246 towards the buffer region 262. In the preferred embodiment, thesample is subject to a prepreparation step which lyses the cells and digests theprotein, resulting in proteinaceous material which has a relatively higher mobilitythan the DNA through the DNA trap 252. Thus, as electrophoresis continues, theprotein materials arrive at the protein trap 260 prior to the arrival of the DNA.After the proteins arrive at the protein trap 260, the electrode and buffer region 262is turned neutral and the electrode and buffer region 264 is biased from neutral topositive. The DNA moving through the DNA trap 250 are then attracted by apositive potential applied to the buffer space 264. In this manner, the proteins areshunted to the protein trap 260 whereas the DNA, most likely at a later time, areshunted to the chamber port 266. The DNA concentration volume is shown in Fig.14A as that portion beyond the gel boundary 258 within the buffer space 264.In one aspect of the invention, the buffer space 264 includes as an input achamber port 266 coupled to one end of the buffer space 264 and a second port orinlet port 268 which is fluidically coupled to the opposite end of the buffer space264, as shown in Figs. 14 and 14A being connected by a bound tube 270 to thebuffer space 264. In operation, the inlet port 268 and chamber port 266 mayreceive the same or different liquid or gas, such as a buffer, a reagent or an airslug. By operation of the materials provided to the inlet port 268 and chamber port266, a hydraulic or pneumatic ram may result. For example, if the buffer space 264contains DNA which has been eluted from the DNA trap 252 into the buffer space264, that material may be forced into the connector 272 by forcing fluid, e.g.,buffer, into the inlet port 268 causing the DNA to move into the connector 272.Advantageously, air slugs may be used to separate various fluidic materials.Additionally, fluid may be withdrawn from the inlet port 266, 268 to cause themovement of other materials in a direction generally opposite to the normalprocessing flow direction.The structure shown in Figs. 14 and 14A additionally includes the connector272 being in communication with the complexity reduction region 274. An exteriorcontainment vessel 276 defines the outer edge of the complexity reduction regionW0 98/ 102771015202530CA 02264688 1999-03-05PCT/US97/1352534274. One or more probe areas 278, having the structure and function describedpreviously with respect to Figs. 10-12, may be utilized. Similarly, one or moredump areas 280 may be disposed within the complexity reduction region 274 asdescribed in connection with Figs. 10-12. Preferably, a volume reduction region282 connects the complexity reduction region 274 to the diagnostic region 284. Thereduced volume region 282 serves a concentrating function. The structure andfunction of the diagnostic portion 284 may include any known diagnostic, butpreferably includes an electronically enhanced hybridization/dehybridization devicesuch as described in "Active Programmable Electronic Devices for MolecularBiological Analysis and Diagnostics", U.S. Serial No. 08/ 146,504, filedNovember 1, 1993, incorporated herein by reference. Preferably, a connector 288provides fluid communication to a waste region 286. The waste region 286preferably is a fully contained volume so as to avoid biological contamination.While the methods and devices herein have generally been described as aserial system, e. g., a sample preparation section, a complexity reduction section andan assay section, some or all of the stages may be performed in a parallel ormultiplexed format. In one embodiment, two or more parallel sets, each comprisinga sample preparation region, a complexity reduction region and an assay may beused. As an alternative embodiment, two or more sets of sample preparationsections may provide output to a smaller number, e.g., one, complexity reductionregion, preferably followed by an assay. Alternatively, two or more sets of asample preparation section and a complexity reduction region may provide theiroutputs to a smaller number, e.g., one, assay. Other variations and combinationsconsistent with the invention will be apparent to those skilled in the art.Further, while the description in the patent refers often to DNA, it will beunderstood that the techniques may be applied to RNA or other chargedmacromolecules, when consistent with the goals and functions of this invention.When the inventive