WO2001021310A9 - Dispositif de traitement rapide d'echantillons d'adn a manipulation des liquides, thermocyclage, et purification integres - Google Patents

Dispositif de traitement rapide d'echantillons d'adn a manipulation des liquides, thermocyclage, et purification integres

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Publication number
WO2001021310A9
WO2001021310A9 PCT/US2000/025851 US0025851W WO0121310A9 WO 2001021310 A9 WO2001021310 A9 WO 2001021310A9 US 0025851 W US0025851 W US 0025851W WO 0121310 A9 WO0121310 A9 WO 0121310A9
Authority
WO
WIPO (PCT)
Prior art keywords
capillaries
capillary
capillary cassette
fluid
cassette
Prior art date
Application number
PCT/US2000/025851
Other languages
English (en)
Other versions
WO2001021310A3 (fr
WO2001021310A2 (fr
Inventor
Patrick Cahill
Douglas Smith
Ulrich Thomann
Marcy Engelstein
Original Assignee
Genome Therapeutics Corp
Patrick Cahill
Douglas Smith
Ulrich Thomann
Marcy Engelstein
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 Corp, Patrick Cahill, Douglas Smith, Ulrich Thomann, Marcy Engelstein filed Critical Genome Therapeutics Corp
Priority to AU75979/00A priority Critical patent/AU7597900A/en
Priority to EP00965231A priority patent/EP1214149A2/fr
Priority to CA002385029A priority patent/CA2385029A1/fr
Priority to JP2001524729A priority patent/JP2004500552A/ja
Publication of WO2001021310A2 publication Critical patent/WO2001021310A2/fr
Publication of WO2001021310A3 publication Critical patent/WO2001021310A3/fr
Publication of WO2001021310A9 publication Critical patent/WO2001021310A9/fr

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    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L9/00—Supporting devices; Holding devices
    • B01L9/06—Test-tube stands; Test-tube holders
    • B01L9/065—Test-tube stands; Test-tube holders specially adapted for capillary tubes
    • 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
    • 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
    • B01D61/24—Dialysis ; Membrane extraction
    • B01D61/28—Apparatus therefor
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D63/00—Apparatus in general for separation processes using semi-permeable membranes
    • B01D63/02—Hollow fibre modules
    • B01D63/026—Wafer type modules or flat-surface type modules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
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    • B01J19/0093—Microreactors, e.g. miniaturised or microfabricated reactors
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    • B01L3/021—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids
    • B01L3/0217—Pipettes, i.e. with only one conduit for withdrawing and redistributing liquids of the plunger pump type
    • B01L3/022—Capillary pipettes, i.e. having very small bore
    • B—PERFORMING OPERATIONS; TRANSPORTING
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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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    • B01D2313/54—Modularity of membrane module elements
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    • B01D2319/06—Use of membranes of different materials or properties within one module
    • 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
    • B01J2219/00364—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
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    • 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
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    • 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
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    • 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
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    • 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
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    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0829—Multi-well plates; Microtitration plates
    • B—PERFORMING OPERATIONS; TRANSPORTING
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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
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    • 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
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    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
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    • 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
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    • 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
    • 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
    • B01L3/5027—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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • 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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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.
  • the capillaries are filled, mixed and handled individually as they are moved through several functional "stations" on a conveyor belt type of arrangement (Friedman and Meldrum, 1998).
  • 96 capillaries are attached to a Hydra dispenser (Robbins Scientific) so that the samples can be moved up and down past heating elements to perform PCR (Hunicke-Smith, 1997).
  • copper heating elements were moved up and down with respect to the position ofthe samples (Stanford Technology Lab, 1998).
  • a significant drawback in standard 5-10 ⁇ l sequencing reactions is that at least 50% ofthe sample is wasted, never being loaded on the gel.
  • a capillary cassette having a frame defining an interior chamber, a plurality of capillaries, each having a first .and second end, wherein at least one ofthe first end and the second end is mounted to the frame, such that each ofthe capillaries is fluidly coupled to an external surface ofthe frame.
  • the present invention provides a sample handling cassette including a frame having a first end and a second end, with a passage through the frame extending from the first end to the second end, a first flat membrane layer disposed along opposed sides ofthe frame and defining, along with the frame a sample handling chamber.
