EP1173613A2 - Procedes de detection de micro-organismes a l'aide de sondes immobilisees - Google Patents

Procedes de detection de micro-organismes a l'aide de sondes immobilisees

Info

Publication number
EP1173613A2
EP1173613A2 EP00921605A EP00921605A EP1173613A2 EP 1173613 A2 EP1173613 A2 EP 1173613A2 EP 00921605 A EP00921605 A EP 00921605A EP 00921605 A EP00921605 A EP 00921605A EP 1173613 A2 EP1173613 A2 EP 1173613A2
Authority
EP
European Patent Office
Prior art keywords
target
nucleic acid
immobilized
target molecule
capture probe
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP00921605A
Other languages
German (de)
English (en)
Inventor
T. Christian Boles
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Exact Sciences Corp
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
Publication of EP1173613A2 publication Critical patent/EP1173613A2/fr
Withdrawn legal-status Critical Current

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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • Stains can be used as well for the detection of microbial contamination. Gram staining as well as Wright ' s staining or acridine orange are often employed to detect bacterial contamination. However, these stains may not be sensitive enough to register a positive signal for bacterial contaminating species contained within a blood bag.
  • a sample of blood, or one of its products, can be cultured in order to determine whether there is contamination.
  • the primary disadvantage of using a culture system is that it generally takes between 24 to 48 hours to obtain a result. As a result, the blood cannot be tested in a time-effective manner.
  • Another disadvantage to this technique is that there is no endpoint in which a negative finding can be detected.
  • the method should be sensitive, rapid and amenable to various environments, such as a hospital, physician's office, or in the field where there is often a lack of sophisticated laboratory equipment.
  • the present invention relates to the discovery that nucleic acids, modified nucleic acids and nucleic acid analogs can be covalently attached (immobilized) 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 capture probes. These immobilized capture probes can be used to analyze a variety of molecules, for example, microbiological target molecules.
  • One specific binding reaction encompassed by the present invention is hybridization.
  • capture probes are typically nucleic acids comprising nucleotide sequences that are substantially complementary to the nucleotide sequences of the target nucleic acid, for example, a microbiological nucleic acid molecule, so that specific hybridization results.
  • nucleic acid analogs such as peptide nucleic acids (PNA) can be covalently attached to the electrophoretic medium for use as capture probes.
  • PNA peptide nucleic acids
  • matrix refers to the immobilized polymeric components of the electrophoretic medium which provide the molecular sieving properties of the medium, and also provide the means for immobilization of the capture probes.
  • suitable matrix materials include gel-forming polymers such as cross-linked polyacrylamide, agarose and starch.
  • Non- 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 present invention specifically relates to methods of detecting the presence or absence of microorganisms in a test sample by detecting the presence or absence of microbiological target molecules, in which electrophoresis is used to move solution-phase target molecules into contact with a capture probe that is immobilized on a suitable electrophoresis matrix.
  • the methods of the present invention are applicable to analysis of any chemical entity originating from a microorganism 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.
  • Such entities include, for example, DNA or RNA molecules, nucleic acid binding proteins, and aptamer binding partners (aptamers are nucleic acids that are selected to bind to specific binding partners such as peptides, proteins, and polysaccharides; Jenison, et al, Science, 263:1425-1429 (1994)).
  • aptamers are nucleic acids that are selected to bind to specific binding partners such as peptides, proteins, and polysaccharides; Jenison, et al, Science, 263:1425-1429 (1994)).
  • methods described herein can be used for analysis of target nucleic acids from microbiological organisms using immobilized capture probes, where specific binding involves interactions between target nucleic acids and the capture probe.
  • the test sample can be from any source and can contain any molecule that can form a binding complex with a capture probe.
  • samples from biological sources containing microorganisms obtained using known techniques, from body tissue (e.g., skin, hair, internal organs), body fluids for example, whole blood, platelets, erythrocytes, plasma, urine, semen, sweat, or cell, tissue and organ culture systems.
  • body tissue e.g., skin, hair, internal organs
  • body fluids for example, whole blood, platelets, erythrocytes, plasma, urine, semen, sweat, or cell, tissue and organ culture systems.
  • the test sample is treated in such a manner, known to those of skill in the art, so as to render the target molecules contained in the test sample available for binding.
  • a bacterial cell lysate is prepared and that 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.
  • the test sample containing the putative microbial target molecule is then introduced into a suitable electrophoretic medium. Capture probes are immobilized within the electrophoresis matrix by direct attachment to the medium, or by attachment to particles that are suspended and trapped within the matrix. In either case, the capture probes are immobilized, that is, they do not migrate under the influence of the applied electric field.
  • the test sample containing the microbial target molecule can be detectably labeled before, during, or after the electrophoresis step. Detecting the presence of microbial target molecule/capture probe complexes immobilized in the matrix is indicative of the presence of the microorganism in the test sample, from which the target molecule originated.
  • test sample Once the test sample 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 target molecule, of the test sample, if present, to bind to one, or more, capture probes, resulting in target molecule/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.
  • 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 matrix can be provided in a disposable container (e.g., a cassette). Positional arrangements of immobilized capture probes useful for the methods described herein can also be provided in a number of different formats.
  • the matrix can contain one, or more, capture probes, homogeneously distributed throughout the entire matrix or, alternatively, in one, or more, discrete regions of the matrix. Also, two, or more, regions of similar (or different) immobilized capture probes, or combinations of probes, can be positioned such that a sample migrates through a sequence of capture probe regions when migrating through the matrix.
  • multiple regions of immobilized capture probes can be positioned such as to form two, or more, migration paths, each of which passes through one, or a sequence of, immobilized capture probe regions.
  • Nucleic acid capture probes complementary to one, or more, putative mutant target molecules contained within a test sample are immobilized within an electrophoretic medium forming a capture layer.
  • the test sample is applied to an electrophoretic medium containing the capture layer and subjected to electrophoresis.
  • complementary single-stranded nucleic acids i.e., putative mutant target molecule
  • the capture probes can be designed specifically to detect sequence mutations within a target molecule.
  • the putative mutant target molecule as it enters the capture layer will hybridize to an immobilized probe designed to be complementary to the mutant target molecule. As a result of this hybridization, the mutant target will be immobilized to the gel.
  • the wild-type target molecule, or target molecules not possessing the specific mutation desired to be detected, will pass through the capture layer. It is conceivable that more than one mutation site can be detected. In this embodiment, multiple capture layers containing immobilized probes specific for a particular mutation site can be formed within an electrophoretic medium.
  • a method of detecting the presence or absence of a microorganism in a test sample by detecting the presence or absence of one, or more, microbiological target molecules using an adapter molecule is disclosed.
  • the microbial target molecule is mixed with an adapter molecule that has at least one nucleotide sequence region specific for the target molecule.
  • the adapter/target complex is then subjected to electrophoresis.
  • the complex will continue to migrate in the electrophoretic medium until it comes in contact with an immobilized capture probe which is specific for the adapter molecule.
  • the adapter molecule has a second nucleotide sequence region which is specific for a particular class of capture probes.