methods and apparatus are used for RNA at the time of lysis,the user would preferably add RNAse inhibitors and RNAse free DNAse to removeDNA. The remainder of the purification process would follow as before. Theisolation of poly A RNA, which includes most of the mRNA, and removal ofribosomal RNA is preferably performed in the complexity reduction chamber.W0 98ll02771015202530CA 02264688 1999-03-05PCT/US97/1352535Oligo dT probes could optionally be used to bind the polyA RNA during electronichybridization and unhybridized RNA, mostly ribosomal RNA, would be removed.To release the poly A RNA, electronic dehybridization could preferably beemployed. Similarly, a specific sequence of mRNA could be isolated by usingprobes complementary to that sequence in the complexity reduction device.Electronic hybridization would be performed as for DNA targets and theunhybridized, irrelevant RNA would be removed. The desired mRNA specieswould be eluted from the probes by electronic dehybridization.The sources and control systems for supply of the potential, current and/orpower to any of the electrodes of these inventions may be selected among thoseknown to persons skilled in the art. The voltage and/or current may be suppliedwith either fixed current or fixed voltage, with the other variable permitted to float,optionally subject to limits or maximum values. The control system may be analogor digital, and may be formed of discrete or integrated components, optionallyincluding microprocessor control, or a combination of any of them. Softwarecontrol of the systems is advantageously utilized.Experimental Data -- Examnle 1 - Comparison of Fig. 13 Device to OiagenThe relative performance of a device as shown in Fig. 13 was compared tothe prior art Qiagen system. For the comparison, the preparation and operation ofthe Fig. 13 device was as follows.First, 50 mL of a stationary phase suspension culture of Staphylococusaureus cells was pelleted and resuspended in 1000 pl of 0.5 X TBE, 50 mM EDTA,and lysed by vortexing in the presence of glass beads. Then, RNA and protein werefragmented by digesting with 50 pg/ml RNAse and 250 ng/mL proteinase K for 40minutes at 50°C. Next, the sample density was increased by the addition of 5 [1,]of 40% sucrose to 20 pl sample, which is roughly equivalent to 109 cells, to achievea final concentration of 8%. Prior to loading the sample, the device was filled with50 mM histidine and then, 25 pl of sample was then loaded in the sample solutionzone 236. In other experiments, volumes as high as 100 pl have been used withsimilar results. Next, the electrodes were connected to a power supply and currentwas sourced at 2.5 mA. In other experiments, currents as low as 1 mA have beenW0 98/102771015202530CA 02264688 1999-03-05PCT/US97/1352536used with similar results except that transport was slowed. After 3 minutes, thepower was turned off, the barrel was removed, and a cellulose acetate membranewith a 25 kD molecular weight cutoff was inserted as second membrane 236. Thesolution was also removed and fresh 50 mM histidine was added to the device.After reassembly, the power was turned on and the sample was eluted into chamber230 formed by the cellulose acetate membrane 228 which supports the gel and the25 kD cutoff cellulose acetate membrane 236. To remove the eluted sample, thepower is turned off, the barrel was removed, a pipette/syringe is used to puncturethe 25 kD cutoff membrane and the sample solution was withdrawn. The total timefor all of the electrophoretic steps was less than 5 minutes. The sample wasanalyzed by agarose gel electrophoresis and spectrophotometry. Gel electrophoresisshowed that the DNA was approximately 20 kbp in length and that the yield wasapproximately 20%.For comparison, a crude sample prepared by the same method of lysis anddigestion was processed in the Qiagen device. The Qiagen device was operated inaccordance with its instructions. A comparison to results obtained with the Qiagendevice is as follows.Optical density readings were performed and the ratio of the optical densityat 260 nanometers to 280 nanometers was determined. The device of Fig. 13provided a ratio of from 1.6 to 1.8 on various operations, in contrast, the Qiagendevice providing a result of less than 1.3 with the same material (the Qiagenliterature does state that a ratio of 1.7 to 1.9 can be obtained). Thus, in the actualtesting on this sample, the device of Fig. 13 provided purified DNA. Secondly, thetransport