EP1567656A2 - Methode et appareil de mise en oeuvre de reactions inferieures a un microlitre a l'aide d'acides nucleiques ou de proteines - Google Patents

Methode et appareil de mise en oeuvre de reactions inferieures a un microlitre a l'aide d'acides nucleiques ou de proteines

Info

Publication number
EP1567656A2
EP1567656A2 EP03713412A EP03713412A EP1567656A2 EP 1567656 A2 EP1567656 A2 EP 1567656A2 EP 03713412 A EP03713412 A EP 03713412A EP 03713412 A EP03713412 A EP 03713412A EP 1567656 A2 EP1567656 A2 EP 1567656A2
Authority
EP
European Patent Office
Prior art keywords
capillary
reaction
nucleic acid
dna
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03713412A
Other languages
German (de)
English (en)
Inventor
Stevan Bogdan Jovanovich
Oscar Salas-Solano
Jeng-Thun Li
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Integenx Acquisition Corp
Original Assignee
Amersham Biosciences SV Corp
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
Priority claimed from US10/262,476 external-priority patent/US6927045B2/en
Application filed by Amersham Biosciences SV Corp filed Critical Amersham Biosciences SV Corp
Publication of EP1567656A2 publication Critical patent/EP1567656A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • 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
    • 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
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00281—Individual reactor vessels
    • B01J2219/00286—Reactor vessels with top and bottom openings
    • 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
    • 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
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00511—Walls of reactor vessels
    • 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
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • 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
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00686—Automatic
    • B01J2219/00691—Automatic using robots
    • 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
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00702—Processes involving means for analysing and characterising the products
    • 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
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00722—Nucleotides
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0832—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
    • B01L2400/0403—Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409—Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5025—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures for parallel transport of multiple samples
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/06—Libraries containing nucleotides or polynucleotides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • This invention is in the field of biotechnology, and relates to methods and apparatus for preparing and performing small scale reactions that use nucleic acid or protein.
  • Molecular pathology relates to the diagnosis, and often formulation of a prognosis, for human diseases by identifying mutations in particular genes.
  • Pharmacogenomics refers to understanding how allelic differences that exist in all human populations affect the therapeutic response, and susceptibility to side effects, of individuals to drugs. 5 [0007] As the need to sequence genes from individual patients grows, so will the demand for point of care sequencing capability. There will need to be a shift from large, centralized, high throughput DNA sequencing facilities that only exist at well-funded academic research centers and genomics companies to small, less complicated, middle-throughput gene sequencing systems that can be installed in the majority of hospitals and clinics.
  • Confocal spatial filtering results in a higher signal-to-noise ratio because superfluous reflections and fluorescence from surrounding materials are eliminated before signal detection at the photomultiplier tube (PMT). Accordingly, sensitivity at the level of subattomoles per sequencing band is attainable. Confocal imaging is also particularly important in microchip analysis systems using capillary o electrophoresis, where the background fluorescence of a glass or plastic microchip may be much higher than that of fused silica capillaries. Capillary array electrophoresis systems will solve many of the initial throughput needs of the genomic community for DNA analysis. However, present methods for low volume sample preparation still present a significant barrier to increased throughput and reduced cost.
  • the sequencing community has also developed higher throughput methods for 5 preparing DNA templates, polymerase chain reaction (PCR) reactions, and DNA sequencing reactions.
  • Sample preparation has been increasingly multiplexed and automated using 96- and 384-well microtiter, multi-channel pipettors, and laboratory robotic workstations. In general, these workstations mimic the manipulations that a technician would perform and have minimum working volumes of about a microliter, although stand- o alone multi-channel pipettors are being used to manipulate smaller volumes.
  • a typical full-scale sample preparation method for DNA shotgun sequencing on capillary systems begins by lysing phage plaques or bacterial colonies to isolate subcloned DNA. Under some circumstances it may be desirable to PCR-amplify the subcloned DNA insert to exponentially increase its concentration in the sample.
  • exonuclease I Exol
  • SAP arctic shrimp alkaline phosphatase
  • SAP converts dNTPs to dNPs and reduces the dNTP concentration from 200 ⁇ M, as used for the PCR reaction, to less than 0.1 ⁇ M for use with fluorescent sequencing.
  • the reaction is performed at 37°C and then heated to 65°C irreversibly denature the Exol and SAP. o [0013] Because PCR amplification can produce excess template DNA for cycle sequencing, the Exol/SAP treated PCR sample can be diluted five-fold before cycle sequencing. This reduces the concentration of contaminants into a range that causes less interference with capillary electrophoresis analysis. Cycle sequencing reagents are added, typically with fluorescently labeled dye primers or terminators and the reaction is thermal 5 cycled to drive linear amplification of labeled fragments.
  • the samples are post-processed, typically by ethanol precipitation or spin filtration, resuspended in formamide, another denaturant, or water, and the sample is electrokinetically injected into the capillary electrophoresis system.
  • This workflow has resulted in a dramatic improvement in the performance of the o MegaBACETM system, and similar work flows currently appear to be the methods of choice for other capillary electrophoresis systems as well.
  • 5,785,926 discloses a system for transporting small volumes of sample.
  • at least one capillary tube is used to transport small amounts of sample.
  • a precision linear actuator connected to a computer controlled motor acts as a pneumatic piston to aliquot and dispense liquid using the tube.
  • the sample amount is monitored by an optical sensor that detects the presence of liquid within the capillary o segment.
  • the system includes a fluid station containing liquids to be deposited and a positioning device for positioning the transport capillary.
  • U.S. Pat. No. 5,897,842 discloses a system for automated sample preparation using thermal cycling.
  • a reaction mixture is pumped into a capillary tube.
  • One end of the tube is sealed using pressure from an associated pump while the other end 5 is sealed by pressing the tube against a barrier.
  • the pump also serves to move fluid within the tube. Once the ends are sealed, the tube is exposed to thermal cycles.
  • a robotic transfer device moves the tubes between the sample preparation station where the pump loads the components of the reaction mixture into the tubes and the thermal cycling station.
  • U.S. Pat. No. 5,846,727 discloses affinity-capture methods wherein template DNA is immobilized inside a glass capillary tube that serves as a reaction chamber for thermal 5 cycling.
  • the capillary is first prepared by immobilizing biotin molecules to the inner surface of the capillary, followed by charging the column with avidin or streptavidin which binds tightly the biotin.
  • Template DNA to be sequenced is covalently linked to a biotin moiety by PCR, and is then exposed to the avidin inside the capillary. This results in immobilization of the template to the capillary wall through a biotin-avidin-biotin linkage.
  • the template DNA must stay bound to the inner surface of the o capillary. Because the DNA is not free in solution, additional time is required for reaction components to diffuse to the walls where they can interact with the DNA. Furthermore, when it is desired to recycle the capillary, it is necessary to remove the template DNA via denaturation of the avidin, washing and then recharging of the avidin in the capillary, all of which add to time and reagent costs.
  • Capillary array electrophoresis systems and capillary electrophoresis microchip analytical systems can detect subattomoles of DNA sequencing reaction products. This o extraordinary sensitivity comes at the cost of reduced tolerance, compared to slab gels, for deviations from the ideal amount of template DNA in the sequencing reactions. For example, if there is too little template DNA in the sequencing reaction, there will be poor yield of fluorescently labeled primer extension products. This results in weak signal strength when the reaction products are scanned by the laser. This prevents the software that 5 analyzes the chromatogram from adequately performing spectral separation, resulting in shorter than average sequence read lengths; the reaction will have to be repeated or the sequence information will be lost.
  • Too much template DNA causes problems as well, due to overloading of the capillary. While there is adequate yield of fluorescently labeled reaction product, if the o template is in excess, it competes with sequencing products for entry into the capillary during electrokinetic injection. The presence of the large template DNA molecules can result in an overall reduction, or sudden drop in capillary current, which can manifest itself in a variety of ways. Overloading can cause weak signal strength, late appearance of interpretable fluorescence intensity peaks in the chromatogram, and poor resolution of the 5 reaction products because the fluorescence emission is broad and diffuse. All these effects lead to shorter reads and lower sequencing data quality.
  • a system for performing small scale reactions comprising: a capillary cassette having a substrate and a plurality of capillaries extending through said substrate, wherein each of said capillaries has first and second open ends on opposing o sides of said substrate; a pair of membranes orientated and spaced such that they may temporarily seal the opposed ends of said capillaries; a thermal cycler having an internal chamber of sufficient capacity to hold said capillary cassette and said membranes; and an automated transfer device positioned to contact and move the capillary cassette to a location where the ends of the capillary may be sealed by the pair of membranes and the 5 capillary cassette with ends sealed by said membranes may be located within the internal chamber of the thermal cycler.
  • wash station has a wash solution tank and an upper wash manifold that may be moved to a position above said wash solution tank, wherein a wash fluid may be introduced into said wash solution tank and drawn by suction into the wash manifold when the capillary cassette is inserted into said wash station. 5 [0044] 12. The system of paragraph 11 , wherein said wash station further includes a plurality of wash fluid bottles each containing a wash fluid and a selector valve allowing selection of a wash fluid from one of said bottles to fill said wash solution tank. [0045] 13. The system of paragraph 1 , further comprising an electronic control which may be programmed to send electronic instructions to components of the system. o [0046] 14.
  • a system for nanoscale reaction preparation comprising: a capillary cassette including a substrate and a plurality of capillaries extending through said substrate, each capillary having an internal volume of between 10 nl and about 1 uL, 0 wherein each of said capillaries has a first and a second open end on opposing sides of said substrate, wherein the length of the capillary extending through substrate on one side of the substrate is matched to be shorter than the depth of a microplate well; a multiwell plate having a plurality of wells into which the capillaries of the capillary cassette may be inserted; a dispenser that dispenses fluid contained within the capillaries of the capillary cassette into wells of said multiwell plate when said capillary is transported to the dispenser; an automated transfer robot having a transfer head to carry articles selected from the group 5 comprising capillary cassettes, multiwell plates, and multiwell plates with capillaries of a capillary cassette inserted into the wells of the multiwell plates; a pair of opposing membrane
  • a system for preparing nanoscale reactions comprising: a substrate having integrally associated elongate submicroliter volume reaction containers 5 having two opposing ends; a reaction mixture contained within said reaction containers; a pair of membranes disposed to temporarily seal said opposing ends of said reaction containers; a thermal cycler having an internal chamber of sufficient dimension to receive said substrate with associated elongate reaction chambers sealed by said membranes.
  • said substrate has capillaries extending o through said substrate, wherein said capillaries act as the reaction chambers.
  • a method to prepare nanoscale thermal cycling reaction mixtures comprising; combining compounds to form a reaction mixture; introducing said reaction mixture into a plurality of reaction containers disposed on a substrate, each reaction container having an internal volume less than one microliter and having a first and second open end; temporarily sealing the ends of reaction containers by pressing a pair of opposing membranes against a first and second set of reaction container ends; exposing the sealed reaction containers to temperature cycles to effect a reaction in the reaction mixture; and 5 dispensing the reaction containers onto a substrate.
  • the method of paragraph 36 wherein the steps of combining compounds to form a reaction mixture includes the steps: immobilizing a biomolecule sample on an interior surface of the reaction container; metering an amount of reaction reagents into the capillaries of the capillary cassette by placing one end of the capillaries of a capillary cassette into contact with the reaction reagents wherein the capillaries fill by capillary action, 5 whereby the reaction reagents and the immobilized biomolecule combine to form the reaction mixture.
  • the method of paragraph 36 wherein the steps of combining compounds to form a reaction mixture include the steps: coating an interior surface of each capillary in a capillary cassette with a layer of desiccated reaction reagents; and metering an amount of nucleic acid sample in solution into the capillaries of the capillary cassette by placing one 5 end of the capillaries of a capillary cassette into contact with the nucleic acid sample in solution, whereby the capillaries fill by capillary action, whereby the solution allows the layer of reaction reagents to dissolve, forming the reaction mixture.
  • the steps of combining compounds to form a reaction mixture include the steps: coating an interior surface of each capillary in a capillary cassette with a layer of desiccated reaction reagents; and metering an amount of nucleic acid sample in solution into the capillaries of the capillary cassette by placing one 5 end of the capillaries of a capillary cassette into contact with the nucleic acid sample in solution, whereby the capillaries fill by
  • step of dispensing the reaction containers onto a substrate is effected by: placing the substrate with associated reaction o containers in a centrifuge; positioning a substrate at a radially distal end of one open end of said reaction containers; and applying centrifugal force such that liquid reaction mixtures contained within said reaction containers are dispensed onto said substrate.
  • step of dispensing the reaction containers onto a substrate is effected by: displacing the contents of the reaction containers 5 onto a substrate using air displacement.
  • a thermal cycling device for exposing reaction mixtures to temperature cycles, the device comprising: a housing enclosing a continuous interior circuit passageway, said housing having a section that may be temporarily opened to allow access to the interior of o the housing; a blower disposed within said circuit passageway to direct air flow in one direction in the internal circuit passageway; a heating element disposed in said internal circuit passageway such that air circulating within said passageway passes through said heating element; a sample holding compartment having two membranes positioned in opposing orientation within said sample holding compartment, wherein said membranes may be biased against opposing ends of containers inserted into the sample holding compartment; housing air vent which may be opened to rapidly exhaust heated circulating 5 air; and a housing air intake for drawing air into said interior circuit passageway when the vent exhausts heated circulating air.
  • the thermal cycling device of paragraph 47 further comprising a temperature monitoring device disposed in the internal passageway proximate to a sample holding compartment. 0 [0081] 49.
  • the thermal cycling device of paragraph 47 further comprising at least one air diffuser disposed in the internal passageway between the blower and the sample holding compartment, said diffuser promoting uniform temperature in the air circulating in the internal passageway.
  • at least one of the 5 membranes within the sample holding compartment is spring biased.
  • thermal cycling device of paragraph 47 further comprising insulation affixed to the surfaces of the interior circuit passageway.
  • the thermal cycling device of paragraph 47 further comprising an electronic control which sends instruction to components of the thermal cycling device. o [0085] 53.
  • a method for performing reactions comprising, a) introducing reaction mixtures into a reaction container set, each container in the set having two opposing ends and an internal volume between 10 to 1000 nl; b) temporarily sealing the ends of the reaction chambers by pressing a deformable membrane against the opposing o ends of said reaction containers; c) effecting a reaction within said reaction containers; d) dispensing reaction mixtures onto discrete locations on a substrate; and e) combining said reaction mixtures with at least 1 .mu.l of a liquid reagent mixture.
  • the method of paragraph 56 further comprising the step of: g) combining reacted mixtures of step f with a reaction reagent set to form a second reaction mixture set; 5 h) introducing said second reaction mixture set into a second reaction container set, each reaction container having two opposing ends and an internal between 10 and 1000 nl; i) temporarily sealing the ends of the set of reaction containers by pressing deformable membranes against the opposing ends of said reaction containers; j) effecting a reaction within said second reaction container set; and k) dispensing reacted mixtures from said o second reaction container set.
  • step f occurs under isothermal reaction conditions.
  • reaction mixture of step a is a PCR mixture
  • liquid reagent mixture of step e contains exonuclease I and shrimp alkaline 5 phospotase, and the second reaction mixture.
  • steps c and j include exposing the reaction container sets to temperature cycles.
  • a method of obtaining substantially the same quantity of nucleic acid from a first and a second sample comprising: saturably binding nucleic acid from said first sample directly on an inner surface of a first capillary tube by contacting said inner surface with a solution comprising a nucleic acid and a chaotropic agent for a time sufficient for the nucleic o acid to become saturably bound to said inner surface; and saturably binding nucleic acid from said second sample directly on an inner surface of a second capillary tube by contacting said inner surface with a solution comprising a nucleic acid and a chaotropic agent for a time sufficient for the nucleic acid to become saturably bound to said inner surface, wherein said inner surfaces of said first and second capillary tubes are capable of saturably binding substantially the same quantity of nucleic acid from each of said first and second samples, respectively. 5 [0098] 66. The method of paragraph 65, wherein the quantity of nucleic acid saturably
  • the method of paragraph 65 further comprising, prior to said binding steps, the step of size-selecting a nucleic acid to be saturably bound. 5 [0102] 70. The method of paragraph 65 further comprising, after said binding steps, the step of using the nucleic acid of either of said first or second capillary tubes in an enzymatic reaction.
  • chaotropic agent is selected from the group consisting of: urea, sodium perchlorate, potassium perchlorate, sodium bromide, 5 potassium bromide, sodium iodide, potassium iodide, sodium thiocyanate, potassium thiocyanate, guanidine thiocyanate, sodium isothiocyanate, potassium isothiocyanate, guanidine hydrochloride, guanidine isothiocyanate, lithium chloride, sodium trichloroacetate, dimethylsulfoxide, tetra-amine halides, tetraethylamine chloride, and potassium trichloroacetate. 0 [0112] 80.
