WO2000066995A2 - Dispositif permettant de traiter rapidement un echantillon d'adn par manipulation de liquide, thermocyclage et purification - Google Patents

Dispositif permettant de traiter rapidement un echantillon d'adn par manipulation de liquide, thermocyclage et purification Download PDF

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Publication number
WO2000066995A2
WO2000066995A2 PCT/US2000/011371 US0011371W WO0066995A2 WO 2000066995 A2 WO2000066995 A2 WO 2000066995A2 US 0011371 W US0011371 W US 0011371W WO 0066995 A2 WO0066995 A2 WO 0066995A2
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Prior art keywords
capillary tube
fluid
capillary
modular
anay
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PCT/US2000/011371
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English (en)
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WO2000066995A3 (fr
WO2000066995A9 (fr
Inventor
Patrick Cahill
Douglas Smith
Ulrich Thomann
Marcy Engelstein
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Genome Therapeutics Corporation
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Priority to EP00928488A priority Critical patent/EP1177043A2/fr
Priority to CA002369363A priority patent/CA2369363A1/fr
Priority to JP2000615584A priority patent/JP2002543418A/ja
Priority to AU46724/00A priority patent/AU4672400A/en
Publication of WO2000066995A2 publication Critical patent/WO2000066995A2/fr
Publication of WO2000066995A3 publication Critical patent/WO2000066995A3/fr
Publication of WO2000066995A9 publication Critical patent/WO2000066995A9/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
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    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/508—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above
    • B01L3/5085—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates
    • B01L3/50857—Containers for the purpose of retaining a material to be analysed, e.g. test tubes rigid containers not provided for above for multiple samples, e.g. microtitration plates using arrays or bundles of open capillaries for holding samples
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/18—Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/24—Dialysis ; Membrane extraction
    • B01D61/28—Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/02—Hollow fibre modules
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • 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
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    • 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
    • B01L3/502715—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 characterised by interfacing components, e.g. fluidic, electrical, optical or mechanical interfaces
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    • 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
    • C12Q1/6869—Methods for sequencing
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01D2313/13—Specific connectors
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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
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    • B01J2219/00364—Pipettes
    • B01J2219/00367—Pipettes capillary
    • B01J2219/00369—Pipettes capillary in multiple or parallel arrangements
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00389—Feeding through valves
    • B01J2219/004—Pinch valves
    • B01J2219/00403—Pinch valves in multiple arrangements
    • 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
    • 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
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    • 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
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    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—Geometry, shape and general structure cylindrical, tube shaped
    • B01L2300/0838—Capillaries
    • 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
    • B01L2400/0475—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure
    • B01L2400/0487—Moving fluids with specific forces or mechanical means specific mechanical means and fluid pressure fluid pressure, pneumatics
    • 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
    • B01L2400/06—Valves, specific forms thereof
    • B01L2400/0633—Valves, specific forms thereof with moving parts
    • B01L2400/0655—Valves, specific forms thereof with moving parts pinch valves
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
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    • 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
    • B01L3/50273—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 characterised by the means or forces applied to move the fluids
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • C—CHEMISTRY; METALLURGY
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    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • G—PHYSICS
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    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178—Special arrangements of analysers
    • G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks

Definitions

  • the invention relates to devices and methods for high speed, low volume automated sample handling of biological samples, which are useful in the field of genomics for a variety of processes, including DNA sequencing, genetic analysis, and gene expression analysis.
  • the invention further relates to devices and methods for setting up and executing assays for high throughput compound screening for pharmaceutical applications.
  • kits such as Quantum Prep TM SEQueaky Kleen 96 (catalog number 732-6260, Bio-Rad Laboratories, Hercules, CA) and Multiscreen 96 (catalog number MAHVS45, Millipore Corp., Bedford, MA) accomplish the same task as ethanol precipitation, but still do not remove primers, and require a subsequent centrifugation or filtration step. Hence, multiple processing steps are employed in an effort to purify a DNA sample. Effective procedures for complete salt and template removal using spin columns and ultrafiltration have also been reported (Ruiz-Martinez et al, 1998; and Salas-Solano et al, 1998), but the materials are costly and the procedures are not readily amenable to automation.
  • the invention relates to an integrated, capillary-based sample handling system for capillary-based aspiration, incubation, purification and delivery of a biological sample that is capable of processing many samples in parallel.
  • the invention further provides integration of pipetting, mixing, temperature treatment, and sample purification that is easy to operate and can be re-used many times.
  • the invention is a device for integrated processing of a biological sample.
  • the device comprises a first capillary tube sized for holding the biological sample, and having a first end and a second end; a second capillary tube sized for holding the biological sample, and also having a first end and a second end; and a connector coupled to the second end of the first capillary tube and the first end of the second capillary tube for mechanically and fluidly connecting the first and second capillary tubes together.
  • the device comprises a first capillary tube sized for holding the biological sample, and having a first end and a second end; a first regulating element coupled to the first end of the first capillary tube for selectively providing a fluid seal thereat; a second capillary tube sized for holding the biological sample, and also having a first end and a second end; and a second fluid regulating element disposed between the second end of the first capillary tube and the first end of the second capillary tube for selectively providing a fluid seal between the first and second capillary tubes.
