+

WO2002015949A2 - Dispositif pour l'identification d'une sequence nucleotidique dans un echantillon d'adn - Google Patents

Dispositif pour l'identification d'une sequence nucleotidique dans un echantillon d'adn Download PDF

Info

Publication number
WO2002015949A2
WO2002015949A2 PCT/US2001/026555 US0126555W WO0215949A2 WO 2002015949 A2 WO2002015949 A2 WO 2002015949A2 US 0126555 W US0126555 W US 0126555W WO 0215949 A2 WO0215949 A2 WO 0215949A2
Authority
WO
WIPO (PCT)
Prior art keywords
flat plate
nucleotide
sample
chamber
sample chamber
Prior art date
Application number
PCT/US2001/026555
Other languages
English (en)
Other versions
WO2002015949A8 (fr
WO2002015949A3 (fr
WO2002015949A9 (fr
Inventor
James Benn
Jean-Francois Manchec
Original Assignee
Genome Therapeutics Corporation
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 Genome Therapeutics Corporation filed Critical Genome Therapeutics Corporation
Priority to AU2001286756A priority Critical patent/AU2001286756A1/en
Publication of WO2002015949A2 publication Critical patent/WO2002015949A2/fr
Publication of WO2002015949A8 publication Critical patent/WO2002015949A8/fr
Publication of WO2002015949A3 publication Critical patent/WO2002015949A3/fr
Publication of WO2002015949A9 publication Critical patent/WO2002015949A9/fr

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/405—Concentrating samples by adsorption or absorption
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • B01L3/50255—Multi-well filtration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6818—Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • 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
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/025—Align devices or objects to ensure defined positions relative to each other
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/02—Adapting objects or devices to another
    • B01L2200/026—Fluid interfacing between devices or objects, e.g. connectors, inlet details
    • B01L2200/027—Fluid interfacing between devices or objects, e.g. connectors, inlet details for microfluidic devices
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/04—Closures and closing means
    • B01L2300/041—Connecting closures to device or container
    • B01L2300/044—Connecting closures to device or container pierceable, e.g. films, membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/06—Auxiliary integrated devices, integrated components
    • B01L2300/0627—Sensor or part of a sensor is integrated
    • B01L2300/0654—Lenses; Optical fibres
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/18—Means for temperature control
    • B01L2300/1838—Means for temperature control using fluid heat transfer medium
    • B01L2300/1844—Means for temperature control using fluid heat transfer medium using fans
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/04—Moving fluids with specific forces or mechanical means
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0406—Moving fluids with specific forces or mechanical means specific forces capillary forces
    • 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
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6823—Release of bound markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples
    • G01N1/4005—Concentrating samples by transferring a selected component through a membrane
    • G01N2001/4016—Concentrating samples by transferring a selected component through a membrane being a selective membrane, e.g. dialysis or osmosis