  • the method of paragraph 65 further comprising the step of removing the solution, wherein said removing step occurs after said contacting step.
  • the method of paragraph 80 further comprising the step of washing the inner surface of either of said first or second capillary tubes, wherein said washing step occurs after said removing step. 5
  • the method of paragraph 81 further comprising the step of drying the inner surface of either of said first or second capillary tubes, wherein said drying step occurs after said washing step.
  • a method of performing an enzymatic reaction in a capillary tube using a normalized quantity of a nucleic acid comprising: performing said enzymatic reaction in a o capillary tube using a normalized quantity of said nucleic acid, said nucleic acid having been saturably bound from an excess thereof directly on an inner surface of said capillary tube by contacting said inner surface with a solution comprising a nucleic acid and a chaotropic agent for a time sufficient for the nucleic acid to have become saturably bound to said inner surface; and said excess of nucleic acid having been removed therefrom. 5 [0116] 84. The method of paragraph 83 further comprising the step of introducing into said capillary tube an enzymatic reaction mixture after said excess of nucleic acid has been removed therefrom.
  • a method of performing an enzymatic reaction in a capillary tube using a normalized quantity of a nucleic acid comprising: introducing an enzymatic reaction mixture o into a capillary tube having a normalized quantity of a nucleic acid, wherein said reaction mixture comprises an oligonucleotide primer, a DNA polymerase, and at least one dideoxynucleotide triphosphate (ddNTP), said nucleic acid having been saturably bound from an excess thereof directly on an inner surface of said capillary tube by contacting said inner surface with a solution comprising nucleic acid and a chaotropic agent for a time sufficient for the nucleic acid to have become saturably bound to said inner surface; and said excess of nucleic acid having been removed therefrom; and performing said enzymatic reaction in said capillary tube using said normalized quantity of nucleic acid.
  • ddNTP dideoxynucleotide triphosphate
  • ddNTPs dideoxynucleotide triphosphates included in said enzymatic reaction mixture are selected from among the group consisting of: A only; C only; G only; T only; A,C; A,G; A,T; C,G; C,T; G,T; A,C,G; A,C,T; A,G,T; C,G,T and A,C,G,T.
  • ddNTP dideoxynucleotide triphosphates
  • fluorophore is selected from among the group consisting of: fluorescein, 5-carboxy-fluorescein, 6-carboxy- rhodamine, N,N,N',N'-tetramethyl-5-carboxyrhodamine and 5-carboxy-X-rhodamine, o rhodamine 110, rhodamine-6-G, tetramethyl rhodamine and rhodamine X.
  • nucleic acid is selected from among the group consisting of: DNA, double stranded DNA, single stranded DNA, DNA produced by polymerase chain reaction, DNA produced by reverse transcription reaction, DNA isolated from a eukaryotic cell, DNA isolated from a prokaryotic cell, DNA o isolated from an archaea cell, DNA isolated from a fungal cell, DNA isolated from a plant cell, DNA isolated from a virus, DNA isolated from a bacteriophage, genomic DNA, plasmid DNA, episomal DNA, RNA, messenger RNA, double stranded RNA, single stranded RNA, RNA isolated from a eukaryotic cell, RNA isolated from a prokaryotic cell, RNA isolated from an archaea cell, RNA isolated from a fungal cell, RNA isolated from a plant cell, RNA isolated from a virus, genomic RNA, DNA-RNA hybrid, nucleic acid obtained from frozen glycerol stocks of
  • nucleic acid is DNA; and further comprising the step of preparing said DNA by polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • said spatially addressable array of capillary tubes is an array having a number of capillaries selected from among the group consisting of: 2, 4, 8, 12, 16, 24, 32, 48, 64, 96, 128, 192, 288, 384, 480, 576, 672, 768, 864, 960 and 1536 capillaries.
  • enzymatic reaction is selected from among the group consisting of: oligonucleotide ligation assay genotyping (OLA) reaction, minisequencing reaction, TaqManTM genotyping reaction, InvaderTM assay reaction, dye labeled oligonucleotide ligation reaction, pyrosequencing reaction, rolling circle amplification (RCA) reaction and single-base extension (SBE) reaction.
  • OLA oligonucleotide ligation assay genotyping
  • minisequencing reaction TaqManTM genotyping reaction
  • InvaderTM assay reaction dye labeled oligonucleotide ligation reaction
  • pyrosequencing reaction rolling circle amplification (RCA) reaction
  • SBE single-base extension
  • a method of performing an enzymatic reaction in a capillary tube using a normalized quantity of a nucleic acid effective to detect a single nucleotide polymorphism comprising: performing said enzymatic reaction in a capillary tube using a normalized quantity of said nucleic acid, said nucleic acid having been saturably o bound from an excess thereof directly on an inner surface of said capillary tube by contacting said inner surface with a solution comprising a nucleic acid and a chaotropic agent for a time sufficient for the nucleic acid to have become saturably bound to said inner surface; and said excess of nucleic acid having been removed therefrom, wherein said enzymatic reaction is selected from among the group consisting of: oligonucleotide ligation assay genotyping (OLA) reaction, minisequencing reaction, TaqManTM genotyping reaction, InvaderTM assay reaction, dye labeled oligonucleotide lig
  • a method of performing an enzymatic reaction in a capillary tube using a normalized quantity of an enzyme comprising: performing said enzymatic reaction in a capillary tube using a normalized quantity of said enzyme, said enzyme having been saturably bound from an excess thereof directly on an inner surface of said capillary tube by contacting said inner surface with a solution comprising an enzyme for a time sufficient for the enzyme to have become saturably bound to said inner surface; and said excess of enzyme having been removed therefrom.
  • a method of performing an enzymatic reaction in a capillary tube using a normalized quantity of an enzyme comprising: performing said enzymatic reaction in a capillary tube using a normalized quantity of said enzyme, said enzyme having been specifically and saturably bound from an excess thereof on a modified inner surface of said capillary tube by contacting said modified inner surface with a solution comprising an enzyme for a time sufficient for the enzyme to have become specifically and saturably bound to said modified inner surface; and said excess of enzyme having been removed therefrom.
  • a method of performing a protein-based reaction in a capillary tube using a normalized quantity of a protein comprising: performing said protein-based reaction in a capillary tube using a normalized quantity of said protein, said protein having been saturably bound from an excess thereof on an inner surface of said capillary tube by contacting said inner surface with a solution comprising a protein for a time sufficient for the o protein to have become saturably bound to said inner surface; and said excess of protein having been removed therefrom.
  • a method of performing a protein-based reaction in a capillary tube using a normalized quantity of a protein comprising: performing said protein-based reaction in a capillary tube using a normalized quantity of said protein, said protein having been 5 specifically and saturably bound from an excess thereof on a modified inner surface of said capillary tube by contacting said modified inner surface with a solution comprising a protein for a time sufficient for the protein to have become specifically and saturably bound to said modified inner surface; and said excess of protein having been removed therefrom.
  • 158. wherein said protein is a 0 noncatalytic protein.
  • said noncatalytic protein is selected from among the group consisting of: antibody, antigen-binding fragment of an antibody, IgG, IgE, IgM, protein G, protein A and streptavidin.
  • a method of obtaining substantially the same quantity of protein l o from a first and a second sample comprising: saturably and specifically binding protein from said first sample directly on a modified inner surface of a first capillary tube by contacting said inner surface with a solution comprising a protein for a time sufficient for the protein to become saturably and specifically bound to said modified inner surface; and saturably and specifically binding protein from said second sample directly on a modified inner surface of
  • a second capillary tube by contacting said inner surface with a solution comprising a protein for a time sufficient for the protein to become saturably and specifically bound to said modified inner surface, wherein said modified inner surfaces of said first and second capillary tubes are capable of saturably and specifically binding substantially the same quantity of protein from each of said first and second samples, respectively.
  • FIG. 1 is a schematic of an integrated system for the preparation of cycle sequencing reaction products, which system can advantageously use the methods of the present invention
  • FIG. 2 is a flow chart illustrating the steps in production of cycling reactions, the first step of which can advantageously be improved by use of the methods of the present invention;
  • FIG. 3A is a perspective view of a capillary cassette that is used in a high throughput embodiment of the present invention;
  • FIG. 3B is a perspective view of the capillary cassette of FIG. 3A inserted into a capillary cassette holder in a system for high throughput application of the methods of the 5 present invention
  • FIG. 3C is a flexible capillary cassette that advantageously can use the methods of the present invention.
  • FIG. 3D illustrates the capillary cassette of FIG. 3C bent to a curved orientation such that the capillary ends are in a curved pattern; o [0202]
  • FIG. 3E is a microchip device containing channels, functionally equivalent to capillary tubes, for sample preparation, including the direct reversible immobilization of nucleic acid, according to the present invention;
  • FIG. 4A illustrates a dispense head for dispensing liquid from the capillary cassette of FIG. 3, for use in the present invention
  • FIG. 4B shows an internal cross section of an air displacement dispense head of FIG. 4A;
  • FIG. 4C shows the dispense head of FIG. 4A with the dispense head closed;
  • FIG. 5A illustrates a top view of a centrifuge that can be used to dispense fluid from the capillary cassette of FIG. 3A; o
  • FIG. 5B illustrates a cross-section of a rotor arm of FIG. 5A holding a swinging microplate bucket containing a capillary cassette inserted into a microtiter plate;
  • FIG. 6 shows a schematic of an air-based thermal cycling device with the capillary cassette and holder shown in FIG. 3B inserted into the temperature cycling device, for performing parallel reactions that advantageously can use the template capture and 5 normalization methods of the present invention;
  • FIG. 7A shows an internal cross section of an air-based thermal cycler with integrated capillary cassette sealing membranes, which can advantageously be used with the template capture methods of the present invention
  • FIG. 7B shows a perspective detail of the air-based thermocycler of FIG. 7A, with o the lid raised to illustrate the chamber into which the capillary cassette is inserted;
  • FIG. 7C shows a cross section of the cassette compartment with the capillary cassette inserted into the internal chamber of the thermal cycler of FIG. 7A;
  • FIG. 8A is a front view of a capillary cassette wash station useful in high throughput performance of the methods of the present invention;
  • FIG. 8B is a side view of the capillary cassette wash station of FIG. 8A with the wash manifold lowered and the wash tank raised; 5 [0214] FIG. 8C is a further view of the capillary wash station of FIGS. 8A and 8B with the wash manifold raised and the wash tank lowered;
  • FIG. 8D is an interior cross-section of the wash manifold
  • FIG. 8E is a schematic plumbing diagram of the wash station
  • FIG. 8F is a top perspective view of the wash tank; 0 [0218]
  • FIG. 9 shows a histogram of the percent success versus read length window for the sequencing analysis of example 1 ;
  • FIG. 10 is an electropherogram of the reaction products of example 2.
  • FIG. 11 shows a histogram of the percent success versus read length window for the sequencing analysis of example 3; 5 [0221] FIG. 12A shows a scanned gel image of electrophoretically separated PCR products prepared at full volume;
  • FIG. 12B show a scanned gel image of electrophoretically separated PCR products prepared at a nanoscale volume (500 nL);
  • FIG. 13 is an electropherogram of analysis of sequencing mixtures prepared by o performing PCR at 500 nL volumes, a cleanup reaction at full volumes, followed by cycle sequencing reactions performed at 500 nL;
  • FIG. 14 is a graph comparing signal strength of an isothermal reaction for products prepared in tubes, capillaries, and capillaries using surface binding;
  • FIG. 15 is a flowchart explaining the methodology for preparing capillary tubes in 5 which nucleic acid is reversibly directly immobilized
  • FIG. 16 illustrates an embodiment of the method of the present invention
  • FIG. 17A shows the results of sequencing PCR products mixed with the reaction mixture prior to sequencing
  • FIG. 17B shows the results of first mixing the PCR template with sodium thiocyanate, binding the DNA to the inner surface of the capillary, washing the o DNA with 80% ethanol, followed by sequencing;
  • FIG. 18 represents the retained mass of DNA following a template capture protocol
  • FIG. 19 shows a plot of read length versus starting DNA mass for samples prepared by premixing DNA and sequencing reagents (A ) compared to samples prepared by template capture (•);
  • FIG. 20 shows products of PCR reactions after template binding of the indicated 5 starting amount o M13mp18, electrophoresed through a 1.5% agarose gel, stained with SYBR Green dye and imaged with a Fluorimager apparatus;
  • FIG. 21 represents the relative signal intensity obtained with increasing template concentration;
  • FIG. 22 represents the relative signal intensity obtained with increasing template o concentration, showing peak height increasing with increasing template concentration;
  • FIGS. 23A and 23B show a trace that had a Phred 20 score of 561 bases obtained by nanoscale direct cycle sequencing from glycerol stocks;
  • FIG. 24 are MegaBACETM traces from four nanoscale single base extension reactions, without template capture, demonstrating heterozygosity in trace 2; 5 [0235]
  • FIG. 25 shows the results of quantitative analysis of nanovolume PCR products (FIG. 25B) in comparison with that of full volume PCR products (FIG. 25A);
  • FIG. 26 shows the results of full volume SBE reactions and a negative control;
  • FIG. 27 presents the MegaBACETM traces of (A) a full volume single base extension reaction; and (B) a nanovolume single base extension reaction, from full volume o PCR and Exol/SAP treatment of the PCR product;
  • FIG. 28 presents the MegaBACETM traces of (A) a full volume single base extension reaction; and (B) a nanovolume single base extension reaction, from nanovolume PCR and template capture of the PCR product;
  • FIG. 29 presents the MegaBACETM traces of (A) a nanovolume single base 5 extension reaction, from nanovolume PCR and template capture of the PCR product, but with no subsequent cleanup of the SBE products;
  • (B) a nanovolume SBE reaction with CIAP cleanup and injected into MegaBACETM with MegaBACETM loading solution;
  • C a nanovolume SBE reaction with CIAP cleanup and injected into MegaBACETM with deionized water; and
  • D a nanovolume SBE reaction with Sephadex cleanup and injected into o MegaBACETM with deionized water;
  • FIG. 30 shows the results of a validation experiment comparing full volume and nanovolume SBE;
  • FIG. 31 illustrates a representative peptide profile of cytochrome C, after protease digestion by trypsin in a capillary cassette. The profile was generated by MegaBACETM analysis. Trypsin is either in solution (FIG. 31A) or immobilized on magnetic beads (FIG. 31 B); 5
  • FIG. 32 presents a representative first run of a peptide profile of cytochrome C, after protease digestion by trypsin that is covalently coated onto an internal surface of a capillary in a multi-capillary cassette.
  • FIG. 33 presents a representative second run of a peptide profile of cytochrome C, after protease digestion by covalently surface-coated trypsin.
  • the capillary surface was modified by either aminoalkylsilane reagent or streptavidin modification.
  • the profile was generated by MegaBACETM analysis;
  • FIG. 34 presents a representative third run of a peptide profile of cytochrome C, 5 after protease digestion by covalently surface-coated trypsin.
  • the capillary surface was modified by either aminoalkylsilane reagent or streptavidin.
  • the profile was generated by MegaBACETM analysis;
  • FIG. 35 presents a representative HPLC profile of cytochrome C, after protease digestion by covalently surface-coated trypsin.
  • the capillary surface was modified by either o aminoalkylsilane reagent or streptavidin modification;
  • FIG. 36 presents the relationship between Asp-N concentration and the amount of polypeptides digested, represented here by signal intensity of the Cy3 emission from the digested peptides.
  • a capillary segment could be used o both to meter reagents and as a reaction container for performing temperature cycling reactions.
  • the length of the capillary and the internal diameter (I.D.) of the bore of the capillary tube define the volume of the interior of the capillary tube segment.
  • Capillaries with a 50-150 urn I.D. are commonly available.
  • the small internal diameter of the capillary tubes allows creation of a reaction container having an interior volume less than one microliter. With the present invention, capillaries having volumes from 10-500 nanoliters are adaptable to the preparation of DNA cycle sequencing reactions or any other reaction. 5 [0249]
  • the process carried out by the present automated system is shown in the flow chart of FIG. 2.
  • the process begins by the assembly of the reaction mixture, box 52, by combination of reagents and a sample nucleic acid.
  • the combined reagents are then introduced into the capillaries of a capillary cassette, box 54.
  • the ends of the capillaries are next sealed, box 56.
  • the sealed capillary segments are exposed to thermal cycles, box 58, o which effect the cycling reaction.
  • the capillaries of the capillary cassette are then dispensed onto a substrate, box 60.