  • the device comprises a first capillary tube sized for holding the biological sample and adapted for thermal processing of the sample, the first capillary tube having a first end and a second end; a second capillary tube sized for holding the biological sample, the second capillary tube also having a first end and a second end; and a fluid regulating element coupled to the second end of the first capillary tube and the first end of the second capillary tube for mechanically and fluidly connecting the first and second capillary tubes together.
  • the invention is a device for integrated processing of a biological microsample.
  • the device comprises a first capillary tube sized for holding the biological microsample and adapted for thermocycling the sample, the first capillary tube having a first end and a second end; a second capillary tube sized for holding the biological sample and adapted for equilibrium dialysis of the sample, the second capillary tube also having a first end and a second end; and a connector coupled to the second end of the first capillary tube and the first end of the second capillary tube for mechanically and fluidly connecting the first and second capillary tubes together.
  • Another embodiment of the invention is a device for dialysis of a biological microsample.
  • the device comprises a capillary tube sized for holding the microsample and comprising separation means for purifying the microsample by molecular size discrimination.
  • the invention is a device for equilibrium dialysis of a biological sample.
  • the invention is a device for flow dialysis of a biological sample.
  • the invention is a device for exchange dialysis of a biological sample.
  • the invention is a modular device for integrated handling of biological sample.
  • the modular device which comprises a first modular array of capillary tubes for holding the biological sample, the first modular array having a first array end and a second array end; a second modular array of capillary tubes for holding the biological sample, the second modular array being coupled to the second array end of the first modular array, wherein the second modular array is removably and replaceably coupled to the first modular array; and an array of fluid regulating elements coupled to the second array end of the first modular array and a first end of the second modular array for selectively providing a fluid seal between the first and second modular arrays of capillary tubes.
  • the invention is a modular device for integrated processing of a biological microsample.
  • the device comprises a first modular array of capillary tubes sized for holding the biological microsample and adapted for thermocycling the sample, the first modular array having a first array end and a second array end; a second modular array of capillary tubes sized for holding the biological microsample and adapted for dialysis of the sample, the second modular array also having a first array end and a second array end, wherein the second modular array is coupled to the second array end of the first modular array, and wherein the second modular array is removably and replaceably coupled to the first modular array; and an array of connectors coupled to the second array end of the first modular array and the first end of the second modular array for selectively providing a fluid seal between the first and second modular arrays of capillary tubes.
  • Another embodiment of the invention is a modular device for dialysis of a biological microsample.
  • the device comprises a modular array of capillary tubes sized for holding the microsample, each of the tubes comprising separation means for purifying the microsample by molecular size discrimination.
  • the invention is a system for automatic and integrated processing of a biological sample.
  • the system comprises a first capillary tube for holding the biological sample, the first capillary tube having a first end and a second end; a support assembly for supporting the capillary tube at its first end; a second capillary tube for holding the biological sample, the second capillary tube also having a first end and a second end; a first fluid regulating element disposed between the second end of the first capillary tube and the first end of the second capillary tube for selectively providing a fluid seal between the first and second capillary tubes; and a plate handling assembly positioned for selectively disposing a fluid reservoir beneath the second capillary tube during operation.
  • the system comprises a first capillary tube for holding the biological sample, the first capillary tube having a first end and a second end; a support assembly for supporting the capillary tube at its first end; a first fluid regulating element disposed between the support element and the first end of the first capillary tube for selectively providing a fluid seal between the support element and the first capillary tube; a second capillary tube for holding the biological sample, the second capillary tube also having a first end and a second end; a second fluid regulating element disposed between the second end of the first capillary tube and the first end of the second capillary tube for selectively providing a fluid seal between the first and second capillary tubes; and a plate handling assembly positioned for selectively disposing a fluid reservoir beneath the second capillary tube during operation.
  • the system comprises a first modular array of capillary tubes for holding the biological sample, the first modular array having a first array end and a second array end; a support assembly coupled to the first array end of the first modular array for supporting the array at its first end; a second modular array of capillary tubes for holding the biological sample, the second modular array coupled to the second array end of the first modular array, wherein the second modular array is removably and replaceably coupled to the first modular array; and a plate handling assembly positioned for selectively disposing a fluid reservoir beneath the second modular array of capillary tubes during operation.
  • the invention is a method for processing a biological sample.
  • the method comprises providing a first capillary tube sized for holding the biological sample, providing a second capillary tube sized for holding the biological sample; mechanically and fluidly coupling a second end of the first capillary tube and a first end of the second capillary tube together; supporting the first capillary tube at a first end; and positioning a fluid reservoir beneath the second capillary tube during operation.
  • the invention is a method for integrated processing of a biological microsample.
  • the method comprises temperature treating the microsample, and purifying the microsample, wherein the temperature treating and purifying steps are carried out in a device hereinabove described that integrates these steps, such that integrated processing of the microsample is achieved.
  • the invention is a method of purifying a biological microsample.
