WO2000060118A2 - Analyse electrophoretique de molecules cibles au moyen de molecules adaptateurs - Google Patents

Analyse electrophoretique de molecules cibles au moyen de molecules adaptateurs Download PDF

Info

Publication number
WO2000060118A2
WO2000060118A2 PCT/US2000/008529 US0008529W WO0060118A2 WO 2000060118 A2 WO2000060118 A2 WO 2000060118A2 US 0008529 W US0008529 W US 0008529W WO 0060118 A2 WO0060118 A2 WO 0060118A2
Authority
WO
WIPO (PCT)
Prior art keywords
adapter
target
molecule
capture probe
nucleotide sequence
Prior art date
Application number
PCT/US2000/008529
Other languages
English (en)
Other versions
WO2000060118A3 (fr
Inventor
Lawrence Weir
T. Christian Boles
Ezra S. Abrams
Original Assignee
Mosaic Technologies, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mosaic Technologies, Inc. filed Critical Mosaic Technologies, Inc.
Priority to JP2000609607A priority Critical patent/JP2003508015A/ja
Priority to CA002371727A priority patent/CA2371727A1/fr
Priority to AU40539/00A priority patent/AU4053900A/en
Priority to EP00919931A priority patent/EP1235930A2/fr
Publication of WO2000060118A2 publication Critical patent/WO2000060118A2/fr
Priority to US09/968,084 priority patent/US20020197614A1/en
Publication of WO2000060118A3 publication Critical patent/WO2000060118A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • nucleic acid base pairing is an extremely high affinity and specific interaction. For this reason, nucleic acid hybridization assays have been devised for a variety of diagnostic purposes.
  • hybridization assays can be extraordinarily sensitive, detecting femtogram amounts of a specific molecule.
  • several technical limitations have prevented widespread use of hybridization analysis in commercial diagnostic techniques.
  • hybridization probes require stringent procedures for separating unhybridized (or improperly hybridized) and hybridized probe. This separation can be facilitated by the use of solid phase hybridization formats, in which either the sample nucleic acid or the probe that is complementary to the desired target is immobilized on a solid support. Hybridized and unhybridized species can be separated by washing the support.
  • a second limitation of hybridization assays is that efficient hybridization of samples containing low concentrations of target nucleic acids frequently requires lengthy incubations (up to several hours) under carefully controlled conditions.
  • use of solid phase assays exacerbates this problem, since immobilized nucleic acids virtually always hybridize with slower kinetics than nonimmobilized ones.
  • a number of workers have sought methods to perform solid phase hybridizations with better kinetics and efficiency.
  • high molecular weight polymers such as dextran sulfate or polyethylene glycol improves solid phase assay performance, albeit modestly.
  • the present invention relates to the discovery that nucleic acids, modified nucleic acids and nucleic acid analogs can be immobilized (e.g., covalently attached) to an electrophoretic medium and that electrophoresis can be used to separate, purify or analyze target molecules that specifically bind to (e.g., associate with), or are specifically bound by, the immobilized nucleic acids, modified nucleic acids or nucleic acid analogs.
  • the immobilized nucleic acids, modified nucleic acids or nucleic acid analogs are referred to herein as universal capture probes (also referred to herein as "capture probes").
  • capture probes are specific for only one class of adapter molecule. Therefore, the gel which possesses a specific class of capture probe, or probes, can only be used for that specific molecule from which the capture probe has been designed.
  • the present invention specifically relates to a solid phase hybridization system for detecting the presence or absence of a target molecule based on a "universal capture” electrophoresis gel.
  • the universal capture gel (also referred to herein as “universal gel”) comprises an electrophoretic medium (also referred to herein as an electrophoretic matrix) within which a capture probe is immobilized. More specifically, the universal capture gel described herein is an electrophoretic gel comprising universal capture probes copolymerized within the gel. The capture probes can be immobilized throughout the gel, or in discrete layers of the gel (e.g., forming capture layers).
  • the universal capture gel can be pre-cast in a variety of formats such as a slab gel or capillary gel and prepared and stored prior to use.
  • the universal capture gel system of the present invention has three components.
  • the first component is a universal capture probe.
  • the capture probe comprises a nucleotide sequence region which is complementary to an adapter molecule.
  • the capture probe is a nucleic acid molecule, either DNA or RNA, usually from about 18 to about 24 nucleotides in length, which is immobilized within the gel by, for example, covalent attachment.
  • the second component is an adapter molecule.
  • the adapter molecule is typically a nucleic acid molecule usually from about 15 to about 40 nucleotides in length.
  • the adapter molecule usually comprises two nucleotide sequences joined together to form two nucleotide sequence regions as described below.
  • the third component is an electrophoretic medium suitable for performing electrophoresis, for example, an acrylamide gel.
  • a key component of the present invention is the adapter molecule.
  • An adapter molecule encompassed by the present invention has at least two nucleotide sequence regions. One region is complementary to a capture probe nucleotide sequence, and, therefore, specifically hybridizes with the capture probe. This region is referred to herein as the "capture probe-specific" nucleotide sequence region. The other region is complementary to a target molecule nucleotide sequence (e.g., bacterial rRNA), and, therefore, specifically hybridizes with a nucleotide sequence region contained within a target molecule. This region is referred to herein as the "target- specific" nucleotide sequence region.
  • the universal capture gel of the present invention is constructed to have at least one class of capture probes immobilized within the electrophoretic medium which can hybridize with any adapter molecule having a nucleotide sequence region complementary to the capture probe.
  • multiple discrete regions within the electrophoretic medium contains separate classes of capture probes, and adapter molecules that are specific for a particular class are used such that multiple target molecules can be captured and isolated within these discrete regions of the electrophoretic medium.
  • Adapter molecules can be designed to analyze any target molecules comprising a nucleotide sequence contained within a test sample, for example, bacterial, viral, fungal, plant, animal (including, but not limited to, vertebrates like mammals such as human) target molecules and combinations thereof.
  • universal gel hybridization complexes comprising an adapter molecule hybridized to a target molecule.
  • the adapter molecule comprises a capture probe-specific nucleotide sequence region which is complementary to a capture probe, and a target-specific nucleotide sequence region which is complementary to a target molecule.
  • This adapter/target hybridization complex described herein is typically formed in a solution phase prior to introduction into the universal capture gel.
  • the present invention further encompasses methods of using the universal capture gel described herein. In one embodiment of the invention, a method of detecting the presence, or absence, of a target molecule in a test sample using a universal capture gel is described.
  • a universal capture gel comprising one class of capture probes immobilized throughout the electrophoresis medium.
  • a test sample which is being analyzed for the presence, or absence, of a target molecule is contacted with (e.g., mixed with) an adapter molecule that has at least one nucleotide sequence region that is complementary to the target molecule to be detected.
  • An adapter/target complex (also referred to herein as a universal gel hybridization complex) is formed under conditions suitable for the adapter molecule's nucleotide sequence region specific for the target molecule to hybridize with the target molecule and form a stable complex. This adapter/target complex is subjected to electrophoresis through the universal capture gel.
  • the complex migrates through the gel until the complex contacts an immobilized capture probe specific for the adapter molecule. Once the adapter/target complex and complementary capture probe hybridize, a tripartite hybridization complex is formed comprising the capture probe and the adapter/target complex, which is immobilized within the universal capture gel. The detection of this immobilized tripartite complex is indicative of the presence of the target molecule in the original test sample.
  • a universal capture gel with one class of immobilized capture probes is used for detecting one, or more, target molecules in a test sample.
  • Multiple classes of adapter molecules are used in which they all share one nucleotide sequence region that is complementary to the capture probe immobilized in the electrophoretic medium, but differ with respect to their target nucleotide sequence complementarity region.
  • a test sample which is being analyzed for the presence, or absence, of a target molecule is contacted with (e.g., mixed with) an adapter molecule that has at least one nucleotide sequence region that is complementary to the target molecule to be detected.