  • the substrate is then transferred to an analytical system for analysis of the reaction mixture, box 62. Details of this process and the structure of the apparatus for carrying out this process are detailed herein. [0250] In reference to FIG.
  • an automated system for assembly of reaction 5 mixtures, temperature cycling to effect the chemical reaction, and dispensing the volume of the completed reaction mixture onto a substrate for subsequent analysis.
  • an automated robot 102 may move the length of stage 114 and may rotate such that automated robot 102 may be moved in relation to other components of the automated system.
  • the automated robot 102 may be rotated to allow the transfer head 104 on o automated robot 102 to access objects on all sides adjacent to stage 114.
  • the assembly of a reaction mixture would begin by the transfer head 104 picking up a capillary cassette from cassette hotel 106.
  • Capillary cassette 15 is shown in FIG. 3A.
  • the capillary cassette is comprised of a number of capillary tubes 12 extending through a substrate 10. It is preferred that the 5 capillary cassette have at least one row of eight capillary tubes and that the capillary tubes have equal spacing.
  • the capillary cassette shown has substrate 10 with 96 capillary tubes arranged in an 8 by 12 array, with spacing of the tubes matching the spacing of the wells of a 96 well microplate.
  • the length of capillary tubes 12 extending from either side of substrate 10 is unequal. It is preferred that the shorter end of capillary tube segments 12 be shorter o than the depth of a microplate well.
  • the capillary tubes may be made of any material compatible with the assay and preparation to be performed, but preferred capillary materials include, but are not limited to, glass and silica capillaries, although plastic, metals and other materials may also be used. Capillary tubes of various dimensions may be used, such as 75 urn ID capillary tubes or 150 5 urn I.D./360 urn O.D. capillary tubes.
  • the capillary tubes 12 extend through a substrate 10 and preferably are arranged in a uniform pattern.
  • the capillary tubes are of equal length and extend through the substrate in a substantially parallel orientation such that each of the two opposing ends of the capillary tubes 12 are coplanar and the planes defined by the ends of the capillary tubes 0 12 are substantially parallel to the substrate 10.
  • the spacing of the capillary tubes may be uniform and selected to match the center-to-center spacing of wells on a microplate. For example on a standard 96 well microplate the capillary tubes would be arranged with a 9 mm center to center spacing, on a 384 well microplate the capillary tubes 12 would be arranged with a 4.5 mm center to center spacing.
  • the capillary tubes 12 are preferably secured within the substrate such that the length of capillary tubes 12 extending from one side of the substrate 10 are shorter than the length of the capillary tube on the opposite side of substrate 10.
  • the length of the capillary tubes 12 on the shorter side of the substrate may be matched to the depth of wells in a o microplate, such that the length of the shorter side is a shorter length than the depth of a well in a microplate.
  • the capillary cassette substrate 10 may be made of a fiberglass board or other o rigid or semi-flexible material.
  • the capillary tubes 12 may be inserted through evenly spaced holes in the substrate and secured with adhesive.
  • the length and width of the substrate are similar to the length and width of a standard 96 well microplate. This simplifies adapting automated systems designed for manipulation of microplates to handle the capillary cassette.
  • Coatings that may be 5 used include bovine serum albumin (BSA), glycerol, polyvinyl alcohol and Tween 20.
  • BSA bovine serum albumin
  • glycerol glycerol
  • polyvinyl alcohol polyvinyl alcohol
  • Tween 20 a coating for the interior of the capillary tube.
  • covalent modification of the interior surface with silanization or Griganard reaction may be desired.
  • covalent modification of capillary tubes interior surfaces that reduce electroendoosmosis may also be useful in reducing charge surface l o effects between a capillary interior surface and components of a reaction mixture.
  • the automated system allows for the combination of reaction reagents and sample DNA using the capillary cassette.
  • a capillary cassette would be taken 20 by transfer head 104 from the cassette hotel 106 and brought into contact with the samples contained in a sample plate at location a.
  • the sample plate is dispensed from sample plate hotel 108.
  • the sample would be drawn into the capillary tubes of the capillary cassette by capillary action.
  • the internal volume of the capillary tube is defined by the length of the capillary tube and its internal diameter.
  • the capillary cassette of FIG. 3A acts as a fixed
  • each capillary tube segment will meter a discrete amount of liquid that may be subsequently dispensed.
  • reaction mixtures 3 o liquids to form a reaction mixture.
  • analytical instruments such as a capillary array electrophoresis system and the amplification of reaction mixture products enabled by cycling reactions allow for nanoscale reactions and analysis. Very small-scale reaction are able to reliably produce reaction mixture products of sufficient quantity for analysis on a capillary array electrophoresis system, a capillary electrophoresis chip, a mass spectrometer, or other analysis instrumentation. Significantly less reaction reagents are required if a nanoscale reaction mixture is enabled.
  • the automated system may be used in various ways to prepare reaction mixtures. A few of the many such methods for use of the system in production of reaction mixtures follow.
  • One method to prepare the reaction mixture is to use the pipettor to separately meter the components of a reaction mixture.
  • the nucleic acid sample and PCR reagents would be separately metered and dispensed into a single container in which the liquids are combined.
  • the automated robot 102 moves transfer head 104 containing a capillary cassette to location a where a sample plate is located.
  • the ends of the capillary tubes of the capillary cassette are dipped into the wells.
  • the capillary tubes fill by capillary action, metering precise amounts of the samples.
  • the wells of sample plate contain the nucleic acid sample to be PCR amplified.
  • the DNA sample should be sufficiently dilute such that 5-20 ng of DNA is contained in the 10-10,000 nL volume metered by each capillary tube segment in the capillary cassette.
  • FIG. 4A shows a 16 channel capillary cassette transferring fluid samples from a multiwell plate 36 into a capillary cassette 15.
  • the capillary tube segments 12 on capillary cassette 15 are extended into the wells of multiwell plate 36,
  • the wells of multiwell plate 36 are conical and liquid in the well will flow to the bottom central area of each well. This allows a small amount of liquid within the well to be positioned such that a capillary inserted into the center of the well and above the bottom of the well will contact the liquid.
  • the capillary tube segments of the capillary cassette then fill by capillary action with the liquid in the wells.
  • the capillary cassette have, capillary tube segments which have the same center to center spacing as the wells of the multiwell plate containing the DNA samples.
  • the capillary cassette has the same number of capillary tube segments as the number of wells in a multiwell plate holding samples.
  • the capillary cassette meters a precise amount of liquid defined by the interior volume of the capillary tubes held in the capillary cassette.
  • the metered amount of reaction reagents may be the same volume as o the volume of sample dispensed or it may be different, depending on the requirements of the application.
  • the reaction reagents are dispensed from each capillary tube segment onto locations on the mixing substrate containing the nucleic acid sample.
  • the present reaction mixture assembly may be used for assembly of numerous 5 types of reactions.
  • the same basic method used to assemble the PCR reaction mixture may be adapted to assembly of a cycle sequencing mixture, rolling circle amplification reaction mixture, enzymatic assays, chemical reactions, or other reaction mixtures.
  • [0263] When dispensing the contents into a microplate some care must be taken to mix the sample and reaction reagents in a manner which avoids splattering. A number of o different methods have been envisioned to dispense liquid from the capillary cassette.
  • the first method to dispense the contents of the capillary cassette while avoiding 5 splattering uses a centrifuge to dispense the fluid by centrifugal force.
  • the centrifugal force is applied evenly to all of the capillaries in the capillary cassette such that capillaries independently dispense into the microplate wells.
  • the dispensed liquid is drawn by centrifugal force to the bottom of wells in the multiwell plate.
  • FIG. 5A the centrifuge 42 is shown having a swinging microplate bucket 43 that o may contain a multiwell plate with an inserted capillary cassette.
  • the swinging microplate buckets are held on rotor 41.
  • FIG. 5B shows a cross-section of swinging microplate bucket 43.
  • the capillary tubes 12 of the capillary cassette are inserted into wells 36a of multiwell plate 36.
  • the cassette is inserted such that the portions of the capillary tubes 12 extending from the substrate 10 are shorter than the depth of the wells 36a.
  • the capillary 5 tube 12 extending from substrate 10 do not reach the bottom of the wells 36a of multiwell plate 36.
  • Microplate swinging bucket 43 is comprised of an arm 45 and a platform 44. An upper end of arm 45 fits onto latch head 42 on rotor 41.
  • Microplate 36 is positioned on platform 44 of microplate swinging bucket 43.
  • platform 44 rotates on latch head 42 such that the multiwell plate faces the side wall of the centrifuge o and the centrifugal force on the liquid in the capillary tubes dispenses the liquid into the bottom of the wells 36a of the multiwell plate 36.
  • the centrifugal force will draw the liquids within the well to the well center, causing the sample to locate at a more precise location.
  • the liquid will be displaced from the capillary at fairly low centrifuge speeds. 5 [0267]
  • a low speed centrifuge may optionally be included in the automated system at the dispensing device location 122.
  • Automated robot 102 uses transfer head 104 to pick up a microtiter plate dispensed onto location b by microtiter plate hotel 110.
  • Transfer head 104 transfers the microtiter plate to the stage having the low speed centrifuge.
  • a capillary cassette is filled with samples or reaction reagents as described and is transferred o onto the microtiter plate on the stage of the low speed centrifuge.
  • the plate and cassette are then spun in the centrifuge, dispensing the liquid from the capillaries into the wells of the microtiter plate. Once the liquid has been dispensed and the centrifuge has stopped rotating, the capillary cassette may by removed by the transfer head and transferred to the cassette washer 118.
  • the transfer head 104 can then pick up a clean capillary cassette 5 from the capillary cassette hotel 106.
  • the clean capillary cassette can be used to meter a second liquid reaction component that is similarly dispensed into the microtiter plate using the centrifuge.
  • the centrifuge includes a sensor associated with the rotor used in conjunction with a rotor braking system to stop the rotor in a position that transfer head 104 can access.
  • a sensor could be magnetic, optical, mechanical, or o use other known means of sensing rotor position.
  • a second method of dispensing the liquid contained in the capillary tube segments of a capillary cassette is through the use of an air displacement device.
  • a microtiter plate dispensed from microtiter plate hotel is transferred by transfer head 5 104 to the dispensing device location 122.
  • an air dispenser such as the one pictured in FIG. 4A-C is located.
  • a capillary cassette is retrieved by transfer head 104, and filled with either sample from a sample multiwell plate or with reaction reagents.
  • the capillary cassette is then moved to the dispensing device location 122 and brought into contact with air displacement head.
  • the substrate of the capillary cassette is o placed on a receiving platform on the air displacement head.
  • the air displacement head may be joinable to automated transfer robot 102.
  • the air displacement head 301 is shown with a capillary cassette 15 held on bottom plate 302.
  • the bottom plate 302 is attached to a manifold assembly by hinge 318.
  • Capillary cassette substrate 10 is held on foam rubber pad 304 that 5 is secured onto bottom plate 302.
  • An array of holes 325 extend through foam rubber pad 304 and bottom plate 302, which are spaced to allow the capillary tubes 12 to extend through foam rubber pad 304 and bottom plate 302 when the capillary cassette is positioned on bottom plate 302.
  • the manifold assembly of the air displacement head is comprised of an upper housing 306, chamber unit 310 and a set of clamps 314. Clamps 314 secure 0 membrane 312 to the lower surface of the chamber unit 310.
  • Membrane 312 forms a seal to the top surface of the capillary cassette 15 when the manifold assembly is closed over the cassette.
  • Membrane 312 has holes 316 corresponding to capillary positions in the cassette when the capillary cassette 15 is placed on bottom plate 302. When the top manifold of air displacement head 301 is closed against bottom plate 302, capillary tubes 12 are positioned 5 extending through capillary tube receiving holes 316 on membrane 312.
  • FIG. 4B illustrates a cross sectional view of displacement head 301.
  • Upper housing o 306 is constructed of metal, acrylic or other rigid material.
  • Gas input coupler 303 is disposed on upper housing 306.
  • a pressurized gas or vacuum line 305 is attached to gas input coupler 303, a vacuum or pressure force may be introduced into upper chamber 307.
  • held between upper housing 306 and chamber unit 310 is a gas impervious elastic membrane 308. The area between elastic membrane 308 and upper housing 306 defines upper chamber 307.
  • Membrane 312 is pressed against substrate 10 of a capillary cassette inserted into displacement head 301.
  • Substrate 5 10 is secured within displacement head 301 by bottom plate 302.
  • Rubber pad 304 provides a deformable surface that exerts uniform force pressing substrate 10 against membrane 312.
  • Membrane 312 has an array of holes 316 that allow the capillaries 12 of the capillary cassette to extend through membrane 312.
  • the substrate seals holes 316 enclosing lower chamber 313.
  • elastic membrane 308 will be pressed into lower chamber 313.
  • Membrane 308 is located between upper chamber 307 and lower chambers 313. Membrane 308 serves both as seal for the upper end of chambers 313 and the chamber displacement actuator when pressure is applied to the upper chamber 307 through coupler 303. The degree of displacement is dependent on the 5 pressure applied and the elasticity of membrane 308. The resulting air displacement will act to dispense liquid from capillary tubes 12 that extend through the capillary cassette 10 and into the lower chamber 313. By regulating the amount of pressure applied through line 305, a consistent displacement force will be applied to each capillary tube. Given the submicroliter volume of the capillary tube segments, fluctuations in the amount of o dispensing pressure should not adversely affect displacement from the tubes.
  • FIG. 4C illustrates the closed air displacement head 301.
  • Upper housing 306 is pulled toward bottom plate 302 by latch 322 in order to compress membrane 312 against the top of the capillary cassette substrate thereby forming a seal.
  • Clamps 314 secure membrane 312 onto chamber unit 310.
  • Air displacement head 301 is mounted on arm 320.
  • Arm 320 may extend from automated transfer robot 102 shown in FIG. 1 or be positioned at dispense location 122. Pressurized gas may be introduced into upper housing 306 through gas input couple 303.
  • This displacement head provides an individual displacement chamber for each of the capillaries dispensed. Although a 16 capillary cassette is depicted, the displacement o head may be constructed to dispense capillary cassettes having an array of 96 capillaries or greater capillary densities. The dispensing force applied to each capillary is sufficiently small to allow dispensing directly onto a substrate with the sample dispensed at a discrete location.
  • Air displacement or centrifugal displacement may be used to dispense liquid from the capillary tube segments in a capillary cassette. It may also be possible to dispense liquid 5 from the capillary tubes using a bank of syringe pumps, applying pressure through a gas permeable/liquid impermeable (hydrophobic) membrane, using electrokinetic dispensing, or other known dispensing means. The air displacement head may also be used to dispense finished reaction mixtures onto a substrate for subsequent analysis.
  • a second method to assemble the reaction mixture is to have the regents required for the reaction stored as a dehydrated coating either on the interior of a capillary or on a substrate, such as within a well of a multiwell plate. If the reaction reagents were 5 dehydrated onto the interior of capillary tube segments in a capillary cassette, introducing a sample into the capillary would cause rehydration, mixing and formation of the reaction mixture. In a similar manner, if the wells of a microplate were coated with the dehydrated reaction reagents, adding a nucleic acid sample into the wells would bring the reaction reagents into solution, forming an assay mixture.
  • the sample could be metered with a o capillary cassette and dispensed from the capillary cassette by one of the methods set out above.
  • the sample would bring the dehydrated reaction reagents into solution and mix with the sample containing nucleic acid by diffusion.
  • This provides a method to assemble the reaction mixture in a very simple manner, potentially without the need to dispense the capillary tubes with a centrifuge or air displacement device. This could both simplify the 5 reaction processing system and shorten the reaction assembly time.
  • a dehydrated reagent mixture is commercially available, sold as Ready- to-Go® (Amersham Pharmacia Biotechnology, Piscataway, N.J.).
  • the stabilized, dehydrated reagents may be coated onto the interior surface of capillary segments or the interior of the wells of a multiwell plate.
  • the Ready-to-Go® product uses a carbohydrate o matrix to stabilize the reaction reagents (DNA polymerase, buffer reagents, dNTPs) in a desiccated state. Bringing the reagents in the Ready-to-Go® mixture into solution with the liquid nucleic acid sample and primers in solution produces the final reaction mixture required for the reaction.
  • the combination of the stabilized Ready-to-Go® compounds, the template DNA, primers, and sufficient water produces a final reaction mixture. It is contemplated that reagents for chain termination sequencing reactions and other reactions could also be stored in a desiccated state. 5 [0276]
  • the coating could be applied to surfaces by a number of different methods including vapor phase coating, filling a capillary (by capillary action, pressure filling, etc.) with the Ready-to-Go® mixture and emptying the bulk phase (under vacuum, pressure or other forces), or dipping a substrate (such as a bead) into the reagents and subsequently drying the bead. 0
  • a third method of assembly of the reaction mixture is to capture material from the sample on the surface of a substrate, such as the interior of a capillary tube segment.