  • the method comprises introducing the microsample into a capillary tube which comprises separation means for purifying the sample by molecular size discrimination, and allowing the microsample to reside in the capillary tube for a time sufficient such that purification of the microsample is achieved.
  • the step of purifying the microsample includes dialysis.
  • the step of purifying the microsample includes flow dialysis.
  • the step of purifying the microsample includes exchange dialysis.
  • the invention relates to purifying and cleaning methods that remove contaminants quickly and efficiently from a DNA reaction mix.
  • the DNA reaction mix comprises DNA templates produced in host cells.
  • the capillaries are designed to prevent leakage, e.g., by sealing, but at the same time to allow penetration, e.g., by a syringe.
  • This arrangement allows for the application of positive or negative pressure.
  • this arrangement permits aspiration of samples from microtiter plates into the capillaries, and subsequent dispensing back into plates for further processing.
  • multiple samples are thermocycled in a single capillary at the same time.
  • DNA and sequencing reagents are metered and mixed in a single capillary.
  • the invention provides a guide cap located above the seals at the top and bottom of each capillary tube. This arrangement serves to guide a syringe needle into the opening of the capillary.
  • the capillary tubes are made of glass, fused silica or TEFLON ® .
  • FIG. 1 is a schematic perspective view of a capillary based sample handling system in accordance with the teachings of the present invention.
  • FIG. 2 is a schematic perspective view of one capillary stage for achieving purification of the biological sample of the system of FIG. 1, in accordance with the teachings of the present invention.
  • FIG. 3 is a schematic perspective of a modular array of capillary tubes suitable for employment with the sample handling system of FIG. 1, in accordance with the teachings of the present invention.
  • FIG. 4 depicts an electrophoretic gel comparing sequencing reaction clean-up results obtained using an embodiment and method of the present invention (Dialysis) to results obtained using ethanol precipitation, as further described in Example I.
  • FIG. 5 depicts an electrophoretic gel comparing results obtained using an embodiment and method of the present invention with either ET primer mix or Big Dye Terminator mix as sample, as further described in Example II.
  • FIG. 6 is a schematic perspective view of a capillary based sample handling system employing a single capillary tube according to the teachings of the present invention.
  • 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.), 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.
  • the methods of the invention can be carried out with a biological sample of just about any volume, 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 from 10 ⁇ l to 0.05 ⁇ L, preferably 0.1 ⁇ L to 3 ⁇ L.
  • cassette refers to a structure or “module” capable of accommodating an array of capillary tubes handling a plurality of samples, e.g., 96 or more samples.
  • dialysis is art-recognized and is understood to refer to the separation of substances in solution by means of their unequal diffusion through a membrane.
  • equivalent dialysis refers to dialysis which occurs without exchange or flow of dialysate.
  • 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.
  • integrated processing refers to a process comprising at least two distinct steps which are "integrated” in the sense that the steps are carried out in a single operation.
  • a process includes, but is not limited to, template purification, polymerase chain reaction (PCR), DNA sequencing, polynucleotide ligation, cloning, ligase chain reaction (LCR), single nucleotide extension reaction, exonuclease treatment, and oligonucleotide hybridization reactions.
  • Process steps associated with these processes include, for example, the aspiration, mixing, incubation, purification, temperature treating, such as heating or cooling, and delivery of the biological sample alone or in a biologically compatible carrier fluid in a selected manner.
  • membrane element refers to a material which may used to separate substances in solution by means of unequal diffusion, e.g., by size exclusion.
  • exemplary membrane elements are semipermeable; i.e., the membrane elements are capable of permitting dialysis to take place.
  • 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 from a desired component(s). Such operations include, but are not limited to, filtration, ultrafiltration, dialysis/equilibrium dialysis, chromatography, etc.
  • 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 fiber materials.
  • temperature processing refers 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, etc.
  • continuous and discontinuous heating regimens e.g., denaturation, annealing, incubation, precipitation, etc.
  • ultrafiltration refers to any method of dialysis wherein the sample is under positive pressure relative to the dialysate.
  • the invention described herein includes an integrated capillary-based sample handling system for automated capillary-based processing of the present invention.
  • the system is capable of processing many samples in parallel, if desired, using standard micro-titer plates as reagent sources.
  • the use of capillary tubes in connection with the present invention has the advantage that only a small fraction of the liquid volume is exposed to the atmosphere, so that evaporation is minimized. This promotes the processing of the sample, while concomitantly eliminating or reducing sample loss.
  • the capillary tubes of 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.
  • FIG. 1 is a schematic depiction of the capillary based sample handling system 10 in accordance with the teachings of the present invention.
  • the illustrated system 10 employs a support assembly 12 that is configured for supporting and/or mounting a capillary based processing assembly 14.
  • the support assembly 12 positions the capillary based processing assembly 14 over a platen surface 30 that is sized and dimensioned for supporting a reservoir plate 32.
  • a controller 36 is coupled to selected components of the sample handling system 10, such as the support assembly and various portions of the processing assembly 14, as well as to a temperature regulating source 40.