  • a universal gel hybridization complex is formed under conditions suitable for the adapter molecule's nucleotide sequence region specific for the target molecule to hybridize with the target molecule and form a stable complex.
  • This adapter/target complex is subjected to electrophoresis through the universal capture gel. The complex migrates through the gel until the complex contacts an immobilized capture probe specific for the adapter molecule.
  • a tripartite hybridization complex comprising the capture probe and the adapter/target complex, which is immobilized within the universal capture gel.
  • the detection of this immobilized tripartite complex is indicative of the presence of the target molecule in the original test sample.
  • multiple classes of capture probes are immobilized within the electrophoretic medium in discrete regions, with each discrete region possessing only one class of capture probe comprising a nucleotide sequence complementary to nucleotide sequence of a specific class of adapter molecule.
  • Each class of adapter molecule in turn comprises a nucleotide sequence complementary to a specific target molecule, or class of target molecule (e.g., rRNA common to bacteria).
  • the adapter molecules and target molecules are contacted with one another under conditions suitable for hybridization.
  • the different adapter/target complexes are then subjected to electrophoresis through the universal capture gel. When the individual adapter/target complex comes in contact with an appropriate immobilized capture probe, then the complex becomes hybridizes to the capture probe forming a tripartite hybridization complex which is immobilized within the gel.
  • a method for purifying at least one target molecule from a test sample is disclosed.
  • a test sample from which at least one target molecule is to be purified is contacted with (e.g., mixed with) an adapter molecule that has at least one nucleotide sequence region that is complementary to the target molecule to be detected.
  • a universal gel hybridization complex is formed under conditions suitable for the adapter molecule's nucleotide sequence region specific for the target molecule to hybridize with the target molecule and form a stable complex.
  • This adapter/target complex is subjected to electrophoresis through the universal capture gel. The complex migrates through the gel until the complex contacts an immobilized capture probe specific for the adapter molecule.
  • a tripartite hybridization complex comprising the capture probe and the adapter/target complex, which is immobilized within the universal capture gel.
  • a modified adapter molecule can be used to displace either the target molecule alone, or the target/adapter complex from the immobilized capture probe.
  • FIG. 1 is a schematic representation of a capture complex being comprised of an immobilized capture probe, adapter molecule and target molecule.
  • FIG. 2 is a photograph of a gel showing the results of an experiment using the universal capture gel to detect two different RNA target molecules.
  • FIG. 3 is an schematic representation of myosin mRNA capture using an adapter molecule.
  • FIG. 4 is the nucleotide sequence of RNA 1.
  • FIG. 5 is the nucleotide sequence of RNA 2.
  • the present invention relates to a universal capture gel system.
  • This universal capture gel system is comprised of three components.
  • the first component of the universal capture gel system is a universal capture probe (or simply, capture probe).
  • the capture probe is a short sequence of a nucleic acid, modified nucleic acid or nucleic acid analog, usually about 5 to about 100 nucleotides in length, which is immobilized within an electrophoretic medium and together, they form a universal gel.
  • the capture probes can be immobilized within a discrete layer, or layers, of the gel forming one or more capture layers. Alternatively, the capture probes can be immobilized throughout the electrophoretic medium.
  • the capture probe hybridizes with an adapter molecule that comprises at least one nucleotide sequence region that is complementary to the capture probe nucleotide sequence. Therefore, a heterogeneous set of adapter molecules specific for different target molecules can all bind to just one class of capture probe, if the heterogenous set of adapter molecules share at least one complementary nucleotide sequence region to a single class of capture probes.
  • the capture probe typically forms a stable complex with an adapter molecule.
  • a "stable complex” as defined herein is a complex with a bound lifetime that is long in comparison to the duration of the electrophoresis.
  • the appropriate length and sequence of the capture probe and adapter necessary to achieve adequate stability is a function of the chemical nature of the adapter and capture probe, the length and sequence of the potential duplex formed by them, and the conditions of electrophoresis. For example, for DNA adapter and capture probes, using electrophoresis in 1 x TBE (89 mM Tris-borate, pH 8.3, 2 mM EDTA) at 25°C and 10 V/cm, the minimum useful duplex length is around 15 base pairs.
  • the second component of the universal capture gel system is the adapter molecule.
  • the adapter molecule is typically a polynucleotide from about 10 to about 100 nucleotides in length and comprises two nucleotide sequence regions.
  • the two nucleotide sequence regions can be immediately adjacent to each other or can have a short region of intervening nucleotides between the two regions.
  • the two regions can comprise subregions within one larger nucleic acid.
  • the two regions within the adapter can be contiguous or non-contiguous within a larger nucleic acid molecule.
  • the two regions can have different chemical compositions or modifications.
  • one region can be DNA and the other region can be PNA.
  • One region is a capture probe-specific nucleotide sequence region complementary to a nucleotide sequence region of the capture probe.
  • Another region is a target-specific nucleotide sequence region complementary to a nucleotide sequence region of the target molecule.
  • the adapter molecule of the present invention specifically hybridizes with both a capture probe and a target molecule.
  • the third component of the universal capture gel comprises one, or more, capture probes, or classes of capture probes, immobilized in a matrix suitable for electrophoresis.
  • the test sample can be from any source containing nucleic acids and can contain any molecule comprising a nucleotide sequence that can form a hybridization complex with a capture probe.
  • samples from biological sources containing cells obtained using known techniques, from body tissue (e.g., skin, hair, internal organs), body fluids (e.g., blood (e.g., platelets, erythrocytes), plasma, urine, semen, sweat) or cell and/or tissue culture systems.
  • Other sources of samples suitable for analysis by the methods of the present invention are microbiological samples, such as bacteria, viruses and yeasts, plasmids, isolated nucleic acids and agricultural or food samples, such as recombinant plants and plant cells.
  • the test sample is treated in such a manner, known to those of ordinary skill in the art, so as to render the target molecules contained in the test sample available for hybridization.
  • the target molecule is a nucleic acid present in a bacterial cell
  • a bacterial cell lysate is prepared, and a crude bacterial cell lysate (e.g., containing the target nucleic acid as well as other cellular components such as proteins and lipids) can be analyzed.
  • the target nucleic acids can be isolated (rendering the target nucleic acids substantially free from other cellular components) prior to analysis. Isolation can be accomplished using known laboratory techniques.
  • the target nucleic acid can also be amplified (e.g., by polymerase chain reaction or ligase chain reaction techniques) prior to analysis.
  • a suitable sample preparation involves steps taken to lyse the cell or cells, thereby releasing their nucleic acids.
  • the RNA is liberated from the constraints of the cell wall and cell membrane.
  • the target is a viral, fungal, parasitic, plant or animal molecule
  • either the DNA or RNA can be a target molecule and must be released from the interior compartments of the organism.
  • additional steps can be taken in order to further purify the target nucleic acid desired from contaminating molecules, such as cellular debris after lysis. Methods of purifying nucleic acids are well known to those of ordinary skill in the art.
  • a single-stranded target molecule, a single-stranded adapter molecule and a single- stranded immobilized probe are used.
  • This embodiment is especially useful for analysis of RNA targets. It is also useful for capture of specific targets from complex samples where renaturation of the target is not rapid. Highly concentrated targets, such as PCR products, may require denaturation immediately prior to electrophoresis because of rapid renaturation. For example, for analysis of PCR products that are 100-250 base pairs in length, it is convenient to bring the sample to 75% formamide (volume/volume) and heat at greater than 75° for 5 minutes immediately prior to electrophoresis.
  • Target molecules can have a size of from about 10 to about 100,000 nucleotides in length.
  • a double-stranded target is placed in a complex with a single-stranded adapter molecule.
  • adapter molecules can be designed that will associate with double-stranded nucleic acids to form a triple-stranded structure.
  • the third strand locates in the major groove of the duplex and forms Hoogsteen base pairing interactions with the bases of the duplex (Hogan and Kessler, U.S. Patent No. 5,176,966 and Cantor, et al, U.S. Patent No. 5,482,836).