  • the 5 material captured can be nucleic acid, enzymes, other biopolymers, or chemicals.
  • the desired material from the sample may be attached onto the surface by a number of methods. These include covalent attachment, binding by antibodies, DNA hybridization, hydrophobic interactions, electric field, magnetic field, or other chemical or physical forces.
  • the remaining liquid in which the sample was o suspended may evacuated from the capillary or microchip by chemical reaction or physical force. Air displacement or centrifugal dispensing method may be used to empty the capillary, as can a vacuum. The sample material would remain on the surface of the substrate.
  • the sample material may be substantially purified.
  • the reaction reagents may then be combined with the sample material, producing the reaction mixture.
  • one method to immobilize a nucleic acid sample is to attach the nucleic acid directly to a surface. This may be done by non-covalent modification (such as surface treatment with NaSCN, DMSO, etc.) or covalent linkage.
  • non-covalent modification such as surface treatment with NaSCN, DMSO, etc.
  • covalent linkage There are a number of different covalent attachment methods for DNA known in the art. For example, an amino group can be attached to the deoxyribose base of DNA and incorporated during a synthetic o reaction, such as during PCR amplification of a DNA plasmid insert.
  • the glass or silica of a capillary interior could be silanized and the amino group on the modified DNA would covalently bond to the silanized interior of the capillary.
  • other chemistries are available to covalently immobilize DNA onto a surface. Once the DNA is bound to the surface of a capillary or other substrate, the liquid in which the DNA was suspended may be eliminated from the capillary and the capillary may be filled with reaction reagents.
  • An alternative method of attaching a nucleic acid to the interior of the capillaries of 5 a capillary cassette is through affinity chemistry.
  • affinity chemistry procedure labels a biomolecule with biotin and then binds the biotinylated biomolecules to avidin or streptavidin.
  • the avidin/streptavidin may be used to link the biotinylated molecules.
  • Nucleic acid labeled with biotin may be subsequently attached to a surface, such as the interior of a capillary tube. This may be accomplished by binding streptavidin to the interior of the 0 capillary.
  • reaction mixture assembly example 1 namely the reaction reagents and the template sample could be separately metered and dispensed into a 384 well microtiter plate.
  • the liquids are combined to form a reaction mixture.
  • the reaction mixture is metered into the capillary tube segments of a capillary cassette.
  • the PCR reaction may be effected by temporarily sealing the ends of the capillary tube segments and exposing the capillary cassette to thermal cycles, as 5 described below.
  • the results of the PCR reaction are exponentially amplified copies of the subcloned plasmid DNA insert containing the biotin labeled primer.
  • the PCR amplified DNA containing the biotin labeled primer may then be immobilized on the walls of the capillary tubes of a capillary cassette.
  • the immobilization capillary cassette would have capillary tubes with avidin or streptavidin coated onto the o interior surface of each capillary tube.
  • the chemistry for attachment of avidin/streptavidin may be that disclosed in, for example, L. Amankwa et al., "On-Line Peptide Mapping by Capillary Zone Electrophoresis," Anal. Chem., vol. 65, pp. 2693-2697 (1993).
  • the capillary is filled with (3-aminopropyl) trimethoxysilane (3-ATPS), incubated for 30 minutes, and air dried.
  • the dried capillaries in the capillary cassette are next filled with sulfosuccinimidyl-6- (biotinamido)hexonate (NHS-LC biotin) which is again incubated followed by air drying.
  • Avidin or streptavidin in phosphate buffer at 7.4 pH is added to each capillary tube.
  • the 5 avidin binds to the biotin immobilized on the interior of each capillary.
  • the double stranded amplified biotinylated PCR products suspended in a buffer (e.g.
  • Tris-HCI, or EDTA with either NaCl or LiCI at 1-3M added for therapeutic binding are added to the capillary tube and incubated for 5-10 min.
  • biotin rather than avidin or streptavidin, is covalently attached first to the capillary wall. This aids in the regeneration of the capillary cassette for subsequent binding reactions. After completing the cycle sequencing reaction, it would be difficult to remove the amplified biotinylated DNA without also denaturing the avidin protein. 5
  • the amplified DNA may be easily removed by filling the capillary with phenol or formamide solution at 65-90 degrees C. This denatures the avidin protein without removal of the biotin bound to the interior surface of the capillary. This mixture is then dispensed. The capillary cassette may then again be filled with the avidin containing solution and reused for binding subsequent biotinylated o amplified template DNA.
  • the contents of the capillary tube may be dispensed in one of the methods described and the DNA would remain bound to the surface of the capillary. This removes debris and other impurities from the DNA presenting a rapid and effective method of DNA purification.
  • the capillary may be 5 rinsed with a buffer for additional purification.
  • the defined area of the interior surface of the capillary provides a known amount of binding sites for the DNA attachment. This provides a simple method for normalization of DNA concentrations. The normalization of DNA concentrations is important in improving the success rate of CAE analysis of cycle sequencing reactions.
  • the capillary cassette may then be dipped into wells or a reagent o reservoir containing the reagents for cycle sequencing.
  • the cycle sequencing reaction can be performed by temporarily sealing the ends of the capillary tubes by pressing each end of the capillary tubes against a deformable membrane.
  • the capillary cassette may then be exposed to thermal cycles that effect the cycle sequencing reaction.
  • the capillary tube segments of the capillary cassette Prior to filling, may be coated with a variety of compounds. Coating the interior surface of the capillary tube segments with 5 bovine serum albumin (BSA) or polyvinyl alcohol has been shown to improve performance of some reactions, such as preparation of chain termination sequencing reactions.
  • BSA bovine serum albumin
  • the thermal cycling device has integrated membranes that seal the ends of the capillaries and exposes the capillary cassette to thermal cycles.
  • the means for sealing the ends of the o capillaries in the capillary cassette is incorporated into the thermal cycling device.
  • the capillary cassette 15 is held on lip 280 within internal passageway 256 between deformable membranes 264a and 264b.
  • deformable membrane 264a is mounted on upper platform 261.
  • Lid 262 is secured on upper platform 261.
  • Platform 261 is attached by pivot 286 to base 265.
  • Pneumatics 284a, 5 284b are attached at an upper end to upper platform 261 at pivot 263.
  • Screw 282 acts as a stop for upper platform 261 when upper platform 261 is lowered onto housing 270, enclosing internal passageway 256.
  • Diffuser 258 promotes temperature uniformly of air circulating in internal passageway 256.
  • Thermocouple 260 measures temperature of the circulating air.
  • the function of pivot 277 and bottom membrane platform 200 is described in o conjunction with FIG. 7C.
  • FIG. 7C shows a cross section of the capillary cassette holding chamber with capillary cassette 15 inserted into the internal passageway 256.
  • the capillary cassette could be inserted into this area by automated robot 102 of FIG. 1 after the capillary tube segments have been filled with the samples and reaction mixture.
  • Capillary cassette 15 is positioned such that substrate 10 rests on ledge 280.
  • Capillary cassette is positioned such that the ends of capillary tube segments 12 are 5 depressed against top deformable membrane 264a and bottom deformable membrane 264b when upper platform 261 is lowered over the capillary cassette and lower platform 271 is raised.
  • Lid 262 seals against housing 270 when upper platform 261 is lowered to provide a flush seal.
  • Screw 282 acts as a stop for upper platform 261 to prevent the platform from lowering so far that capillary tube segments are bowed or damaged.
  • Base platform 266 is o secured to post 273 and secured to housing 270.
  • the lower end of pneumatics, 284b is secured at a lower pivot 271 a to lower platform 271.
  • a motor 250 turns shaft 251 that rotates squirrel cage blower
  • Blower 253 produces air movement through diffuser 254 to flow into internal passageway 256.
  • the blower generates sufficient circulation flow that the air flowing through internal passageway 256 circulates at 2,000 feet per minute.
  • Diffuser 254 ensures that the heat of the air blown by blower 253 is uniform throughout passageway 256.
  • Cone o 255 on diffuser 254 aids in mixing the flowing air, promoting temperature uniformity through passageway 256.
  • Diffuser 254 acts to ensure an even flow of air through passageway 256 in the region of the capillary cassette and reduces non-uniformity from wall loss effects in internal passageway 256.
  • the internal passageway 256 is defined by outer housing 270.
  • Outer housing 270 is preferably of rectangular cross section and comprised of sheet metal, plastic or other 5 durable materials.
  • the internal surface of outer housing 270 at all locations except for inlet 278 is lined with thermal foam insulation 272. Insulation 272 prevents excess heating of outer housing 270 and helps retain heat and aids temperature uniformity of the air circulating through internal passageway 256.
  • first diffuser 254 After flowing through first diffuser 254 the air flows through second diffuser 258. Diffusers 254 and 258 promote uniform air flow and i o temperature uniformity through internal passageway 256.
  • Past first diffuser 254 internal passageway 256 transitions to match the dimensions of the capillary cassette.
  • thermocouple 260 that is vertically disposed at the center of internal passageway 256 just beyond second diffuser 258.
  • Thermocouple 260 acts to monitor the temperature within internal passageway 256.
  • Thermocouple 260 may be a temperature-
  • thermocouple 260 may be selected such that it accurately reflects the internal temperature of a capillary tube.
  • the air circulating through internal passageway 256 passes thermocouple 260 and flows past the capillary tube segments 12 of capillary cassette 15. The ends of the capillary
  • deformable membrane 264a mounted on upper platform 261 that has been lowered to form an air tight seal with housing 270.
  • the lower ends of capillary tube segments 12 are sealed by deformable membrane 264b.
  • Deformable membrane 264b is mounted on platform 200 that is secured on a bottom surface by shoulder screws 268. Shoulder screws 268 extend through housing 270 and
  • Membrane 264a is mounted on upper platform 261 preferably such that membrane 264a extends into internal passageway 256 at least far enough that membrane 264a is even with insulation 272. As the air travels past capillary tube.segments 12, the length of the capillary tube segments 12 below substrate 10 are rapidly heated and cooled to the temperature of the air rapidly moving through internal passageway 256.
  • Door 274 controlled by motor 276 is used in conjunction with thermocouple 260 5 and heating element 252 to control the temperature within internal passageway 256.
  • thermocouple 260 5 When door 274 is closed, the air circulating within internal passageway will not be exchanged with outside air. As the air continuously passes over heating element 252 the air is rapidly heated until the air comes to the selected temperature.
  • thermocouple 260 senses that the temperature is at a selected temperature, heating element 252 may be kept at a lower o heat output such that the internal temperature is maintained.
  • door 274 may be moved to orientation 274a by motor 276 with the door 274 moved into internal passageway 256, allowing all air which has passed capillary cassette 15 to be exhausted from internal passageway 256 to the outside.
  • a filter or exhaust duct could be 5 mounted about door 274 to prevent compounds in the circulating air from being exhausted to the environment.
  • the rapidly circulating air will be quickly exhausted to outside of the thermal cycler while ambient air is drawn in through air intake 278. Air drawn into internal passageway 256 through intake 278 flows through heater 252. The area through which the air moves is restricted by block 259 positioned above heater 252 within internal chamber o 256.
  • thermocouple 260 Again the temperature of the air within internal passageway 256 is monitored by thermocouple 260 and when the desired temperature drop has occurred, door 274 will be brought toward housing 270, reducing air volume drawn through air intake 278.
  • this thermal cycler may perform 5 precise air temperature varying sequences. Additional heat is added when needed by heating element 252 and heat is exhausted by opening door 274, with the temperature result of either action monitored by thermocouple 260. Exhausting circulating air through door 274 allows air within internal passageway to drop in temperature at a rate greater than 10 degrees per second.
  • the rapid temperature change combined with the rapid transfer of heat to or from the capillaries allows for efficient temperature cycling reactions.
  • the denaturing of nucleic acid strands and the annealing of primer to template strands each may take place in one to five seconds.
  • the extension of the primer will require less time to effect since the rapidly circulating air and the thin walls of the capillaries rapidly bring the internal volume of the capillaries to the selected temperature.
  • the thin walls of the capillaries and the small capillary volume enable a rapid 5 temperature change and heat transfer throughout the internal capillary volume. This greatly reduces the time required for each cycle of the reaction, allowing more efficient use of the thermal cycler and greater throughput in sample preparation.
  • a 30 cycle PCR amplification may be performed in under 30 minutes. It should be possible to reduce this time to less than 8 minutes.
  • upper platform 261 may be raised and capillary cassette 15 removed from internal passageway 256.
  • capillary cassette 15 removed from internal passageway 256.
  • the liquid within each capillary tube segment will expand somewhat and some liquid will leak from the capillary and be carried away by the rapidly flowing air.
  • loss is only a few percent of the volume of the capillary tube segment and 5 should not present either a contamination problem or cause enough reaction product loss to materially affect subsequent analysis.
  • disposable materials such as a thin film can be placed over the deformable membranes.
  • the disposable materials can be individual sheets or rolls of material that o advance after each use to prevent the capillary openings from touching a section of material previously used.
  • the portion of capillary tube segments 12 located between substrate 10 and deformable membrane 264a will receive only poor air flow and will be less likely to rapidly reach the denaturation temperature. However since this length is short, the failure of this area to as rapidly reach the denaturation temperature should not adversely 5 affect the ability of the remaining portion of the capillary from producing sufficient reaction product for subsequent analysis.
  • An alternative device for sealing the ends of the capillary is a capillary cassette holder that seals the ends of capillary tube segments of a capillary cassette.
  • the capillary cassette holder is comprised of a pair of parallel deformable o membranes 14a, 14b each secured onto platforms 16a, 16b.
  • the deformable membranes may be silicon rubber seals, Teflon®, plastics or other resilient, deformable material.
  • a pair of parallel posts 9 extend from bottom platform 16a to top support platform 24 where the posts are secured by internally threaded nut 18. Post 9 passes through platform 24 and nut 18 is retained on an annular lip of platform 24. Shoulder screws 20 extend through holes in support 24 and are secured to top platform 16b.
  • the substrate 10 5 of capillary cassette 15 may be designed to have holes which conform to the spacing and dimension of posts 18 such that capillary cassette 15 may be more easily and securely held within holder 23.
  • FIG. 3E shows a chip substrate 70 comprised of two bonded substrate layers 72, 74. One layer 72 has grooves 76 extending the length of the chip. The affixed top substrate 72 encloses a capillary dimension passage 5 76 with opposite open ends. A liquid reaction mixture may be introduced into the enclosed passage.
  • the ends of these passages may be sealed by pressing the ends against a deformable membrane, as was done with the capillary cassettes. Temperature cycling may require longer times because of greater mass material comprising the chip, but cycling times should still be more rapid than conventional cycling. o [0298] For isothermal reactions, such as rolling cycle amplification, temperature cycling is not required to effect the reaction. Once an isothermal reaction mixture is combined and introduced into a capillary cassette, incubation of the cassette at a reaction temperature will allow the reaction to occur. With reference to FIG. 1 , the automated transfer device may transfer a capillary cassette into incubator 124 where the capillary cassette is incubated at a 5 selected temperature. A set of deformable membranes may be used to seal the ends of the capillaries during incubation. As was seen in other system components, incubator 124 may be used at the same time as other system components.
  • thermocycling device may be any device that can expose the capillary tube segments of the capillary cassette to temperature cycles.
  • Thermal cycling devices that use water, electric field, heating blocks, or other means may be used.
  • air based thermal cycling devices are rapid and adaptable to the low volume cycling of the present invention.
  • FIG. 6 A thermal cycling device that uses air as the temperature transfer medium is shown in FIG. 6.
  • the reaction mixture is contained in capillary tube segments that have a 5 high surface to volume ratio and small material thickness. This allows very rapid transfer of heat through the walls of the capillary and throughout the liquid reaction mixture. An equilibrium temperature is reached rapidly throughout the liquid in the capillary.
  • the use of air as a heat transfer medium enables the rapid ramping of temperature in the reaction chamber. Rapid circulation of the air ensures rapid and more uniform heating or cooling of o the capillary segments and their contents.
  • the capillary cassette 15 sealed within holder 8 is inserted through opening 215 in housing 202 of the air based thermal cycler.
  • the holder 8 is supported by housing surface 215 of the thermal cycling chamber 210.
  • the capillary tubes 12 mounted to substrate 10 are exposed to the air of thermal cycling chamber 210 such that 5 the air may freely flow around capillary tube segments 12.
  • Thermocouple 216 monitors the temperature of the air moving past capillary tubes 12.
  • paddle 208 driven by motor 206 rapidly circulates air within chamber 210.
  • the air is rapidly circulated past the capillaries 12 of capillary cassette 15.