  • the support assembly 12, platen 30, and control 36 can form part of a conventional fluid dispensing unit, such as a Hydra dispenser, manufactured by Robbins Scientific, U.S.A., and which can be modified in an appropriate manner obvious to the ordinarily skilled artisan in light of the teachings herein to operative ly mount the capillary based processing assembly 14.
  • a fluid dispensing unit such as a Hydra dispenser, manufactured by Robbins Scientific, U.S.A.
  • Those of ordinary skill will also recognize that other fluid dispensing and sample handling units presently employed and in practice, whether in modular or discrete forms, can be employed to perform the features and functions described herein, provided they are adapted to mount the capillary based processing assembly 14 of the present invention.
  • the illustrated processing assembly 14 includes multiple, discrete components that are functionally and operatively connected in a selected manner so as to perform multiple processing steps upon a biological sample.
  • the capillary based processing assembly 10 includes multiple, operatively connected capillary tubes that are coupled in a selected fashion to perform multiple processing steps in a single, vertically integrated unit.
  • the illustrated assembly 14 includes a fluid coupling 44 that is connected at one end to the support assembly 12, and at an opposite end to a fluid regulating device 48.
  • the fluid coupling 44 operates in connection with the controller 36 and support assembly 12 to regulate fluid movement within the processing assembly 14.
  • the fluid coupling 44 is coupled to a pump mechanism, which forms part of the support assembly 12, to either introduce fluid into or discharge fluid from a proximal end of the processing assembly 14.
  • the fluid coupling 44 can comprise any suitable self contained fluid actuating mechanism, such as a plunger-type or a syringe-type mechanical coupling, or can be any other suitable fluid conduit that serves as a fluid and mechanical coupling between the support assembly 12, which can comprise a fluid movement or pump mechanism, and the remainder of the processing assembly 14.
  • the fluid coupling can also optionally include a guide for a syringe/syringe needle at the top and/or bottom of the capillary tube 50.
  • the illustrated fluid regulating device 48 can be a valve-type structure, such as a fluid valve or pinchable siiicone connector, that is adapted for selectively placing the fluid coupling 44 and selected fluid communication with the remainder of the processing system, such as the capillary tube 50.
  • the fluid regulating device 48 can either be operated manually, or can be part of an overall automated sample handling system, such as the automated sample handling system 10 of the present invention, by coupling via any suitable communication pathway to the illustrated controller 36.
  • the capillary tube 50 can be a commercially standard and available capillary element that is sized and dimensioned for fluidly retaining microliters of a biological fluid.
  • the illustrated capillary tube 50 is adapted for holding a biological sample in a biologically compatible carrier medium.
  • suitable biological samples include DNA, proteins and other like biological components
  • the biologically compatible earner medium can be any known buffer or processing fluid typically employed in biological processing techniques.
  • the illustrated capillary tube 50 is further coupled at a proximal end 50A to a second fluid regulating device 48.
  • the fluid regulating device 48 is preferably similar to the fluid regulating device coupled to the distal end 50B of the capillary tube 50.
  • the combination of the pair of fluid regulating devices 48, (48 coupled to either end of the tube 50 can fluidly isolate the chamber of the capillary tube 50 from the remainder of the processing system 14). This configuration allows a selected processing regimen as implemented by the controller or by hand to occur within the confines of the capillary tube independent of any other activities being performed in any other section of the processing assembly 14.
  • a temperature regulating source 40 which can include a heating element or a cooling source, is thermally coupled to the capillary tube 50 when the processing assembly 14 is disposed in a selected position.
  • the temperature regulating source 40 is preferably a heating source that heats the capillary tube 50.
  • the biological sample and associated carrier fluid if desired and contained within the chamber of the capillary tube 50, can undergo a thermocycling process according to a user selected regimen.
  • the temperature regulating source 40 can comprise a series of resistive heating elements, or can be a heating source that passes heated air across the outer surface of the capillary tube 50.
  • the illustrated capillary tube 50 can be made of glass, or is coated with a polyimide coated fused silica.
  • Suitable capillary tubes for use in accordance with the teaching of the present invention are within the purview of one of ordinary skill, when considering the particular temperature ranges, capillary size, heating duration, and specific contents and quantities of the biological sample and carrier fluid. The ordinarily skilled artisan in light of all these parameters would be able to determine the appropriate capillary tube size and length.
  • the particular temperatures in which to heat the contents of the capillary tube are also within the purview of one of ordinary skill when considering the particular type of processing that is desired to be carried out within the capillary tube, and the type and quantity of the biological sample and carrier fluid.
  • a second capillary tube 52 has a distal end 52A coupled to the intermediate fluid regulating device 48, and has a proximal end 52B coupled to a third fluid regulating device 48.
  • the fluid regulating devices 48 coupled to the proximal and distal ends 52A and 52B of the capillary tube 52, also serve to isolate fluidly the capillary tube 52. Consequently, a second or additional processing regimen can occur within the capillary tube 52 independently, contemporaneously and concurrently of any particular processing regimen that occurs within the other capillary tube 50.
  • a fluid tip 56 having a central lumen is coupled to the third fluid regulating device 48 in accordance with known techniques.