Definitions

  • the present invention relates to devices and methods for detecting the presence, absence or mutation of a particular nucleotide at a specific location on a strand of DNA.
  • a single nucleotide position on a strand of DNA may be responsible for a polymorphism or an allelic variation.
  • the usefulness of detecting such variations includes but is not limited to, genotyping, DNA family planning, diagnostics (including infectious disease), prenatal testing, paternal determination, pharmacogenetics, and forensic analysis.
  • the present invention provides a flat plate nucleotide detection cell for detecting the presence, absence or mutation of a nucleotide sequence in a target DNA sample.
  • the flat plate nucleotide detection cell has a sample chamber, a membrane provided along a portion of said sample chamber for effecting separation of a sample in the sample chamber and an optical detection window providing optical access to the sample chamber or another chamber in the detection cell.
  • the flat plate nucleotide detection cell is used to detect a single nucleotide polymorphism (SNP) in a strand of DNA.
  • SNP single nucleotide polymorphism
  • a target DNA sample is mixed with labeled nucleotides and other chemistries, and undergoes a chemical reaction. If the DNA sample has the SNP, the resulting mixture will contain labeled nucleotides that have undergone a change in molecular weight.
  • the illustrative flat plate nucleotide detection system may be utilized with any suitable technique for detecting a nucleotide sequence in a DNA sample.
  • the membrane in the flat plate system is utilized to effect the separation of the smaller molecular weight labels from the sample.
  • the flat plate system of the invention provides one or more optical detection windows to allow direct detection of a label in an interior chamber of the flat plate, without necessitating transfer of the sample to a separate detection system.
  • a flat plate nucleotide detection cell comprises an upper flat plate, at least one sample chamber formed along a bottom surface of the upper flat plate, a membrane provided along a portion of the sample chamber, and an optical window.
  • the optical window is provided in the upper channel plate, and permits light to pass between the sample chamber and a detector.
  • a flat plate nucleotide detection cell comprising an upper flat plate, a sample chamber, a membrane, a lower flat plate forming a filtrate chamber, and optical window provided in the lower channel plate.
  • the optical window permits light to pass between the filtrate chamber and a detector.
  • a system for detecting the presence of a nucleotide sequence in a DNA sample comprises a flat plate detection cell having an interior chamber, a membrane provided along a portion of the interior chamber and an optical window providing access to the interior chamber.
  • the system further includes an optical detector positioned relative to the optical window to monitor the interior chamber.
  • a method of detecting the presence of a nucleotide sequence in a DNA sample using the flat plate detection cell comprises providing a flat plate detection cell, injecting an admixture containing a DNA sample into the flat plate detection cell to effect separation of the admixture and monitoring either/or a sample chamber or a filtrate chamber for the presence of a label through an optical window in the flat plate detection cell.
  • Figure 1 is a cross-sectional view of a flat plate detection system of a first embodiment of the invention, with an optical detection window for detecting the sample chamber.
  • Figure 2 is a cross-sectional view of a flat plate detection system of a second embodiment of the invention, with an optical window for detecting the dialysate chamber.
  • Figure 3 is a cross-sectional view of a flat plate detection system of a third embodiment of the invention with a plurality of optical windows for detecting the sample chamber and the dialysate chamber.
  • Figure 4 provides dialysis results for two samples using a flat dialysis plate.
  • Figure 5 is a bottom view of a dialysis chamber of the embodiments of Figures 1, 2 and 3;
  • Figure 6 is an exploded perspective view of a fourth embodiment of the invention
  • Figure 7 is a view of a top surface of a needle guide of the fourth embodiment of the invention
  • Figure 8 is a cross-sectional view of a portion of the needle guide illustrated in Figure 7;
  • Figure 9 illustrates a bottom surface of the needle guide illustrated in
  • Figure 10 shows a top surface of an upper channel plate according to the fourth embodiment of the invention.
  • Figure 11 shows a cross-sectional view of a portion of the upper channel plate illustrated in Figure 10;
  • Figure 12 provides a bottom surface view of the upper channel plate illustrated in Figure 10;
  • Figure 13 is an upper surface view of a lower channel plate according to the fourth embodiment of the invention.
  • Figure 14 is a detailed view of a portion of the upper surface of the lower channel plate illustrated in Figure 13;
  • Figure 15 provides a bottom surface view of the lower channel plate illustrated in Figure 13 according to the fourth embodiment of the invention.
  • Figure 16 provides an upper surface view of a manifold according to the fourth embodiment of the invention.
  • Figure 17 provides a bottom surface view of the manifold illustrated in Figure 16.
  • biological sample refers to a sample comprising one or more cellular or extracellular components of a biological organism.
  • Such components include, but are not limited to, nucleotides (e.g. , DNA, RNA, fragments thereof and plasmids), peptides (e.g., structural proteins and fragments thereof, enzymes, etc.), and carbohydrates, etc.
  • the biological samples described herein may also include transport media, biological buffers and other reagents well know in the art for carrying out the processes described above.
  • biological samples in accordance with the invention typically have microliter ( ⁇ L) volumes and therefore can be referred to as microsamples, e.g., biological microsamples.
  • the methods of the invention are advantageously practiced with biological samples having volumes ranging between about 10 ⁇ l and about 0.05 ⁇ L, and preferably between about 0.1 ⁇ L and about 3 ⁇ L.
  • dialysis is art-recognized and is understood to refer to the separation or filtering of substances in solution by means of their unequal diffusion through a membrane, including the following forms of dialysis.
  • equivalent dialysis refers to dialysis which occurs without exchange or flow of dialysate, e.g. dialysis solution.
  • Flow dialysis refers to dialysis which occurs with a flow (or counterflow) of dialysate.
  • Exchange dialysis refers to dialysis which includes at least one change of the dialysate surrounding the membrane.
  • membrane as used herein refers to both dialysis membranes and ultrafiltration membranes, as appropriate, to accomplish dialysis or ultrafiltration.
  • the membrane is a material of any suitable composition and size which may used to separate or filter substances in solution by means of unequal diffusion, e.g., by size exclusion.
  • dialysis membranes and ultrafiltration membranes typically are semipermeable, the term "membrane" as used herein is not so limited. Dialysis membranes and ultrafiltration membranes are closely related and are interchangeable as used herein. In most applications, ultrafiltration membranes are generally designed to withstand elevated pressures.
  • purification is intended to encompass, in its various grammatical forms and synonyms (e.g., purification, purifying, clean up, etc.) any operation whereby an undesired component(s) is/are separated or filtered from a desired component(s). Such operations include, but are not limited to, filtration, ultrafiltration, dialysis/equilibrium dialysis, chromatography, and the like.
  • purification is achieved by molecular size discrimination among the components of the biological sample. Purification by molecular size discrimination can be achieved using any number of materials of varying porosity well known in the art including, but not limited to, filters, membranes, and semipermeable ultrafiltration filter materials.
  • sequence refers to one or more nucleotides in a target DNA sample.
  • temperature processing temperature treating
  • thermal processing are used interchangeably herein to refer to the application of a variety of temperature conditions to the sample, depending on the particular process underway and include, but are not limited to, continuous and discontinuous heating regimens, e.g., denaturation, annealing, incubation, precipitation, and the like.
  • continuous and discontinuous heating regimens e.g., denaturation, annealing, incubation, precipitation, and the like.