  • a docking port is provided including a guide cap having a concave end and, optionally, a fluid-tight seal.
  • a method of performing dialysis on a biological sample involving the use of a capillary cassette.
  • a method of performing temperature processing on a biological sample involving the use of a capillary cassette is provided.
  • a biological sample handling system for use with a liquid handling machine involving the use of a capillary cassette and a needle bed.
  • a hotel for processing multiple biological samples is provided including a housing and a fluid management system.
  • the hotel ofthe present invention can process a plurality of capillary cassettes.
  • a template preparation module is provided involving an apparatus management device and a capillary cassette.
  • Figure 1 is a perspective view of a capillary cassette according to a variation of a first embodiment ofthe invention
  • Figure 2 is a perspective view of a frame according to the first embodiment of the invention.
  • Figure 3 is a close-up perspective cross-sectional view of a docking port according to the first embodiment ofthe invention
  • Figure 4 is a cross-sectional view of a docking port according to the first embodiment ofthe invention
  • Figure 5 is a cross-sectional view of a portion of a sample handling cassette according to a second embodiment ofthe invention
  • Figures 6 and 7 are perspective views of a capillary cassette used in conjunction with a liquid handling machine according to a third embodiment ofthe invention.
  • Figure 8 is a perspective cross-sectional view ofthe third embodiment of the invention.
  • Figures 9 and 10 are a cross-sectional perspective view of a sample handling cassette according to the second embodiment ofthe invention used with a needle bed and microtiter plate according to the third embodiment ofthe invention;
  • Figure 11 is a perspective view of a template preparation module in accordance with a fourth embodiment ofthe invention;
  • Figures 12 and 13 are close-up perspective views ofthe template preparation module according to the fourth embodiment ofthe invention;
  • Figure 14 is a perspective view of a hotel according to a fifth embodiment ofthe invention.
  • Figure 15 is a perspective view of a hotel according to a sixth embodiment ofthe invention.
  • Figure 16 is a close-up perspective view of a capillary cassette and hotel according to the sixth embodiment ofthe invention.
  • the present invention addresses a need in the art for small-volume DNA purification methods, preferably with re-usable components, that can be easily integrated with capillary-based sample handling, and that eliminate the need for centrifugation.
  • the invention also performs capillary-based clean-up devices and sample handling alternatives to air-driven thermocycling, and methods for efficiently handling sample amounts that are just sufficient for each separation, in order to achieve significant cost savings.
  • 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, R A, 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.
  • biological samples in accordance with the invention preferably have microliter ( ⁇ L) volumes and therefore can be referred to as microsamples, e.g. , biological microsamples.
  • cassette refers to a structure or “module” capable of handling a plurality of samples, for example, 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.
  • equilibrium 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 ofthe dialysate surrounding the membrane.
  • frame refers to any suitable structure for providing mechanical support to a capillary.
  • the term "hotel” refers to a unit for housing one or more cassettes and provides a platform for sample processing.
  • the hotel is adapted for sample thermocycling by the inclusion of means for circulating a fluid, for example, water or air, to provide temperature control.
  • the hotel also provides a platform for sample purification.
  • the hotel is adapted for dialysis ofthe sample by the inclusion of means for circulating a dialysis fluid.
  • the hotel advantageously provides a washing platform by the inclusion of means for circulating a liquid (e.g., water or a chemical cleaning solution) such that cassettes can be washed to prevent sample carryover, or can be regenerated.
  • filter and "membrane element” refer to a material which may used to separate substances in solution by means of unequal diffusion, for example. , by size exclusion.
  • exemplary membrane elements and filters are semipermeable; i.e., the membrane elements or filters 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 ofthe 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.
  • purification of a biological 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 ofthe invention provides at least one capillary comprising a membrane element in operative contact with a dialysate, for example, water.
  • a dialysate for example, water.
  • the capillary may be exposed successively to at least two dialysates.
  • the capillary cassette 10 may further include one or more ports for inflow and/or outflow of dialysate.
  • the invention further includes microdialysis-based sample clean up and plasmid clean up.