  • the design of the adapter molecule is therefore subject to the constraints governing those chemical interactions. However, the frequency of sequences capable of forming triplex structures in naturally occurring nucleic acids is high enough that many target nucleic acids can be specifically captured using this adapter molecule design strategy.
  • adapter molecules can be designed that will associate with double-stranded nucleic acids by formation of a displacement loop structure. Such adapter molecules bind to only one strand of the duplex nucleic acid and displace the adapter polynucleoide-homologous duplex strand of the duplex locally. This displacement can only be achieved if the adapter molecule-target strand interaction is much more favorable than the interaction between the target strands.
  • Such adapter molecules can be made using modified bases and techniques described in Wetmur, Critical Reviews in Biochemistry and Molecular Biology, vol 26, pp 227-259 (1991), backbone modifications (Moody, et al, Nucleic Acids Res., vol. 17, pp.
  • the adapter molecule is typically from about 10 to about 100 nucleotides in length. Preferably, the adapter molecule contains from about 10 to about 50 nucleotides.
  • An adapter molecule comprises at least two distinct nucleotide sequence regions.
  • the capture probe-specific nucleotide sequence region from about 5 to about 50 nucleotides in length, is complementary to a nucleotide sequence defined by the target molecule desired to be captured.
  • the target-specific nucleotide sequence region, from about 5 to about 50 nucleotides in length, of the adapter molecule is complementary to a nucleotide sequence contained within a capture probe.
  • the adapter is synthesized using standard automated methods for oligonucleotide or peptide (for PNA) synthesis wherein both sequence regions form part of a single longer adapter molecule.
  • the two sequence regions can be synthesized separately and subsequently linked using chemical crosslinkers well known to those skilled in the art. (Wong, Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, FL (1991)). The latter strategy can be especially useful for chimeric adapters where the two regions are chemically different, that is, a PNA capture probe-specific domain with a DNA target-specific domain.
  • the adapter molecule is a DNA molecule.
  • the adapter molecule can be an RNA molecule.
  • the adapter molecule can be either a single, double, or partially double stranded nucleic acid.
  • the adapter molecule is a single-stranded nucleic acid.
  • the adapter molecule brings together in a hybridization complex the target molecule and the capture probe, that is, it serves as a connector between the target molecule and capture probe, as shown in Figure 1. Only target molecules with nucleotide sequence complementary to all, or a portion, of the nucleotide sequence of the adapter molecule will be captured during electrophoresis through the universal capture gel. Any contaminating molecule (e.g., a molecule that does not comprise a nucleotide sequence complementary to the adapter molecule) will migrate through the capture layer of the gel and will go undetected. Hence, this adapter technology can also be useful in purification schemes.
  • the adapter molecule allows for versatility with respect to the adapter/capture probe complex.
  • the same capture probe can be used to detect multitude of test molecules because of the adapter molecule.
  • the nucleotide sequence region of the adapter molecule that hybridizes to a particular capture probe will remain constant (i.e., a non-variable nucleotide sequence), while the nucleotide sequence region of the adapter molecule that hybridizes to a particular target molecule can vary (i.e., a variable nucleotide sequence) depending upon the specific target molecule desired to be captured. Therefore, a set of adapter molecules can be produced which have the ability to bind to the same capture probe, while possessing specificity for different target molecules.
  • only one class of capture probes is required in order to detect one, or many, target molecules using a universal capture gel.
  • only one type of electrophoretic medium need be produced for analyzing multiple target molecules independently given that the same class of capture probes can be used for a variety of target molecules. For example, if there are ten target molecules to be analyzed the practitioner can use ten electrophoretic gels that are the same, that is, contain the same capture probes immobilized within the gel. It is the adapter molecule which provides for this versatility of target molecule analysis which can employ only one type of gel comprising one class of capture probes.
  • the target molecule from the test sample is brought in contact with an appropriate adapter molecule in solution under conditions suitable for hybridization and the target molecule of a test sample will hybridize with the adapter molecule forming a universal gel hybridization complex (i.e., adapter/target complex).
  • hybridization can be achieved by heating the solution containing the adapter molecule and target molecule to 90°C and allowing to cool to room temperature.
  • Hybridization of adapter molecule to a target molecule can be accomplished in several ways.
  • target and adapter molecules can be hybridized together prior to electrophoresis.
  • an RNA target is mixed in solution with a DNA adapter molecule (adapter concentration is form about 0.05 micromolar to about 1 micromolar) in from about 0.05 M to about 1 M monovalent salt, heated to around 90°C and allowed to slow cool (over from about 30 minutes to about 1 hour) down to room temperature.
  • the adapter can be hybridized to the capture probe prior to loading the target on the universal gel.
  • an adapter molecule is loaded in about 2-fold molar excess over the amount of the capture probe immobilized within the gel (or gel lane) and subjected to electrophoresis through the universal gel under conditions suitable for adapter hybridizing to the immobilized capture probe.
  • the capture probes therefore, become saturated with adapters, and remain stably immobilized on the capture layer.
  • the target molecules can then be loaded onto the gel and subjected to electrophoresis through the capture layer where they can hybridize to the single-stranded target-specific region of the adapter molecule.
  • pre-electrophoresis of the universal gel with an excess of adapter changes the specificity of the capture layer from a an adapter-specific sequence to a target- specific sequence. If different adapters are pre-electrophoresed in different lanes of the gel, the same universal capture layer can have different target specificities in adjacent lanes.
  • hybridization between adapters and targets can occur in the gel during electrophoresis using sequential loading of target and adapter. Typically, the target has lower electrophoretic mobility than the adapter. If the target is loaded before the adapter molecule, then a concentrated layer of adapter will overtake and pass the slower moving target during electrophoresis. As the adapters pass the target band, they can hybridize with the target.
  • the target-adapter complexes can be captured on a universal capture layer.
  • the methods of the present invention are also useful for analysis of nucleic acid binding proteins.
  • the adapter molecules that are selected mimic, in some manner, the protein's natural binding substrate.
  • sequence-specific and non-sequence-specific nucleic acid binding proteins can be analyzed.
  • the adapter molecule is designed to contain a sequence which is recognized by the target binding protein.
  • mixtures of adapter molecules can be used to ensure that any observed binding is not dependent on any particular nucleic acid sequence.
  • Electrophoretic analysis is performed under conditions which allow the protein to retain its native structure, thereby permitting the protein to bind to the adapter molecule during electrophoresis.
  • the presence of the protein within the gel region containing an immobilized capture probe specific for the adapter molecule (where the adapter/target complex binds) can be detected by staining with colored or fluorescent dyes, autoradiography (if the sample has been radioactively labeled), silver staining, as well as other various other standard methods well known to those of ordinary skill in the art of protein electrophoresis.
  • a double-stranded adapter molecule (or partially double-stranded) containing a sequence known (or suspected) to be recognized by the protein target is used.
  • the test sample is subjected to electrophoresis through the region containing a capture probe specific for the adapter molecule. Following electrophoresis, the position of the protein within the gel is determined. The presence of protein in the gel region containing the adapter/target/capture probe complex indicates the presence of a DNA binding- protein in the sample. Control experiments demonstrating that binding does not occur with a DNA adapter molecule, which lacks the specific sequence of interest, can be used to demonstrate the sequence specificity of the binding. Single stranded adapter molecules may also be useful.
  • single- stranded RNA adapter molecule can be used for detection and purification of proteins that bind to specific RNA sequences.
  • Single-stranded DNA adapter molecules may be useful for detecting regulatory proteins of viruses that contain single-stranded DNA genomes, or proteins that bind specifically to single-stranded DNA segments within replication origins.