  • the fluid tip 56 can serve to either transfer fluid to or receive fluid from the reservoir 32 disposed on the platen 30 of the sample handling system 10.
  • the illustrated reservoir 32 can be a standard 96-well micro-titer plate, and hence can be employed in connection with the illustrated system 10 to perform multiple, parallel processing of biological samples.
  • the fluid tip is preferably any suitable mechanical coupling that fluidly connects the processing assembly with an external fluid source, such as the fluid reservoir, according to one
  • the illustrated processing assembly 14 provides for a single, stacked, and integrated processing sub-assembly 14 that allows for single or multiple biological samples to be aspirated throughout the axial length of the assembly, and can isolate, in connection with the fluid regulating devices, selected portions of the assembly to perform different processing regiments in parallel.
  • the illustrated control 36 is programmed to control, according to user selected information, the fluid regulating elements 48 to selectively connect or disconnect either of the capillary tubes 50 and 52 relative to each other.
  • the controller 36 can dispose in an open position the illustrated fluid regulating devices 48 to effectively form a single axial fluid lumen along about the entire axial length of the assembly 14; that is, from the support assembly to the exit port of the fluid tip 56.
  • the controller 36 can actuate one or more of the fluid regulating devices 48 to fluidly separate or isolate one or more regions of the processing assembly from the remaining regions.
  • the illustrated controller 36 also controls the support assembly 12.
  • the controller provides selected control data that instructs the support assembly 12 to position the processing assembly 14 at selected positions to either retrieve or introduce fluid to and from the reservoir 32.
  • the axial movement imparted by the support assembly 12 to the processing assembly 14 is indicated by the arrow 58.
  • the illustrated controller 36 further provides control signals to the temperature regulating source 40 to selectively actuate the source to provide heating or cooling of a particular region of the processing assembly 14.
  • the illustrated temperature regulating source 40 is positioned for regulating the temperature of the first capillary tube 50, those of ordinary skill will readily recognize that the support assembly 12 can position other portions of the processing assembly 14, such as the second capillary tube 52, in a position to be heated or cooled by the source 40. Alternatively, multiple sources 40 can be used.
  • the illustrated support assembly 12 and/or controller can move the processing assembly 14 in the horizontal direction, as indicated by anow 58A.
  • a significant advantage of the illustrated sample handling system 10 is that it provides for an automated, high speed sample handling system that can process low volumes of a biological sample in an automated format, without the need for centrifugation which typically requires large volumes of sample.
  • the illustrated processing assembly of the system 10 preferably employs reusable components, thereby significantly extending the useful life of the major system components.
  • the integrated processing assembly 14 also provides for an efficient, compact and relatively simple processing assembly for performing one or more processing regimens in parallel, and if desired, independently of each other.
  • a significant advantage of employing multiple capillary tubes in the processing assembly 14 is that the sample volumes provided by each capillary tube allows the processing of significantly smaller sample portions, since relatively small volumes of the overall earner fluid disposed within the capillary tubes are subject to evaporation.
  • the capillary tubes preferably have internal volumes that accommodate fluid sizes of less than about 1 microliter.
  • An advantage of employing the novel submicroliter capillary tubes, in tandem, as illustrated in Figure 1, is that it allows the use of minimal amounts of expensive sequencing reagents and relatively small volumes of biological samples in an automated sample handling format.
  • the illustrated processing assembly 14 can be used to perform purification procedures on polymerase chain reaction (PCR) products, preparing sequencing ladders, and injecting the sequencing ladders into appropriate microtiter plates, or aspirating the biological products into particular zones of the processing assembly 14.
  • PCR polymerase chain reaction
  • the illustrated processing assembly 14 can be employed to purify a biological sample disposed within one or more regions of the processing assembly.
  • the capillary tube 52 which is coupled to a fluid regulating device 48 at either end, can house a separation element, such as a semipermeable microfiber, for processing a biological sample, such as DNA.
  • the capillary 52 and illustrated microfiber 60 can be employed to perform equilibrium dialysis within the confines of a capillary electrophoretic system.
  • DNA sequencing products are purified to remove excess salt, nucleotides, primers, and templates from the biological sample.
  • the illustrated microfiber 16 can be employed to perform the filtration process upon the DNA, to exclude the desired products, while concomitantly allowing undesired components to pass therethrough when the processing assembly is exposed to a pressure or vacuum condition at a proximal end.
  • the DNA sample is cycled through the microfiber by the pressure formed within the system, thereby resulting in relatively small components being filtered out of the hollow fibers and hence the sample.
  • the use of a capillary tube with one or more microfibers disposed therein, provides for the ability to perform equilibrium dialysis upon very small volumes of between about 10 to 0.05 microliters.
  • an additional significant advantage of the present invention is that the capillary tubes 50 and 52 can form part of an anay of capillary tubes 70, e.g., a cassette of capillary tubes.
  • the illustrated array of capillary tubes allow the illustrated sample handling system 10 to perform multiple, parallel processes in a vast array of processing assemblies 14.