  • 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 optionally using a membrane element, such as one or more microfibers inserted within one ofthe capillaries. 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 Georgia Pacific Scientific, 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 Sequencing Ready Reaction Kit, part # 4303154 PE Applied Biosystems Foster City CA, on a reaction sample size of between 0.05-10 ⁇ l.
  • the sample volume is drawn up into a hollow fiber filter which has been cut out of a Spectrum cartridge cat # 132229 Spectrum Labs Website, CA using a 10 ⁇ l syringe from Hamilton, Reno, Nevada (see FIG. 2). Purification is then achieved according to any ofthe various methods described herein.
  • the invention also relates to purifying and cleaning methods that remove contaminants quickly and efficiently from a DNA reaction mix.
  • Current 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 ofthe 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 ofthe different DNA lengths.
  • One major source of contaminants in the DNA sample is the result of by-products ofthe 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.
  • the present invention provides for effective removal of contaminants from a thermocycling reaction. Once the reaction mixture is thermocycled, 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 involves processes including, 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 ofthe biological sample alone or in a biologically compatible carrier fluid in a selected manner.
  • a first embodiment ofthe present invention involves a capillary cassette 10 based on a standard microtiter plate configuration, such as an 8 x 12 array of elements on 9 mm centers, to allow immediate integration with existing laboratory automation devices and capillary electrophoresis (CE) sequencing instruments.
  • the footprint of this embodiment will be ofthe same dimensions as a 96-well plate, but the height may be increased to accommodate an appropriate length of capillary tubing.
  • This embodiment enables the optional use of an existing 96 channel pipetting device, such as the Robbins Hydra, to perform the liquid handling aspects ofthe process.
  • the capillary cassette 10 ofthe present invention may optionally be formed with any number of channel pipetting devices. Other devices may include 384 channels or more.
  • the capillary cassette 10 ofthe present invention includes a plurality of capillaries 12. Each capillary 12 has a first end 14 and a second end 15, each of which is securely mounted within a frame 16.
  • Frame 16 may be formed so as to accommodate 96 capillaries 12, or may optionally be formed to accommodate a subset ofthe capillaries 12 within the capillary cassette 10, as shown in Figure 1.
  • Open space 18 is preferably provided between capillaries 12, so as to allow fluids, such as liquids or gasses, to pass within or through an interior of capillary cassette 10.
  • the assembled capillary cassette 10 thus defines an interior chamber 30, with capillaries located therein, and through which air or water can be introduced and circulated over the capillaries to achieve thermocycling or dialysis.
  • the capillaries preferably have internal volumes that accommodate fluid sizes of less than about 1 microliter.
  • the methods of the invention are advantageously practiced with biological samples having volumes ranging down to approximately 0.05 ⁇ L, preferably 0.1 ⁇ L to 3 ⁇ L.
  • An advantage of employing the novel submicroliter capillaries is that minimal amounts of expensive sequencing reagents and relatively small volumes of biological samples may be used in an automated sample handling format.
  • the invention can be used, for example, 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.
  • PCR polymerase chain reaction
  • the capillary cassette 10 ofthe present invention is formed with a first and second end 20, 22. Both the first and second end 20, 22 ofthe capillary cassette 10 may be open or closed by an optional end plate 24.
  • the end plate 24 may further optionally be provided with ports to facilitate the entry and exit of fluids, such as gasses or liquids, passed within an interior chamber 30 of capillary cassette 10.
  • a sealing gasket 26 is preferably optionally provided between frame 16 and the optional end plate 24.
  • An optional sealing gasket 26 is also provided between frames 16, if more than one frame 16 is used, as shown in Figure 1.
  • holes are formed in the frames to accommodate one or more fasteners, such as pins 28, that are optionally provided to mount the plurality of frames 16 to one another.
  • the pins 28 may also secure optional end plates 24 to the frames 16.
  • four pins 28, each having threads and associated nuts are used, spaced along a perimeter of frames 16, as shown in Figure 1.
  • screws, rivets or other compressive fasteners may be used in combination with or in place ofthe pins 28.
  • the pins 28, or their alternative, are preferably formed of stainless steel.