  • ultrafiltration refers to any method of purification, separation or filtration wherein the sample is under positive or negative pressure.
  • the nucleotide detection system for detecting the presence or absence of a sequence in a target DNA sample comprises a flat plate detection cell 10, as shown in Figure 1.
  • the flat plate detection cell 10 comprises a top flat plate 11 including a syringe docking port 20 fluidly coupled with a sample chamber 30 for holding a sample and a vent hole 40.
  • the syringe docking port 20 is used to direct a sample into the sample chamber 30.
  • the vent hole 40 provides a vent for the sample chamber 30.
  • a dialysis or ultrafiltration membrane 50 for separating a sample by means of size exclusion is provided along a portion of the sample chamber 30.
  • the flat plate detection cell 10 of Figure 1 further includes an optically transparent portion in the flat plate 11 forming an optical window 52 to the sample chamber 30.
  • the optical window 52 allows viewing and detection of a sample or other contents of the sample chamber.
  • a detector 53 such as a fluorescent reader, may be disposed relative to the optical window 52 to detect the contents of the sample chamber 30 through the optical window 52. In this manner, the contents of the sample chamber can be monitored and detected directly, at any time during processing of a sample, without necessitating transfer of the sample to a separate detection system.
  • the flat plate detection cell 10 is generally opaque, excluding the optical window, which is substantially transparent.
  • the optical window 52 may be formed of any suitable material for optically connecting an enclosed chamber within the flat plate detection cell 10 with a detector, such as styrene, polycarbonate or any material that is substantially transparent to selected wavelengths of light.
  • the upper flat plate 11 is comprised entirely of an optically transparent material, and the opacity of the non- window portions of the flat plate is provided using paint or masks to block light.
  • the syringe docking port 20 preferably includes a needle guide 60, a seal 70 and a needle stop 80.
  • the syringe docking port 20 includes an entry portion 22 opposite a distal end.
  • the optional needle guide 60 defines an insertion axis 90 for guiding a syringe holding a sample through the syringe docking port 20, which is preferably perpendicular to the membrane 50.
  • the needle guide 60, formed near entry portion 22, is preferably funnel shaped so as to guide a syringe along a path intersecting the insertion axis 90.
  • the needle guide 60 is preferably formed of polyethylene.
  • the optional seal 70 is preferably formed to provide a fluid-tight seal within the syringe docking port 20.
  • the seal 70 is designed to be repeatedly pierced by a syringe 21 while maintaining the ability to provide a fluid-tight seal.
  • the seal 70 is preferably pierced upon manufacture. Alternatively, the seal 70 may be manufactured without piercing and later pierced by a sharp needle during use.
  • the seal 70 may be formed of silicone, rubber, silicone rubber, or other elastic material, although silicone rubber is preferred.
  • the optional needle stop 80 can be formed near the distal end of the syringe docking port 20 to prevent a needle from piercing the membrane 50, preferably by preventing the needle from entering the sample chamber 30.
  • the needle stop 80 is formed of a non-reactive material, such as polyethylene.
  • the flat plate detection cell 10 includes the sample chamber 30 formed in the upper flat plate 11.
  • the sample chamber 30 is preferably formed with the distal end of the syringe docking port 20 fluidly coupled to one end of the sample chamber 30.
  • An optional vent hole 40 is also fluidly coupled to the sample chamber 30, preferably near an opposite end of the sample chamber 30.
  • An optional seal 45 or valve may be provided in or in fluid communication with the vent hole 40 to provide for the control of pressure within the sample chamber 30.
  • the dialysis/ultrafiltration membrane 50 is preferably provided along a portion of the sample chamber 30. According to an alternate embodiment, the vent hole is eliminated from the flat plate detection cell 10.
  • the flat plate detection cell 10 of the invention may be used to separate biological samples less than one microliter by the use of the membrane 50.
  • the membrane 50 is selected to have a molecular cutoff to retain molecules of interest and allow unwanted molecules to pass through the membrane, out of the sample, by means of dialysis or ultrafiltration.
  • a dialysis solution is provided on the opposite side of the membrane 50 from the sample chamber 30.
  • the dialysis solution may be stationary or may have a flow.
  • a positive or a negative pressure differential is applied to the sample chamber 30 to force the sample through the membrane 50.
  • the flat plate detection cells 10 of the illustrative embodiment is utilized to detect a single nucleotide polymorphism (SNP) in a target DNA sample.
  • SNP single nucleotide polymorphism
  • a target DNA sample is mixed with labeled nucleotides and other chemistries, and undergoes a chemical reaction. If the DNA sample has the SNP, the resulting mixture will contain labeled nucleotides that have undergone a change in molecular weight.
  • a needle containing the resulting mixture of the DNA sample and the labeled nucleotides is introduced into the syringe docking port 20.
  • the needle guide 60 guides the needle onto insertion axis 90 and into seal 70.
  • the needle stop 80 prevents the needle from being inserted too far.
  • the needle introduces the sample into the sample chamber 30 preferably through needle stop 80.
  • the vent hole 40 allows for the escape of air from the sample chamber 30 as the sample is introduced.
  • the portion of the sample chamber 30 having the membrane 50 is exposed to a dialysis solution.
  • the membrane 50 in the flat plate system is utilized to effect the separation of the smaller molecular weight labels from the sample.
  • the sample chamber 30 may be monitored through the optical windows 52 to detect the presence of a retained label in the sample chamber, using a detector 53.
  • the presence of the label in the sample chamber, or on the membrane itself, indicates the presence or absence of the SNP, depending on the particular assay used.
  • the needle previously used to insert the sample, or a different needle removes the sample from the sample chamber through needle stop 80.
  • the needle may optionally be removed from syringe docking port 20 during dialysis and reinserted upon completion of dialysis to effect removal of the sample.
  • the sample may also be separated using ultrafiltration, wherein a pressure differential is applied to the sample in the sample chamber 30 to effect separation of the sample.
  • the flat plate nucleotide detection cell 55 of Figure 2 includes a lower plate 56 including a filtrate chamber 57 below the membrane 50 for collecting the filtrate that passes through the membrane 50.
  • the lower plate 56 further includes an optical detection window 54 to facilitate direct detection of the filtrate in the filtrate chamber 57 without necessitating transfer of the filtrate to a separate detection system.
  • a detector 53 such as a fluorescent reader may be disposed adjacent to the optical detection window 54 to monitor the filtrate chamber 57 and detect the presence of a label in the filtrate and identify the presence of a SNP in a target DNA sample.
  • the membrane 50 is substantially transparent.
  • the filtrate chamber 57 and lower optical window 54 are shifted relative to the sample chamber 30. In this manner, only the filtrate side of the membrane will be detected through the optical window 54 and the sample side will be blocked from view.
  • the optical window 54 is located in a portion of the filtrate chamber 57 that is does not overlap with the sample chamber 30.
  • the invention is not limited a filtrate chamber that is offset from a sample chamber and it is within the scope of the invention to align the filtrate chamber with the sample chamber.
  • Figure 3 illustrates a third embodiment of the flat plate nucleotide detection cell
  • optical windows 52, 54 in both the top flat plate 10 and the bottom flat plate 56 to facilitate detection of both the sample chamber 30 and the filtrate chamber 57 using detectors 53 disposed relative to each optical window 52, 54, to detect the presence of a label in either or both chambers.
  • the sample chamber 30 and filtrate chamber 57 are shifted relative to each other. In this manner, the contents of the sample chamber 30 can be detected independent of the contents of the filtrate chamber 57 and vice versa.