  • a higher complex is formed between the adapter/target complex and the appropriate immobilized capture probe. Detection of this tripartite complex is indicative of the presence of the microbial target molecule in the test sample, and therefore, the presence of a microorganism in the test sample.
  • the instant invention discloses a method of detecting the presence or absence of a microorganism in a test sample by detecting the presence or absence of one, or more, microbiological target molecule using a signal polynucleotide displacement reaction.
  • a complex is formed between a capture probe and a signal polynucleotide.
  • the capture probe can be immobilized through an electrophoretic medium, or immobilized in one, or more, discrete regions in the medium.
  • the target molecule is introduced to the electrophoresis medium and undergoes electrophoresis.
  • the signal polynucleotide In the presence of the target molecule, the signal polynucleotide is displaced either by the concentration of the target molecules, or it can be displaced due to the capture probe having higher affinity for the target than for the signal polynucleotide. Detecting the presence of the liberated signal polynucleotides indicates the presence of the microbial target molecule in the test sample, and therefore, the presence of a microorganism in the test sample.
  • a method for detecting the presence or absence of a target molecule in a test sample using a displacement assay is disclosed.
  • a test sample containing one, or more target molecules is subjected to electrophoresis in a medium containing immobilized capture probes.
  • These capture probes are composed of two components.
  • the first component is a single-stranded nucleic acid molecule that is detectably labeled. This signal molecule is typically shorter than the second component.
  • the second component is a tether nucleic acid molecule.
  • This tether molecule contains a nucleotide sequence region which is complementary to a nucleotide sequence region contained within the target molecule.
  • the signal molecule hybridizes to a portion of this complementary region of the tether molecule. Together the signal and tether form a probe complex.
  • the tether molecule is immobilized within an electrophoretic medium. As the target molecule migrates under electrophoresis it can come into contact with the probe complex. The target molecule will displace the signal from the tether and hybridize to the tether molecule. The signal molecule will continue to migrate until the electric field is terminated. Detection of the single-stranded signal molecule is indicative of the presence of one, or more target molecules in the test sample. In another embodiment, a method for detecting the presence or absence of a target molecule within a test sample using a reverse displacement assay is disclosed.
  • a test sample containing one, or more, target molecules is subjected to electrophoresis.
  • the electrophoretic medium contains immobilized capture probes. These capture probes comprise two components.
  • the first component is a signal nucleic acid molecule which is detectably labeled.
  • the second component is a tether nucleic acid molecule.
  • the signal molecule is complementary to a nucleotide sequence contained within the target molecule.
  • the tether molecule and signal molecule form a hybrid, this hybrid is referred to as a probe complex.
  • the signal molecule is displaced from the complex and hybridizes with the target molecule. This new hybrid can continue to migrate until the electric field is terminated. Detection of this hybrid is indicative of one, or more, target molecules in the test sample.
  • FIG. 1 is a schematic representation showing the different stages involved in detecting bacterial target molecules.
  • FIG. 2 is a schematic representation illustrating the preparation of potential bacterial target molecules from a platelet-containing source.
  • FIG. 3 is a schematic representation illustrating a sandwich assay.
  • FIG. 4 is a schematic representation illustrating a strand displacement assay.
  • FIG. 5 is a photograph of a gel showing the results of a sandwich assay for a target molecule.
  • FIG. 6 is a photograph of a gel showing the results of a single displacement assay for a target molecule.
  • FIG. 7 is a photograph of a gel showing the results of a displacement assay using a capture probe immobilized in a discrete layer within the medium and two target molecules complementary to the capture probe, and one noncomplementary target molecule.
  • FIG. 8 A and B illustrate the analysis of mutations using solid phase technology
  • FIG. 8A is a gel obtained from gel analysis of detecting mutations within target molecules
  • FIG. 8B are profiles for 1 1 different target molecules used in this experiment.
  • FIG. 9 is a photograph of a gel showing the results of an experiment for detecting mutations in PCR product, specifically, in the HIV reverse transcriptase gene.
  • Fig. 10 is a photograph of a gel showing the results of an experiment using a multiple capture layer gel.
  • the present invention relates to electrophoretic methods for analyzing specific binding reactions in which capture probes are immobilized in (e.g., covalently attached) an electrophoretic matrix. Specifically, the present invention pertains to methods for detecting the presence or absence of a microorganism in a test sample by detecting the presence or absence of one, or more, microbial target molecules, including bacterial, viral, fungal, and parasitic target molecules.
  • the capture probes are nucleic acids, modified nucleic acids or nucleic acid analogs
  • FIG. 1 is a schematic representation illustrating some of the stages involved in detecting the presence or absence of a microorganism by detecting the presence or absence of one, or more, microbial target molecules in a test sample.
  • Suitable sample preparation involves steps taken to lyse the microorganism, or microorganisms, thereby releasing their biochemical contents, including proteins, peptides and nucleic acids.
  • the host cell can be subjected to lysis in order to liberate the microbial target molecules, for example, a viral invasion of a mammalian host cell demonstrated by rhinoviruses or adenoviruses infecting epithelial cells of the respiratory system.
  • the microbial target molecule can be specific for a particular microbe, for example, E.
  • coli or can be representative of a class of microorganisms, for example, bacteria (using 16S rRNA which is common to most bacteria in order to detect bacterial contamination).
  • the presence of one, or more, microbial target molecules is indicative of the presence of microbial contamination in a test sample.
  • deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are contemplated to be within the scope of the present invention.
  • RNA ribonucleic acid
  • RNA ribonucleic acid
  • RNA ribonucleic acid
  • a bacterial target preferably the RNA is liberated from the constraints of the cell wall and cell membrane.
  • bacterial DNA for example plasmid DNA, is also contemplated to be within the scope of the present invention.
  • both DNA and RNA molecules can be subjected to analysis by first being liberated from the host cell or viral coat depending upon the stage of viral infection.
  • additional steps can be taken in order to purify further the target molecule desired from a contaminating microorganism, such as cell lysis followed by semi -purification of the lysate.
  • the desired microbial target molecule is double-stranded DNA
  • the preparation can be treated, for example, employing S 1 nuclease, to eliminate single-stranded nucleic acid species from the test sample to be analyzed.
  • Figure 2 is a schematic representation illustrating the preparation of microbial target molecule specifically bacterial target molecules from a platelet-containing source.
  • ELECTROPHORETIC MATRICES Any matrix suitable for electrophoresis can be used for the methods of the present invention. Suitable matrices include acrylamide and agarose, both commonly used for nucleic acid electrophoresis. However, other materials can 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 Polysciences, 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 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 an example is the composite acrylamide-agarose gel.
  • These gels typically contain from 2-5% acrylamide and 0.5%- 1% agarose.
  • 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.
  • 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.
  • soluble polymers For many applications gel-forming matrices such as agarose and cross-linked polyacrylamide will be preferred. However, for capillary electrophoresis (CE) applications it is convenient and reproducible to use soluble polymers as electrophoretic matrices.
  • soluble polymers that have proven to be useful for CE analysis are linear polymers of polyacrylamide, poly(N,N- dimethylacrylamide), poly(hydroxyethylcellulose), poly(ethyleneoxide) and poly(vinylalcohol) as described in Quesada (Current Opinion in Biotechnology, 8:82-93 (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. Serial No.