  • frames 16 accommodating subsets ofthe total number of capillaries 12 within capillary cassette 10 provides the ability to replace a portion ofthe capillaries 12 within the capillary cassette 10 in the event of a capillary failure. Therefore, all capillaries 12 within capillary cassette 10 need not be discarded. Only the capillaries 12 sharing a frame 16 with the failed capillary are discarded.
  • FIG. 2 illustrates a frame 16 according to a variation ofthe first embodiment ofthe invention.
  • a docking port 40 may be provided according to the first embodiment ofthe invention.
  • the docking port 40 may optionally be mounted to frames 16 so as to be fluidly connected to the first end 14 and the second end 15 of capillary 12.
  • a docking port 40 may only be provided to a single end of capillary 12, or may be omitted entirely.
  • the docking port 40 includes a guide cap 42, optionally configured with a concave surface facing away from the capillary 12 so as to guide a needle 44 to be co-axially aligned with the capillary 12.
  • the needle 44 will be a blunt syringe needle.
  • the guide cap 42 is optionally securely mounted to the frame 16.
  • the guide cap 42 may be press fit within a portion ofthe frame 16.
  • an adhesive is preferably used for mounting the guide cap 42 to the frame 16 within a portion of the frame 16.
  • a sealing element 46 is provided within docking port 40.
  • the sealing element 46 is preferably a rubber o-ring gland seal from Apple Rubber Corp., press fit within a cavity 47 of guide cap 42.
  • the dimensions ofthe sealing element 46 and cavity 47 are formed so as to allow a blunt needle 44 to be inserted through the sealing element 46 without damaging the sealing element 46, while simultaneously providing a fluid-tight seal to the corresponding end ofthe capillary 12.
  • the use of a blunt needle 44 aids in reducing damage to the sealing element 46, and provides for extensive repetitive use ofthe docking port 40.
  • DT-mix DNA and Big Dye Terminator Cycle Sequencing Ready Reaction Mix
  • the capillaries are made of glass, fused silica, polyimide coated fused silica, or TEFLON .
  • the frames and end plates are preferably fabricated as injection molded parts. Assembly of individual capillaries in the apparatus ofthe present invention may be achieved in a variety of ways. In one embodiment, capillaries may be cut to size, assembled into cast grooves, and then secured in place. Capillaries may be secured using a waterproof and temperature resistant glue.
  • a significant advantage of employing multiple capillaries is that the sample volumes provided by each capillary tube allows the processing of significantly smaller sample portions, since relatively small volumes ofthe overall carrier fluid disposed within the capillaries are subject to evaporation.
  • This sample conservation advantage significantly reduces the sample volumes necessary to achieve selected processing ofthe sample, while concomitantly affording sample outputs that have sequencing ladders with improved signal strength and resolution.
  • the amount of fluorescently labeled DNA that can be detected on current sequencing machines is much lower than the amounts that are typically processed; 0.5-1 ⁇ l samples are sufficient.
  • FIG. 5 illustrates a second embodiment ofthe invention involving a variation ofthe construction ofthe sample handling chamber.
  • the second embodiment ofthe invention uses a flat membrane 61 in place of capillaries.
  • a flat membrane 61 is provided along both sides of a frame 62.
  • a sample handling chamber 64 is formed by a cutout ofthe frame 62 and two flat membranes 61 layers.
  • a first flat membrane layer 66 is provided along a forward surface ofthe frame 62 as shown in Figure 5.
  • a second flat membrane layer 68 is mounted along a back side ofthe frame 62 as illustrated in Figure 5.
  • a hollow fiber 70 may be mounted within the first or second embodiment ofthe invention.
  • the second embodiment ofthe invention provides additional durability and economy. By omitting capillaries, there is no glass to break during rough handling ofthe frame 62. Furthermore, by the use ofthe flat membrane 61 to form a plurality of sample handling chambers 64, each frame can be quickly and easily formed.
  • the capillary cassette 10 is used in conjunction with a liquid handling machine 80.
  • Hydra liquid handling machines manufactured by Robbins Scientific, U.S.A., are convenient because ofthe 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.
  • the Robbins Hydra is also preferred because ofthe Teflon seals on the syringe plungers, availability of 384-channel models, and lower overall cost.