  • Suitable matrices include acrylamide and agarose, both commonly used for nucleic acid electrophoresis. However, other materials may be used as well. Examples include chemically modified acrylamides, starch, dextrans and cellulose-based polymers. Additional examples include modified acrylamides and acrylate esters (for examples see Poly sciences, Inc., Polymer & Monomer catalog, 1996-1997, Warrington, PA), starch (Smithies, Biochem. J., 71 :585 (1959); product number S5651, Sigma Chemical Co., St.
  • nucleic acids, modified nucleic acids or nucleic acid analogs as capture probes.
  • Methods of coupling nucleic acids to create nucleic acid-containing gels are known to those of ordinary skill in the art.
  • Nucleic acids, modified nucleic acids and nucleic acid analogs can be coupled to agarose, dextrans, cellulose, and starch polymers using cyanogen bromide or cyanuric chloride activation.
  • Polymers containing carboxyl groups can be coupled to synthetic capture probes having primary amine groups using carbodiimide coupling.
  • Polymers carrying primary amines can be coupled to amine-containing probes with glutaraldehyde or cyanuric chloride.
  • composite matrices containing a mixture of two or more matrix forming materials may be useful to use.
  • An example is the composite acrylamide-agarose gel. These gels typically contain from 2-5% acrylamide and 0.5%>- 1 % agarose. In these gels the acrylamide provides the chief sieving function, but without the agarose, such low concentration acrylamide gels lack mechanical strength for convenient handling. The agarose provides mechanical support without significantly altering the sieving properties of the acrylamide. In such cases, the nucleic acid can be attached to the component that confers the sieving function of the gel, since that component makes the most intimate contacts with the solution phase nucleic acid target.
  • gel-forming matrices such as agarose and cross- linked polyacrylamide will be preferred.
  • soluble polymers such as linear polymers of polyacrylamide, poly(N,N- dimethylacrylamide), poly(hydroxyethylcellulose), poly(ethyleneoxide) and poly(vinylalcohol) as described in Quesada (Current Opinion in Biotechnology, vol. 8, pp.82-93, (1997)).
  • These soluble matrices can also be used to practice the methods of the present invention. It is particularly convenient to use the methods found in the application U.S.
  • Nucleic acids may be attached to particles which themselves can be incorporated into electrophoretic matrices.
  • the particles can be macroscopic, microscopic, or colloidal in nature. (See Polyciences, Inc., 1995-1996 particle Catalog, Warrington, PA).
  • Cantor, et al, U.S. Patent No. 5,482,863 describes methods for casting electrophoresis gels containing suspensions or particles.
  • the particles are linked to nucleic acids using methods similar to those described above mixed with gel forming compounds and cast as a suspension into the desired matrix form.
  • a linear gel may be formed by techniques including formation within a linear support, such as a trough or tube, where the gel is formed by polymerization within the support, alternatively, by subdividing a two-dimensional gel into a number of strips by partitions or formation of channels.
  • quantities of one, or more, copolymerizable capture probes can be added to the gel material, optionally in spatially defined positions, such as by spatially positioned dropper techniques, either before or during gel polymerization to provide one, or more, capture probes within the polymerized gel.
  • a sequence of gel monomers and mixtures of gel monomers and polymerizable capture probes may be introduced into the tube sequentially such as to provide a spatially distinguished set of components and concentrations which are then polymerized in situ to preserve the components' spatial relationships.
  • the tube walls can be made of elastic material which laterally contracts during shrinkage of the gel.
  • progressive polymerization may be induced from one end of the tube while adding more liquid material to the other end to compensate for shrinkage.
  • Such progressive polymerization may be induced by means including diffusion of a polymerization catalytic agent, or by progressive application of polymerization inducing electromagnetic or other radiation from one end of the tube to the other, such as by movement of, or progressive exposure to, the radiation source.
  • a linear format gel may be produced by taking a linear slice from a two-dimensional gel, or a linear core from a three-dimensional gel, produced as described below.
  • a two-dimensional gel may be formed by techniques including formation on a surface of a support, or formation between two support surfaces.
  • a layer of gel monomer is applied and quantities of coplymerizable capture probes may be applied to the layer, optionally in a spatially significant manner, before or during polymerization, which are then polymerized in situ to preserve their spatial positions in the gel.
  • Application of quantities of polymerizable capture probes may be effected by known means including positional programmable dropper techniques.
  • Gel shrinkage during polymerization may be adjusted for by means including permitting contraction of the gap between support surfaces and by permitting lateral contraction with more material added from the side to compensate.
  • a two- dimensional gel may be subdivided into a number of strips, by the use of partitions before, during or after gel formation, or by formation in channels, or by being sliced into narrower sections after formation.
  • Three-dimensional gels may be formed by a number of techniques. Multiple linear strips or two-dimensional layers may be repetitively constructed as above, each optionally containing localized capture probes, with each strip or layer being polymerized onto an underlying layer such that a three-dimensional volume results. Alternatively, a number of two-dimensional gels, optionally with capture probes localized in place, may be formed as above and assembled together to provide a three-dimensional structure.
  • Electrophoretic matrices useful for the methods described herein can be provided in a number of different formats.
  • the matrix can be provided in a format where its physical length significantly exceeds its breadth or depth, for example, contained within a tube or formatted as a narrow strip.
  • the matrix can be provided in a format where its length and breadth significantly exceed its depth, for example, as a relatively thin layer on a surface or formatted as a slab.
  • the matrix can be provided essentially as a solid body, where its length, breadth and depth are of the same order, for example, as an actual or approximately rectilinear, polygonal, spherical, ellipsoid solid or similar physical form.
  • the electrophoretic matrix can comprise a homogeneous or heterogeneous matrix material.
  • suitable matrix materials include gel- forming polymers such as cross-linked polyacrylamide, agarose, starch and combinations thereof.
  • gel-forming polymers such as linear polyacrylamide, poly(N,N-dimethylacrylamide), poly(hydroxyethylcellulose), poly(ethyleneoxide) and poly(vinlyalcohol), as commonly used in capillary electrophoresis applications, can also serve as suitable matrices.
  • the capture probes of the present invention comprise a nucleic acid with a polynucleotide sequence substantially complementary to at least one nucleotide sequence region of one, or more, classes of adapter molecules, wherein the adapter molecules hybridize to the capture probe.
  • the complementarity of nucleic acid capture probes need only be sufficient enough to specifically bind the adapter molecule.
  • Probes suitable for use in the present invention comprise RNA, DNA, nucleic acid analogs, and chimeric probes of a mixed class comprising a nucleic acid with another organic component, for example, peptide nucleic acids.
  • Capture probes can be single-stranded or double-stranded nucleic acids.
  • the capture probe will be modified in such a manner as to allow it to be immobilized within an electrophoretic medium, such as modifying the 5 '-terminus with an acrylamide moiety (AcryditeTM, Mosaic Technologies, Boston, MA).
  • nucleic acid includes DNA (deoxyribonucleic acid) or RNA(ribonucleic acid).
  • Nucleic acids referred to herein as “isolated” are nucleic acids separated away from the components of their source of origin (e.g., as it exists in cells, or a mixture of nucleic acids such as a library) and may have undergone further processing. Isolated nucleic acids include nucleic acids obtained by methods known to those of ordinary skill in the art. (Ausubel, F.M., et al, (eds), Current Protocols in Molecular Biology, John Wiley & Sons (Pub.), vol. 1, ch. 2 through 4 (1991). These isolated nucleic acids include substantially pure nucleic acids by one, or a combination of, biological, chemical and recombinant methods from which nucleic acids have been isolated.
  • Modified nucleic acids include nucleic acids containing modified sugar groups, phosphate groups or modified bases.
  • nucleic acids having modified bases include, for example, acetylated, carboxylated or methylated bases (e.g., 4-acetylcytidine, 5-carboxymethylaminomethyluridine, 1- methylinosine, norvaline or allo-isoleucine).
  • Probes containing modified polynucleotides may also be useful.
  • polynucleotides containing deazaguanine and uracil bases can be used in place of guanine and thymine-containing polynucleotides to decrease the thermal stability of hybridized probes (Wetmur, Critical reviews in Biochemistry and Molecular Biology, vol. 26, pp. 227-259 (1991)).
  • 5-methylcytosine can be substituted for cytosine if hybrids of increased thermal stability are desired (Wetmur, Critical reviews in Biochemistry and Molecular Biology, vol. 26, pp. 227-259 (1991)).