  • Halogen bulb 220 acts as a heat source to heat the air within the o thermal cycling chamber 210.
  • the circulating air is held at a selected temperature for a selected period of time.
  • the thermocouple 216 transmits the temperature of the capillary tube segment 12 to microprocessor 218.
  • the microprocessor instructs actuator 222 to open door 226 allowing air to pass through vent 224.
  • As air passes through vent 224 additional air is drawn into the 5 reaction chamber through air inlet 203 by fan blade 204.
  • Fan blade 204 is driven by motor
  • thermocouple 216 The venting of hot air and replacement with cooler ambient temperature air, combined with the rapid circulation of air by fan 208, a relatively small thermal cycling chamber 210 and precise measurement of sample temperatures by thermocouple 216 enables rapid temperature ramping. The time required for effecting the thermal cycles is greatly reduced. 0 A typical thermal cycling reaction requires different temperatures for denaturing of nucleic acid strands, annealing of a primer, and extension of a polymerase. The denaturing and annealing steps occur rapidly in a capillary tube where the small internal volume of liquid will rapidly come to equilibrium, while the extension of the DNA molecule takes less than 10 seconds for a 500 base extension.
  • the time required for each thermal cycle of the three temperatures may be reduced to less than 15 seconds by using the rapid heat transfer of the air based thermal cycling apparatus.
  • the use of the capillary cassette in combination with an air based thermal cycler allows additional advantages.
  • the capillary cassette holder temporarily seals the capillary, allowing rapid and simplified sealing of each capillary tube segment.
  • the capillary cassette o contains a number of capillary tubes in parallel arrangement, allowing for more efficient use of the thermal cycler and allowing greater sample throughput.
  • the capillary cassette 15 contained within holder 8 is removed through opening 215.
  • the capillary cassette 15 is released from the holder and is subsequently dispensed.
  • the thermal cyclers of FIGS. 6 and 7A-C were illustrated as being used with 5 capillary cassettes. The same devices are adaptable to other containers with opposing ends.
  • a chip-like substrate with a plurality of passageways extending through the chip has, like a capillary cassette, evenly spaced opposed open ends.
  • Several chips could be placed into a thermal cycler with the open ends temporarily sealed and exposed to thermal cycles. The rapid temperature changes may be a bit slower o due to increased material thickness.
  • Other containers with opposing open ends may also be used with either temperature cycling device.
  • the prepared reaction mixture is dispensed into a substrate for analysis by an analytical system.
  • the capillary cassette may be dispensed by air displacement, centrifugal force, vacuum or any other displacement method.
  • the substrate into which the reaction mixture is displaced may be the wells of a multiwell plate, locations on a planar substrate, or o wells that lead into an analytical chip.
  • the reaction mixture though small, still may produce enough reaction products that dilution is necessary.
  • Dispensing Completed Reaction Mixture Example 1 Direct Dilution
  • the capillary cassette may be removed from air thermal cycler 116 by transfer head 104.
  • the 5 capillary cassette may be moved by transfer head 104 to be placed in a plate dispensed from finished sample hotel 112.
  • the plate located at position c, may be a multiwell plate such as a 384 well microplate.
  • the wells of the plate contain a dilution liquid, such as formamide, water, TBE, or other selected buffers.
  • the reaction mixture may be dispensed from the capillary tube segments of the capillary cassette by positive displacement, o centrifugation, or other dispensing means.
  • the reaction may also be dispensed into a solution for further chemical or biochemical reaction.
  • Ethanol precipitation may be effected in a dispensing means similar to the means of direct dilution.
  • Transfer head 104 of FIG. 1 would again take the capillary cassette from air thermal cycler 116 and place the short ends of the capillaries in a multiwell plate located at position c.
  • the wells of the plate would contain an alcohol, such as 90% ethanol chilled to 4.degree. C.
  • the reaction mixture would be dispensed from the capillary o cassette into the ethanol by centrifuge. Air displacement or other dispensing methods can also be used.
  • the multiwell plate can be moved into the centrifuge by transfer head 102 and a low speed centrifugation performed to collect the precipitated nucleic acid in the bottom of the multiwell plate.
  • the alcohol could then be removed by aspiration or other means.
  • the precipitated DNA could then be resuspended in 5 formamide, water or other suitable diluent.
  • the plate may be transferred by transfer head 104 to analytical stage 120.
  • Analytical stage 120 may feed the sample plate directly into an analytical device, for example a capillary array electrophoresis system, such as MegaBACETM produced by Amersham Biosciences, Sunnyvale Calif.
  • the o analytical stage could direct the product to other systems for further processing. It is also possible to dispense the samples onto a substrate for mass spectrometry analysis, calorimetric analysis, or other analytical methods. Dispensing Completed Reaction Mixture Example 3: Dispense Directly into Analytical System
  • the samples were dispensed into multiwell plates. These plates could then be moved manually or robotically onto a stage for analysis by an analytical system.
  • the capillary cassette could be dispensed directly into the wells of an analytical device, such as an electrophoresis chip.
  • an analytical device such as an electrophoresis chip.
  • a capillary cassette having 16 capillaries disposed in the substrate in two parallel rows of eight o capillaries may dock with 16 wells in an analytical microchip.
  • Such a microchip would have an array of analytical lanes in fluid communication with a sample port.
  • the capillary cassette may be designed such that the spacing of the capillaries matches the spacing of the sample reservoir inlets. For example, the capillary cassette illustrated in FIG.
  • FIG. 3C includes capillaries 12 extending through flexible strip 11.
  • Flexible strip 5 11 may be used alone or in combination with other such strips.
  • the orientation of the capillaries in an essentially straight line may be altered by bending strip 11 to form an arc.
  • FIG. 3D illustrates strip 11 bent to allow capillaries 12 to mate with input ports that are disposed on a substrate in a circular pattern.
  • the liquid in capillaries 12 may then be electrokinetically injected or otherwise dispensed from capillaries 12 into ports of an o analytical chip if an appropriate electrode array or other dispensing methods are used.
  • the capillary cassette could be dispensed by air displacement or other dispensing means preferably selected to minimize splattering and 5 bubble formation. Prior to dispensing the prepared reaction mixture into the wells for analysis, a small amount of a dilutant could be added to each analytical microchip well. When the capillary cassette is dispensed, the diluent will dilute the samples in the sample wells.
  • the sub-microliter volume reaction mixtures prepared in the capillary cassette such as a DNA sequencing reaction product mixture, can readily be integrated with the analytical o microchip for sequencing or other analysis methods.
  • capillary cassette washer 410 is comprised of wash manifold 412 and wash tank stage 416. Between wash manifold 412 and wash tank stage 416 is capillary cassette platform 414. Extending from wash tank stage 416 is leg 419. In o this wash system, a wash liquid is pumped from one or more of containers 452, 454, 456,
  • FIG. 8E provides a schematic of the working of the wash station.
  • Nitrogen tank 460 provides a pressure source to direct fluid flow.
  • Opening manual valve 462 allows gas to flow o through regulator 466 and through filter 468.
  • Regulator 466 regulates the pressure from the pressure source.
  • Pressure sensor 464 monitors gas pressure from the nitrogen source and indicates if gas pressure is below a selected pressure.
  • the pressurized gas flows through filter 468 into line 470.
  • Pressurized gas line 470 branches into the top of sealed wash bottles 471 , 472, 473, and 474.
  • the pressurized nitrogen pumps the wash liquid within each 5 wash bottle into respective fluid lines 471a, 472a, 473a and 474a respectively through an intake filter 476 on each of said respective fluid lines.
  • Each of the sealed wash solution bottles may contain a different wash solution, such as water, alcohol, a buffer or other wash solution.
  • wash bottles Although four wash bottles are illustrated, the system is adaptable for use with more or fewer wash fluids.
  • exchange of wash bottles simply requires venting o nitrogen pressure on bottles 471 , 472, 473, 474 at valve 462, the removal of the cap from the selected bottle and replacement of the cap with attached pressure and fluid lines into a new or refilled wash fluid bottle.
  • Each of the fluid lines 471 a, 472a, 473a and 474a terminate in selector valve 478.
  • the selector valve routes one of the selected fluids from the input line into valve output line 480.
  • the valve output line then transports the pressurized liquid into wash tank 440.
  • the capillary tubes in the capillary cassette function as a conduit for transport of 5 fluid from the wash tank 440 into the wash manifold interior 425.
  • Vacuum source 496 provides a vacuum force once valve 492 is open.
  • vacuum valve 498 When vacuum valve 498 is open, a vacuum force is directed into waste bottle 490 creating negative pressure within line 490a.
  • valve 495 When valve 495 is open, suction will be applied through suction line 490a, suction line 495a and suction lines 424a.
  • suction is applied through suction ports 424 by suction lines o 424a the negative pressure through interior wash manifold 425 will draw liquid up through the capillary tube segments extending into wash manifold interior 425. The liquid will travel through suction passageways 424, into suction lines 424a, past valve 495, through suction lines 495a and 490a and into waste bottle 490.
  • FIG. 8D illustrates a view of the wash manifold.
  • the bottom of the wash manifold 5 contains holes 426 into which the capillaries are inserted.
  • Wash manifold interior 425 is comprised of lanes joined at a first end to suction passageways 424 and at a second end to purge passageways 423.
  • suction is applied through line 424a fluid will be drawn through capillaries into the lanes comprising interior 425, through passageways 424 and into line 424a.
  • the purge valve is opened, air will pass through line 423a, through o passageway 423, into interior 425, and into passageway 424, clearing interior 425 of any liquid remaining in interior 425.
  • wash tank 440 is lowered relative to the capillary cassette platform such that the ends of the capillary tube segments are not in contact with the liquid in wash tank 440.
  • the liquid within wash tank 440 is drained through drain 484 5 which transmits the fluid into drain line 484a when valve 485 is opened and suction is applied through suction line 490a.
  • the fluid within wash tank 440 will then drain into waste bottle 490.
  • wash fluid supply line 480 and the wash tank distribution manifold 480a are purged to empty the line of any o previous liquid. This is effected by opening one of the valves in selector valve 478 and flowing wash fluid through supply line 480 and through bleed lines 482. Opening valve 487 allows a vacuum force to be transmitted through line 490a through line 488 providing suction which in conjunction with fluid pressure is used to purge the distribution manifold through bleed lines 482.
  • valve 487 is closed and the wash tank is raised and filled.
  • the fill level of wash tank 440 is controlled by the selected wash fluid fill time and wash fluid pressure.
  • Overflow port 5 486 acts as a safety drain to drain off fluid overfill. If the fluid level within wash tank 440 is too high, liquid will flow from wash tank 440 into overflow port 486 and into line 486a. When valve 487 is open, the suction force from line 490a and 488 will draw overflow liquid from overflow port 486 into waste bottle 490. Restriction flow valve 441 limits liquid fluid flow through lines 482. 0 [0317] FIG. 8F shows the top perspective of wash fluid tank 440. An input line introduces a wash solution into wash fluid distribution manifold 480a. This manifold supplies wash fluid ports 481 that fill tank 440. The spacing of wash fluid ports 481 aids in uniform filling across the width of tank 440.
  • the fill time and fluid pressure regulate the amount of fluid filling tank 440. If excess fluid enters tank 440 it will drain from overflow port 486. 5 [0318] To empty the tank, the tank is lowered by the pneumatics as described, and drain 484 is opened. The shape of tank 440 directs fluid to drain 484 when the end of tank 440 containing drain 484 is lowered. This configuration is designed for efficient filling, emptying and purging of tank 440 and associated fill lines. [0319] Again with reference to FIG. 8E, once a wash cycle has been completed, any liquid o remaining within wash manifold interior 425 may be eliminated by opening valve 491 while suction is applied through the manifold. Opening valve 491 causes a pulse of air to be drawn in through vent 493.
  • the air is introduced into wash manifold interior 425 through purge lines 423a and is removed by suction lines 424a. If the manifold is in contact with capillaries, the relatively narrow bores of the capillaries in the capillary cassette provide a 5 limited capacity for drawing air through the wash manifold. By opening valve 491 , a much greater amount of air may be drawn through the manifold through purge lines 423a which have a much greater capacity for drawing air. This will result in a sudden rush of air drawn through the manifold. This acts to clear the wash manifold of any liquid remaining within the wash manifold interior 425. Preferably manifold interior 425 is purged before and after o raising the wash manifold.
  • the wash station 410 is shown in side view.
  • the capillary cassette platform 414 is mounted on support legs 445.
  • the reservoir section, shown in internal cross section has at a back lower end of the reservoir, drain outlet 484. Upwardly positioned from the drain outlet at the back wall of the tank is overflow outlet 486. Disposed at the front of the reservoir is reservoir bleed outlet 446.
  • Each outlet is associated with a respective tube and valve, as described in conjunction with FIG. 8E.
  • Each tube 5 carries liquid flowing from an associated outlet when the associated valve is opened and vacuum source applied.
  • Capillary cassette platform 414 is held in a fixed position by support legs 445. Extending downward from the front of capillary cassette platform 414 is hinge 418 with pivot 432. Attached to a lower end of hinge 418 is wash tank stage 416. Extending from below 0 wash tank stage 416 is leg 419 that is attached at a lower end by pivot 443 to pneumatic cylinder 429. At the back end of the stationary capillary cassette platform 414, the wash manifold is attached at pivot 420. When pneumatic cylinder 429 is extended from the lower end, wash tank stage 416 will be lowered in an arc away from stationary capillary platform. This occurs when no pressure is applied to 429 and gravity causes the wash tank stage to 5 pivot down. When pneumatic cylinder 429 is extended from the upper end by applied pressure, wash manifold 412 will be raised in an arc away from capillary cassette platform
  • wash manifold 412 Disposed above capillary cassette platform 414 is wash manifold 412.
  • the wash manifold has a purge passageway 423 disposed at a front end and a suction passageway o 424 disposed toward the back end.
  • the respective lines carrying air to the manifold or removing gas or liquids from the manifold are described in conjunction with FIG. 8E.
  • pneumatic cylinder 429 is shown fully extended from a lower connection pivot 443 on leg 419, through hole 333 in capillary cassette platform 414, to an upper connection at pivot 428 on wash manifold 412.
  • the extended height of the wash 5 manifold is limited by plate 430 that is secured to the top of manifold 412.
  • cutout 434 The dimensions of cutout 434 are such that capillary cassette 15, when placed on capillary cassette platform 414 has associated capillary tube segments 12 extending through capillary cassette platform 414 while the four edges of capillary cassette substrate 10 are retained on the capillary cassette platform 414 on the edge of cutout 434. Alignment 5 pins may be added to capillary cassette platform 414 to properly position the capillary cassette.
  • an electronic controller implements a sequence of steps.
  • the electronic controller instructs associated controlled devices of the wash station to carry out a programmed wash sequence.
  • the programmed sequence o begins with the capillary cassette being placed on the capillary cassette stage by the robotic transfer device.
  • the wash manifold lowers onto the capillary cassette such that the shorter end of capillary tube segments extend into the wash manifold and the opposite end of the capillary tube segments are within the wash liquid in the wash tank once filled.
  • the substrate provides a partial seal between the wash manifold and cassette such that when 5 suction is applied to the capillary tube segments by the wash manifold, fluid will be drawn up into the wash manifold through the capillary tube segments.
  • the wash solution supply line is purged with the first selected solution to clear the previous solution from the line.
  • the purge solution is removed through distribution manifold to drain 484 and bleed lines 482 to wash waste line 488 and 490a then into waste bottle 490.
  • the o wash tank 440 is then raised and filled with the selected wash solution.
  • a vacuum is applied to the wash manifold causing the solution in the wash tank to be drawn up through all of the capillary tube segments in the capillary cassette. After the programmed wash duration, the wash tank is drained and lowered. The vacuum force is continued through the wash manifold, drawing air through the capillary tube segments. 5 Once the capillary tube segments are dried, the vacuum line of the wash manifold is turned off. The wash solution supply line is purged with the next wash solution and the steps of raising and filling the wash tank, drawing the wash solution through the capillary tube segments and emptying the wash tank are repeated for each selected solution. The specified sequence may repeat these steps for any number of wash solutions.
  • the manifold 5 vacuum is again applied and the purge valve 491 is opened and air is drawn through vent 493.into purge line 423a into purge inlet 423. This ensures that any remaining liquid is removed from the wash manifold interior.
  • the vacuum is then shut off.
  • the washed and dried capillary cassette may then be moved by the transfer robot to a capillary cassette hotel or other location.
  • the components of the system could be integrated in a combined system that allows several elements of the complete system of FIG. 1 to operate at the same time.
  • electronic control device 123 may be used to send instructions to the components of the integrated system.
  • the electronic control device may be a computer that sends electronic signals to various system components to effect a programmed set of instructions.