  • the optical windows 52, 54 may be positioned in any location suitable for providing an optical connection to an interior chamber formed by one or more of the flat plates 10, 56.
  • the illustrative flat plate nucleotide detection system may be utilized with any suitable technique for detecting a nucleotide sequence in a DNA sample.
  • the sample chamber 30 is loaded with a labeled probe mixture, and a target DNA sample.
  • the sample chamber 30 is pre-loaded with labeled probe and the target DNA sample is injected into the pre-loaded sample chamber 30 through the syringe docking port 20.
  • the chamber 30 is then temperature-cycled between approximately 30 and 95 deg C to effect extension of the primers, and change the molecular weight of the probe.
  • the mixture is then brought into contact with the membrane 50, which is used to separate, by means of size, those labeled species that have changed molecular weight and those that have not changed molecular weight.
  • a positive identification of a DNA sequence will result in an increase in molecular weight.
  • extended primers will be trapped in the sample chamber 30 and will not be able to pass through the membrane 50, which is selected to have a molecular cutoff to retain the extended primers having an increased molecular weight.
  • Either dialysis or pressure driven ultrafiltration is used to drive the labeled species that did not increase in molecular weight across the membrane 50.
  • excess water can be injected, under pressure, into the sample chamber 30, to exit through the membrane 50, carrying with it those labeled species that did not increase in molecular weight, leaving trapped on the membrane 50 those species that did increase in molecular weight.
  • the membrane 50 is selected to have a molecular cutoff to allow passage of the lower- weight labeled species.
  • the presence of the increased molecular- weight species is then directly detected by means of optical imaging and fluorescence detection of the chamber and /or the membrane using the optical detection window 52 in the device illustrated in Figures 1 and 3.
  • the sample can be detected directly and immediately after separation, or at any time during the sample processing, without necessitating transfer of the sample to a separate detection system.
  • a positive identification of a DNA sequence will result in a decrease in molecular weight of the labeled primer or nucleotide.
  • the labeled primer or nucleotide passes through the membrane 50, which is selected to have a molecular cutoff to allow passage of the labeled primer or nucleotide, and is detected on the opposite side of the membrane 50 from the original sample through an optical window 54 located on the lower flat plate 57. Either dialysis or ultrafiltration is used to drive these species across the membrane 50.
  • the illustrative embodiment may be used in conjunction with any suitable technique for detecting a single nucleotide polymorphism (SNP) within a DNA molecule, such as the exonuclease assay described in U.S. Patent Number 5,391,480, the contents of which are incorporated by reference, wherein a labeled primer extends during thermocycling, thus increasing in molecular weight.
  • Other suitable assays for use with the illustrative embodiment involve the single base extension of a primer by adding a labeled nucleotide upon match of a single nucleotide polymorphism (SNP) site, disclosed in U.S. Patent Number 5,888,819 and U.S. Patent Number 6,004,744. The contents of both patents are incorporated herein by reference.
  • the flat plate detection system is used with a
  • the SNP assay described in U.S. application number 60/266,035 provides a method for detecting the presence or absence of a first nucleotide, at a position within a DNA molecule in a sample by forming an admixture of a primer and a strand of DNA of the sample and imposing conditions such that a hybridization product is formed between the primer and the DNA strand.
  • the primer comprises a sequence of DNA, which hybridizes with the strand of DNA adjacent to the first nucleotide position and has a second nucleotide opposite the first nucleotide position.
  • the second nucleotide has an associated label (e.g., a fluorescent label, a radioactive label or a mass-tag) and hybridizes to the first nucleotide in the event that the second nucleotide is complementary to the first nucleotide.
  • the second nucleotide does not hybridize to the first nucleotide in the event that the second nucleotide is not complementary.
  • a proofreading polymerase is applied to the hybridization product under conditions in which the second nucleotide is preferentially excised to form a labeled nucleotide product in the event that the second nucleotide is not hybridized to the first nucleotide, and in which the second nucleotide is preferentially incorporated into a primer extension product in the event that the second nucleotide is hybridized to the first nucleotide.
  • the presence or absence of a label in excised nucleotides and extension products may then be detected using the flat plate detection system of the illustrative embodiment.
  • the admixture is injected, e.g., from a syringe 21, through the syringe docking port 20, into the sample chamber 30 of the flat plate detection cell 10 and into contact with a first side of the membrane 50.
  • the membrane 50 of the corresponding flat plate detection cell is selected to have a molecular weight cut-off such that the labeled nucleotide excision product may pass through (or passes through quickly), the primer may not pass through (or passes through slowly), and the extension product may not pass through.
  • a dialysis solution is applied to a second side of the membrane opposite the first side of the membrane to effect separation of the components.
  • a pressure is applied to the sample to effect separation of the components through ultrafiltration.
  • the filtrate chamber 57 in the flat plate detection cell 10 may then be directly observed through the optical detection window 54 to determine the presence of the label in the filtrate.
  • Any suitable detection means may be utilized, including direct fluorescence measurement, or mass spectrometry.
  • the sample on the first side of the membrane 50 may also be monitored through an optical detection window 52 in the upper flat plate 11 for the presence of a label after providing sufficient time for separation of the various components of the sample to occur.
  • the presence of a label in the filtrate chamber 57 in concentrations greater than a background amount after a first predetermined time period is indicative of the absence of the first nucleotide
  • the presence of a label remaining in the sample chamber 30 in concentrations greater than a background amount after a second predetermined time period that is greater than the first predetermined time period (extension product) is indicative of the presence of the first nucleotide.
  • Detection methods well known to those skilled in the art may be employed to determine the presence or absence of a label in the extension product through one or more of the optical windows 52, 54.
  • the reaction products are fractionated by the membrane 50 to separate excised nucleotides, primers, and extension products. These components (or mixtures thereof) are then independently tested for the presence or absence of a label.
  • the second nucleotide in an assay used to detect the presence or absence of a particular nucleotide has a fluorescent label and the presence or absence of the fluorescent label in excised nucleotides and extension products is detected by direct fluorescence or by using fluorescence polarization through one or more of the optical windows 52, 54.
  • the fluorescence polarization measurements may be performed using any suitable instruments available in the art including the FluoroMax-2 instrument (available from Instruments S.A., Edison, NJ), FP777 spectrofluorimeter equipped with a microcomputer-assisted polarization measurement module and a Peltier temperature regulation system (available from Jasco, Tokyo), and the Analyst HT microplate reader (available from Molecular Devices Corp.).
  • FluoroMax-2 instrument available from Instruments S.A., Edison, NJ
  • FP777 spectrofluorimeter equipped with a microcomputer-assisted polarization measurement module and a Peltier temperature regulation system available from Jasco, Tokyo
  • the Analyst HT microplate reader available from Molecular Devices Corp.
  • a radioactive label is used and the detector 53 comprises a radiometric detector for detecting the presence or absence of the radioactive label in the sample chamber 30 or the filtrate chamber 57.