  • 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 can 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 can 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 can be induced from one end of the tube while adding more liquid material to the other end to compensate for shrinkage.
  • Such progressive polymerization can 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 can 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 can 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 can 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 can be effected by known means including positional programmable dropper techniques.
  • Gel shrinkage during polymerization can 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 can 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 can be formed by a number of techniques. Multiple linear strips or two-dimensional layers can 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, can be formed as above and assembled together to provide a three-dimensional structure.
  • the capture probes of the present invention comprise a nucleic acid with a polynucleotide sequence substantially complementary to a microbial target molecule wherein the target molecule hybridizes to the capture probe.
  • the complementarity of nucleic acid capture probes need only be sufficient enough to specifically bind the target molecule and demonstrate the presence, or absence, of the microbial target molecule in a test sample, and therefore, the presence or absence of the microorganism in the test sample.
  • Probes suitable for use in the present invention comprise RNA, DNA, nucleic acid analogs, modified nucleic acids and chimeric probes of a mixed class comprising a nucleic acid with another organic component, e.g., peptide nucleic acids.
  • Capture probes can be single-stranded or double-stranded nucleic acids.
  • the length of a capture probe will be at least 5 nucleotides in length, more typically between 5 and 50 nucleotides, and can be as long as several thousand bases in length.
  • 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 can have undergone further processing. Isolated nucleic acids include nucleic acids obtained by methods known to those of skill in the art.
  • nucleic acids include substantially pure nucleic acids (i.e., nucleic acids free from protein,' carbohydrate or lipid associations), nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods and recombinant nucleic acids which are isolated.
  • Modified nucleic acid 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 can 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,
  • Nucleic acid analogs as used herein include molecules which lack a sugar- phosphate backbone, but retain the ability to form complexes via base-pairing. Such nucleic acid analogs are known to those of skill in the art.
  • 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, 254:1497-1500, (1991)).
  • PNA peptide nucleic acid
  • the peptide backbone is capable of holding the bases at the proper distance to base pair with standard DNA and RNA single strands.
  • 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 skilled 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.
  • substantially complementary means that the polynucleotide sequence of the capture probe need not reflect the exact polynucleotide sequence of the microbial target molecule, but must be sufficiently complementary to hybridize with the target 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 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 its 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, e.g., moderate, or high stringency, can be determined empirically, depending on part of the chemical characteristics of the probe and microbial target molecule.
  • the capture probe employed should be directed at a microbial-specific molecule.
  • the probe can be directed against either the gag, pol, or env viral nucleotide sequence, or fragments thereof.
  • multiple probes can be used in that three classes of probes are employed where each class is directed against one of the viral nucleotide sequences, or fragments thereof, listed.
  • the probe can be directed against a specific bacterial DNA sequence, or a bacterial RNA sequence (e.g., a rRNA sequence).
  • the probe preferably will not react with human RNA, but should react with RNA from the bacterial specie that has contaminated the platelets.
  • RNA from the bacterial specie that has contaminated the platelets.
  • a single-stranded microbial target molecule and a single-stranded immobilized capture probe are used.
  • This embodiment is especially useful for analysis of microbial RNA target molecules. It is also useful for capture of specific targets from complex samples where renaturation of the target is not rapid. Highly concentrated microbial targets, such as PCR products, can 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 greater 70°C for five minutes immediately prior to electrophoresis.
  • a double-stranded microbial target is captured by a single-stranded immobilized capture probe.
  • capture probes 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 probe 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 microbial target nucleic acids can be specifically captured using this probe design strategy.
  • capture probes can be designed that will associate with double- stranded nucleic acids by formation of a displacement loop structure. Such probes bind to only one strand of the duplex nucleic acid and displace the probe- homologous duplex strand of the duplex locally. This displacement can only be achieved if the probe-target strand interaction is much more favorable than the interaction between the target strands.
  • Such probes can be made using modified bases and techniques described in Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991), backbone modifications (Moody, et al, Nucleic Acids Res., 17:4769-4782 (1989)) and nucleic acid analogs (Nielson, et al, Science, 254:1497-1500 (1991)).
  • PNA peptide nucleic acid
  • substantially all of the medium is modified with capture probe, or probes.
  • capture probe or probes.
  • the choice of capture probe and electrophoresis conditions are made such that the binding between capture probe and target molecule is transient and rapidly reversible on the time scale of an electrophoretic analysis. Under these conditions, target molecules undergo many cycles of binding, release, and rebinding to the capture probes during the electrophoresis run. This reversible binding has the effect of reducing the electrophoretic mobility of the target measured relative to its mobility in the absence of capture probe. If binding to the capture probe is strong, the mobility of the target is substantially reduced. If binding to the capture probe is weak, target mobility is only slightly reduced.
  • 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 probe.
  • microbial target nucleic acids that are complementary to the capture probe hybridize to the capture probe contained within a gel layer and are 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 sample nucleic acids is detected within the capture layer by appropriate labeling strategies described herein.
  • sample volume is not important.
  • all sample nucleic acids 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 microorganisms need to be detected.
  • the presence and/or identification of any bacteria in culture or in a blood sample (e.g., platelets) that is to be used for transfusion can employ this embodiment. Therefore, a general test for any bacteria might use a collection of conserved bacterial gene sequences common to most, if not all, bacteria as capture probes. Since identification of the specific bacteria may not necessarily be important, the collection of probes could comprise a broad spectrum of multiple probes specific to various bacteria which ensures that any and all bacteria will be detected in the same capture layer.
  • Multiple capture layers can 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. During the assay, the test sample is subjected to electrophoresis through all of the layers, and complementary sample target nucleic acids are captured at each layer. The amount of hybrid in each layer directly reflects the sample composition with respect to the capture probes used. For example, if there are different, discrete layers of immobilized capture probes each layer being specific for a particular species of bacteria, then when a test sample is subjected to electrophoresis, if a complementary species of bacteria is present, then it will be detected by a specific class of immobilized capture probe specific for that particular species of bacteria.
  • an electrophoresis medium is constructed such that there are three discrete layers comprising immobilize capture probes. Each layer is specific for one of three bacteria, for example, layer one contains capture probes specific for E. coli, layer two contains capture probes specific for S. aureus, and layer three contains capture probes specific for S. pyogenes.