  • Elements of the system could operate simultaneously, increasing system efficiency.
  • automated robot 102 could retrieve a capillary cassette from cassette hotel 106, o place the capillary cassette in a sample plate at stage a. An amount of sample from the plate is drawn into the capillary tubes by capillary action.
  • the capillary cassette could then be moved and placed on top of a microtiter plate such that the short ends of the capillary tube segments are in the wells of the microtiter plate.
  • the robot 102 could then transfer the combined microtiter plate/capillary cassette to dispense location 122 for dispensing.
  • thermocycler 116 could also be sending electronic signals to thermocycler 116.
  • the vent door, heating element, and thermocouple of thermocycler 116 could be linked to electronic control device 0 123, allowing electronic control device 123 to effect a selected temperature cycling procedure by regulating the temperature at which air is cycling within the thermal cycler. This precise monitoring allows the temperature cycling procedure to be effected in a minimum amount of time.
  • the electronic control device 123 could electronically instruct the thermal cycler to shut off the thermocycler fan and heating element and open the lid pneumatically to allow a capillary cassette to be removed from the interior of the thermal cycler.
  • the cassette washer 118 could also be cleaning a capillary cassette. Again the electronic control device 123 could instruct the cassette washer 118 to perform a wash sequence in which a capillary cassette is cleaned with a selected sequence of wash liquids and air-dried. o [0330] Electronic control device 123 enables each element of the system to be used with maximum efficiency. A single set of instructions to electronic control device 123 could allow assembly of the reaction mixture, thermal cycling of the reaction mixture to effect the desired reaction, dispensing of the completed reaction mixture onto an analytical substrate, movement of the analytical substrate to a stage for processing by an analytical instrument, 5 and cleaning of used capillary cassettes.
  • the invention provides methods and apparatus for performing o nucleic acid reactions in reduced volume, and for normalizing the amount of nucleic acid template present in such reactions.
  • the present invention is based, in part, upon the novel use of the saturable, yet reversible, binding of nucleic acids by certain materials to control the mass of nucleic acid delivered as template to a subsequent reaction, without a required antecedent 5 determination of the concentration of nucleic acid in the solution from which the nucleic acid is to be captured.
  • the internal surface of a capillary is used to effect nucleic acid capture, permitting nucleic acid template to be captured directly in the chamber in which subsequent reaction is to be performed.
  • the present invention is described herein with particular reference to its use for performing DNA sequencing reactions, especially in the context of a high-throughput sample processing system employing capillary electrophoresis, for which the methods and apparatus of the present invention are particularly advantageous.
  • this invention can be used in the course of performing many types of biochemical and chemical reactions using DNA, as well as RNA, as the substrate.
  • the present invention provides methods for reversibly immobilizing nucleic acid directly on the inner surface of a reaction chamber, such as a o glass capillary tube, or the functional equivalent thereof. After immobilization and other processing steps, the nucleic acid is ready to be used in a chemical, biochemical or enzymatic reaction performed inside the capillary tube. Alternatively, the nucleic acid can be eluted and expelled from the capillary so as to dispense a controlled amount of nucleic acid for subsequent use.
  • nucleic acid binding is an inherent property of glass surfaces, it will be 5 appreciated that the capture surface can be modified to alter its binding capacity or binding selectivity.
  • major binding forces are hydrophobic forces, charge-charge (electrostatic) forces, and hydrogen bonding.
  • vinyl groups can be added to the capture surface by reaction in the solution phase
  • propyl amine groups can be added by CVD
  • other amines preferably o tertiary amines
  • oligo d(T) can be covalently linked to aminated surface, increasing capture of poly(A) mRNA.
  • a spacer of the general form Cn can be added between the silicon surface and the functional groups. For each of these, the characteristics and/or binding capacity can be altered by changing the concentration of the functional groups.
  • An additional advantage of the present invention is that it is useful for reducing the 5 number of processing steps associated with, and the quantity of nucleic acid and reagents needed for, carrying out a reaction with nucleic acid, especially in the context of a high- throughput sample processing system.
  • a DNA sequencing reaction it is necessary to combine template DNA with a reaction mixture comprising sequencing primer, DNA polymerase, dideoxynucleotides, dNTPs, buffers, salts and water, prior to performing o thermal cycling that activates the reaction. Typically, this involves preparing a 20 ⁇ l reaction by aliquoting the reaction mixture into a tube, followed by the addition of 200 ng template DNA.
  • a capillary tube is filled with 5 a DNA solution, resulting in the reversible immobilization of 5 ng of the template inside the capillary.
  • the capillary is then filled with 500 nl of reaction mixture, which causes the template to elute from the inside of the tube into the mixture.
  • the capillary is then sealed and thermocycled, with subsequent analysis of the reaction products by a high sensitivity capillary electrophoresis system.
  • the capillary serves o simultaneously as a pipettor that is filled by capillary action, and as a reaction chamber, it is unnecessary to separately aliquot, with dedicated pipetting systems, either template DNA solution, or the reaction mixture. It is only necessary to provide a stock of each into which the capillary is dipped to fill it. This saves processing steps and materials such as disposable pipettor tips. It also saves reagent that would otherwise be carried over during 5 processing steps, and not introduced into a reaction.
  • FIG. 15 is a flowchart
  • FIG. 16 is a schematic that shows the steps associated with embodiments of the instant invention, whereby nucleic acid is reversibly immobilized to the inner surface of a reaction chamber, such as a glass capillary tube.
  • Reaction chambers prepared in this way can then be used to carry out a sequencing reaction with nucleic acid, to effect another type of enzymatic or biochemical reaction with nucleic acid, or for dispensing a predetermined quantity of nucleic acid onto a substrate, such as a microtiter dish well, or into an analysis instrument, such as a capillary electropheresis device.
  • a substrate such as a microtiter dish well
  • an analysis instrument such as a capillary electropheresis device.
  • step 1 the nucleic acid sample is prepared from a suitable source, after which, in step 2, the nucleic acid 80 is dissolved in a solution 81 containing chaotropic ions.
  • step 3 the reaction chamber is filled with the nucleic acid-chaotrope solution and incubated, in step 4, for sufficient time to allow reversible binding of the nucleic acid 80 to the inner surfaces 82 of the reaction chamber 12.
  • step 5 the nucleic acid-chaotrope solution is removed, followed by washing, step 6, and drying, step 7, of the reaction chamber. At this point the reaction chamber is useable.
  • Part 12 refers to a capillary tube, or more broadly, a reaction chamber, including capillary tubes and structures equivalent in function thereto.
  • Part 80 refers to DNA, or more broadly, nucleic acid, including DNA and RNA and derivatives thereof.
  • the process begins by obtaining nucleic acid, FIG. 15, step 1 , from a suitable source.
  • the nucleic acid may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or derivatized forms of these molecules.
  • Nucleic acids can be isolated and purified according to methods well known in the art (see Current Protocols in Molecular Biology, John Wiley & Sons, Inc., 2000, Edited by Fred M. Ausubel et al., ISBN 0-471 -50338-X) from a variety of living organisms or self-replicating systems that rely on living cells.
  • Cells can be eukaryotic cells, including human and non-human mammalian cells, non-mammalian animal cells, plant cells and fungal cells. Additionally, eukaryotic cells can be free living single celled organisms, such as amoebae or other parasites. Cells can also be prokaryotic cells including bacteria and archaebacteria. Nucleic acids can also be obtained from viruses, including RNA and DNA viruses, and viruses that infect animal cells, plant cells, fungal cells, and bacterial cells. Nucleic acids can also be produced according to chemical synthetic methods well known in the art.
  • the nucleic acid, FIG. 16 80 is resuspended and/or dissolved into a solution containing a chaotropic agent, FIG. 15, step 2, and FIG. 16 82.
  • the chaotropic agent is desirably at sufficiently high concentration (e.g., about 0.5 M to 8.0 M) to effect the reversible binding of the nucleic acid, but not so high as to cause the nucleic acid, or the chaotrope itself to precipitate out of the solution under all of the conditions to which the solution is subjected in carrying out the invention.
  • a chaotropic agent is a substance that affects the partitioning of molecules from a nonaqueous to an aqueous phase due to the disruptive effect that the substance has on the local structure of water.
  • Chaotropic agents are salts of chaotropic ions, and are highly soluble in aqueous solutions. At sufficiently high concentration in aqueous solutions the chaotropic ions provided by such salts cause nucleic acids to lose secondary or tertiary structure, and double-stranded nucleic acids to melt (i.e., strand-separate).
  • chaotropic ions have these effects by disrupting hydrogen-bond networks existing in water, causing the denatured form of the nucleic acids to be more thermodynamically stable as compared to the structure of more highly ordered structures (e.g. the double helix) that exist in a typical aqueous environment.
  • nucleic acids will reversibly bind certain substances, such as silica.
  • chaotropic ions e.g. about 0.5 M to about 8.0 M
  • the mechanism of nucleic acid binding to silica may involve chaotropic ion disruption of the water structure at the surface of the negatively charged silica, allowing a cation (e.g.
  • Na+ or K+ mediated salt bridge to form between it and the negatively charged phosphate backbone of the nucleic acid strand.
  • a chaotropic agent may be used singly or as a mixture of two or more chaotropes.
  • the salt bridge is not a permanent bond and can be disrupted when the ionic concentration in the proximity of the bond is lowered. In this way, nucleic acid can be eluted from silica or similar material with water or other suitable low ionic strength aqueous buffer.
  • Chaotropic ions include guanidinium, iodide, perchlorate and trichloroacetate.
  • Chaotropic salts include sodium perchlorate, potassium perchlorate, sodium bromide, potassium bromide, sodium iodide, potassium iodide, sodium thiocyanate, potassium thiocyanate, guanidine thiocyanate, sodium isothiocyanate, potassium isothiocyanate, 5 guanidine hydrochloride, guanidine isothiocyanate, lithium chloride, sodium trichloroacetate, and potassium trichloroacetate.
  • Other substances with chaotropic properties include dimethylsulfoxide (DMSO), urea, and the tetra-amine halides, including tetraethylamine chloride.
  • the reaction chamber will typically be of very small volume, desirably from about 1 - 1000 nanoliters (nl), more desirably from about 10 - 500 nl, most desirably from about 100 5 - 500 nl.
  • the reaction chamber is configured so that solutions can be introduced into it passively, by taking advantage of capillary action.
  • Capillary action is the phenomenon by which the elevation of a liquid rises where it is in contact with a solid, such as the sides of a tube, and is most marked in capillary tubes, i.e., tubes of very small o diameter. Capillary action depends on the forces created by surface tension and by wetting of the sides of the tube. If the forces of adhesion of the liquid to the solid (wetting) exceed the forces of cohesion within the liquid (surface tension), the liquid will rise up the tube, i.e., it will rise above the hydrostatic level.
  • the solution can be introduced into the reaction chamber actively, such as by pumping using positive or negative atmospheric 5 pressure.
  • a capillary tube serves as the reaction chamber. If the bore of the capillary is of known and uniform areal cross section, then the volume of the tube is easily calculated, being linearly proportional to o its length. Thus, a capillary tube reaction chamber of given total volume is obtainable by cutting the tubing to the desired length given by the calculation. In accordance with the laws of fluid dynamics however, care must be taken that the density of the solution is not so great, its surface tension so low, and the diameter of the tubing insufficiently small, that the column of solution cannot overcome gravity, and thereby fails to fill the tube.
  • one end of the tube is dipped into the nucleic acid-chaotrope solution, FIG. 16 83, that is usually provided in volume excess over the total volume of any 5 tube to be filled. In this manner, the tube is filled in one step, reducing the chance of bubble formation at the inlet.
  • the opposite end of the capillary must be open, or otherwise able to allow air to escape from the filling tube.
  • capillary tube should be understood to represent not only that structure commonly referred to as a capillary tube, but also any structure that is functionally equivalent thereto.
  • a tunnel, channel or groove can be formed that is configured so that fluid can fill it by capillary action, or by the direct 5 application of some force, e.g. positive or negative pressure, or centrifugal force.
  • the tunnel, channel or groove can be formed mechanically, chemically, thermally, or by other means known to the skilled artisan.
  • a channel or tunnel can be formed by removing material from a matrix, e.g., using a drill bit, laser, or chemical etching [0353]
  • a groove or channel 78 in the surface of a substrate 72 o such as a glass slide of any shape and dimension, can be cut with a saw, or formed by laser ablation or chemical etching to create a structure called a chip or microchip 70.
  • grooves in a silicon wafer can be formed by photolithographic methodologies known in the art, and grooves in glass slides can be etched using hydrofluoric acid.
  • a groove or similar depression 78 is formed in the surface of a substrate 72, it will 5 usually be advantageous to cover it with a cover 74 to form an enclosed space. Covering the groove or depression 78 ensures that there is maximal surface area for the fluid to interact with, thereby promoting the capillary action, minimizes the opportunity for contaminants to contact the reactants, and creates a vapor barrier to ensure that during any elevation in temperature of the reaction, such as during thermal cycling, the tendency of the o reaction to vaporize is minimized.
  • Covers 74 which can be comprised of material identical to, or different from, that of the substrate 72 in which the groove is cut, can be applied using a variety of means known in the art.
  • the cover 74 can be glued to the substrate using an epoxy, cyanoacrylate or other type of glue.
  • the cover can be welded by melting it and underlying material until they fuse, through the application of heat or light.
  • the cover 74 can also be fixed in place mechanically, such as with a clamp, or even magnetically.
  • the material of which the reaction chamber is comprised is advantageously a material to which template DNA, or other nucleic acid, reversibly and saturably binds in the presence of a sufficiently high concentration of chaotropic ions.
  • the reaction chamber is comprised of glass, especially when configured as capillary tubing.
  • High quality glass capillary tubing is readily available in a range of interior dimensions from a variety of 0 manufacturers, including Polymicro Technologies (Phoenix, Arizona, USA).
  • a polymer material such as a polyimide.
  • a polyimide coating provides a protective layer that protects the capillary tubing from abrasions and breaking by bending.
  • Polyimide also creates a hydrophobic layer on the 5 outer surface of the capillary which can help prevent the adherence of aqueous reaction mixtures when the capillary is filled by dipping it into a reaction mix; this helps prevent wastage of reagents.
  • Other potential coatings are acrylates, silicones, fluoropolymers, and aluminum.
  • glass may be used including alkali-borosilicate glass, alumina- o silicate glass, barium flint glass, barium-borate glass, borosilicate glass, borate glass comprising B203, germinate glass comprising Ge02, chalcogenide glass, silicate glass comprising Si02, silica glass, fused silica glass, synthetic fused silica glass, quartz (crystalline Si02), fused quartz (amorphous Si02), doped synthetic fused silica (doped with trace elements such as germanium, fluorine, boron, phosphorous, and titanium), lanthanum 5 glass, optical glass, phosphate glass, and soda-lime glass.
  • alkali-borosilicate glass alkali-borosilicate glass
  • alumina- o silicate glass barium flint glass
  • barium-borate glass barium-borate glass
  • borosilicate glass borate glass comprising B203
  • germinate glass comprising Ge02
  • the reaction chamber can be comprised of a metal or metalloid, materials that, like glass, can be fashioned into capillaries or wafers.
  • Suitable pure and alloyed metals include magnesium, aluminum, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, gallium, zirconium, niobium, molybdenum, palladium, gold, o silver, cobalt, niobium, indium, rhodium, tin, steel, stainless steel, and bronze.
  • Suitable pure and alloyed metalloids include silicon, germanium, arsenic, and gallium arsenide.
  • the reaction chamber can also be comprised of carbon in its multiple allotropes, including graphite, diamond, C60 and related allotropes comprising, for example, nanotubes, or comprised of organic compounds such as plastic.
  • carbon in its multiple allotropes, including graphite, diamond, C60 and related allotropes comprising, for example, nanotubes, or comprised of organic compounds such as plastic.
  • reaction chamber such as glass capillary, FIG. 16 12
  • nucleic acid-chaotrope solution 83 the solution is incubated for such time and under such conditions that at least a portion of the DNA in the solution reversibly binds to the inner surface, FIG. 16 82, of the chamber or tube, FIG. 15, step 4.
  • i o irreversible binding can be effected.
  • nucleic acid-chaotrope solution is removed from the chamber by a variety of means including application of positive or negative air pressure, or
  • Washing is performed to purify the bound nucleic acid by removing excess, unbound nucleic acid, chaotropic agent, and any impurities that may have contaminated the nucleic acid. It is important to remove the chaotropic agent because these ions can severely interfere with most subsequent chemical and biochemical reactions, even at very low
  • washing can be performed in a variety of ways.
  • a capillary tube can be filled by capillary action, after which the washing solution is expelled in similar manner by which the nucleic acid-chaotrope solution was removed.