  • the detector 53 comprises a radiometric detector for detecting the presence or absence of the radioactive label in the sample chamber 30 or the filtrate chamber 57.
  • Figure 4 illustrates dialysis results of a DNA sample that is dialyzed and detected using a flat dialysis plate, such as that described in copending U.S. application Attny. Docket No. GEN-007CP, filed August 24, 2001.
  • a labeled primer was used to make extension products from wild-type DNA and mutant DNA carrying a single base substitution at a single site.
  • five microliters of the wild type sample were placed in a first flat dialysis plate, and five microliters of the mutant type sample were placed into another flat dialysis plate.
  • Both flat dialysis plates contained Spectrum Brand 100k MWCO CE Dialysis membranes. The dialysis side of each of the plates was loaded with five microliters of distilled water.
  • the top row of Figure 4 illustrates the results from the wild-type sample.
  • the bottom row of Figure 4 illustrates the results from the mutant-type sample.
  • the first column shows an initial reading of the sample before any dialysis has taken place, indicating that the labels were present on the sample side of the membrane in both the wild (label incorporated) and mutant (label clipped) samples.
  • the second and third columns show the results of detection of the dialysis solution after 15 and 20 minutes of dialysis, respectively. Where the labels were incorporated in the extension product of the wild-type samples, no clipped labels were present to dialyze across the membrane, thus none were detected by the fluorescent reader. Where the labels were clipped due to the presence of a mutation, they diffused across the membrane from the sample into the dialysis solution, and were detected by the fluorescent reader.
  • the fourth and fifth columns show the original samples after dialysis for 45 and 60 minutes.
  • extension products were made from the labeled primers. Where the labels were retained on the primers, they were incorporated into the extension products, and where they were clipped off by the exonuclease reaction, they were not incorporated.
  • the primers that did not participate in the reaction, and the single labeled nucleotides were all substantially dialyzed out of the sample, resulting in only extension products remaining in the sample chamber. Fluorescence was detected in the extension products that retained the labels, and no fluorescence was detected in the extension products that did not retain the labels.
  • the ability to differentiate between the wild type (incorporated labels) from the mutant type (clipped labels) was demonstrated in two ways.
  • the first way was by detecting labeled nucleotides in the dialysis solution, where clipping had taken place, and the second was by detecting incorporated labels in the dialyzed sample solution, where no clipping had taken place.
  • the same experiment as described above can be more rapidly and efficiently performed in a flat plate dialysis system of the invention, such as that illustrated in Figure 3.
  • the built in optical windows 52 and 54 enable detection without the need for transferring the sample and/or dialysate to another device for detection.
  • Figure 5 illustrates a top view of the sample chamber 30.
  • the sample chamber 30 shown in Figure 5 is preferably formed with a diameter of less than 1 mm and greater than 0.1 mm, preferably having a volume of less than 1 microliter. A diameter of approximately 0.5 is preferred.
  • the sample chamber is preferably formed in the shape of an elongated tube cut along its longitudinal axis, thereby forming a flat portion along substantially all its length.
  • the sample chamber may be formed in a serpentine shape, such as an S shape as is shown in Figure 5, or may be straight.
  • the sample chamber 30 shown in Figure 5 shows a lower portion 67 of a guide channel 65 (shown in Figure 8) in fluid communication with the sample chamber 30, near an end of the sample chamber 30.
  • a vent hole 40 is also illustrated in fluid communication near an opposite end of the sample chamber 30.
  • An optical window 52 may be located along any point of the sample chamber to allow for detection of the sample in the sample chamber.
  • Optional washing of the flat plate detection cell 10, or any part thereof, may be performed after separation of the sample and detection of the sample chamber and/or filtrate chamber.
  • an alcohol-based solution is used. Washing may be performed with or without disassembly of the flat plate detection cell 10.
  • the flat plate detection cell 10 is easily optionally multiplied into an array of multiple flat plate detection cells, each having a corresponding optical window or a set of optical windows, allowing each flat plate detection cell 10 to use a portion of a single, continuous membrane 50.
  • the invention is capable of processing and detecting many samples in parallel, if desired, using standard micro-titer plates as reagent sources.
  • the system can be used to retrieve, mix and dispense fluids by integration with air or liquid-filled volumetric devices, such as piezoelectric elements, movable pistons or syringe-type plungers.
  • a syringe needle docking system comprising at least one syringe 21, may be used to automate the insertion and removal of samples.
  • the syringe needle docking system may optionally include automated syringe needle movement and automated syringe plunger actuation.
  • the dimensions of the sample chamber 30 provide for the use of small sample volumes while providing a large surface area for the sample to be in contact with the membrane 50.
  • the sample chamber 30 diameter is between 1.0 mm and 0.1 mm. Specifically, approximately 0.5mm is preferred.
  • a large surface area along the membrane 50 allows for more rapid separation of a sample. This large surface area is provided without need for additional components, such as those disclosed in U.S. Patent 5, 679,310 to Manns.
  • the flat plate detection system 100 shown in Figure 6 preferably includes a needle guide 200, a seal 300, an upper channel plate 400, a membrane 500, a lower channel plate 600 and a manifold 700.
  • a plurality, such as 96 or 384 or more, flat plate detection cells are provided in the flat plate detection system 100, as described below.
  • a plurality of the flat plate detection cells in the flat plate detection system 100 include one or more optical windows to allow direct detection of the sample chamber and/or a filtrate chamber.
  • any number of flat plate detection cells may be provided with one or more optical windows to provide access to the sample chamber 30 and/or the filtrate chamber 57 of a selected number of flat plate detection cells in the flat plate detection system 100.
  • a needle guide 200 is provided with a plurality of holes. For each flat plate detection cell 10, an entry portion 22 and a vent hole 40 are preferably provided within needle guide 200. For each flat plate detection cell 10 having an optical window 52 to the sample chamber, an optical window 52 is preferably provided within the needle guide as well. Alignment holes 210 are also preferably provided to aid in mounting of the various components of the flat plate dialysis system 100 to each other.
  • a cross-section of the needle guide 200 shown in Figure 7 is provided in Figure 8. Entry portions 22 are fluidly coupled via an upper portion 66 of the guide channel 65, preferably to an annular seal receiving portion 220.
  • the bottom surface of needle guide 200 preferably provides an annular seal receiving portion 220 for each flat plate detection cell 10.
  • Figure 9 also illustrates a vent hole 40 corresponding to each annular seal receiving portion 220.
  • the optional seal 300 is preferably provided between the needle guide 200 and the upper channel plate 400. The seal 300 is preferably configured so as to mate with the annular seal receiving portion 220 to provide a fluid-tight seal along the guide channel 65.
  • the upper channel plate 400 is described with reference to Figure 10.
  • Figure 10 illustrates a pattern of holes similar to those provided in the needle guide 200 in that a pair of two holes is provided for each flat plate detection cell 10.
  • the upper channel plate 400 differs from the needle guide 200 in that the upper channel plate 400 preferably provides a needle stop, analogous to needle stop 80 of the first embodiment of the invention.
  • An upper portion 66 of the guide channel 65 corresponding to a flat plate detection cell 10 is shown in Figure 7.
  • a corresponding vent hole 40 is also provided, as shown in Figure 7.
  • Optical windows 52 are provided in the upper channel plate 400 at selected locations to facilitate detection of a corresponding sample chamber.