  • the system 10 can accommodate the use of multiple arrays of the capillary tubes 50 and 52.
  • the capillary tubes 50 can comprise part of an array, and the capillary tube 52 can also form part of a second array of capillary tubes.
  • the illustrated fluid regulating devices 48 can be interposed between the modular capillary tube arrays, to form a modular and stackable assembly of processing tubes for use in connection with the illustrated system 10.
  • the illustrated controller 36 can provide instruction data to a suitable robotic assembly, such as a pipetting robot, to selectively stack together or remove one or more of the modular arrays of capillary tubes 50 and 52 from the processing assembly 14 of the illustrated system 10.
  • a suitable robotic assembly such as a pipetting robot
  • This modular aspect of the illustrated system 10 allows for the removable and replaceable use of modular anays of capillary tubes, in order to aspirate or introduce biological samples to a standard 96-well microtiter plate.
  • the stackable aspect of the modular array of capillary tubes allows multiple selected processes to be conducted within one or more of the capillary tubes.
  • the system can disconnect one or more of the modular anays with the pipetting robot for downstream processing or storage.
  • the sample handling system 10' can employ a single capillary tube 51 for processing a biological sample.
  • the sample handling system 10' can be used in applications in which it may be desirable to effect a single processing regimen upon the biological sample.
  • processing regimens can include sample purification and thermocycling, as well as any other of the sample processing steps described herein.
  • a membrane element such as the microfiber 60 illustrated in Figure 2, can be provided within the capillary tube 51.
  • purification of a sample may be achieved by a variety of methods, including dialysis, filtration, ultrafiltration and chromatography.
  • the invention further provides various configurations to achieve purification, depending on the method of purification selected.
  • the apparatus of the invention provides at least one capillary comprising a membrane element in operative contact with a dialysate, e.g., water.
  • a dialysate e.g., water
  • the dialysate is contained in a cassette.
  • the capillary may be inserted successively into at least two cassettes containing a dialysate.
  • the present invention includes dialysis techniques, which may be used effectively to "clean up" polymerase chain reaction (PCR) and cycle sequencing reactions.
  • PCR polymerase chain reaction
  • the typical PCR or sequencing reaction generally utilizes sample volumes of approximately 10 ⁇ L or less, significantly smaller than the sample volumes in conventional dialysis techniques.
  • the present invention addresses this disparity by using a membrane element, such as one or more microfibers inserted within one of the capillary tubes.
  • the microfiber performs the same separation functions as the much larger dialysis operations, but with much smaller sample volumes and without the use of centrifugation.
  • the microfibers can be generated or manufactured by removing one or more hollow fibers from commercially available filtration cartridges. Typical cartridges contain many hundreds of fibers, since the cartridge is solely designed to perform dialysis on large sample volumes, e.g., 1 mL or more. Many types and sizes of hollow fiber filtration cartridges are available through such suppliers as Millipore Corp. Bedford, MA or Spectrum Labs Websites Website, CA.
  • these cartridges are used as ultrafiltration devices, where the dialysis membrane acts as a filter, excluding the desired products while allowing the undesired components to pass through when pressure or vacuum is applied to the system.
  • the present invention achieves proper filtration or separation of components from small volumes of a biological sample by employing one fiber for each biological sample. In this way, dialysis on sample volumes of 10 to 0.05 ⁇ L volumes is achieved.
  • the present invention achieves appropriate purification of a sample by first performing a standard Big Dye Terminator Cycle
  • Capillary ultrafiltration material allows the removal of unwanted reaction components that are small in molecular size compared to the desired DNA products by dialysis against water in an adjacent chamber.
  • each zone of, or processing regimen performed by, the sample handling device can be isolated from the others by the illustrated valves 48 to prevent 'wandering' of the sample during each step of the procedure.
  • a linear or rectangular anay of such devices interfaces conveniently with a microtiter plate 32 disposed on the system platen 30.
  • the dialysis and thermocycling sections of the array are enclosed within separate sealed containers to provide controlled liquid or air flow across the devices. This anangement allows sub-microliter quantities of liquids to be aspirated by negative pressure on the system.
  • the samples are specifically moved to various parts of the system by controlled pressure changes.
  • a volume of 0.5 ⁇ l occupies a height of approximately 1 cm.
  • a number of commercial devices are available on the market that may be adapted in accordance with the teachings of the present invention for small volume aspiration and delivery utilizing capillary sample holders. These include syringe pumps from several manufacturers, 96- and 384- channel sample liquid dispensers from Robbins Scientific, Inc. (Hydras), "Nanomovers” from Precision Scientific, and several other possible designs.
  • the Hydra systems are convenient because of the ability to simultaneously, accurately, and coherently aspirate and deliver selected volumes from parallel channels. These systems offer ease of integration with physical plate-handling systems and PC-based programming systems through an RS232 port.
  • a two-temperature air-circulation system with appropriately placed valves may be used to enable a wide range of air temperatures to be quickly attained.
  • the heating source 40 can be employed to heat a sample disposed in the capillary tube 50 and isolated from other sections of the processing assembly 14 by a pair of fluid regulating devices 48.