  • a reaction chamber can be filled and emptied by pumping of the wash solution. Sufficient volume of washing solution is used to essentially eliminate the presence of all contaminants.
  • the wash solution is removed from the chamber or tube, [0366]
  • the composition of the washing solution is chosen so that it does not remove by 5 elution any substantial portion of the nucleic acid that has become bound to the inner surface of the chamber or tubing, and is typically a solution of an alcohol with pure water.
  • Suitable alcohols include the lower molecular mass alcohols methanol, ethanol and isopropanol.
  • concentration of alcohol is high enough that elution of nucleic acid minimized, and is preferably at least 50%, more preferably at least 60%, and most o preferably at least 70% volume by volume.
  • ethanol is used at concentration greater than about 70% - 80% volume by volume.
  • the washing solution can also comprise a salt, preferably in the form of a buffer, such as an acetate buffer, or a tris-EDTA buffer (containing, e.g., 10 mM Tris-HCI and 1 mM ethylenediamine-tetraacetic acid (EDTA), pH 8.0).
  • the salt can have the effect of buffering 5 pH so that the pH is in the range of about 6.5 - 8.5, and also stabilizing the binding interaction between DNA and the inner surface of the chamber or tube during washing.
  • Drying can be effected by subjecting the chamber or tube to a high enough vacuum so that the liquid vaporizes and is carried away.
  • a dry gas such as air, nitrogen or argon, can be forced at pressure through the chamber or tube to promote the evaporation of the liquid. The drying gas can be warmed to further promote 5 evaporation.
  • reaction chamber now bearing reversibly immobilized nucleic acid, can be used immediately to perform a biochemical reaction with the nucleic acid, or stored, under appropriate conditions, for future use.
  • Reaction chambers prepared according to the steps discussed above can be advantageously used to normalize the o amount of a nucleic acid to be used in parallel reactions, dispense predetermined amounts of DNA or RNA onto a substrate, and to perform nanoscale DNA sequencing reactions, as well as many other types of reactions with DNA and RNA.
  • these particular applications should not be seen as limiting the scope of uses to which such reaction chambers can be put.
  • Reaction chambers in the form of capillary tubes can be processed as illustrated in FIG. 15 and used singly, but it will frequently be advantageous to combine multiple capillary tubes in parallel fashion, so as to be able to increase sample throughput, particularly in an automated system.
  • capillary tubes can be conveniently organized into a o capillary cassette; the greater the density of capillary tubes per cassette, the greater the potential sample throughput.
  • An apparatus such as that described in copending U.S. Application Serial No. 09/577,199, can be used to automate the processing steps illustrated in FIG. 1 , as well as any subsequent steps associated with carrying out reactions with the immobilized nucleic acid, including capillary filling, emptying, washing, drying, and or 5 thermal cycling. Used in this way, the cassette becomes an automated, fixed-volume parallel pipettor, allowing all the capillary tubes to be filled simultaneously from the wells of a sample plate by capillary action.
  • Capillary cassette 15 is shown in FIG. 3A.
  • the capillary cassette is comprised of a number of capillary tubes extending through a substrate 10. It is preferred that the capillary o cassette have at least one row of eight capillary tubes and that the capillary tubes have equal spacing.
  • the capillary cassette shown has substrate 10 with 96 capillary tubes arranged in an 8 by 12 array, with spacing of the tubes matching the spacing of the wells of a 96 well microplate.
  • the capillary tubes 12 extend through a substrate 10 and preferably are arranged 5 in a uniform pattern.
  • the capillary tubes are of equal length and extend through the substrate in a substantially parallel orientation such that each of the two opposing ends of the capillary tubes 12 are coplanar and the planes defined by the ends of the capillary tubes 12 are substantially parallel to the substrate 10.
  • the spacing of the capillary tubes may be uniform and selected to match the center-to-center spacing of wells on a microplate. For o example on a standard 96 well microplate the capillary tubes would be arranged with a 9 mm center to center spacing, on a 384 well microplate the capillary tubes 12 would be arranged with a 4.5 mm center to center spacing.
  • the capillary tubes 12 are preferably secured within the substrate such that the length of capillary tubes 12 extending from one side of the substrate 10 are shorter than the length of the capillary tube on the opposite side of substrate 10.
  • the length of the 5 capillary tubes 12 on the shorter side of the substrate may be matched to the depth of wells in a microplate, such that the length of the shorter side is a shorter length than the depth of a well in a microplate.
  • the capillary cassette substrate 10 may be made of a fiberglass board or other rigid or semi-flexible material.
  • the capillary tubes 12 may be inserted through evenly spaced holes in the substrate and secured with adhesive.
  • the length and width of the substrate are similar to the length and width of a standard 96 well microplate. This simplifies adapting automated systems designed for manipulation of o microplates to handle the capillary cassette.
  • the present invention is useful for precisely controlling the amount of nucleic acid to be used for a variety of applications. 5 [0375] If during the binding reaction occurring in the reaction chamber, the nucleic acid- chaotrope solution is allowed to stay in contact with the inner surface of the chamber or tube for sufficient time, and if the nucleic acid is at high enough concentration in the solution, it is possible to saturate the available binding sites on the inner surface of the chamber or capillary with nucleic acid. This is known as saturable binding.
  • nucleic acid in solution prior to incubation exceeds the binding capacity of the inner surface of the chamber, a fixed, maximal quantity of nucleic acid will be immobilized, regardless of the amount of nucleic acid initially in the solution.
  • concentration of nucleic acid in solution exceeds a minimum, it is not necessary to know the actual concentration; the amount of nucleic acid bound will be determined solely by the binding capacity of the 5 reaction chamber. Accordingly, if the nucleic acid in a capillary tube that was saturably bound is eluted into a known volume of liquid, the concentration and amount of nucleic acid in the liquid is knowable with a high degree of accuracy.
  • nucleic acid-chaotrope solution wherein both the nucleic acid and chaotrope are at sufficiently high concentration to support saturable binding in reasonable 5 time.
  • the binding capacity, or amount of nucleic acid that can be saturably bound to the inner surface is determined empirically. For example, a known amount of test o nucleic acid is labeled with a radionuclide, such as 35 S, 33 P or 32 P, according to methods known in the art. After labeling, the specific activity of the labeled nucleic acid is determined to establish a ratio of disintegrations per minute per mass unit, or concentration unit of nucleic acid.
  • the labeled nucleic acid is then dissolved in a solution containing chaotropic ions at a predetermined concentration.
  • a standard reaction chamber representative of a general supply, is then tested. For example, a predetermined length of glass capillary tubing is cut and filled with the labeled nucleic acid-chaotrope solution. After sufficient time 5 for saturable binding to occur, the capillary is emptied and washed. Then, the amount of radioactivity retained inside the tube is measured, and, with knowledge of the specific activity of labeling, converted to an amount of nucleic acid. This factor can then be used to calculate the amount of nucleic acid that will be retained in any length of capillary tubing cut from the same lot, so long as similar conditions for binding are used in any subsequent 0 experiment.
  • An advantage of using the present invention to accurately obtain a predetermined quantity of nucleic acid is to normalize quantities of nucleic acid for subsequent use. This advantage is especially significant if it is necessary to process many samples. For example, in the current state of the art, it is not practical, when preparing different template DNAs for 5 sequencing, to ensure that the concentration of the templates is the same. Thus, according to prior methods it was necessary to normalize the different template DNA samples, by separately determining the DNA concentration in each prep, and diluting the DNA to the proper concentration for each and every sample. This is especially important for capillary electrophoresis because of the sensitivity of that technology to overloading of the capillaries o with template DNA.
  • the present invention allows very rapid normalization to minimize differences in starting template concentration.
  • Template DNA is that DNA for which the sequence of constituent bases is to be determined.
  • Template DNA can be single stranded, or double stranded, wherein two complementary DNA strands are hybridized together, and knowledge of the sequence of one strand can be used to infer the sequence of bases in the other strand according to o the rules of Watson-Crick base pair complementarity.
  • Template DNA is typically obtained directly from self-replicating genetic systems, grown in a host, into which the DNA fragment to be sequenced was cloned.
  • the template can be obtained from any source, e.g., genomic DNA, by amplifying a particular DNA sequence using the polymerase chain reaction, or a functionally equivalent 5 linear or exponential amplification process.
  • Self-replicating genetic systems include episomal elements, such as plasmids containing an origin of replication, or bacteriophage (e.g. lambda or M13), both of which can replicate inside bacteria, such as E. coli, after transformation or infection, respectively. Plasmids harboring template DNA are obtained by breaking open the bacteria in which they o have replicated to sufficiently high copy number, and isolating the plasmid from the supernatant. Bacteriophage released into bacterial culture supernatant after lysing the host bacteria are collected, and the DNA isolated by breaking open the bacteriophage particles.
  • episomal elements such as plasmids containing an origin of replication, or bacteriophage (e.g. lambda or M13), both of which can replicate inside bacteria, such as E. coli, after transformation or infection, respectively. Plasmids harboring template DNA are obtained by breaking open the bacteria in which they o have replicated to sufficiently high copy number, and isolating the plasmid from the super
  • the plasmids because of their small mass, easily pass into the bore of the o capillary as it fills, thereby interacting with the glass walls to establish salt-bridges and become immobilized.
  • the intact genomic DNA being of extremely large molecular mass, is excluded from the small bore of the capillary, and is thus separated by size exclusion from the plasmids.
  • template DNA can also be obtained without the need for cloning 5 steps by amplifying a DNA fragment directly from an appropriate source, such as a virus, a prokaryotic cell, including bacteria, or eukaryotic cell, including mammals, other animals, or plants.
  • an appropriate source such as a virus, a prokaryotic cell, including bacteria, or eukaryotic cell, including mammals, other animals, or plants.
  • FIG. 16 80 After the template DNA, FIG. 16 80, is reversibly immobilized directly to the inner surface 82 of a glass capillary tube 12, in accordance with the methods of the present o invention, the capillaries are filled with the sequencing reaction mixture 84 that effects the
  • DNA sequencing reaction The reaction is carried out according to techniques well known in the art, whereby the products of the DNA sequencing reaction are labeled with fluorescent dyes. Well established in the art is the Sanger dideoxynucleotide chain termination technique. Briefly, a primer complementary to sequence in the template DNA 5 molecule is permitted to hybridize to the template. Then DNA polymerase extends the primer by reading the sequence of bases in the template, by adding dNTPs to the 3' end of the growing primer. However, dideoxynucleotide triphosphates that lack the hydroxyl group characteristic of the corresponding dNTP prevent the further addition of bases to the growing strand. As a result the chain terminates. The pattern of terminated chains in a o chromatogram permits the experimenter to infer the sequence of bases in the template.
  • the terminated reaction products are fluorescently labeled either by conjugating a fluorophore to the primer that is extended, or alternatively, by conjugating a fluorophore to all the dideoxy terminators that, when incorporated into growing DNA chain, result in termination of primer extension.
  • the o acceptor dyes for example, rhodamine 110, rhodamine-6-G, tetramethyl rhodamine, and rhodamine X, then emit light at their characteristic wavelengths. The fluorescence is detected by the instrument allowing identification of which nucleotide caused the termination event.
  • Use of the energy transfer system results in more efficient excitation of the acceptor dyes than direct excitation by the laser, resulting in greater sensitivity.
  • a donor dye to be conjugated to a primer is 5-carboxy-fluorescein (FAM), and examples of acceptor dyes to be conjugated to primers are rhodamine 110 (R110) for cytosine, 6-carboxyrhodamine (REG) for adenine, N,N,N',N'- o tetramethyl-5-carboxyrhodamine (TAMRA) for guanine, and 5-carboxy-X-rhodamine (ROX) for thymine.
  • R110 cytosine
  • REG 6-carboxyrhodamine
  • TAMRA N,N,N',N'- o tetramethyl-5-carboxyrhodamine
  • ROX 5-carboxy-X-rhodamine
  • the capillary, FIG. 16 12, containing the immobilized template DNA 80 is filled by 5 capillary action by dipping it into a reservoir 85 filled with the reaction mixture.
  • the reaction mixture 84 contains all the components at the appropriate concentration to effect the sequencing reaction, including water, salts, buffers, primer, DNA polymerase, dNTPs and dideoxy terminators. Without wishing to be bound by theory, at present it is hypothesized that as the aqueous mixture ascends the capillary, the immobilized DNA likely rehydrates.
  • the salt- bridge causing the DNA to be immobilized is disrupted by the water molecules and the DNA is eluted from the inner surface of the capillary, and diffuses into the reaction mixture.
  • the DNA desorbs during the thermocycling reactions. Whatever the mechanism, physical mixing of the DNA into the mixture is not necessary for performance of the reaction.
  • the ends are sealed to prevent vaporization of the liquid contained inside, followed by thermal cycling to activate multiple rounds of the sequencing reaction, so as to generate the fluorescently labeled product to be analyzed.
  • Sealing of the capillary and thermal cycling may be effected in multiple ways, as will be apparent to the skilled artisan. If, as will often be the case, it is desirable to perform multiple sequencing reactions in parallel, the experimenter can use a high-throughput apparatus, such as that disclosed in the copending application U.S. Serial No. 09/577,199, which is hereby incorporated by reference in its entirety.
  • the disclosed apparatus provides means both for sealing multiple capillary tubes arranged into a cassette format, and for effecting thermal cycling of the sequencing reaction mixtures contained in the capillaries.
  • the reaction products are expelled from the capillary tubes, typically in preparation for analysis by capillary electrophoresis.
  • reaction product is expelled onto a substrate, or into some form of holder for liquid, such as a well of a microtiter dish, from which a capillary electrophoresis system may sample the product for analysis.
  • a capillary electrophoresis system may sample the product for analysis.
  • the reaction product may be expelled directly from the reaction capillary into the electrophoresis capillary.
  • Reaction product may be expelled from the reaction capillaries by the application of centrifugal force, electrokinetically, by the application of positive or negative air pressure, or by other means known in the art.
  • reaction product can be expelled onto a substrate adapted for other types of analytical process, such as a MALDI (matrix-assisted laser desorption/ionization) or SELDI (surface-enhanced laser desorption/ionization) substrate for mass spectrometric analysis.
  • MALDI matrix-assisted laser desorption/ionization
  • SELDI surface-enhanced laser desorption/ionization
  • a laser scans a window in the capillaries carrying the products and excites the fluorophores.
  • Light emission by the fluorophores is captured and converted into intensity and light frequency data that is stored in a computer memory.
  • the computer After scanning and reading is complete, the computer assembles a chromatogram representing all the reaction products detected by the scanning system.
  • the data in the chromatogram is processed by computer software that interprets the chromatogram to infer the sequence of nucleotide bases in the starting template DNA.
  • the sequence output is then stored in a computer data file, either in random access memory or on a dedicated long term memory device, such as floppy disk, ZIP disk, JAZ disk, hard disk, CD-ROM, computer tape, etc.
  • a computer data file can be stored on a computer server that can be accessed from remote client computers.
  • the file is transferred it is represented as a data signal associated with a carrier wave carried through copper or fiberoptic telephone lines, cable television lines, or by radio waves.
  • the wash solution is an aqueous wash solution of low ionic strength such that any remaining immobilized nucleic acid will tend to be eluted and carried away. Double 5 distilled water is effective.
  • the wash solution may be heated to increase the effectiveness of washes, and the number of washes and/or volume of wash solution per wash cycle can be varied as necessary to maximize washing effectiveness. Capillaries can be filled with wash solution by capillary action and then emptied using the same methods by which reaction product is expelled. If washing is to be effected by electrokinetic pumping, then the o wash solution must contain some minimum concentration of ions.
  • a mechanical pump can be used to drive wash solution through the capillaries.
  • the washing can also be accomplished by a mechanical capillary cassette washer as disclosed in commonly owned and copending U.S. patent application serial no. 09/577,199, filed May 23, 2000, the disclosure of which is incorporated herein by reference 5 in its entirety.
  • the recycling process can comprise steps effective at destroying traces of nucleic 5 acid.
  • Such means include filling the capillary with a solution containing an exonuclease and incubating for such time as is necessary to digest any nucleic acid.
  • Other means include chemical degradation of the nucleic acid, such as by washing with highly acidic or basic solutions; contact with bleach; irradiating the capillary with ionizing radiation; or baking to high temperature. After destroying residual nucleic acids, the capillaries would typically be o washed using standard solutions.
  • SNPs single nucleotide polymorphisms
  • the methods and apparatus 5 of the present invention make possible "deep" sequencing, in which the same gene or genetic locus is sequenced from a plurality of individuals, differences in the sequence identifying polymorphisms that exist in the sequenced population. Of these, some SNPs will be demonstrated to be associated with significant phenotypes, such as predisposition, presence, or progressive potential of disease.