  • a cross-section of a portion of the upper channel plate 400 is provided in Figure 11.
  • a guide channel 65 is shown having an upper portion 66 and a lower portion 67.
  • the lower portion 67 of the guide channel 65 preferably has a needle stop formed by a reduced diameter so as to prevent a needle from traveling within the lower portion 67 of the guide channel 65.
  • a vent hole 40 is also provided within the upper channel plate 400.
  • the vent hole 40 may be provided with a varying diameter.
  • An optical window 52 is also provided in the upper channel plate 400.
  • Figure 11 also illustrates a cross-section of the sample chamber 30 in fluid communication with the lower portion 67 of the guide channel 65 and the vent hole 40 and in optical communication with the optical window 52. It is within the scope of the invention to provide an optional seal valve in or in fluid communication with the vent hole 40. Such a seal may be provided to facilitate elevated or reduced pressure within the sample chamber 30.
  • Figure 12 illustrates a bottom surface of the upper channel plate 400.
  • a sample chamber 30 is provided for each flat plate detection cell 10.
  • a vent hole 40, optical window 52 and a lower portion 67 of the guide channel 65 are illustrated in Figure 12 and correspond to those shown in Figure 10.
  • Alignment holes 410 are preferably provided within the upper channel plate 400 to correspond to the alignment holes 210 of the needle guide 200. It is within the scope of the invention to integrally form the needle guide 200 and the upper channel plate 400 in a unitary piece.
  • the membrane 500 is provided between the upper channel plate 400 and the lower channel plate 600.
  • the membrane 500 may optionally be bonded to upper channel plate 400 or may be mounted by a compressive force applied to keep the upper channel plate 400 and the lower channel plate 600 together. Bonding may be performed by ultrasonic welding, heat bonding or a variety of adhesives.
  • an upper surface of the lower transfer plate 600 provides filtrate chambers 57 to correspond to the sample chambers 30 of the upper channel plate 400 shown in Figure 9.
  • the filtrate chambers 57 are generally offset from the sample chambers 30 to facilitate detection of either or both chambers through one or more of the optical windows 52, 54.
  • Each filtrate chamber 57 may be provided with a first port 630 and a second port 640.
  • An optical window 54 may also be provided along the filtrate chamber 57.
  • a detailed view of the filtrate chamber 57 is provided in Figure 14.
  • Figure 15 shows a bottom surface view of the lower channel plate 600. Alignment holes 610 are optionally provided within the lower channel plate 600 to correspond to the alignment holes in other components of the flat plate dialysis system 100.
  • a manifold 700 is provided under the lower channel plate 600.
  • Manifold 700 provides on an upper surface, a first and a second trough 730, 740.
  • First and second troughs 730, 740 are fluidly coupled to first and second ports 630, 640, respectively, of the lower channel plate 600.
  • First trough 730 fluidly communicates with a first external port 735.
  • Second trough 740 preferably does not communicate with an external port.
  • Both first and second troughs 730, 740 allow fluid communication among first and second ports 630, 640 along a row of flat plate dialysis cells 10 within the flat plate dialysis system 100.
  • alignment holes 710 are provided within the manifold 700 to correspond to alignment holes of the other components of the flat plate detection system 100.
  • Figure 17 provides a view of a bottom surface of the manifold 700.
  • the lower channel plate 600 and the manifold 700 are both optional components of the flat plate dialysis system 100. Processing of a sample, such as conducting dialysis or thermocy cling, can be performed in the dialysis chamber 30 by passing a dialysis solution along the dialysis membrane 500 with or without the filtrate chamber 57 of the lower channel plate 600.
  • the flat plate detection system 100 is adapted to be used with a syringe needle docking system or a multi-channel pipettor system, such as a 96 or 384 or more channel pipettor. Pipettor syringes are provided to align with the entry portions 22 shown in Figures 6, 7 and 8.
  • the needles of the pipettor syringes are inserted into the needle guide 200 each along an insertion axis 90, shown in Figure 8.
  • the needles travel along the guide channel 65.
  • the guide channel 65 is provided with a larger diameter along an upper portion 66 and a narrower diameter along lower portion 67.
  • the lower portion 67 of the guide channel 65 preferably does not allow the needle to pass within it.
  • a fluid-tight seal is provided by the seal 300 preferably seated within the annular seal receiving portion 220, illustrated in Figures 3, 5 and 6.
  • the needles deposit a biological sample to be analyzed and the accompanying reagents necessary for effecting a reaction through the lower portion 67 of the guide channel 65 into the sample chamber 30.
  • the sample flows freely into the sample chamber 30 due to vent hole 40 allowing the release of air contained within the sample chamber 30.
  • an optional seal 45 or valve may be provided within or in fluid communication with vent hole 40 to regulate the flow through vent hole 40.
  • the components of the flat plate detection cell 10, 55, 58 and flat plate detection system are preferably formed of non-reactive plastic.
  • the optical windows 52, 54 are formed of a suitable transparent material, such as styrene or polycarbonate and the non- window portions of the flat plate detection cell are substantially opaque.
  • components such as the needle guide 200, upper channel plate 400, lower channel plate 600 and manifold 700 may preferably be formed of hydrophobic materials, such as polystyrene, polycarbonate, TEFLON , or DELRTN .
  • Optional coatings of TEFLONTM or silane may also be used to enhance hydrophobic properties of these materials.
  • the membrane 50, 500 may preferably be formed of cellulose, cellulose ester, TEFLONTM, polysulfone and polyethersulfone.
  • the membrane 50, 500 is selected to have a molecular cutoff suitable for separating a DNA sample mixed with labeled nucleotides.
  • a further variation of the invention allows the use of alignment holes 210, 410, 610 and 710 for the passage of a temperature-controlled solution so as to vary the temperature of the dialysis chamber 30 and/or filtrate chamber 57.
  • Thermocy cling may be achieved, for example, by blowing air of different temperatures, although a liquid medium could also be used for heat transfer.
  • additional holes or passages may be provided to allow for the distribution of a temperature controlled fluid to effect the temperature of dialysis chamber 30 or filtrate chamber 57 and the dialysis membrane 500.
  • compression bolts may be provided within the alignment holes 210, 410, 610, 710 of the invention to compress the flat plate dialysis system. Screws may also be used in place of or in combination with compression bolts.
  • Other devices such as C-clamps or large hose clamps may be used to hold the needle guide 200, upper channel plate 400, dialysis membrane 50, lower channel plate 600 and manifold 700 together. Any of the above-described items may also be used with a subset of components of the flat plate SNP detection system.
  • Another variation of the invention involves the use of a beveled corner on each of the needle guide 200, the upper channel plate 400, lower channel plate 600 and manifold 700, or any subset thereof, to aid in alignment of these components of the flat plate dialysis system, as shown in Figure 6.
  • the present invention can be used with a conventional fluid dispensing unit, such as a Hydra dispenser, manufactured by Robbins Scientific.
  • a conventional fluid dispensing unit such as a Hydra dispenser, manufactured by Robbins Scientific.
  • Those of ordinary skill will also recognize that other fluid dispensing and sample handling units, whether in modular or discrete forms, can be employed to work with the invention.
  • the present invention provides benefits over current systems for detecting sequences in DNA samples.
  • the invention integrates the processing, separation and detection systems and processes into a single device, and prevents unnecessary transfer of the sample between separate systems.
  • the accuracy of the detecting process is improved while the time, cost and complication involved in detecting a nucleotide sequence are significantly reduced.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Dispersion Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Separation Using Semi-Permeable Membranes (AREA)