  • the test sample is then subjected to electrophoresis. If the test sample is contaminated with E. coli and S.
  • 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
  • test sample 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.
  • sample nucleic acids are present at high copy numbers, and so this problem does not present a significant obstacle.
  • hybridization methods described herein can also encompass three- dimensional arrays, such as can be particularly useful for multiplexed parallel assays, for example, high throughput and/or cost-effectiveness.
  • Such assays can be provided in the format of three-dimensional solids, where multiple samples can 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 can be produced such that each sample encounters the same sequence of capture probes during migration through the array, alternatively, different sequences of capture probes can be positioned for this purpose, such as to analyze different sample mixtures or to analyze differing sets of components within one, or more, sample mixtures DETECTION OF MUTATIONS
  • the invention pertains to a method of detecting mutations within the nucleotide sequence of a target molecule. For example, one may desire to determine whether a bacteria, virus, parasite, etc., has mutated. Detecting mutations within a microorganism's genome can lead to significant information concerning that microbe. For example, it is well established that bacteria can become resistant to antibiotics by receiving genetic material which conveys resistance. Certain viruses are notorious for possessing fast mutation rates, such as retroviruses. Viruses responsible for the common cold (e.g., adenovirus) can mutate quite readily between hosts.
  • adenovirus e.g., adenovirus
  • the test sample contains one, or more, putative mutant target molecules, that is, target nucleic acids that have at least one mutation site contained within their nucleotide sequence.
  • the mutation site can contain one, or more, base mutations within the nucleotide sequence.
  • the capture probe immobilized within an electrophoretic medium contains a nucleotide sequence region which is complementary to a particular mutation site contained within a mutant target molecule.
  • the capture probe can be found throughout the matrix in a uniform concentration.
  • the capture probes are immobilized in one, or more, discrete regions of the electrophoretic medium. Multiple mutant target molecules containing different mutation sites can be detected using immobilized capture probes containing nucleotide sequence regions corresponding to, or complementary with, the different mutation sites contained within the various mutant target molecules. (Kenney, M. et al, BioTechniques, 25 (3):516-521 (1998), the teachings of which are herein incorporated in its entirety). It is also conceivable that one mutant target molecule can contain more than one mutation site.
  • These mutation sites can be detected by multiple capture probes designed to be complementary to one specific mutation site contained within the mutant target molecule.
  • the multiple capture probes can be immobilized in different capture layers specific for the detection of one specific mutation site within the electrophoretic medium.
  • the electrophoretic mobility of the test sample nucleic acid is influenced by the extent of complementarity with the immobilized probe. Target molecules with complete complementarity to the probe will become immobilized via the capture probe to the electrophoretic medium, and those target molecules with less complementarity will pass through the capture layer. (Kenney, M. et al, BioTechniques, 25 (3):516-521 (1998)). Samples can be conveniently prepared and labeled by PCR amplification using labeled nucleotide triphosphates prior to solid phase hybridization analysis.
  • the longer single-stranded nucleic acid portion of the probe is complementary to a nucleotide sequence region contained within the target.
  • the tether is from about 5 to about 100 nucleotides in length.
  • the other component of the probe is a shorter single-stranded, detectably-labeled nucleic acid, hereinafter referred to as the "signal" nucleic acid, which is complementary to a specific subsequence of the unlabeled tether component (the same subsequence that is complementary to a nucleotide sequence region contained within the target molecule).
  • the signal is from about 5 to about 50 nucleotides in length.
  • the sequence of the signal nucleic acid is identical in sequence to (or, very similar to) a portion of the target.
  • the signal and tether single strands form a hybrid in which the signal nucleic acid is completely base-paired with the tether, and the tether contains a single-stranded region which is available for hybridization with the target.
  • the probe is incubated with a test sample containing one, or more, target molecules, and the targets hybridize to the single-stranded portion of the tether component.
  • the target Since the target is homologous to the entire length of the tether, the target will initiate a homologous strand exchange reaction with the signal nucleic acid, and will displace it from the tether.
  • the strand exchange reaction proceeds rapidly in the direction of signal nucleic acid displacement because the target is longer and forms a more stable duplex with the tether.
  • One skilled in the art can find experimental conditions under which the amount of displaced labeled signal nucleic acid accurately reflects the amount of target in the sample. (See U.S. Patent No. 4,766,062; U.S. Patent No. 4,766,064; Vary, Nucleic Acids Res., 15:6883-6897 (1987); Vary et al, Clinical Chemistry, 32:1696-1701 (1986)).
  • a simplified process for performing microbiological assays using a displacement method is disclosed.
  • the tether component of the displacement probe complex is immobilized within an electrophoresis matrix.
  • target molecules are contacted, under conditions suitable for hybridization, with the probe complex (comprising the hybrid signal and tether nucleic acids) by subjecting them to electrophoresis through the matrix containing capture probes.
  • the probe complex comprising the hybrid signal and tether nucleic acids
  • the target molecule displaces the signal component of the probe complex and hybridizes to the immobilized tethered component of the complex.
  • the displaced signal probe component is detected and quantified to determine the presence and number of target molecules in the original test sample.
  • reverse displacement is described for the detection of one, or more target molecules in a test sample.
  • the displacement probe complex is composed of two nucleic acids: a "signal" nucleic acid and a "tether" nucleic acid.
  • the signal is complementary to the target nucleic acids.
  • the tether nucleic acid is complementary to a specific subsequence of the signal nucleic acid.
  • the tether nucleic acid is therefore identical, or substantially similar in sequence, to a specific subsequence of the target.
  • the signal nucleic acid is detectably labeled.
  • a target nucleic acid When a target nucleic acid is subjected to electrophoresis through an electrophoretic medium containing immobilized displacement probe complexes and comes into contact, under conditions suitable for hybridization, with the probe complex, then the signal nucleic acid will hybridize with the target, and the signal nucleic acid is displaced from the tether nucleic acid by homologous strand exchange.
  • the product of the reverse displacement reaction is a hybrid between the signal nucleic acid and the target. This hybrid will migrate from the capture probe layer, and can be detected and quantified as it migrates away from the immobilized displacement probe layer.
  • One principle advantage of these displacement methods is that the target molecules do not need to be detectably labeled.
  • the target molecule displacement reaction results in the migration of a detectably labeled probe molecule in the electrophoretic matrix.
  • labeling probes many different methods have been described, both direct, such as radioactivity, fluorescent moieties, chemiluminescent moieties, direct enzyme conjugates; and indirect, such as affinity labels for use with secondary or tertiary labeled molecules.
  • the reverse displacement assay is not label- specific and any practicable label can be used.
  • the target-dependent probe displacement can be detected by changes in the properties of the tether nucleic acid or by changes in the properties of the signal -tether complex that are altered by the displacement reaction.
  • probe displacement can be accomplished by other means, including, but not limited to, detecting the value or change in value of any chemical or physical property of a substance.