  • Modifications to the ribose sugar group can reduce the nuclease susceptibility of immobilized RNA probes (Wagner, Nature, vol. 372, pp. 333-335 (1994)). Modifications that remove negative charge from the phosphodiester backbone can increase the thermal stability of hybrids (Moody et al. Nucleic Acids Res., vol. 17, pp.4769-4782 (1989); Iyer et al J. Biol. Chem., vol. 270, pp.l4712-14717 (1995)).
  • substantially complementary means that the polynucleotide sequence of the capture probe need not reflect the exact polynucleotide sequence of the adapter molecule, but must be sufficiently complementary in order to hybridize with the adapter molecule under specified conditions.
  • non-complementary bases, or additional polynucleotides can be interspersed in sequences provided that the sequences have sufficient complementary bases to hybridize therewith.
  • the degree of complementarity required is from about 90 to about 100%.
  • Specified conditions of hybridization can be determined empirically by those of ordinary skill in the art. For example, conditions of stringency should be chosen that significantly decrease non-specific hybridization reactions. Stringency conditions for nucleic acid hybridizations are explained in, for example, Current Protocols in Molecular Biology, Ausubel, F.M., et al, eds., vol. 1, suppl, 26, 1991, the teachings of which are herein incorporated by reference in their entirety. Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of nucleic acid hybrids. Stringent conditions, for example, moderate, or high stringency, can be determined empirically, depending in part on the characteristics of the probe and adapter molecule.
  • the length of a capture probe will be at least 5 nucleotides in length, usually between 5 and 50 nucleotides, and can be as long as several thousand bases in length.
  • the capture probes immobilized in the universal capture gel are from about 18 to about 24 nucleotides in length.
  • Nucleic acid analogs as used herein include molecules which lack sugar- phosphate backbones, but retain the ability to form complexes via basepairing. Such nucleic acid analogs are known to those of ordinary skill in the art. Nucleic acid analogs can also be useful as immobilized probes.
  • a useful nucleic acid analog is peptide nucleic acid (PNA), in which standard DNA bases are attached to a modified peptide backbone comprised of repeating N-(2-aminoethyl)glycine units (Nielsen et al., Science, vol. 254, pp. 1497-1500 (1991)). The peptide backbone is capable of holding the bases at the proper distance to base pair with standard DNA and RNA single strands.
  • PNA peptide nucleic acid
  • PNA-DNA hybrid duplexes are much stronger than equivalent DNA-DNA duplexes, probably due to the fact that there are no negatively charged phosphodiester linkages in the PNA strand.
  • PNAs are very resistant to nuclease degradation.
  • PNA nucleic acid analogs are useful for immobilized probe assays. It will be apparent to those of ordinary skill in the art that similar design strategies can be used to construct other nucleic acid analogs that will have useful properties for immobilized probe assays.
  • Detection of the specific binding reaction for example, detection of the immobilized adapter/target complex bound to the capture probe, can be accomplished in a number of different ways.
  • the target molecule can be detectably labeled prior to the hybridization reaction with the adapter molecule.
  • Suitable labels for direct target labeling can be intensely absorbing (e.g., brightly colored), radioactive, fluorescent, phosphorescent, chemiluminescent or catalytic.
  • Direct target labeling of nucleic acid samples using modified polynucleotides can be accomplished by a number of enzymatic methods well known to those practiced in the art (reviewed in Sambrook, et al, "Molecular Cloning: A Laboratory Manual", 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, NY 1989).
  • the target molecule can be labeled indirectly using a ligand which can be recognized by a second specific binding entity which is either labeled itself or can produce a detectable signal.
  • biotinylated polynucleotides An example of such an indirect system is labeling using biotinylated polynucleotides.
  • the sample is labeled enzymatically using standard nucleic acid labeling techniques and biotinylated polynucleotides.
  • the resulting biotin-modified nucleic acids can be detected by the biotin-specific binding of streptavidin or avidin protein molecules.
  • streptavidin or avidin molecules can be conjugated to fluorescent labels, such as fluorescein or reporter enzymes, such as alkaline phosphatase or horseradish peroxidase, which can be used to produce chemiluminescent or colorimetric signals with appropriate substrates (for review see Keller and Manak, "DNA Probes", 2nd ed., Macmillan Publishers, London, 1993; Pershing, et al, eds "Diagnostic Molecular Microbiology: Principles and Applications", American Society for Microbiology, Washington, D.C., 1993).
  • fluorescent labels such as fluorescein or reporter enzymes, such as alkaline phosphatase or horseradish peroxidase
  • reporter enzymes such as alkaline phosphatase or horseradish peroxidase
  • Another useful detection system is the digoxigenin system which uses an anti-digoxigenin antibody, conjugated to alkaline phosphatase, which recognizes digoxigenin-dUTP incorporated into nucleic acids. (Current Protocols in Molecular Biology, ed. Ausubel, F.M., vol.l, ⁇ 3.18.1 to 3.19.6, (1995), the teachings of which are incorporated herein by reference in its entirety).
  • Detectably labeled hybridization probes can also be used as indirect target labels.
  • target nucleic acids can be indirectly labeled prior to electrophoresis by hybridization with a detectably labeled probe, hereafter termed a "sandwich" probe.
  • the sandwich probe is designed to hybridize with a region of the target which does not overlap the region recognized by an appropriate adapter molecule.
  • the sandwich probe is designed to remain associated with the target during electrophoresis, and cannot bind directly to the capture probe nor to the adapter molecule.
  • Sandwich probes can also be used to label target molecules after electrophoretic capture.
  • the unlabeled target molecule is subjected to electrophoresis in complex with an appropriate adapter molecule and the adapter/target hybridizes to the capture probe first.
  • the sandwich probe is subjected to electrophoresis through the capture layer.
  • the captured adapter/target complex now acts as a new "capture" probe for the sandwich probe.
  • the captured target sandwich probe complex can now be detected through the sandwich probe label. Blotting techniques can also be adapted for detection of target bound capture probes.
  • a detection surface is juxtaposed to the separation medium having bound sample components, and the sample components then migrate to the detection surface, optionally assisted by, for example, chemical means such as solvent or reagent changes, where the transferred sample components are detected by known means such as optical detection of intercalating dyes, or by detection of radioactivity from hybridized radioactive species, or other known means.
  • a variety of optical techniques can be used to detect the presence of sample components bound to the capture probes. For example, if the capture probes are arranged in a linear array, the position and intensity of each signal may be measured by mechanically or optically scanning a single detector along the array of detectable signals. Alternatively, a linear array of detectable signals may be detected by a linear array detector, such as by juxtaposition of the array detector to the array of detectable signals or by optically imaging all or part of the signal array onto the array detector.
  • the capture probes with detectable signals are arranged as a two- dimensional array
  • a number of detection schemes may be employed.
  • a single detector may be used to measure the signal at each point by mechanical or optical scanning, or by any combination.
  • a linear optical detection array may be used to detect a set of signals by juxtaposition or optical imaging, and multiple sets of such signals may be detected by mechanically or optically scanning the signal array or detector.
  • the two-dimensional array of capture probes may be optically detected in whole or in part by a two-dimensional optical area detector by juxtaposition to, or optical imaging of, the array of optical signals from the immobilized capture probes.
  • detection of individual signals may be arranged by the above techniques, optionally assisted by first physically taking one, or more, sub-sections of the array.
  • optical schemes such as confocal microscopic techniques may be employed whereby one or a number of detectable signals are imaged and detected with minimal interference from others, and other signals are subsequently detected after optical adjustment.
  • METHODS OF UNIVERSAL CAPTURE GEL USE The methods of the present invention are applicable to analysis of any chemical entity that can be subjected to electrophoresis (e.g., a charged molecule that has detectable mobility when placed in an electrophoretic field) and that binds to, or is bound by, nucleic acids.
  • electrophoresis e.g., a charged molecule that has detectable mobility when placed in an electrophoretic field
  • Such entities include, for example, DNA or RNA samples, nucleic acid binding proteins, nucleic acid analogs, modified nucleic acids, and aptamer binding partners (aptamers are nucleic acids that are selected to bind to specific binding partners such as peptides, proteins, drugs, polysaccharides and small organic molecules, for example, theophylline and caffeine; Jenison, et al, Science, 263:1425-1429 (1994)).