Abstract

L'invention concerne un système et un procédé permettant d'identifier des séquences nucléotidiques spécifiques dans un échantillon d'ADN cible. Le système de détection comprend une cellule de détection à plaque plate équipée d'une chambre à échantillon, d'une membrane et d'une fenêtre optique assurant l'accès optique à l'intérieur de la cellule. Un échantillon d'ADN cible est mélangé avec des nucléotides étiquetés et autres substances chimiques, puis soumis à une réaction chimique. Si la séquence nucléotidique est présente, le mélange renfermera des nucléotides étiquetés dont le poids moléculaire est altéré. L'échantillon ayant subi la réaction est appliqué à la chambre et la membrane du système sépare de l'échantillon les étiquettes de poids moléculaire inférieur. Ensuite, la présence de telle ou telle étiquette dans la chambre à échantillon ou la chambre à filtrat ou sur la membrane elle-même est décelée à travers la fenêtre optique vers la chambre à échantillon ou à travers une fenêtre optique vers la chambre à filtrat pour déterminer la présence ou l'absence de la séquence.
PCT/US2001/026555 2000-08-25 2001-08-24 Dispositif pour l'identification d'une sequence nucleotidique dans un echantillon d'adn WO2002015949A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001286756A AU2001286756A1 (en) 2000-08-25 2001-08-24 Device for identifying the presence of a nucleotide sequence in a dna sample

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US22823900P 2000-08-25 2000-08-25
US60/228,239 2000-08-25
US26603501P 2001-02-02 2001-02-02
US60/266,035 2001-02-02

Publications (4)

Publication Number Publication Date
WO2002015949A2 true WO2002015949A2 (fr) 2002-02-28
WO2002015949A8 WO2002015949A8 (fr) 2002-07-04
WO2002015949A3 WO2002015949A3 (fr) 2002-10-03
WO2002015949A9 WO2002015949A9 (fr) 2003-03-27

Family

ID=26922164

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2001/026555 WO2002015949A2 (fr) 2000-08-25 2001-08-24 Dispositif pour l'identification d'une sequence nucleotidique dans un echantillon d'adn
PCT/US2001/026611 WO2002016652A2 (fr) 2000-08-25 2001-08-24 Dispositif de traitement rapide d'echantillons d'adn, par manipulation, thermocyclage et purification integrees de liquides

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/US2001/026611 WO2002016652A2 (fr) 2000-08-25 2001-08-24 Dispositif de traitement rapide d'echantillons d'adn, par manipulation, thermocyclage et purification integrees de liquides

Country Status (2)

Country Link
AU (2) AU2001286756A1 (fr)
WO (2) WO2002015949A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005079986A1 (fr) * 2004-02-18 2005-09-01 Applera Corporation Essais biologiques a etapes multiples sur des plates-formes d'applications micro-fluidiques modulaires
WO2006092293A1 (fr) * 2005-03-04 2006-09-08 Fraunhofer Ges Forschung Procede d'analyse par separation par osmose et concentration de substances de masse moleculaire elevee a detecter, et microsysteme fluidique servant a la mise en oeuvre de ce procede d'analyse