  • the thermocycler will use a combination of hot and cold air to change sample temperature.
  • Simple air blowers or blowing ambient air and air heated by resistance heaters over the capillaries may be used to change the temperature.
  • the temperature may be measured and controlled by standard PID controllers.
  • the heating rate may be increased as desired by using, for example, superheated air for the first part of the heating cycle, then cooler air to avoid excessive overshoot of the temperature of the capillaries.
  • the in-line processing assembly 14 mounted to the support 12 of the sample handling system 10 integrates sample aspiration, mixing, thermocycling, dialysis and dispensing in a single device. This achieves definite advantages in terms of sample integrity (no evaporation), simplicity and throughput.
  • Optical sensors may be employed in connection with the illustrated system 10 to detect liquid levels at one or more points in the system 10, and provide open loop or feedback control to the controller 36 to adjust, if necessary, the sample or fluid level volumes within one or more sections of the processing assembly.
  • the present system 10 provides an accurate volumetric system that is much simpler to operate and maintain than conventional systems.
  • the invention also relates to purifying and cleaning methods that remove contaminants quickly and efficiently from a DNA reaction mix.
  • Cunent sequencing machines use electrophoresis through a gel to separate and detect different lengths of DNA that have been appropriately labeled. To make these machines provide results faster and more accurately, the shapes of the gel separation media have gone from thick gels to a gel captured by thin capillaries.
  • a major drawback is the contaminants in the DNA being sequenced tend to physically plug the capillary and interfere with the accurate detection of the different DNA lengths.
  • One major source of contaminants in the DNA sample is the result of by-products of the thermocycling reaction that generates the DNA sample. Both regular and dye-labeled nucleotides that are not incorporated into the DNA strings during the reaction become contaminants that degrade the DNA sequencer. Additionally, ionic components of the reaction reagents remaining in the reaction (e.g., salts) also degrade the machines.
  • the present invention provides for effective removal of contaminants from a thermocycling reaction.
  • purification may be achieved by placing the mixture into a hollow membrane element, which is in contact with a solution having a lower concentration of ionic components. The difference in osmotic pressure across the membrane forces contaminants in the product to migrate across the membrane into the aqueous solution, effectively removing them from the product.
  • the invention provides an apparatus and method for purifying DNA molecules produced in host cells.
  • the invention provides an apparatus and method for producing template D ⁇ A in a host cell, lysing the host cell, purifying and sequencing the template D ⁇ A.
  • the system 14 is first rinsed with water, and the syringes are then filled with water except for about 5 ⁇ L.
  • a 1 cm (0.5 ⁇ L) air gap is drawn up, followed by 0.5 ⁇ l DNA (50 ng/ ⁇ L), and 0.5 ⁇ L of Big Dye Terminator Cycle Sequencing Ready Reaction Mix ("DT")(part number 4303154, PE Applied Biosystems, Foster City, CA).
  • DT and DNA are then ejected to mix.
  • 1 ⁇ l of the reaction mixture is aspirated and moved up to the thermocycling zone 50 (maintaining a 1 -2 cm air gap), and then thermocycling is performed.
  • the sample is moved down to the dialysis zone 52 and dialyzed for 20 min.
  • the samples are ejected into a microtiter plate and rinsed with water.
  • a plate handler below changes plates (from DNA source, to DT mix, to output plate) and moves them up and down (to create air gaps).
  • the pipetting, flow-through purification, and thermocycling are fast, so the throughput is very high; e.g. , on the order of 384 samples per hour for a 384 channel system. If gels can be run at a rate of 96 samples every 3h (the maximum proposed speed), 1 sample robot could feed 8 gel systems running at full speed.
  • the sample handling device may also be constructed as a series of independent modules that can be stacked together, or independently attached temporarily to a liquid handling device to effect the liquid handling steps.
  • An advantage of this approach is that a single liquid handling device, which is the most expensive part of the system to build, could be more optimally utilized to process samples in a large number of dialysis and thermocycling modules.
  • the latter modules can then be handled in a similar fashion as plates are handled on a typical integrated automation system, such that the independent units would be advantageously sealed automatically when detached from the liquid handling device, and that plate-to-plate transfers would be replaced by capillary-to-capillary transfers (avoiding contact with the atmosphere).
  • An example of a dialysis module is shown in Figure 3.
  • the individual hollow fiber dialysis tubes were obtained by cutting open hollow fiber filtration cartridges produced by Amicon of Beverly, MA and then removing the fibers. Fibers were stored in sterile distilled water until ready for use. Fibers containing different pore sizes were used in these studies but the bulk of the experiments were canied out using filters with an average pore size of 1 OOKdal.
  • the typical method of removing by-products of the sequencing reaction refened to as "cleaning up” is ethanol precipitation (EtOH prec).
  • EtOH prec ethanol precipitation
  • the method of the present invention was compared to EtOH prec.
  • Figure 4 visual inspection of a 377 gel comparing results obtained with the dialysis procedures of the invention versus a standard EtOH prec. protocol that the dialysis methodology removes the unincorporated dye terminators which are by-products of sequencing reactions and produces better resolution compared to EtOH precip.