  • the liquid handling machine 80 is provided with needles 44 and optionally a needle alignment frame 82.
  • the needle alignment frame 82 is preferably configured so as to align the needles 44 axially with the capillaries 12 ofthe capillary cassette 10.
  • docking ports 40 are provided at a first end 14 ofthe capillaries 12 to assist in alignment ofthe needles 44 with the capillaries 12.
  • a needle bed 90 is provided below the capillary cassette 10 and is provided with a plurality of needles 92.
  • Each needle 92 has a first end 94 oriented toward the capillary cassette 10 and a second end 96 oriented away from the capillary cassette 10.
  • the first end 94 of needles 92 is axially aligned with the second end 15 ofthe capillaries 12 so as to be able to be inserted through docking port 40 located in fluid contact with the second end 15 of capillary 12.
  • a second end 96 ofthe needles 96 may optionally be configured so as to be inserted within a well 102 of microtiter plate 100.
  • the liquid handling machine 80 is preferably configured so as to provide relative vertical movement between the needles 44, the needle alignment frame 82, the capillary cassette 10, the needle bed 90 and the microtiter plate 100.
  • the liquid handling machine 80 is provided with a platen surface 84 to support the microtiter plate 100.
  • Figures 9 provides a cross-sectional perspective view ofthe non-capillary configuration ofthe second embodiment ofthe invention, used with a needle bed 90 and microtiter plate 100.
  • Figure 10 provides a further perspective view showing an exemplary sample handling chamber 64 provided with a hollow fiber 70 and provided with guide caps 40.
  • a first flat membrane layer 66 is provided along a forward side of frame 62 and a second flat membrane layer 68 is provided along a rear surface of frame 62.
  • a two-temperature fluid circulation system with appropriately placed valves may be used to enable a wide range of fluid temperatures to be quickly attained.
  • the heating source 40 can be employed to heat a sample disposed in the capillary 12.
  • Various flow patterns can be created, such as front to back, side to side, and general circulation.
  • thermocycler may optionally use a combination of hot and cold fluid to change sample temperature.
  • Simple blowers or fluid pumps or blowing ambient air and air heated by resistance heaters over the capillaries are another alternative to change the temperature.
  • the temperature may be measured and controlled by standard Proportional Integral Differential (PID) controllers.
  • PID Proportional Integral Differential
  • the heating rate may be increased as desired by using, for example, superheated fluid for the first part ofthe heating cycle, then cooler fluid to avoid excessive overshoot ofthe temperature ofthe capillaries.
  • Optical sensors may optionally be employed in connection with the invention to detect liquid levels at one or more points, and provide open loop or feedback control to adjust, if necessary, the sample or fluid level volumes.
  • the present invention is capable of processing many samples in parallel, if desired, using standard micro-titer plates as reagent sources.
  • the use of capillaries in connection with the present invention is beneficial in that only a small fraction ofthe liquid volume is exposed to the atmosphere, so that evaporation is minimized. This promotes the processing ofthe sample, while concomitantly eliminating or reducing sample loss.
  • the capillaries ofthe 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.
  • DNA sequencing products are purified to remove excess salt, nucleotides, primers, and templates from the biological sample.
  • the illustrated microfiber 70 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 ofthe hollow fibers and hence the sample.
  • the use of a capillary tube with one or more microfibers 70 disposed therein, provides for the ability to perform equilibrium dialysis upon very small volumes of between about 10 to 0.05 microliters.
  • a well plate such as a microtiter plate
  • the needles may then be withdrawn from the top and the bottom ofthe cassette, allowing the gland seals to close, thereby effectively sealing the capillaries in the cassette.
  • the cassettes may be thermocycled or dialyzed in place, or moved to separate hotels for these operations, as described below.
  • cassettes are hard-mounted to 96 channel pipettors, and a needle bed is hard mounted to the bottom ofthe cassette.
  • a needle bed is hard mounted to the bottom ofthe cassette.
  • FIG. 14 illustrates one embodiment of a hotel.
  • a hotel may be used while thermocycling and/or dialysis occurs, to provide a washing function, to prevent sample cross-contamination, and/or regenerate chemically cassettes that fail to perform adequately.