  • Such values or changes in value may include, but not limited to, the following: physical measurements (such as mass or density measurement, mass spectrometry, plasmon resonance), optical detection (such as emission, absorption, fluorescence, phosphorescence, luminescence, chemiluminescence, polarization, refractive index changes, etc.), electrical conductivity, absorption or emission of other electromagnetic energy, radioactivity, and induced changes in solution properties (such as viscosity, turbidity, optical rotation), and alike.
  • physical measurements such as mass or density measurement, mass spectrometry, plasmon resonance
  • optical detection such as emission, absorption, fluorescence, phosphorescence, luminescence, chemiluminescence, polarization, refractive index changes, etc.
  • electrical conductivity such as absorption or emission of other electromagnetic energy, radioactivity, and induced changes in solution properties (such as viscosity, turbidity, optical rotation), and alike.
  • Detection of the specific binding reaction for example, detection of the immobilized microbial target molecule bound to the capture probe, can be accomplished in a number of different ways.
  • the test molecule can be detectably labeled prior to the binding reaction.
  • 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.
  • a ligand which can be recognized by a second specific binding entity which is either labeled itself or can produce a detectable signal.
  • 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
  • digoxigenin system which uses an anti-digoxigenin antibody, conjugated to alkaline phosphatase, which recognizes digoxigenin-dUTP incorporated into nucleic acids.
  • 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 microbial target which does not overlap the region recognized by the capture probe.
  • the sandwich probe is designed to remain associated with the target during electrophoresis, and cannot bind directly to the capture probe.
  • Sandwich probes can also be used to label target molecules after electrophoretic capture.
  • the unlabeled target is subjected to electrophoresis and hybridized to the capture probes first.
  • the sandwich probe is subjected to electrophoresis through the capture layer.
  • the captured target 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.
  • known means such as optical detection of intercalating dyes, or by detection of radioactivity from hybridized radioactive species, or other known means.
  • 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 can be measured by mechanically or optically scanning a single detector along the array of detectable signals. Alternatively, a linear array of detectable signals can 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 can be employed.
  • a single detector can be used to measure the signal at each point by mechanical or optical scanning, or by any combination.
  • a linear optical detection array can be used to detect a set of signals by juxtaposition or optical imaging, and multiple sets of such signals can be detected by mechanically or optically scanning the signal array or detector.
  • the two-dimensional array of capture probes can 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 can 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 can 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.
  • EXAMPLE 1 Gel-based sandwich assay for bacterial contamination in platelet concentrates Platelet concentrates suspected of microbial contamination were prepared by mixing 250 ⁇ L with 250 mL of buffer solution (200 mM sodium phosphate buffer, pH 7.0, 2 mM EDTA, 2% sodium dodecyl sulfate (SDS), 0.1% heparin) containing Escherica coli (E. coli) cells. The E.
  • buffer solution 200 mM sodium phosphate buffer, pH 7.0, 2 mM EDTA, 2% sodium dodecyl sulfate (SDS), 0.1% heparin
  • coli cells were prepared by inoculating 2 mL of liquid LB broth (Maniatis, et al, Molecular Cloning: A Laboratory Manual, 2 nd ed., 1989, Cold Spring Harbor Press, Cold Spring Harbor, NY) with a colony of bacteria from a LB agar plate culture, and grown overnight at 37°C with shaking. The next morning the overnight culture was diluted 100-fold with fresh LB medium and grown to an optical absorbance of 2.4 at 600 nm.
  • liquid LB broth Maniatis, et al, Molecular Cloning: A Laboratory Manual, 2 nd ed., 1989, Cold Spring Harbor Press, Cold Spring Harbor, NY
  • the culture was pelleted in a microcentrifuge (10,000 x g) for 30 seconds and resuspended in ice-cold TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) to a density of 4x10 9 cells per mL. Aliquots of platelet concentrate were spiked with the washed E. coli cells as follows:
  • the adapter probe is a 41-mer DNA polynucleotide in which the 5'-most 21 nucleotides are complementary to bases 65 through 41 of E. coifs 4.5S RNA, and the 3 '-most 20 nucleotides are complementary to the gel immobilized capture probe polynucleotide.
  • the bacterial target polynucleotide used in this example is a nucleotide sequence from E. coifs 4.5S RNA and is as follows:
  • the gel was poured in three sections.
  • the central layer of the gel contained an immobilized capture probe polynucleotide (or simply capture probe, 5'- acrylamide-AGG CCC GGG AAC GTA TTC AC-3' [SEQ ID No. 4]).
  • the acrylamide-modified probe was included in the acrylamide gel mixture during polymerization with the polyacrylamide matrix.
  • the acrylamide group was added to the capture probe using a commercially available acrylamide phosphoramidite (AcryditeTM, Mosaic Technologies, Boston, MA). The concentration of capture probe in the capture layer was 4 ⁇ M.
  • the upper and lower sections of the gel contained no capture probe.
  • the gel was approximately 0.75 mm thick by 10 cm long by 10 cm wide. Electrophoresis was performed at 200 V for 90 minutes at a gel temperature of 34°C (Penguin apparatus, Owl Scientific, Woburn, MA).
  • the gel was removed from the glass electrophoresis plates and washed for 10 minutes in AP buffer (2.4 M diethylamine- HC1 buffer, pH 1 IN mM MgCl 2 , 0.1 mM ZnCl 2 ). The gel was then soaked in 1.5 mL of Attophos chemifluorescent AP substrate (Attophos, Lumigen, Boehringer Mannheim Biochemicals, Indianapolis, IN) for 10 minutes. Fluorescent AP products were imaged using a two dimensional fluorescence scanner (Fluorimager 595, Molecular Dynamics, Sunnyvale, CA).
  • the fluorescence image of the gel is shown in FIG. 5.
  • the position of the capture layer, which extends horizontally across the gel, is indicated by bracket "7".
  • the samples were loaded in the following manner: lane 1 : prehybridized lysate containing 10 7 E. coli cells, lane 2: no sample loaded, lane 3 : prehybridized lysate containing 10 6 E. coli cells, lane 4: prehybridized lysate containing 10 5 E. coli cells, lane 5: prehybridized lysate containing 10 4 E. coli cells, lane 6: prehybridized lysate without added E. coli cells.
  • EXAMPLE 2 Strand displacement detection of bacterial RNA by gel hybridization Total bacterial RNA from Enterobacter cloacae, Pseudomonas aeruginsoa, Serratia marcescens, Bacillus cereus, Escherichia coli, Staphylococcus epidermidis and Staphylococcus aureus was purified by extraction from cultured cells using hot phenol.
  • a 5 mL nutrient broth (Difco, distributed by Fisher Scientific, Pittsburg, PA) culture was grown overnight at 37°C starting with a single isolated colony obtained from a nutrient agar plate. Following this overnight culture, a 100 mL nutrient broth was inoculated with the overnight culture. This new culture was allowed to grow for 4 hours, then the bacteria was harvested by using centrifugation (6000 x g). The bacterial cells were resuspended in 5 mL of sodium acetate buffer (pH 5.2, 20 mM sodium acetate, 1 mM EDTA).
  • RNA was then recovered from the aqueous phase by two consecutive ethanol precipitations using a 10% volume solution of 3 M sodium acetate (pH 5.2, 3 M sodium acetate) and 2.5 volumes of ethanol.
  • the recovered RNA was then dissolved in approximately 1-2 mL TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and stored at -20°C.
  • the concentration of RNA was determined by absorbance at 260 nm assuming 33 ⁇ g/mL per 1 OD 260 unit. The RNA concentrations ranged from 1-3 ⁇ g/ ⁇ L in the final solutions.
  • the electrophoresis gel was poured in three sections.
  • the central layer of the gel contained an immobilized, partially double stranded displacement probe complex formed by hybridizing an immobilizable tether capture probe polynucleotide (5'- acrylamide- GGT AAG GTT CTT CGC GTT GCA TCG AAT TAA ACC ACA TGC TCC ACC GCT TGT G-3' [SEQ ID No. 5]) with a fluorescent signal polynucleotide (5'-Cy3- CAC AAG CGG TGG AGC ATG TGG TTT-3' [SEQ ID No. 6], where Cy3 indicates the position of fluorescent phosphoramidite).
  • an immobilizable tether capture probe polynucleotide (5'- acrylamide- GGT AAG GTT CTT CGC GTT GCA TCG AAT TAA ACC ACA TGC TCC ACC GCT TGT G-3' [SEQ ID No. 5]
  • the acrylamide-modified displacement complex was included in the acrylamide gel mixture during polymerization, and became immobilized in the layer by copolymerization with the polyacrylamide matrix.
  • the acrylamide group was added to the tether polynucleotide using a commercially available acrylamide phosphoramidite (AcryditeTM, Mosaic Technologies, Boston, MA).
  • the concentration of displacement probe complex in the capture layer was 2 ⁇ M.