  • methods described herein can be used for analysis and purification of target nucleic acids using immobilized capture probes, where specific binding involves base pairing interactions between sample nucleic acids and the capture probe, as in nucleic acid hybridization.
  • the methods described herein are also useful for purification of sequence-specific nucleic acid binding proteins, since synthetic nucleic acids of defined sequence can be immobilized in matrices commonly used for protein electrophoresis.
  • the sample containing the adapter/target complex can be detectably labeled before, during, or after the electrophoresis step.
  • the adapter can be labeled prior to hybridizing with the target molecule.
  • the target molecule can be labeled prior to hybridization with the adapter molecule.
  • both can be labeled prior to hybridization with each other. Detecting the presence of adapter/target/capture probe complexes immobilized within the matrix is indicative of the presence of the target molecule that is bound by the capture probe via an adapter molecule.
  • test sample containing an adapter/target complex is introduced into the electrophoretic medium it is subjected to an electrical field resulting in the electrophoretic migration of the test sample through the matrix, under conditions and time sufficient for the adapter/target complex, of the test sample, if present, to bind to one, or more, capture probes, resulting in adapter/target/capture probe complexes immobilized in the matrix.
  • Typical voltage gradients used in nucleic acid electrophoresis procedures range from approximately 1 V/cm to 100 V/cm. Other field strengths can be useful for certain highly specialized applications.
  • a method for detecting a target molecule contained in a test sample using a universal capture gel is disclosed.
  • An adapter/target hybridization complex is formed by mixing an appropriate adapter molecule with a test sample containing a target molecule under conditions suitable for hybridization.
  • An appropriate adapter molecule is an adapter molecule comprising at least one nucleotide sequence region complementary to at least one nucleotide sequence region contained within a target molecule.
  • the adapter molecule comprises at least two defined nucleotide sequence regions. First, the target-specific region is complementary to a nucleotide sequence region contained within a target molecule.
  • This hybridization product, that is, the adapter/target complex can then be introduced into the universal capture gel.
  • the universal capture gel contains capture probes which are immobilized within the medium.
  • the universal capture gel is subjected to an electric field resulting in the electrophoretic migration of the hybridization complex (adapter/target complex) formed above.
  • an appropriate capture probe that is, a capture probe that is complementary to the capture probe-specific nucleotide sequence region (the second defined nucleotide sequence region of an adapter molecule) of the adapter probe, then the adapter/target complex will become immobilized within the medium through the binding of the adapter molecule to the immobilized capture probe. Detection of the new tripartite complex formed by the adapter/target/capture probe complex is indicative of the presence of the target molecule in the test sample.
  • a method of detecting one, or more, target molecules in a test sample using a universal capture gel comprising only one class of immobilized capture probes is disclosed.
  • this universal capture gel multiple target molecules can be detected.
  • Multiple classes of adapter molecules can be used wherein they all share one nucleotide sequence region that is complementary to the capture probe immobilized in the electrophoretic medium.
  • the classes of different adapter molecules differ in that their target-specific nucleotide sequence region is specific for a particular target molecule contained within a test sample. In this embodiment it may not be necessary to isolate the different target molecules in discrete regions of the gel, but only necessary to qualitatively detect the presence of any of these particular target molecules in the test sample and therefore the capture probes can be immobilized throughout the gel.
  • the immobilized capture probes can be immobilized in discrete regions of the electrophoretic medium.
  • the adapter molecules used are first mixed with target molecules from the test sample under conditions suitable for hybridization wherein, the adapter molecule specific for a particular target molecule will hybridize to that target molecule. This hybridization step typically proceeds until all, or any desired number of target molecules, have hybridized to their complementary adapter molecule. Then the hybridization complex preparation containing one, or more, adapter/target complexes, is subjected to electrophoresis through the universal capture gel. The adapter/target complexes will migrate in the electrophoretic medium until they come in contact with an immobilized capture probe which is specific for the adapter molecule.
  • an adapter/target complex comes into contact with an immobilized capture probe a second complex is formed between the adapter/target complex and immobilized probe forming an adapter/target/capture probe tripartite complex.
  • This tripartite complex is immobilized to the electrophoretic medium via the immobilized capture probe. The detection of this tripartite complex is indicative of the presence of the target molecule, or molecules, in the test sample.
  • a method for detecting one, or more, target molecules from a test sample using a universal capture gel using multiple classes of capture probes is disclosed.
  • multiple classes of capture probes are immobilized within the electrophoretic medium in discrete regions. This allows for the isolation and identification of one target molecule from another captured in this embodiment.
  • the capture probes can be immobilized throughout the electrophoretic medium.
  • one or more classes of capture probes are immobilized in discrete regions of the gel.
  • only one class of capture probe is immobilized to any given discrete capture layer within the gel.
  • a capture layer is a layer comprising immobilized capture probes within the electrophoretic medium.
  • adapter molecules which have a complementary nucleotide sequence region to a particular class of capture probe immobilized in the electrophoretic medium.
  • An adapter molecule corresponding to a particular class of immobilized capture probe has a target-specific nucleotide sequence region complementary to a specific target molecule.
  • the adapter molecules and target molecules are first mixed with one another under conditions suitable for hybridization. This first step continues until all, or any desired number of target molecules are hybridized to its corresponding complementary adapter molecule.
  • These adapter/target complexes are then subjected to electrophoresis. When the individual adapter/target complex comes in contact with the appropriate immobilized capture probe, then the complex becomes immobilized to the gel.
  • the location of the immobilized tripartite complex of adapter/target/capture probe depends upon the specificity between the adapter molecule (complexed with a particular target molecule) and the capture probe immobilized in a particular, discrete region of the gel.
  • This universal capture gel may comprise five discrete capture layers containing five separate classes of immobilized capture probes specific for their particular adapter molecule.
  • Five classes of adapter molecules are used where each class is specific for a particular class of capture probe as well as being specific for a particular target molecule.
  • the adapter molecules are mixed with the target molecules of the test sample, then five sets of adapter/target complexes are formed and can be subjected to electrophoresis.
  • Each adapter/target complex will migrate in the electrophoresis medium until it comes in contact with the appropriate immobilized capture probe forming an immobilized tripartite complex. Therefore, there could be five separate capture layers comprising five different tripartite complexes, indicating that the test sample had at least five different classes of target molecules.
  • a sample containing a target molecule is subjected to electrophoresis through a series of discrete matrix layers each of which contain at least one class of capture probes.
  • the capture probes can be throughout the matrix.
  • a target nucleic acid that is bound to an adapter molecule that is complementary to the capture probe can hybridize to that capture probe which is contained within a gel layer and is thus retained in the gel layer, that is, the capture layer.
  • Noncomplementary sample nucleic acids pass through the capture layer.
  • the presence of hybrids between capture probes and complementary adapter/target complex is detected within the capture layer by appropriate labeling strategies described herein.
  • nucleic acid species that have discrete electrophoretic mobilities are not required for analysis by this method. Since the use of specific adapter polynucletides convey specificity with respect to the target molecule. In traditional zonal electrophoresis, all target molecules must migrate as a discrete band for detection.
  • the sample volume is not important.
  • all target molecules that are in complexes with their appropriate adapter molecules pass through the capture layer even though large sample volumes are used. This is a significant advantage over traditional zonal electrophoresis, where the sample volume needs to be as small as possible for maximum detection sensitivity and resolution.