  • the system may also recirculate water, or some other solution (acid, base, or detergent), through the lumen ofthe thermocycling or dialysis capillaries.
  • the water or other solution is heated.
  • the invention provides "hotels" that accept a plurality of cassettes for parallel processing to process large numbers of samples efficiently during the dialysis and thermocycling steps.
  • a hotel can process up to 15-20 cassettes.
  • Each housing ofthe hotel accepts and processes a cassette independently ofthe others.
  • the hotel includes a fixture that accepts an individual cassette, and makes the appropriate fluid connections between the cassette and the dialysis, thermocycling or wash media.
  • the end plates ofthe cassettes incorporate specialized fluid connectors that mate with similar fittings in the hotel.
  • the cassette is optionally seated on a tray that includes means for grasping the cassette and holding it firmly against the housing ofthe hotel during processing.
  • the dialysis hotel recirculates dialysis solution to the cassettes, and contains a reservoir and pump to perform this function.
  • the pump constantly recirculates the solution through all the stations ofthe hotel.
  • a valve opens to allow a stream of this recirculating dialysate to pass through the cassette and back to the reservoir.
  • Figure 15 depicts one embodiment of a dialysis hotel according to the invention.
  • a needle bed 390 may optionally be used to provide the dialysis solution.
  • the thermocycling hotel recirculates hot and cold fluid, such as air or liquid, through a set of closed conduits.
  • the hot air source is maintained at approximately 100 °C, for example, by electric resistance elements, and the cool air source may be ambient air.
  • the cool air source is a refrigeration unit.
  • a proportional mixing valve that controls the temperature ofthe air circulating through the cassette by selectively mixing the hot and cold air sources.
  • a valve may open to allow the air mixture to pass through the cassette.
  • the invention also optionally provides a temperature sensor in the air-flow stream entering (or leaving) the cassette to provide positive feedback to a controller that operates the mixing valve to control the temperature and time profile during thermocycling.
  • a control unit in each station ofthe hotel causes the preset thermocycling protocol to initiate, and to preferably proceed until finished.
  • liquid such as water
  • a washing hotel serves to rinse out the inner surfaces ofthe capillaries after each use to minimize and/or eliminate cross contamination of samples, and also to retain the microporosity characteristics ofthe membrane element.
  • the washing hotel circulates a wash fluid, followed by a water rinse whenever a cassette is introduced to a particular station.
  • the cassettes may be mounted differently in the washing station than in the thermocycling hotels to allow the circulating solutions to be directed to the insides ofthe capillaries rather than the outside.
  • the circulation pumps are preferably capable of developing pressures in the range of about 15 psi to dislodge deposits from the capillary walls. Chemical regeneration may also be performed, by the use of appropriate regeneration chemicals.
  • one embodiment ofthe invention pertains to a system to prepare cleared lysates for plasmid template preparation or "template preparation module.”
  • the system is mounted on a Hydra, and includes heating and filtration functions to process cells from deep-well plates.
  • the unit is based on the transfer Hydra design, but includes a specialized filtration manifold that utilizes a roll of filter material instead of multi-well filter plates.
  • a deep-well plate containing resuspended cells in lysis buffer is placed on the system and the samples are aspirated into a large-volume (100 ⁇ L) thermocycling cassette. The samples are heated at 95-100 °C for 1 minute, and the deep-well plate is removed.
  • FIG. 16 depicts one embodiment of a template preparation module.
  • An apparatus management device 300 is preferably used to manipulate the above-noted components during processing.
  • automated processing may be provided by commercially available hardware and software.
  • Custom automated systems to facilitate key aspects ofthe sequencing and finishing process have been developed and are known to the skilled artisan.
  • Such systems preferably include: 1) a robust platform for silica- bead based template preparation, quantification, and sample reconfiguration utilizing a CRS systems robot, two TECAN liquid handling stations, a fluorometer, a Sagian 96 channel pipettor, and several plate stackers and sealers, 2) a custom platform for automated fabrication and spotting of "paper combs" using a Seiko D-Tran robot and plate handling system designed for microarray spotting and built for GTC by Intelligent Automation Systems, Inc., and 3) a finishing automation system built around a sophisticated storage and retrieval system (Gira), a Tecan liquid handling station, a 96 channel Quadra pipettor, two plate stackers and a plate sealer.