  • the upper and lower sections of the gel contained no capture probe.
  • the gel was approximately 0.75 mm thick by 10 cm long by 10 cm wide.
  • E. coli 16S rRNA nucleotide sequence used in this example was as follows: 5'-CAC AAG CGG TGG AGC ATG TGG TTT AAT TCG ATG CAA CGC GAA GAA CCT TAC C-3' (Genbank Accession # M24386; rRNA nucleotide sequence positions 933-984, [S ⁇ Q ID No. 7]). The 5'-end of this 16S rRNA from E.
  • E. coli 16S rRNA nucleotide sequence has a greater degree of complementarity with the immobilized capture probe than does the fluorescent signal due to its increased base-pairing with the capture probe polynucleotide.
  • the target polynucleotide when the target polynucleotide comes in contact with the capture probe/signal polynucleotide complex, the target polynucleotide will replace the signal polynucleotide and a new complex will be formed between the target polynucleotide and the capture probe polynucleotide.
  • RNA samples were run on a neutral 1 x TB ⁇ (89 mM Tris-borate, pH 8.3, 2 mM ⁇ DTA) 6% polyacrylamide gels (29:1 monome ⁇ bisacrylamide, wt:wt). Approximately 30-40 ⁇ g of total RNA was run in each lane of the gel. Negative and positive control samples comprising human 18S rRNA sequences and E. coli 16S rRNA sequences, respectively, were produced by in vitro transcription of appropriate vectors using T7 RNA polymerase. These in vitro transcripts were also hydrolyzed with NaOH, neutralized, and subjected to electrophoresis in parallel with the authentic bacterial RNA samples.
  • Electrophoresis was performed at 100 V using a gel temperature of 35°C (Penguin apparatus, Owl Scientific, Woburn, MA). Following electrophoresis, the gel was removed from the glass plates and Cy3 dye fluorescence (Amersham Pharmacia Biotech, Piscataway, NJ) was measured using a gel scanner (Molecular Dynamics Fluorimager 595, Sunnyvale, CA).
  • Figure 6 shows the fluorescent image of the gel. Approximately 30-40 ⁇ g of RNA was added to each lane, except for the lanes containing in vitro transcript controls. Approximately 8 picomoles of human, and 4 picomoles of E. coli in vitro transcripts were used for controls (right lanes). As can be observed, the E. coli in vitro transcript and all other bacterial RNAs were able to displace the fluorescent signal polynucleotide, which is now shown in the signal layer, from the displacement complex. In contrast, the human 18S rRNA in vitro transcript was unable to displace the signal because of the lack of nucleotide sequence complementarity with the displacement complex. Thus, the displacement assay shown is capable of detecting bacterial RNA of broad phylogenetic diversity without interference from human rRNA.
  • EXAMPLE 3 Mutation detection using solid phase analysis Polynucleotides containing 5'-terminal Acrydite groups were used in the present example (Research Genetics, Huntsville, AL; Eurogentec, Seraing, Belgium;
  • Lyophilized polynucleotides were dissolved in TE buffer (10 mM Tris-HCl, pH 8.3, 1 mM EDTA) and stored frozen at -20°C. Concentrations were determined from
  • FI and "Ac” represent fluorescein and Acrydite modifications, respectively.
  • the capture probe used in the first experiment designed to demonstrate the efficiency of hybridization (viz, capture) using fluorescein-labeled target molecules was 5'-Ac- GTA CCA TAA CAG CAA GCC TCA-3' [SEQ ID NO. 8], and the target polynucleotides used were as follows: (i) left complementary lane, 5'-Fl- TGA GGC TTG CTG TTA TGG TAC-3' [SEQ ID NO. 9]; (ii) right complementary lane, 5'-Fl- TGA GGC TTG CTT TTA TGG TAC-3' [SEQ ID NO.
  • the target molecule used in the c:t,5 lane is: 5'-Fl-TGA GTC TTG CTG TTA TGG TAC-3' [SEQ ID NO. 13]
  • the target used in the c:t,5;a:a,7;a:c,14 lane is : 5'-Fl-TGA GJC ATG CTG TCA TGG TAC-3' [SEQ ID NO. 14].
  • the underlined positions differ from the fully complementary target molecule.
  • the noncomplementary target molecule in this experiment was the same as that used in the previous experiment.
  • the capture probes were: (i) A', 5'-Ac- GTT TTC AGC TCC ACC TAC CAC AAG T-3 * [SEQ ID NO. 17]; (ii) B', 5'-Ac- GTT TTA GCT CCA ACT ACC ACA AGT T-3' [SEQ ID NO. 18]; (iii) C, 5'-Ac- GTT TTG CAC CTC AAA GCT GTT CCG T-3' [SEQ ID NO. 19]; and (iv) D', 5'-Ac-GTT TTG TTC ATG CCG CCC ATG CAG G-3' [SEQ ID NO.
  • Target molecules were: (i) A, 5'-Fl-AC TTG TGG TAG TTG GAG CTG-3' [SEQ ID NO. 21]; (ii) B, 5'-Fl-AA CTT GTG GTA GTT GGA GCT-3' [SEQ ID NO. 22]; (iii) C, 5'-Fl-AC GGA ACA GCT TTG AGG TGC-3' SEQ ID NO. 23]; and (iv) D, 5'-Fl-CC TGC ATG GGC GGC ATG AAC-3' [SEQ ID NO. 24].
  • Target molecules A and B share a common nucleotide sequence of 19 bases. (See FIG. 10).
  • Acrylamide gels (5%-20% total acryamide, 29:1 monomer to bis-acrylamide) were prepared in 0.5 x TBE buffer (45 mM Tris-borate, pH 8.3, 1 mM EDTA) and polymerized by addition of 7 ⁇ L 10% aqueous ammonium persulfate and 2 ⁇ L N,N,N',N'-tetramethylenediamine (TEMED) per mL of gel solution.
  • Standard minigel systems were used (Mini-PROTEAN® II, 8 x 10 cm plates (Bio-Rad) and PenguinTM, 10 x 10 cm plates (Owl Scientific, Woburn, MA) with spacers ranging in thickness between 0.8-1.5 mm.
  • the gels were cast in three sections. First, the lower unmodified gel was poured and allowed to polymerize. Next, a capture layer containing
  • Acrydite-modified capture probes was polymerized over the unmodified gel. To ensure even polymerization of the layer, the volume of the capture layer was approximately 0.5 cm. Acrydite capture probes (10 ⁇ M final concentration) were mixed with the unpolymerized gel solution before catalyst addition. Following polymerization of the capture layer, the gel surface was rinsed thoroughly with 05. X TBE, and the remainder of the gel cassette was filled with unmodified gel.
  • Non-denaturing acrylamide gels were run in 0.5 x TBE buffer at applied voltages of 100-150 V (gel lengths were 8-10 cm). Gel were pre-run for 15-20 minutes before sample loading. Fluorescein-labeled target molecules were mixed with sucrose-containing buffer and loaded without preheating. Labeled PCR products, usually 5 ⁇ L from a 50 ⁇ L reaction, were brought to 75% formamide (vol/vol) and heated to 90°C for 2 minutes before loading.
  • Plasmids containing mutant (pKRT67/70/215/219, pKRT41/215/219) and wild-type (pKRTwt) subclones of the HIV reverse transcriptase gene (HIV-RT) were used. Plasmid samples were restricted with Hindlll, and 10 6 copies of the restricted material were used as initial PCR targets. Amplification was carried out for 30 cycles consisting of 10 seconds at 94°C, 10 seconds at 50°C and 1 minute at 72°C. The reaction mixture contained 10 mM Tris-HCl (pH 8.3), 50 mM KC1, 2.5 mM MgCl 2 .
  • a 249-bp target molecule containing the codon 41 region of HIV-RT was amplified using primers 5'-GGA TGG CCC AAA AGT TA-3' [SEQ ID NO. 27], and 5'-CCT GCG GGA TGT GGT ATT C-3' [SEQ ID NO. 28].
  • FIG. 7 illustrates the efficiency of hybridization using fluorescein-labeled target molecules.
  • the results demonstrate that the complementary samples (C lanes) were completely immobilized on the capture layers that contain capture probes, while noncomplementary target molecules (N lanes) were not retained.
  • Figure 8 shows a series of target molecules containing different multiple and single base mismatches with the capture probe run in Acrydite gels at various temperatures.
  • the high stringency achieved with Acrydite gel assays is demonstrated by the fact that the near-terminal mutant target smears into (and through) the capture layer because of its weakened binding under conditions where the fully complementary target forms a tight band.
  • Figure 8A shows a typical gel run at 40°C
  • FIG. 8B provides a graphical representation of the entire data set. As seen in FIG. 8B, the mismatches fell into several classes. Most were sufficiently destabilized such that a gel temperature of 40°C prevented capture. A few were still partially retained at 40°C, but were efficiently cleared from the capture layer at 45°C.
  • Figure 9 illustrate the use of Acrydite gels in typing mutations of PCR products. Specifically, two sets of mutations in HIV reverse transcriptase were detected. The codon 41 mutation is a single nucleotide change, while in the codon 215-219, mutation involves substitution of four nucleotides. Fluorescein-labeled PCR products were prepared from plasmid target molecules carrying HIV-RT fragments, denatured with formamide and then loaded onto gels containing capture probes specific for HIV-RT mutants.
  • FIG. 10 illustrates the detection of polymorphisms by using multiple capture layers.
  • Target molecules "A” and “B” share a common 19-base sequence and are both efficiently captured by the first "A”' capture layer. Complementarity of targets “A” and “B” with the “B”' capture layer can be observed from the target spillover caused by a slight overloading of the "A"' capture layer, as is best seen in the lanes where both "A” and “B” targets were loaded simultaneously (FIG. 10, far left and right lanes).
  • Targets "C” and “D” display complete specificity for layers “C” and "D”', respectively.