  • the capture layer can contain single or multiple classes of capture probes. The use of multiple capture probes in a single layer is useful for assays where any one of a number of different organisms need to be detected. Multiple capture layers can be also be used in this embodiment. It is straightforward to cast multiple capture layers sequentially in the same gel apparatus to create a multiplex hybridization assay.
  • the target molecule in a complex with an appropriate adapter molecule is subjected to electrophoresis through all of the layers, and complementary adapter molecules are captured at each layer by an immobilized capture probe specific for that particular class of adapter molecule.
  • the amount of hybrid in each layer directly reflects the sample composition with respect to the capture probes used. Conditions can be identified to ensure that only properly hybridized adapter/target molecule complexes will be retained in each layer. Electrophoretic hybridization with capture probes as long as twenty bases can be carried out using traditional non-denaturing gels and buffer systems at room temperature. Fully complementary hybrids of this size appear to be stable for many hours. However, additional stringency can be achieved by adding denaturants such as urea or formamide to the gel, or running the gel at elevated temperatures.
  • One dimensional probe arrays can be used for analysis that employ limited numbers of capture probes.
  • a two- dimensional array of immobilized probes can be used.
  • the arrays can be formed in a number of ways. Simple two-dimensional arrays can be cast, for example, in conventional slab gel devices using multiple vertical aligned spacers, in effect creating an array of one dimensional arrays. More complex two dimensional arrays can be created in two steps, first, polymerizing the capture probe regions as an array of matrix (for example, polyacrylamide gel) dots on one plate, then "sandwiching" the dots by placing an upper gel plate over the array and filling in the empty spaces between the probe dots with unmodified gel.
  • matrix for example, polyacrylamide gel
  • the sample containing adapter/target complexes is loaded as a band across the entire length of the matrix, for example, at the top edge of the matrix.
  • the entire test sample does not contact all of the capture probes.
  • the sample nucleic acids are present at high copy numbers, and so this problem does not present a significant obstacle.
  • the hybridization methods described herein may also encompass three- dimensional arrays, such as may be particularly useful for multiplexed parallel assays, for example, high throughput and/or cost-effectiveness.
  • Such assays may be provided in the format of three-dimensional solids, where multiple samples containing adapter/target complexes may be applied to a surface or face, then made to migrate through the volume of the solid such that one, or more, regions of capture probe are encountered.
  • the array may be produced such that each sample encounters the same sequence of capture probes during migration through the array, alternatively, different sequences of capture probes may be positioned for this purpose, such as to analyze different test sample mixtures or to analyze differing sets of components within one, or more, test sample mixtures.
  • the electrophoresis methods described herein are especially useful for selectively purifying specific target molecules from a crude, or semi-crude, mixture.
  • a semi-purified mixture (perhaps using the supernatant preparation) of cell extract mixed with an appropriate adapter, or adapters, is placed over a gel containing an immobilized capture probe.
  • the mixture undergoes electrophoresis through the gel.
  • Target molecules are immobilized on the layer containing the capture probes through binding with the appropriate adapter molecule.
  • Non-target molecules with the same charge as the targets are attracted to the electrode of opposite electrical polarity (which will be referred to here as the "attracting electrode") and pass through the capture probe layer, eventually electrophoresing out of the gel.
  • Non-target molecules of the opposite charge migrate out of the sample well toward the non-attracting electrode. Uncharged sample molecules remain in the sample well and do not enter the gel. After allowing sufficient electrophoresis time, in order to be sure that all charged non-target molecules have been removed from the gel, the captured target molecules are eluted from the gel by one of two methods:
  • the eluted target molecules can be concentrated and recovered from the attracting electrode chamber by several methods. For instance, after undergoing electrophoresis, some non-target sample molecules will migrate out of the gel, some non-target molecules will migrate to the attracting electrode chamber which can be flushed out with an appropriate wash solution, finally, the target molecules can be eluted directly into the original attracting electrode chamber.
  • the electrophoresis device can have two attracting electrodes so that one is used to clear the non-target components from the sample while the second is used to elute the target molecules.
  • the electrophoresis device can be constructed with a replaceable attracting electrode chamber so that after removal of non-target components, the attracting electrode chamber can be replaced with a clean chamber in order to perform target elution.
  • This embodiment is particularly well-suited for purification of specific nucleic acids from semi-crude biological samples by hybridization methods.
  • specific target molecules can be isolated from a semi-crude preparation.
  • these methods result in substantial purification of target nucleic acids in one step because charged sample contaminants are eliminated during electrophoresis and uncharged contaminants are eliminated since they cannot enter the matrix.
  • very large samples can be used.
  • the nucleic acids undergo electrophoresis in free solution in virtually the same manner as they do in polymeric matrices. Therefore, large sample volumes can be used.
  • the matrix layer acts like a highly selective filter to select only the desired nucleic acids (formed in a complex with an appropriate adapter molecule) from the sample.
  • large volumes of very dilute samples can be concentrated quantitatively using the methods described herein.
  • target molecules are purified by employing modified adapter molecules.
  • a modified adapter molecule can be used to displace either the target molecule alone, or the target adapter complex from immobilization to the gel matrix.
  • a modified adapter molecule comprising a nucleotide sequence region that is complementary to the unmodified adapter's target- specific nucleotide sequence region.
  • displacement of the target molecule from the complex can be effectuated by subjecting the modified adapter molecule to electrophoresis.
  • the modified adapter molecule When the modified adapter molecule comes into contact with the tripartite complex, the modified adapter molecule will displace the target molecule from the tripartite complex and will bind to the adapter molecule hybridized to the immobilized capture probe. The target molecule can then continue to migrate through the gel and can be retrieved.
  • a modified adapter molecule can comprise a nucleotide sequence region that is complementary to the unmodified adapter's capture probe-specific nucleotide sequence region.
  • This modified adapter molecule can be placed into an electrophoresis medium already containing a tripartite hybridization complex (comprising an adapter molecule/target molecule/universal capture probe).
  • the modified adapter molecule can be subjected to electrophoresis and migrate until it comes into contact with the tripartite complex.
  • the modified adapter can displace the adapter/target complex form the immobilized capture probe and hybridize to the capture probe.
  • the released adapter/target complex can continue to migrate through the gel.
  • RNA 1 and RNA 2 produced using T7 RNA polymerase- based in vitro transcription kit, Promega Corp., Madison, WI, supplemented with fluorescent nucleotide as label, fluorescein-rUTP, Boehringer Mannheim, Indianapolis, IN).
  • RNA 1 and RNA 2 were mixed, independently, with a specific adapter molecule.
  • RNA 1 was transcribed from a PCR product containing the E. coli Ml RNA gene. (See FIG. 4, SEQ ID NO. 3).
  • RNA 2 was transcribed from a plasmid containing a gene fragment from the human nonmuscle mysosin B gene cloned into pGEM3Zf(-), Promege, Madison, WI. (See FIG. 5, SEQ ID NO. 4).
  • This vector contains a T7 RNA polymerase promoter that was used to produce RNA 2 in an in vitro transcription reaction, as described above for RNA 1.
  • Increasing amounts of the appropriate adapter molecule were added to separate reaction vials to a final volume of 20 ⁇ L in 0.1 M NaCl. The samples were heated to 90°C and allowed to cool to room temperature.
  • a ficoll sample loading buffer containing xylene cyanol and bromophenol blue were added to each sample and then 12 ⁇ L were loaded into the gel.
  • the gel was 5% polyacrylamide (29:1, acrylamide:bis) and 0.5 x TBE.
  • a capture layer containing 13V acrydite polynucleotide capture probe (10 ⁇ M, total volume of 600 ⁇ L) was placed in the gel about 1 cm below the sample wells.
  • the capture probe sequence used was: 5 '-acrylamide- AGG CCC GGG A AC GTA TTC AC-3' [SEQ ID NO. 5].
  • the sample were subjected to electrophoresis using 200 V for 35 minutes at room temperature.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)