of the prepared sample on a microelectronic chip constructed as disclosedin "Active Programmable Electronic Devices for Molecular Biological Analysis andDiagnostics", Serial No. 08/146,504, filed November 1, 1993, was better than thetransport on the same device of the Qiagen prepared material. It is believed that theQiagen material has a relatively high residual salt content, and that therefore thetransport on the chip was poorer than in the case of transport of material preparedby the method and structure of Fig. 13. Fig. 15 shows test results for transport ofsamples prepared by the device of this invention as compared to sample preparedby the Qiagen device. Third, sample preparation was significantly faster with theW0 98/ 102771015202530CA 02264688 1999-03-05PCTIU S97/ 1352537Fig. 13 device, approximately 5 minutes (6.5 minutes in one test) in comparison toover two hours for the Qiagen device. Finally, the yields for both methods weresimilar, approximately 20%.Example 2 -- Performance of Fig. 1 DeviceA device was prepared with the same structural components as Fig. 1,though its actual shape was as disclosed here. The purification device wasconstructed from polymethacrylate (PMA) generally as shown in Figure 1. Toallow the insertion of different materials 22, 16 and 24, the device is assembledfrom separate sections of PMA whose ends meet at the lines indicated by the spacerregion 16. The sections are held together by screws which run longitudinally.Electrodes made of Pt wires were attached to electrodes 26, 28. The wiresprotruded into electrode chambers 18, 20. Electrode chambers 18, 20 were eachfilled with 300 pl TAE, 250 mM HEPES. An Elutrap ultrafiltration celluloseacetate membrane from Schleicher and Schuell was inserted between PMA sectionsat position membrane 24 and a cellulose acetate membrane with a 25 kD cutoff fromSialomed was inserted at membrane position 22 to separate the electrode chambers18, 20 from the sample chambers 12, 14. The sample chambers 12, 14 were filled-with 75 pl each of TAE, 250 mM HEPES. Although different membranes havebeen inserted at spacer region 16 to achieve separation of DNA from protein, in thisexperiment, spacer region 16 contained an Immobilon P membrane (PVDF with0.45 pm pores) from Millipore. The membrane was wetted with methanol andsoaked in 1 x TAE, 250 mM, 0.1% Triton x 100 prior to loading into region 16.A crude sample, a 162 pd of a mixture of a protein, 48.5 pg of Bodipy Fluoresceinlabeled BSA, and 0.79 pg of a Bodipy Texas Red labeled 19mer in 1 x TAE, 250mM HEPES was loaded into chamber 12. After the addition of the sample, firstelectrode 26 was biased negative and second electrode 28 was biased positive for2.75 minutes at 10 mA. As a result, the sample was electrophoretically transportedto spacer region 16. The labeled DNA passed through the membrane and wascollected on the other side of the membrane in right central chamber 14. Theamounts of DNA and BSA were determined by fluorirnetry. The results show thatthe yield of DNA was 40% an that 78% of the BSA was removed.W0 98/ 102771015CA 02264688 1999-03-05PCT/U S97] 1352538Example 3 -- Performance of Complexitv Reduction DeviceIn Group A Streptococcal (GAS) experiments, Figures 18 and 19, theconductive polymer is pre-mixed with a 20 ,uM concentration of a 25 merstreptavidin-biotin bound capture probe. In GAS experiments, Figures 18 and 19,40 ML aliquots of target DNA are placed into the sample well in 50 mM histidine.Electrophoretic transport was conducted using a linear stair starting at 250 ;rA/mm’and ending at 25 MA/mmz an the electronic wash was conducted using a250 MA/mmz pulse for 45 s and 120 s, respectively. Dehybridization, Figure 18,was conducted at 400 ,uA/mmz for 150 s in 0.5 X TBE. Results from Figure 18show a purification ratio of approximately 33-fold (10023) and a recovery of 82%of the pure hybridized target. Figure 19 shows purification of a specific target inincreasing ratios of irrelevant DNA with a purification ratio of 3.4 fold in thepresence of a 200,000 fold mass excess of irrelevant material.Although the foregoing invention has been described in some detail by wayof illustration and example for purposes of clarity and understanding, it will bereadily apparent to those of ordinary skill in the art in light of the teachings of thisinvention that certain changes and modifications may be made thereto withoutdeparting from the spirit or scope of the appended claims.