Landscapes

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

Abstract

L'invention porte sur des procédés de détection de la présence ou de l'absence d'un micro-organisme, ce procédé consistant à détecter, par électrophorèse, la présence ou l'absence, dans un échantillon test, de molécules cibles microbiologiques comprenant des sondes de capture immobilisées. L'invention porte également sur des procédés de détection de sites de mutation dans une molécule cible mutante putative.
EP00921605A 1999-04-02 2000-03-31 Procedes de detection de micro-organismes a l'aide de sondes immobilisees Withdrawn EP1173613A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US28609199A 1999-04-02 1999-04-02
US286091 1999-04-02
PCT/US2000/008773 WO2000060120A2 (fr) 1999-04-02 2000-03-31 Procedes de detection de micro-organismes a l'aide de sondes immobilisees

Publications (1)

Publication Number Publication Date
EP1173613A2 true EP1173613A2 (fr) 2002-01-23

Family

ID=23097034

Family Applications (1)

Application Number Title Priority Date Filing Date
EP00921605A Withdrawn EP1173613A2 (fr) 1999-04-02 2000-03-31 Procedes de detection de micro-organismes a l'aide de sondes immobilisees

Country Status (5)

Country Link
EP (1) EP1173613A2 (fr)
JP (1) JP2003503011A (fr)
AU (1) AU4189800A (fr)
CA (1) CA2371732A1 (fr)
WO (1) WO2000060120A2 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9658178B2 (en) 2012-09-28 2017-05-23 General Electric Company Sensor systems for measuring an interface level in a multi-phase fluid composition
US10598650B2 (en) 2012-08-22 2020-03-24 General Electric Company System and method for measuring an operative condition of a machine
US10684268B2 (en) 2012-09-28 2020-06-16 Bl Technologies, Inc. Sensor systems for measuring an interface level in a multi-phase fluid composition