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030077611A1 (en) 2001-10-24 2003-04-24 Sention Methods and systems for dynamic gene expression profiling
US7081339B2 (en) 2002-04-12 2006-07-25 Primera Biosystems, Inc. Methods for variation detection
US7445893B2 (en) 2002-04-12 2008-11-04 Primera Biosystems, Inc. Sampling method for amplification reaction analysis
JP2006507849A (ja) * 2002-11-21 2006-03-09 プリメラ バイオシステムズ 増幅反応分析のためのサンプリング方法および装置
EP2012923A1 (fr) * 2006-04-07 2009-01-14 Corning Incorporated Microplaque fermée à écoulement continu et modes d'utilisation et procédés de fabrication de celle-ci
DE102007011866A1 (de) 2007-03-08 2008-09-11 Friedrich-Schiller-Universität Jena Vorrichtung zur Aufnahme, Behandlung und Aufbewahrung kleinvolumiger Proben
WO2011009073A1 (fr) * 2009-07-16 2011-01-20 Abbott Laboratories Procédé d'amplification d'acides nucléiques
CN106964411B (zh) 2012-03-16 2019-12-10 统计诊断与创新有限公司 具有集成传送模块的测试盒
JP6894617B2 (ja) * 2018-10-29 2021-06-30 Nok株式会社 マイクロ流体チップおよびマイクロ流体デバイス
EP4288206A1 (fr) * 2021-02-05 2023-12-13 Quantoom Biosciences France SAS Dispositif de réactions en plusieurs étapes thermocyclée amélioré

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3591493A (en) * 1968-06-17 1971-07-06 Rashid A Zeineh Method for the treatment of biological fluids and apparatus therefor
GB8629740D0 (en) * 1986-12-12 1987-01-21 Iq Bio Ltd Immunoassay
US5188963A (en) * 1989-11-17 1993-02-23 Gene Tec Corporation Device for processing biological specimens for analysis of nucleic acids
US5010930A (en) * 1989-12-22 1991-04-30 Eastman Kodak Company Pipette and liquid transfer apparatus for dispensing liquid for analysis
US5733442A (en) * 1990-12-07 1998-03-31 Shukla; Ashok K. Microdialysis/Microelectrodialysis system
US5632957A (en) * 1993-11-01 1997-05-27 Nanogen Molecular biological diagnostic systems including electrodes
US5672319A (en) * 1993-04-29 1997-09-30 Danfoss A/S Device for analyzing a fluid medium
WO2000044497A1 (fr) * 1999-01-29 2000-08-03 Genomic Instrumentation Services, Inc. Dispositif de pipettes
DE19950385A1 (de) * 1999-01-29 2000-08-03 Max Planck Gesellschaft Verfahren zur Isolation von Apoptose-induzierenden DNA-Sequenzen und Detektionssystem
DE10005024C2 (de) * 2000-02-04 2002-09-26 Eppendorf Ag Dialysestopfen

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005079986A1 (fr) * 2004-02-18 2005-09-01 Applera Corporation Essais biologiques a etapes multiples sur des plates-formes d'applications micro-fluidiques modulaires
US7250260B2 (en) 2004-02-18 2007-07-31 Applera Corporation Multi-step bioassays on modular microfluidic application platforms
EP2392402A3 (fr) * 2004-02-18 2012-03-21 Life Technologies Corporation Essais biologiques a etapes multiples sur des plates-formes d'applications micro-fluidiques modulaires
WO2006092293A1 (fr) * 2005-03-04 2006-09-08 Fraunhofer Ges Forschung Procede d'analyse par separation par osmose et concentration de substances de masse moleculaire elevee a detecter, et microsysteme fluidique servant a la mise en oeuvre de ce procede d'analyse
US7892857B2 (en) 2005-03-04 2011-02-22 Rudolf Ehwald Assay by osmotically induced separation and concentration of high-molecular detectable substances and a fluid microsystem for carrying out said assay

Also Published As

Publication number Publication date
AU2001286784A1 (en) 2002-03-04
WO2002016652A3 (fr) 2003-07-10
WO2002015949A8 (fr) 2002-07-04
WO2002015949A3 (fr) 2002-10-03
WO2002016652A2 (fr) 2002-02-28
AU2001286756A1 (en) 2002-03-04
WO2002015949A9 (fr) 2003-03-27

Similar Documents

Publication Publication Date Title
CN107338189B (zh) 用于集成的样品制备、反应和检测的设备与方法
EP1608952B1 (fr) Appareil et procede de dosage utilisant des reseaux microfluidiques
CA2565679C (fr) Dispositif et procede pour de detecter des interactions moleculaires
US20060166347A1 (en) Sample preparation devices and methods
US10099216B2 (en) Plurality of reaction chambers in a test cartridge
EP4043546A1 (fr) Appareil et méthodes pour la préparation, la réaction et la détection intégrées d'échantillons
US20020106787A1 (en) Device for repid DNA sample processing with integrated liquid handling, thermocycling, and purification
EP2732053B1 (fr) Systèmes, appareils et procédés d'analyse biochimique à haut débit
US20090093064A1 (en) Method of determining the presence of a mineral within a material
WO2002015949A2 (fr) Dispositif pour l'identification d'une sequence nucleotidique dans un echantillon d'adn
JP2004500552A (ja) 液体処理、サーマルサイクリング及び精製を統合した迅速なｄｎａ試料処理のための装置
US20020086309A1 (en) Device for identifying the presence of a nucleotide sequence in a DNA sample
US11927600B2 (en) Fluidic bridge device and sample processing methods
US6200781B1 (en) Apparatus, system and method for automated execution and analysis of biological and chemical reactions
CA2369363A1 (fr) Dispositif permettant de traiter rapidement un echantillon d'adn par manipulation de liquide, thermocyclage et purification
WO2000066995A9 (fr) Dispositif permettant de traiter rapidement un echantillon d'adn par manipulation de liquide, thermocyclage et purification
WO2000066267A1 (fr) Systeme empechant l'intercontamination dans une plaque multipuits

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 BZ CA CH CN CO CR CU CZ DE DK DM DZ EC 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 MZ NO NZ PH 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 MZ 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 TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
AK Designated states

Kind code of ref document: C1

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC 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 MZ NO NZ PH 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: C1

Designated state(s): GH GM KE LS MW MZ 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 TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

CFP Corrected version of a pamphlet front page
AK Designated states

Kind code of ref document: A3

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC 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 MZ NO NZ PH 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 MZ 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 TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

COP Corrected version of pamphlet

Free format text: PAGES 1/17-17/17, DRAWINGS, REPLACED BY NEW PAGES 1/11-11/11; DUE TO LATE TRANSMITTAL BY THE RECEIVING OFFICE

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载