Abstract

L'invention concerne un gel universel et des procédés d'utilisation destinés à détecter la présence ou l'absence d'au moins une molécule cible dans un échantillon d'essai, consistant à former un complexe d'hybridation de gel universel dans lequel une molécule adaptateur est hybridée à une molécule cible et à une sonde de capture universelle.
PCT/US2000/008529 1997-05-16 2000-03-31 Analyse electrophoretique de molecules cibles au moyen de molecules adaptateurs WO2000060118A2 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2000609607A JP2003508015A (ja) 1999-04-02 2000-03-31 アダプター分子を用いる標的分子の電気泳動分析
CA002371727A CA2371727A1 (fr) 1999-04-02 2000-03-31 Analyse electrophoretique de molecules cibles au moyen de molecules adaptateurs
AU40539/00A AU4053900A (en) 1999-04-02 2000-03-31 Electrophoretic analysis of target molecules using adapter molecules
EP00919931A EP1235930A2 (fr) 1999-04-02 2000-03-31 Analyse electrophoretique de molecules cibles au moyen de molecules adaptateurs
US09/968,084 US20020197614A1 (en) 1997-05-16 2001-10-01 Electrophoretic analysis of target molecules using adapter molecules

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US28538099A 1999-04-02 1999-04-02
US09/285,380 1999-04-02

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
US28538099A Continuation 1997-05-16 1999-04-02

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US09/968,084 Continuation US20020197614A1 (en) 1997-05-16 2001-10-01 Electrophoretic analysis of target molecules using adapter molecules

Publications (2)

Publication Number Publication Date
WO2000060118A2 true WO2000060118A2 (fr) 2000-10-12
WO2000060118A3 WO2000060118A3 (fr) 2002-07-11

Family

ID=23093992

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2000/008529 WO2000060118A2 (fr) 1997-05-16 2000-03-31 Analyse electrophoretique de molecules cibles au moyen de molecules adaptateurs

Country Status (5)

Country Link
EP (1) EP1235930A2 (fr)
JP (1) JP2003508015A (fr)
AU (1) AU4053900A (fr)
CA (1) CA2371727A1 (fr)
WO (1) WO2000060118A2 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1173613A2 (fr) * 1999-04-02 2002-01-23 Mosaic Technologies Procedes de detection de micro-organismes a l'aide de sondes immobilisees
US6586177B1 (en) 1999-09-08 2003-07-01 Exact Sciences Corporation Methods for disease detection
US7452668B2 (en) 1997-05-16 2008-11-18 Exact Sciences Corporation Electrophoretic analysis of molecules using immobilized probes
US8492084B2 (en) 2006-01-24 2013-07-23 Koji Sode Method and apparatus for assaying test substance in sample
US20140332383A1 (en) * 2013-05-10 2014-11-13 The Regents Of The University Of California Free-standing microfluidic gel electrophoresis devices and methods

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2006244394B2 (en) * 2005-05-06 2010-10-21 Gen-Probe Incorporated Methods of nucleic acid target capture
US9081006B2 (en) * 2007-02-07 2015-07-14 Lyzer Diagnostics, Inc. Rapid homogeneous immunoassay using electrophoresis

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994006937A1 (fr) * 1992-09-15 1994-03-31 Boehringer Mannheim Corporation Technique amelioree du deplacement des brins et complexe utilise a cet effet
WO1997031256A2 (fr) * 1996-02-09 1997-08-28 Cornell Research Foundation, Inc. Detection de differences entre sequences d'acide nucleique faisant appel a la reaction de detection de ligase et a des reseaux adressables
WO1998039351A1 (fr) * 1997-03-05 1998-09-11 Mosaic Technologies, Inc. Complexe polymerisable contenant de l'acide nucleique
WO1998051823A1 (fr) * 1997-05-16 1998-11-19 Mosaic Technologies Analyse electrophoretique de molecules au moyen de sondes immobilisees
WO1999066079A1 (fr) * 1998-06-19 1999-12-23 Mosaic Technologies Detection d'organismes non viraux ayant un arn srp

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0347097A (ja) * 1989-07-13 1991-02-28 Hitachi Ltd ハイブリダイゼーション方法、これを用いた遺伝子変異検出方法及びその装置

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994006937A1 (fr) * 1992-09-15 1994-03-31 Boehringer Mannheim Corporation Technique amelioree du deplacement des brins et complexe utilise a cet effet
WO1997031256A2 (fr) * 1996-02-09 1997-08-28 Cornell Research Foundation, Inc. Detection de differences entre sequences d'acide nucleique faisant appel a la reaction de detection de ligase et a des reseaux adressables
WO1998039351A1 (fr) * 1997-03-05 1998-09-11 Mosaic Technologies, Inc. Complexe polymerisable contenant de l'acide nucleique
WO1998051823A1 (fr) * 1997-05-16 1998-11-19 Mosaic Technologies Analyse electrophoretique de molecules au moyen de sondes immobilisees
WO1999066079A1 (fr) * 1998-06-19 1999-12-23 Mosaic Technologies Detection d'organismes non viraux ayant un arn srp

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KENNEY MARY ET AL: "Mutation typing using electrophoresis and gel-immobilized acrydite probes." BIOTECHNIQUES, vol. 25, no. 3, 1998, pages 516-521, XP002159876 ISSN: 0736-6205 *
PATENT ABSTRACTS OF JAPAN vol. 015, no. 189 (C-0831), 15 May 1991 (1991-05-15) & JP 03 047097 A (HITACHI LTD), 28 February 1991 (1991-02-28) *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7452668B2 (en) 1997-05-16 2008-11-18 Exact Sciences Corporation Electrophoretic analysis of molecules using immobilized probes
EP1173613A2 (fr) * 1999-04-02 2002-01-23 Mosaic Technologies Procedes de detection de micro-organismes a l'aide de sondes immobilisees
US6586177B1 (en) 1999-09-08 2003-07-01 Exact Sciences Corporation Methods for disease detection
US8492084B2 (en) 2006-01-24 2013-07-23 Koji Sode Method and apparatus for assaying test substance in sample
US20140332383A1 (en) * 2013-05-10 2014-11-13 The Regents Of The University Of California Free-standing microfluidic gel electrophoresis devices and methods
US9976984B2 (en) * 2013-05-10 2018-05-22 The Regents Of The University Of California Free-standing microfluidic gel electrophoresis devices and methods

Also Published As

Publication number Publication date
CA2371727A1 (fr) 2000-10-12
JP2003508015A (ja) 2003-03-04
EP1235930A2 (fr) 2002-09-04
WO2000060118A3 (fr) 2002-07-11
AU4053900A (en) 2000-10-23

Similar Documents

Publication Publication Date Title
US20020197614A1 (en) Electrophoretic analysis of target molecules using adapter molecules
AU730491B2 (en) Electrophoretic analysis of molecules using immobilized probes
AU760236B2 (en) Denaturing gradient affinity electrophoresis and methods of use thereof
EP1157264B1 (fr) Dispositifs de purification biochimique avec sondes de capture immobilisees et leurs utilisations
US6238927B1 (en) Reverse displacement assay for detection of nucleic acid sequences
WO2000060120A9 (fr) Procedes de detection de micro-organismes a l'aide de sondes immobilisees
EP1411340A2 (fr) Dispositifs biochimiques de purification au moyen de sondes de capture et leurs utilisations
EP1235930A2 (fr) Analyse electrophoretique de molecules cibles au moyen de molecules adaptateurs
JP2003520961A (ja) 核酸を分離および検出するための方法および装置
AU3708300A (en) Methods for purifying dna using immobilized capture probes

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 09968084

Country of ref document: US

ENP Entry into the national phase in:

Ref country code: JP

Ref document number: 2000 609607

Kind code of ref document: A

Format of ref document f/p: F

ENP Entry into the national phase in:

Ref country code: CA

Ref document number: 2371727

Kind code of ref document: A

Format of ref document f/p: F

Ref document number: 2371727

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2000919931

Country of ref document: EP

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY CA CH CN CR CU CZ DE DK DM DZ EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A3

Designated state(s): GH GM KE LS MW SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

WWP Wipo information: published in national office

Ref document number: 2000919931

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 2000919931

Country of ref document: EP