  • the skilled artisan may use known databases and software tools to automate the apparatuses and methods ofthe invention.
  • the invention provides a method of plasmid isolation ("Automated Template Preparation” or "ATP”) which is based on standard alkaline lysis chemistry coupled with reversible capture on silica beads (Engelstein, 1998).
  • ATP Automated Template Preparation
  • This method meets the design criteria for a filtration-based process that can produce templates yielding data of comparable or better quality than Qiagen-generated preparations, and the samples are stable upon storage at 4° C for 6 months.
  • the ATP hardware preferably includes a Tecan Genesis 200 with Robotic Manipulator (RoMa), a CRS T475, a Sagian Multipette 96-channel pipettor, Tecan shakers, Scitec vacuum manifolds and a drying station.
  • the automated microplate heat sealer (Marsh BioMedical, NY) has RS232 capabilities and can function with plates manufactured from several different plastics.
  • the material used to seal the plates is aluminum foil backed with a thin plastic film, which differs depending on the composition ofthe microplates to be sealed.
  • the sealing process actually welds the seal to each well rim, with temperature and thickness ofthe plastic film controlling the strength ofthe seal. This permits both permanent and removable seals to be used in the process.
  • thinner foils are also available which allow the seal to be pierced ("easy pierce seals") by the pipetting robot to gain access to samples.
  • 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.
  • 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 provides a capillary cassette system which allows integration with existing laboratory automation devices and sequencing instruments.
  • the cassette includes open frames comprising a plurality of openings on the top and the bottom portions ofthe frame. In a preferred embodiment, there are twelve openings on the top and bottom portions ofthe frame. At each corner ofthe frame, slots are provided for means, e.g., pins for connecting the frames. In one embodiment, the frames are separated by sealing gaskets.
  • 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 by the use of air gaps and, preferably, sealing elements at each end ofthe capillary.
  • 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 ofthe capillary.
  • the cassette includes open frames to allow for air or water flow through the capillaries for thermocycling and dialysis.
  • cassettes are processed in a hotel, e.g., an air or water temperature controlled station, for thermocycling or dialysis, or as a washing station to regenerate the cassettes.
  • fluid regulating elements could be positioned along the capillaries discussed herein.

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Abstract

L'invention porte dans une exécution sur une cassette capilaire facilitant des dialyses rapides ou le thermocyclage, via des liquides ou des gaz d'échantillons inférieurs au microlitre. Dans une autre exécution la cassette est configurée pour être utilisée avec une machine de manipulation des fluides permettant un traitement automatique à fort débit, les dyalises et le thermocyclage s'effectuant lorsque la cassette est montée sur ladite machine. D'autres exécutions permettent des débits plus élevés en recourant à des hôtels effectuant le traitement simultané de plusieurs cassettes. Les capacités de ces hotels permettent la dyalise, le thermocyclage, le nettoyage et la régénération chimique. Citons à titre d'exemple des applications englobant non exclusivement la PCR, le séquencage de l'ADN, la ligation des oligonucléotides, la LCR, l'extension de nucléotides isolés, le traitement par l'exonucléase, et les essais d'hybridation d'oligonucléotides.
PCT/US2000/025851 1999-09-21 2000-09-21 Dispositif de traitement rapide d'echantillons d'adn a manipulation des liquides, thermocyclage, et purification integres WO2001021310A2 (fr)

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AU75979/00A AU7597900A (en) 1999-09-21 2000-09-21 Device for rapid dna sample processing with integrated liquid handling, thermocycling, and purification
EP00965231A EP1214149A2 (fr) 1999-09-21 2000-09-21 Dispositif de traitement rapide d'echantillons d'adn a manipulation des liquides, thermocyclage, et purification integres
CA002385029A CA2385029A1 (fr) 1999-09-21 2000-09-21 Dispositif de traitement rapide d'echantillons d'adn a manipulation des liquides, thermocyclage, et purification integres
JP2001524729A JP2004500552A (ja) 1999-09-21 2000-09-21 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

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