Families Citing this family (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998051823A1 (fr) 1997-05-16 1998-11-19 Mosaic Technologies Analyse electrophoretique de molecules au moyen de sondes immobilisees
JP2002518026A (ja) * 1998-06-19 2002-06-25 エムティー テクノロジー， インコーポレイテッド Ｓｒｐｒｎａを用いる非ウイルス生物の検出
JP2003284597A (ja) * 2002-03-29 2003-10-07 Fujirebio Inc 標的核酸の測定方法及びそのためのキット
JPWO2005012571A1 (ja) * 2003-07-30 2007-09-27 財団法人理工学振興会 部分二重鎖型核酸分子
EP1508622A1 (fr) * 2003-08-22 2005-02-23 Siegfried Prof. Dr. Scherer Détection du peptide synthase impliqué dans la production de la toxine émétique de Bacillus cereus
JP2007512509A (ja) * 2003-11-05 2007-05-17 エグザクト サイエンシーズ コーポレイション 反復的親和性分離およびその使用
US9589686B2 (en) 2006-11-16 2017-03-07 General Electric Company Apparatus for detecting contaminants in a liquid and a system for use thereof
US9536122B2 (en) 2014-11-04 2017-01-03 General Electric Company Disposable multivariable sensing devices having radio frequency based sensors
US10914698B2 (en) 2006-11-16 2021-02-09 General Electric Company Sensing method and system
US20100134286A1 (en) * 2008-12-01 2010-06-03 General Electric Company Radio frequency based sensors employing analyte recognition element
US9538657B2 (en) 2012-06-29 2017-01-03 General Electric Company Resonant sensor and an associated sensing method
EP2115455B1 (fr) * 2007-02-07 2012-02-22 Lyzer Diagnostics, Inc. Immunodosage homogène rapide par électrophorèse
US7919250B2 (en) * 2007-07-31 2011-04-05 New York University Diagnostic and treatment methods for characterizing bacterial microbiota in skin conditions
CN102933720B (zh) * 2010-05-07 2015-03-18 昆特拜克股份公司 用于生成具有单链悬突的双链核酸的方法
US8542023B2 (en) 2010-11-09 2013-09-24 General Electric Company Highly selective chemical and biological sensors
KR102128248B1 (ko) 2011-06-08 2020-07-01 샤이어 휴먼 지네틱 테라피즈 인크. Mrna 전달을 위한 지질 나노입자 조성물 및 방법
EA030927B1 (ru) 2012-08-22 2018-10-31 Дженерал Электрик Компани Система и способ беспроводного измерения параметров рабочего состояния машины
AU2014215764A1 (en) * 2013-02-05 2015-09-17 Victoria Link Limited Novel bio-sensor for the detection of small molecules
CN107794232B (zh) * 2016-08-31 2022-03-08 广州金羿噬菌体生物技术有限公司 拮抗痤疮丙酸杆菌和革兰氏阳性菌生长的链球菌及其代谢产物
KR101862198B1 (ko) * 2016-09-30 2018-05-30 (주)스파크바이오파마 2차원 겔 전기영동에서의 열 안정성 변화-기반 형광 차이를 이용한 표적 단백질의 규명 방법
CN108374052A (zh) * 2018-03-20 2018-08-07 徐州工程学院 用于生物芯片检测金黄色葡萄球菌的探针组序列
US20220396824A1 (en) * 2019-09-24 2022-12-15 Genepath Diagnostics Inc. Composition and method for improving detection of biomolecules in biofluid samples

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000060118A2 (fr) * 1999-04-02 2000-10-12 Mosaic Technologies, Inc. Analyse electrophoretique de molecules cibles au moyen de molecules adaptateurs

Family Cites Families (4)

* 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
US5632957A (en) * 1993-11-01 1997-05-27 Nanogen Molecular biological diagnostic systems including electrodes
WO1998051823A1 (fr) * 1997-05-16 1998-11-19 Mosaic Technologies Analyse electrophoretique de molecules au moyen de sondes immobilisees
AU1804099A (en) * 1997-12-05 1999-06-28 Mosaic Technologies Slotted electrophoresis gel composition and methods of use thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000060118A2 (fr) * 1999-04-02 2000-10-12 Mosaic Technologies, Inc. Analyse electrophoretique de molecules cibles au moyen de molecules adaptateurs

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO0060120A3 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10598650B2 (en) 2012-08-22 2020-03-24 General Electric Company System and method for measuring an operative condition of a machine
US9658178B2 (en) 2012-09-28 2017-05-23 General Electric Company Sensor systems for measuring an interface level in a multi-phase fluid composition
US10684268B2 (en) 2012-09-28 2020-06-16 Bl Technologies, Inc. Sensor systems for measuring an interface level in a multi-phase fluid composition

Also Published As

Publication number Publication date
WO2000060120A9 (fr) 2002-08-29
JP2003503011A (ja) 2003-01-28
WO2000060120A2 (fr) 2000-10-12
CA2371732A1 (fr) 2000-10-12
WO2000060120A3 (fr) 2001-09-13
AU4189800A (en) 2000-10-23

Similar Documents

Publication Publication Date Title
WO2000060120A2 (fr) Procedes de detection de micro-organismes a l'aide de sondes immobilisees
JP2719225B2 (ja) クラミジア・トラコーマチスの検出方法およびそのためのキット
JP4598722B2 (ja) 特異的配列構成を有する核酸の検出法
US20020197614A1 (en) Electrophoretic analysis of target molecules using adapter molecules
CA2196604A1 (fr) Appareil et procede pour realiser une amplification d'acides nucleiques sur des supports
AU7489498A (en) Electrophoretic analysis of molecules using immobilized probes
JPH08501689A (ja) 改良型の鎖置換アッセイおよびそれに有用な複合体
EP0133288A2 (fr) Méthode de détection des bactéries au moyen d'hybridation d'acide nucléique, sondes marquées et trousse d'essai à cet effet
JP5156726B2 (ja) ハイブリダイゼーションアッセイを使用する真正細菌群の検出、同定および区別
EP0630973A2 (fr) Compositions de diagnostic, éléments, procédés et kits pour tests d'amplification et de détection de deux ou plusieurs ADN's utilisant des amorces à températures de fusion-adaptés
Zhao et al. Recent advances in peptide nucleic acids for rapid detection of foodborne pathogens
JP5403573B2 (ja) 肺炎原因菌検出用プライマーセット
Urdea et al. Application of a rapid non-radioisotopic nucleic acid analysis system to the detection of sexually transmitted disease-causing organisms and their associated antimicrobial resistances.
Vizzini et al. Development and application of DNA molecular probes
JP2000511777A (ja) クラミジア・ニューモニエ検出用核酸プライマーおよびプローブ
WO1992004458A1 (fr) Procede de depistage de la resistance aux medicaments
Pettersson et al. Nucleic acid hybridization—an alternative tool in diagnostic microbiology
JP2000511058A (ja) Ｍｙｃｏｂａｃｔｅｒｉｕｍ Ｋａｎｓａｓｉｉを検出するための組成物および方法
CA2075186A1 (fr) Sondes d'hybridation pour la detection des souches de branhamella catarrhalis
EP1235930A2 (fr) Analyse electrophoretique de molecules cibles au moyen de molecules adaptateurs
CA2692882C (fr) Detection ultrasensible d'une cible par particules de cible prete
KR19990022595A (ko) 나이세리아 종에 대한 핵산 프로브 및 증폭 올리고뉴클레오티드
JP2012161319A (ja) β２アドレナリン多型の検出
WO2021201206A1 (fr) Procédé de détermination de la mutation d'une seule base du gène erm (41) d'un bacille acido-résistant appartenant au complexe mycobacteroides abscessus, ensemble d'amorces et sonde utilisés dans ce procédé
US20030219755A1 (en) Compositions and methods for performing hybridization assays using target enhanced signal amplification (TESA)

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20011101

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE

AX Request for extension of the european patent

Free format text: AL;LT;LV;MK;RO;SI

17Q First examination report despatched

Effective date: 20030327

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: EXACT SCIENCES CORPORATION

RAP1 Party data changed (applicant data changed or rights of an application transferred)

Owner name: EXACT SCIENCES CORPORATION

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20031007