Claims (97)

We Claim:

1. An apparatus for active biological sample preparation adapted to separate desired materials from undesired materials comprising:
a first sample chamber adapted to receive a buffer solution, a second sample chamber adapted to receive a buffer solution, a spacer region disposed between the first sample chamber and the second sample chamber, the spacer region providing a trap having a differential effect on the desired materials versus undesired materials, a first electrode adapted for electrical contact with the buffer solution when located within the first sample chamber, and a second electrode adapted for electrical contact with the buffer solution when in the second sample chamber.

2. The apparatus for active biological sample preparation of Claim 1 wherein the trap is hydrophobic.

3. The apparatus of Claim 1 for active biological sample preparation wherein the trap is a protein trap.

4. The apparatus of Claim 3 wherein the protein trap is PVDF.

5. The apparatus of Claim 3 wherein the protein trap is nitrocellulose.

6. The apparatus of Claim 1 for active biological sample preparation wherein the trap is a DNA trap.

7. The apparatus of Claim 1 for active biological sample preparation wherein the trap is removable from the apparatus.

8. The apparatus of Claim 7 further including a frame adapted to include the trap.

9. The apparatus of Claim 1 for active biological sample preparation wherein the trap is adapted to mate with the spacer region.

10. The apparatus of Claim 1 wherein the trap includes a third electrode proximal to the trap.

11. The apparatus of Claim 10 for active biological sample preparation wherein the forward electrode and trap comprise a metal-coated filter material.

12. The apparatus of Claim 1 further including an incinerator electrode disposed within one or more sample chambers.

13. The apparatus of Claim 1 for active biological sample preparation further including a control system operatively coupled to the electrodes.

14. The apparatus of Claim 1 for active biological sample preparation wherein the spacer region has a volume less than 50% of the volume of the first sample chamber.

15. The apparatus of Claim 14 for active biological sample preparation wherein the spacer region has a volume of 30% or less of the volume of the firstsample chamber.

16. The apparatus of Claim 15 for active biological sample preparation wherein the volume of the spacer region is 25 % or less of the first sample chamber.

17. The apparatus of Claim 1 for active biological sample preparation wherein the thickness of the sample chamber is 20% or less of the distance between the first electrode and the second electrode.

18. The apparatus of Claim 17 for active biological sample preparation wherein the thickness of the sample chamber is 10% or less of the distance between the first electrode and the second electrode.

19. The apparatus of Claim 18 for active biological sample preparation wherein the thickness of the sample chamber is 5% or less of the distance between the first electrode and the second electrode.

20. The apparatus of Claim 1 for active biological sample preparation wherein the width of the sample region is 5 mm or less.

21. The apparatus of Claim 20 for active biological sample preparation wherein the thickness of the sample chamber is 4 mm or less.

22. The apparatus of Claim 21 for active biological sample preparation wherein the thickness of the sample chamber is from approximately 1 to approximately 2 mm.

23. The apparatus of Claim 1 for active biological sample preparation further including a protective layer between the first sample chamber and the first electrode, thereby forming a first electrode chamber.

24. The apparatus of Claim 23 for active biological sample preparation wherein the protective layer is a membrane.

25. The apparatus of Claim 24 for active biological sample preparation wherein the membrane is an ultrafiltration membrane.

26. The apparatus of Claim 24 for active biological sample preparation wherein the membrane is a cellulose acetate membrane.

27. The apparatus of Claim 26 wherein the volume of the first electrode chamber is at least 10 times the volume of the sample chamber.

28. The apparatus of Claim 1 for active biological sample preparation wherein the first sample chamber is disposed vertically above the spacer region.

29. A method for active biological sample preparation of a sample comprising a collection of materials including desired materials and undesired materials having differential charge-to-mass ratios, the separation being achieved in an electrophoretic system including solution phase regions and at least one trapregion having differential effect on desired materials as compared to undesired materials, comprising the steps of:
providing the sample materials to a first solution phase region of the device, electrophoresing the sample within the system to affect net differential migration between the desired material and the undesired material whereby one ofthe desired or undesired materials is located within the trap and the other material is in a solution phase region, removing the desired material from the system, whereby relatively purified desired materials are prepared.

30. The method of Claim 29 wherein the trap selectively traps specific cells.

31. The method of Claim 30 wherein desired materials are eluted from the cell specific trap.

32. The method of Claim 29 wherein the trap serves to trap proteins and pass at least one of DNA and RNA.

33. The method of Claim 29 wherein the trap serves to trap at least one of DNA and RNA.

34. The method of Claim 33 wherein after at least a portion of the DNA
or RNA has contacted the trap, a potential is applied to the materials in the solution phase region so as to repel them from the trap.

35. The method of Claim 29 for active biological sample preparation wherein the sample is first electrophoretically transported through a fluidic region prior to contacting the trap.

36. The method of Claim 35 for active biological sample preparation wherein the desired material is located within the trap.

37. The method of Claim 36 for active biological sample preparation wherein the desired material and trap are removed from the system.

38. The method of Claim 37 for active biological sample preparation wherein the trap is a dipstick.

39. The method of Claim 36 for active biological sample preparation wherein the desired material is removed from the trap.

40. The method of Claim 39 for active biological sample preparation wherein the desired material is removed from the trap into a fluidic region.

41. The method of Claim 39 for active biological sample preparation wherein the fluidic region is said first solution phase region.