  • Table 1 Column one describes the readlength which is the total number of reliable bases produced for that particular sequencing reaction. Columns two and three list the total number of bases which have quality scores that are greater than or equal to the Phred scores 30 and 20, respectively.
  • the increase number of bases for the readlengths for dialysis coupled with the increased number of bases have a higher quality Phred scores indicates that the dialysis method produces superior results.
  • the following experiment demonstrates the kinetics of the dialysis reaction.
  • Ten microliters of solution containing either BD terminator mix or ET primer mix was used as sample.
  • the BD terminator mix simulates the excess fluorescently labeled dideoxy- terminator products found in a standard sequencing reaction
  • the ET primer mix simulates the unincorporated primers found in a PCR reaction.
  • Each sample was dialyzed using the above methods with a fibers having an average pore size of 1 OOKdal for the following time points: 0, 5,10, 15, 30, 60, 120 minutes and one final point at 12 hours or overnight (o/n).
  • the results, as shown in Figure 5, clearly indicate that for the sequencing reactions (i.e., BD terminator mix) dialysis occurs very quickly: equilibrium occurs within 5 to 10 min.
  • Plasmid template purification was performed using 1.1 mm ID diameter tubing having a pore size of lOOKdal. Plasmid subclones containing human BAC inserts were grown in 96 well format and processed by centrifugation, heating at >95°C for 2 min in STET buffer with lysozyme (75uL), and filtration. The cleared filtrate ( ⁇ 30 ⁇ L) was then dialyzed for 30 minutes against 1 mM EDTA. Four microliters of the resulting samples were then added directly to Big Dye terminator sequencing mix and the samples were separated on an ABI 377 automated DNA sequencer (PE Applied Biosystems, Foster City, CA).
  • RapidCycler 1605 (Idaho Technology; Idaho Falls, ID) was tested for small
  • thermocycling using glass, silica, TEFLON , and stainless steel capillaries.
  • thermocycling to be achieved in long TEFLON capillary tubes. Therefore, sealing means, valves, or a pressure control system are a desirable part of a robust flow-through capillary thermocycling system.
  • sequencing reagents are advantageously added to the DNA prior to thermocycling in accordance with the reaction set up protocol.
  • the dye is metered into a Microtiter plate, then the DNA is metered into the same plate.
  • the plate is then mixed and thermocycled.
  • microliter quantities are thermocycled in a capillary.
  • DNA from the dialysis tubing was performed as follows. A standard l/4x Big Dye terminator reaction was canied out, and diluted to the equivalent of l/32x, l/64x, and l/128x. Eleven replicates of the diluted samples were then dialyzed as described in Example II and separated on a MegaBace 100 automated DNA sequencer (Molecular Dynamics Corp., Sunnyvale, CA). The results indicated that the small amounts of sequencing reaction products present in these samples were effectively recovered in approximately 90% of the samples. The readlength and quality statistics are shown in the Table 3 (average of all samples).

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Abstract

L'invention concerne un dispositif automatique, permettant de traiter rapidement des échantillons de la taille du sous-microlitre, par réactions chimiques ou enzymatiques qui impliquent une manipulation de liquide, une ou plusieurs étapes d'incubation à température régulée (notamment le thermocyclage), et une étape de purification basée sur la discrimination de taille moléculaire. On compte, notamment, parmi les applications exemplaires, la réaction en chaîne de la polymérase (PCR), des applications de séquençage d'ADN, la ligation d'oligonucléotides, la ligation répétitive d'oligonucléotides (LCR), l'extension unique de nucléotides, le traitement par un exonucléase, et des dosages d'hybridation d'oligonucléotides.
PCT/US2000/011371 1999-04-29 2000-04-29 Dispositif permettant de traiter rapidement un echantillon d'adn par manipulation de liquide, thermocyclage et purification WO2000066995A2 (fr)

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EP00928488A EP1177043A2 (fr) 1999-04-29 2000-04-29 Dispositif permettant de traiter rapidement un echantillon d'adn par manipulation de liquide, thermocyclage et purification
CA002369363A CA2369363A1 (fr) 1999-04-29 2000-04-29 Dispositif permettant de traiter rapidement un echantillon d'adn par manipulation de liquide, thermocyclage et purification
JP2000615584A JP2002543418A (ja) 1999-04-29 2000-04-29 流体操作、サーモサイクリング、及び精製を一体化した迅速なｄｎａサンプル処理装置
AU46724/00A AU4672400A (en) 1999-04-29 2000-04-29 Device for rapid dna sample processing with integrated liquid handling, thermocycling, and purification

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US7550266B2 (en) 2001-10-24 2009-06-23 Primera Biosystems, Inc. Methods and systems for dynamic gene expression profiling
US7674582B2 (en) 2002-04-12 2010-03-09 Primeradx, Inc. Methods for amplification monitoring and gene expression profiling, involving real time amplification reaction sampling
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AU4672400A (en) 2000-11-17
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CA2369363A1 (fr) 2000-11-09
JP2002543418A (ja) 2002-12-17

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