42. The method of Claim 39 for active biological sample preparation wherein the fluidic region is a second fluidic region.

43. The method of Claim 35 for active biological sample preparation wherein the undesired material is located within the trap.

44. The method of Claim 43 for active biological sample preparation wherein the desired material is removed from the system by fluidics.

45. The method of Claim 43 for active biological sample preparation wherein the desired materials are electrophoresed through the trap prior to the undesired materials being trapped in the trap.

46. The method of Claim 29 for active biological sample preparation wherein the sample is initially placed in a first fluidic region immediately adjacent the trap.

47. The method of Claim 29 for active biological sample preparation wherein the sample is subject to a proteinase step prior to providing the samplematerials to the first solution phase region of the device.

48. The method of Claim 46 for active biological sample preparation wherein the desired material is retained in the trap and the undesired material is electrophoresed through the trap into a second fluidic region.

49. The method of Claim 48 for active biological sample preparation wherein the desired material is eluted from the trap after the undesired materials are electrophoresed through the trap.

50. The method of Claim 49 for active biological sample preparation wherein the desired materials are eluted into the second fluidic region.

51. The method of Claim 29 for active biological sample preparation wherein the sample is subject to a densification step.

52. The method of Claims 29 and 51 for active biological sample preparation further including the step of subjecting the sample to a proteinase.

53. A method for selective isolation of desired charged biological materials from undesired charged biological materials in a electrophoretic system having a solution phase region, the method comprising the steps of:
applying a repulsive potential to a first electrode so as to accelerate motion of the desired charged materials which are relatively closer to the first electrode than to the second electrode, and applying a repulsive potential to a second electrode so as to decelerate motion of the desired charged materials which are relatively closer to the second electrode than to the first electrode, whereby spatial distribution of the desired charged materials between the first and second electrodes is reduced.

54. The method of Claim 53 wherein the desired materials are at least one of DNA, RNA and proteins.

55. The method of Claim 53 wherein after the spatial distribution of the desired materials has been reduced, the repulsive potential on the first electrode is increased.

56. An apparatus for the collection of macromolecular nucleic acids within an electrophoretic system, and for the transport of the nucleic acids comprising:
a first collection material disposed within a collection region, a support member in contact with said collection region, the collection material support member providing an integrated structure for the collection and transportation of nucleic acid.

57. The apparatus of Claim 56 wherein the macromolecular nucleic acid is DNA.

58. The apparatus of Claim 56 wherein the macromolecular nucleic acid is RNA.

59. The apparatus of Claim 56 wherein the carrier apparatus comprises two halves of a frame adapted for mating engagement.

60. The apparatus of Claim 56 wherein the sample material is a layered material.

61. A system for the active biological sample preparation of materials comprising:
an input, a purification chamber, the purification chamber including at least a first electrophoretic solution region, and first and second electrophoresis electrodes, a protein trap region within the first electrophoretic solution phase region, a tap connected to the purification region, a denaturation region adapted to receive the output of the tap from the purification region, a complexity reduction region adapted to receive the output of the denaturation region, and a diagnostic region adapted to receive the output of the complexity reduction region.

62. The system of Claim 61 further including a cell separation region disposed between the input and the purification region.

63. The system of Claim 62 wherein the cell separation is performed by a method selected from the group consisting of:
electroporation, boiling, addition of a reagent for cell membrane disruption and agitation of the sample with rigid structures.

64. The apparatus of Claim 61 further including a third electrode, the third electrode located adjacent the tap and adapted to move materials from the purification chamber to the tap.

65. The apparatus of Claim 64 wherein the electrode is a C shaped electrode.

66. The apparatus of Claim 64 wherein the electrode includes:
first and second portions generally disposed in a direction which is oblique to the axis defined by the first and second electrodes, and a third portion positioned for driving material towards the trap.

67. A system for electrophoretic movement of charged biological macromolecules, comprising:
a first bunching electrode region for causing movement of said charged biological macromolecules in a direction having a component in the same direction to the first direction, a second bunching electrode region for causing movement of said charged biological macromolecules in a direction having a component opposite said first direction having a component opposite said first direction, a third electrode region for causing movement of said charged biological macromolecules in a direction having a component which is generally perpendicular to said first direction, and an electronic control system for activating said electrode regions to effect concentration and net movement of said charged biological macromolecules.

68. The system of Claim 67 for electrophoretic movement of charged biological macromolecules wherein the first bunching electrode region, the second bunching electrode region and the third electrode region form a C shaped electrode.

69. A device for purification of DNA from a sample including proteinaceous material having a charge-to-mass ratio which is greater than that of the DNA, the device comprising:
an upstream reservoir adapted for receipt of a buffer solution, the upstream reservoir including a sample solution reception region for receipt of the DNA and proteinaceous material, a conductive polymer region disposed adjacent to and vertically beneath the sample solution reception region of the upstream reservoir, a collection chamber adapted for receipt of a buffer solution and for receipt of the DNA, and electrodes adapted for electrical contact with the buffer solutions to provide electrophoretic movement of the DNA and proteinaceous material through the conductive polymer and collection chamber.

70. The device of Claim 69 for purification of DNA further including a membrane defining at least a portion of the collection chamber.

71. The device of Claim 70 for purification of DNA wherein the membrane is an ultrafiltration membrane.

72. The device Claim 70 for purification of DNA wherein the membrane comprises a molecular weight cut-off membrane.

73. The device of Claim 72 for purification of DNA wherein the molecular weight cut-off membrane retains the DNA within the collection chamber.

74. The device of Claim 69 for purification of DNA wherein the collection chamber has a volume which is smaller than the volume of the conductive polymer.

75 . The device of Claim 74 wherein the collection chamber volume is less than or equal to half the volume of the conductive polymer.

76. The device of Claim 75 for purification of DNA wherein the volume of the collection chamber is equal to or less than 1/3 the volume of the conductive polymer.

77. The device of Claim 69 for purification of DNA further including a membrane between the conductive polymer and the collection chamber.

78. The device of Claim 77 for purification of DNA wherein the membrane has a pore size which permits transport of both the DNA and proteinaceous materials through the membrane.

79. The device of Claim 69 for purification of DNA further including a lower reservoir adapted to receive a buffer solution and disposed beneath the collection chamber.

80. The device of Claim 69 wherein the conductive polymer is a molecular sieve.

81. The device of Claim 80 wherein the molecular sieve is acrylamide.

82. The device of Claim 69 further including a buffer.

83. The device of Claim 82 wherein the buffer is histidine.

84. The device of Claim 69 wherein the conductive polymer has a thickness in the direction of migration of substantially 10 to 20 mm.

85. A method for purification of DNA from a sample mixture including DNA and other charged materials, including certain materials having a higher charge-to-mass ratio than the DNA, in a device having an upstream buffer chamber, a conductive polymer region disposed below the upstream chamber, and a collection chamber disposed adjacent the conductive polymer region, and electrodes connected for electrophoretic motion of charged materials through the system, the method comprising the steps of:
placing the sample mixture in the upstream buffer chamber above the conductive polymer, activating the electrodes so as to electrophorese the sample mixture into the conductive polymer region for a time sufficient to cause the undesired material to substantially move through the conductive polymer region, and eluting the DNA into the collection chamber.

86. The method of Claim 85 for purification of DNA from a sample mixture further including the step of densification of the sample mixture.

87. The method of Claim 85 for purification of DNA from a sample mixture wherein the densification step includes the addition of sucrose.

88. The method of Claim 85 for purification of DNA from a sample mixture including the further step of lysing of cells prior to the placement of the sample mixture in the upstream buffer chamber.

89. The method of Claim 85 for purification of DNA from a sample mixture further including the step of shearing the sample prior to placement of the sample mixture in the upstream buffer chamber.

90. The method of Claim 85 for purification of DNA from a sample mixture wherein the sample mixture is subject to a proteinase.

91. The method of Claim 90 for purification of DNA from a sample mixture wherein the sample mixture is subject to an RNAse step prior to said proteinase step.

92. The method of Claim 91 wherein the proteinase is proteinase K.

93. The method of Claim 85 for purification of DNA from a sample mixture further including the step of changing the buffer solution prior to the step of eluting the DNA into the collection chamber.

94. The method of Claim 85 for purification of DNA from a sample mixture further including the step of adding a membrane to the collection chamber prior to eluting the DNA into the collection chamber.

95. The method of Claim 85 for purification of DNA from a sample mixture including the step of extracting the DNA from the collection chamber.

96. The method of Claim 95 for purification of DNA from a sample mixture wherein the DNA is extracted from the collection chamber by piercing of the collection chamber.

97. The method of Claim 95 for purification of DNA from a sample mixture wherein the DNA is extracted from the collection chamber through a valve.