  • SNPs are single base changes that occur approximately once every 1000 bases and are the most common form of genetic variation in humans. If such polymorphisms occur in coding sequence or regulatory regions of genes, they can alter the function of the gene or gene product, as compared to the wild type sequence. Depending on the extent to which gene function is modified, the effect on the organism can minimal, or result in 5 deleterious phenotypes, including genetic diseases.
  • SNPs are used as markers for genetic linkage analysis to assist in identifying genes responsible for diseases with a strong o hereditary component.
  • SNP analysis has proved useful for identifying changes in alleleic variants of genes correlated with important phenotypes, such as response to drug compounds or other therapeutic regimes, as well as predisposition to or progressive potential of diseases.
  • SNP analysis is also useful for customizing drug or other therapeutic regimes to individual patients based upon a patient's unique genetic characteristics.
  • This is concept 5 underlies the burgeoning field of pharmacogenetics. For example, a particular polymorphism or set of polymorphisms may be correlated with poor responsiveness to a particular drug. Further research may then show that the polymorphic changes reside in a gene encoding an enzyme responsible for metabolizing the drug, and that the changes alter the kinetic rate of the enzyme. As a result, the drug is metabolized more quickly as o compared to the wild type enzyme.
  • SNP analysis therefore, is useful both for identifying genes that affect therapeutic 5 regimes in human and non-human patients, and identifying those patients who will require a modified therapy compared to the patient population that lacks the SNP marker.
  • the usefulness of SNP analysis is not limited to applications related to medical care alone, however. Indeed, identification of SNPs in the genes of any organism that can be correlated with an interesting phenotype is increasingly useful both for identifying those o genes responsible for a particular phenotype, as well as those genetic alterations that cause the phenotype to be modified.
  • Such knowledge offers an improved understanding of how particular gene products function, as well as insights as to how such functions can be beneficially modified.
  • SNP analysis is most beneficially undertaken in a high throughput manner, for which application of the present invention is particularly well suited.
  • the presence of SNPs in one or a few genes is analyzed from a large number of samples from patients, or another type of non-genetically identical sources, including non-human sources.
  • This approach is typically, but not exclusively, adopted in studies designed to obtain large data sets for correlating particular SNPs with particular phenotypes.
  • This approach will often also be adopted by facilities that analyze SNPs present in genes of large numbers of human or animal patients, which information is to be used for customizing treatment regimes to individual patients.
  • high throughput SNP analysis may be undertaken on a large number of genes obtained from relatively few samples. This approach typically will be advantageous when a comprehensive analysis of SNPs present in a patient is desired. Such information may be necessary to customize treatment regimes in the context of diseases with complex multigene etiologies.
  • oligonucleotide ligation assay genotyping OLA
  • minisequencing TaqManTM genotyping
  • InvaderTM assay dye labeled oligonucleotide ligation
  • pyrosequencing rolling circle amplification
  • RCA rolling circle amplification
  • SBE in part, is based upon the dideoxyterminator approach to DNA sequencing, described above.
  • Template nucleic acid is provided for analysis to determine whether the sequence contains one or more SNPs at particular base positions in the sequence.
  • a primer that specifically recognizes known sequence immediately 5' of a base to be interrogated in the template is then allowed to contact and bind the template via Watson-Crick base pairing.
  • a DNA polymerase which may include a thermostabile version thereof, reads the template strand beginning at the base to be interrogated and enzymatically attaches a complementary dideoxyterminator nucleotide triphosphate (ddNTP), present in the reaction mixture, to the 3' hydroxyl group of the primer.
  • ddNTP complementary dideoxyterminator nucleotide triphosphate
  • Each of the four bases, A, C, G, T, is represented among the dideoxyterminators present in the reaction mixture, and each of the four bases is labeled with a fluorophore that emits excited photons at a wavelength that uniquely identifies which base is present in association with the particular fluorophore.
  • the extended primer is released, thermally or chemically, from the template and the primer is analyzed to detect the fluorophore associated with the dideoxyterminator base attached to primer 3' end. Identification of the fluorophore, based on its emission spectrum, permits unequivocal identification of the base incorporated by the DNA polymerase during single base strand extension, and the base defines the SNP present in the gene at the position interrogated.
  • a subset rather than all four ddNTPs may be included in the SBE reaction mixture, according to the needs and preference of the skilled artisan.
  • Such ddNTP subsets comprise those listed in the following table.
  • Identification of the fluorophore can be accomplished using a variety of techniques according to the knowledge of the skilled artisan. For example, the products of a single base extension reaction can be separated from unincorporated dideoxyterminator nucleotides on a denaturing gel similar to that used for DNA sequencing. After the SBE products have been resolved by gel electrophoresis, the fluorophores associated with the primers in the gel are excited by light of the appropriate wavelengh and fluorescence 5 emission detected and analyzed according to the knowledge of the skilled artisan.
  • unincorporated dideoxynucleotides can be removed prior to analysis of the SBE products by gel electrophoresis.
  • fluorescently labeled dideoxterminator nucleotides incorporated into SBE extension products are detected using fluorescence o polarization (FP) according to the knowledge of the skilled artisan.
  • FP fluorescence o polarization
  • polarized light is used to stimulate emission from the fluorophores. Unincorporated fluorophores are small and therefore emit depolarized light upon fluorescent excitation, whereas fluorophores incorporated into the much larger SBE extended primers emit polarized light.
  • Preferential detection of polarized fluorescent emission can therefore be 5 used to infer the incorporation of particular fluorophores, and therefore bases, into the extended primers.
  • Use of FP permits analysis without prior removal of unincorporated dideoxyterminators.
  • Template can be obtained, according to techniques well known in the art, from a variety of sources, including, but not limited to genomic DNA obtained from eukaroytic cells, prokaryotic cells, or viruses; episomal DNA, including plasmids; and messenger, or other types of RNA. Template can be single stranded DNA or RNA, double stranded DNA or RNA, or DNA-RNA hybrids. If template is substantially comprised of RNA, the DNA 5 polymerase to be used to extend the primer is a reverse transcriptase (RT), including thermostable versions thereof.
  • RT reverse transcriptase
  • the template is a PCR product obtained from genomic DNA.
  • a PCR reaction is performed, using methods well known in the art, using primers that o specifically recognize genomic DNA which serves as the template for PCR. Thereafter, the primers that o specifically recognize genomic DNA which serves as the template for PCR.
  • DNA fragment generated by PCR serves as the template for SBE.
  • Amplified template from genomic DNA or other nucleic acid can also be obtained by a linear amplification process, or an exponential amplification process functionally equivalent to PCR.
  • SBE reactions have traditionally been performed in large, so-called “full volume” reaction volumes, as described in greater detail in Example 22, below. According to these 5 methods, PCR is performed in multiple microliter reaction volumes using genomic DNA template to generate the template to be used in subsequent SBE reactions. Thereafter, the PCR products are treated with Exol and SAP to degrade single stranded DNA and excess dNTPs, respectively. Subsequently, SBE is performed using a portion of the template generated by PCR, after which the SBE reaction products are treated with CIAP and then 0 analyzed by capillary electrophoresis, e.g., using MegaBACETM.
  • Full volume reactions are performed in volumes of up to about 10, 15, 20, 25, 50, 75, 100 or 200 microliters, and as in volumes as low as about 100, 75, 50, 25, 20, 15, 10, or 5 microliters.
  • the full volume methods just described have proved efficacious, they are 5 also wasteful of reagents and other materials because the mass of SBE product necessary to obtain high quality data is very small relative to the actual amount generated using the full volume approach. Additionally, the full volume approach demands considerable time to effect the various thermal cycling steps in PCR and SBE and to transfer fluid volumes between steps.
  • the advantages of the present invention as applied to SBE and other techniques of SNP detection are an effect of performing one or more enzymatic reactions in o nanoliter volume (also called “nanovolume”) reactions using the capillaries of the instant invention.
  • nanovolume reactions reduces the quantity of reagents used, which translates to saved costs as compared full volume reactions.
  • Nanovolumes also reduces the time necessary to proceed from one temperature to another during thermal cycling of reactions because the total mass of the reaction mixture is lower, and the surface area per unit volume of the reaction is greater when using capillary tubes as compared to the reaction tubes used for full volume reactions. Both effects increase the rate of heat 5 transfer and thereby reduce the time necessary to perform an entire series of thermal cycles.
  • template capture i.e., the reversible binding of template to the internal surface of the capillary in the presence of a chaotrope, permits elimination of one or more steps necessary to perform SBE, further reducing reagents, costs, and time associated with performing the assay.
  • nanoliter volume reactions are performed in volumes of up to about 25, 50, 100, 250, 500, 750, 1000, 1500, 2000, 2500, 5000, or more nanoliters, and in volumes as low as about 2500, 2000, 1500, 1000, 750, 500, 250, 100, 50, 25, 10, or fewer nanoliters.
  • template dissolved in 5 chaotrope solution is withdrawn into a capillary of the present invention by capillary action, or other method as described herein, and contacts the inner surface until a predetermined approximate mass of such template is caused to bind reversibly thereto. After binding is complete excess template in chaotrope is removed and the bound template washed as explained elsewhere herein. After a futher optional drying step, SBE reaction mixture, o containing all ingredients necessary to effect SBE, including buffers, salts, water, SBE primer, fluorescently labeled ddNTPs, and DNA polymerase, is drawn into the capillary by capillary action, or other method described herein.
  • SBE template may be reversibly bound to the inner surface of a capillary in the presence of chaotrope until an amount of template, as determined by the skilled artisan, is bound which is sufficient to yield detectable SBE product after conducting the reaction. That is, it is not necessary that a predetermined approximate mass of SBE template be reversibly bound inside the capillary for the o usefulness of the present invention to be realized.
  • SBE product is typically expelled from the capillary, as described elsewhere herein, for subsequent processing, including removal of unincorporated ddNTPs, e.g., by treatment with calf intestinal alkaline phosphatase (CIAP), according to methods known in the art.
  • CIAP treatment removes phosphate groups from ddNTPs, rendering the dephosphorylated ddNTPs uncharged.
  • electrophoresis e.g., using MegaBACETM
  • the 5 treated ddNTPs are not induced to move by the strong electric field that causes the charged SBE products to enter the sieving gel. This approach facilitates separation of unincorporated ddNTPs from the SBE products.
  • CIAP treatment may be effected in full volume reactions, or alternatively, in nanovolume reactions.
  • Full volume CIAP treatment is conveniently performed in the wells 0 of microtiter plates, e.g., 96, 384, 1536, or higher numbers of wells per plate.
  • nanovolume CIAP treatment is performed within a capillary of the present invention after having mixed the SBE product with CIAP reaction mixture, e.g., within a well of a microtiter plate.
  • excess unincorporated 5 ddNTPs may be removed by contacting the SBE reaction products with a gel filtration media for sufficient time to separate ddNTPs from SBE products.
  • Gel filtration media is chosen with properties that ensures that ddNTPs can enter the pores of the media whereas SBE products are substantially excluded. In this manner o ddNTPs are contained in the total volume, whereas SBE product is contained within the void volume.
  • media suitable for use in the present invention include, but are not limited to superdex, superose, sephacryl, and sephadex.
  • Electrophoretic methods coupled with a microfluidic platform can also be used to resolve extension products of SBE. Such methods are discussed in more detail in U.S. Patent Nos. 6,316,201 ; 6,306,659; 6,306,590; 6,303,343; 6,287,774; 6,274,337; 6,267,858; 6,235,471 ; 6,235,175; 6,174,675; 6,153,073; 6,107,044; 6,068,752; 6,042,710; 5,976,336; 5,965,410; 5,958,694; and 5,948,227, each of which is incorporated o herein by reference in its entirety.
  • SBE products can be analyzed using mass spectrometric techniques, including matrix-assisted laser desorption/ionization (MALDI) or surface-enhanced laser desorption/ionization (SELDI).
  • MALDI matrix-assisted laser desorption/ionization
  • SELDI surface-enhanced laser desorption
  • SBE template can be drawn into the 5 capillary of the instant invention having already been mixed with the SBE reaction mixture, in which case template normalization does not occur.
  • template for SBE is prepared by PCR, according to methods well known in the art.
  • PCR may be effected in full volume reactions. After PCR is completed, the o reaction can be treated to remove primers and dNTPs, as described in further detail below.
  • the PCR products are mixed with chaotrope and used to fill a capillary of the instant invention for template normalization of the SBE template, followed by the extension reaction, as described above.
  • a portion of the PCR products, after treatment, are added to SBE reaction mixture and used to 5 fill a capillary of the instant invention for subsequent performance of the extension reaction, as described above.
  • PCR may also be performed in a capillary of the instant invention using nanoliter volume reactions, in which case PCR may be preceded by template normalization of the genomic DNA, or other PCR template, to be used in the reaction, similarly as described o above for SBE template.
  • PCR template may be added to the PCR reaction mixture prior to filling the capillary, in which case template normalization does not occur.
  • the reaction product typically is expelled from the capillary, as described elsewhere herein, and treated to remove primers and dNTPs, as described in further detail below. As in the case of full volume PCR, treated PCR products may then be 5 mixed with chaotrope and used for template normalization of the SBE template, or added to
  • PCR product is typically expelled from the capillary, as described elsewhere herein, and treated to remove excess o unincorporated PCR primers and dNTPs by, for example, using a single stranded Dnase, e.g., exonuclease I (Exo I), and a phosphatase, e.g., shrimp alkaline phosphatase (SAP), respectively, according to methods known in the art.
  • PCR product, as SBE template may then be normalized, or added directly to SBE reaction mixture, and used in SBE in a capillary of the present invention, as described above.
  • Exol/SAP treatment may be effected in full volume reactions, or alternatively, in nanovolume reactions.
  • Full volume Exol/SAP treatment is conveniently performed in the wells of microtiter plates, which plates may comprise 96, 384, 1536, or higher numbers of wells per plate.
  • nanovolume Exol/SAP treatment is performed within a capillary of the present invention after having mixed the PCR product with Exol/SAP reaction mixture, e.g., within a well of a microtiter plate.
  • the PCR product treatment step is exluded.
  • the PCR products are added directly to chaotrope, after which the solution is used to fill a capillary of the instant invention until such time that a predetermined approximate mass of template, or a mass of template sufficient to yield detectable SBE products, is reversibly bound to the inner surface of the capillary. Thereafter, excess unbound PCR product (i.e., SBE template), primers, and dNTPs are removed by washing, as described elsewhere herein. After an optional drying step, SBE reaction mixture is then drawn into the capillary for subsequent performance of the extension reaction, as described elesewhere herein.
  • a new capillary may be used.
  • the same capillary from one or more previous steps may be reused, with, or without first having washed the interior of the capillary, or otherwise treat the capillary, to remove or inactivate traces of reagents, reactants or products deposited therein from the previous step. Methods of washing or treating capillaries of the instant invention are discussed elsewhere herein.
  • a plurality of the capillaries of the present invention are provided arranged in a spatially addressible array to facilitate high-throughput processing of multiple samples in parallel.
  • the number and pattern of capillaries in an array and the dimensions of an array of capillaries corresponds to the number, pattern and dimensions of wells in one or more types of microtiter plates such that an capillary array and wells of a plate can be mated, preferably in the context of an automated or semi-automated robotic work flow system.
  • arrays are rectangular, but may be circular, triangular.etc.
  • the number of capillaries in an array may include 2, 4, 8, 12, 16, 24, 32, 48, 64, 96, 128, 192, 288, 384, 480, 576, 672, 768, 864, 960, 1536 capillaries, or higher number of capillaries.
  • microarrays are finding increased use in basic and applied research and are typically comprised of a rectangular array of spots of DNA on a glass slide, with a different, known DNA sequence at each spot.
  • the experimenter then takes a labeled sample, either RNA or DNA and detects hybridization 5 events between the labeled nucleic acid and the DNA spotted to the array. In this way, the experimenter can infer the identity and/or partial or complete sequence of the labeled nucleic acid.
  • PCR is often used to generate the DNA to be spotted.
  • Taq and other thermostable polymerases introduce a certain number of erroneous base pairs per thousand as it amplifies the template. If errors have been introduced, they must be detected, and the amplified product discarded. Usually, this requires numerous 5 processing steps separate from those associated with spotting the PCR product.
  • use of an embodiment of the present invention greatly increases the efficiency of sequence determination and confirmation.
  • the DNA sample to be spotted is usually dissolved at a predetermined concentration in a solution comprising chaotropic ions, for example sodium thiocyanate. o
  • the DNA is so dissolved because it is to be immobilized to the surface of the glass microarray slide in a manner similar to that by which nucleic acid is immobilized inside capillary tubes.
  • chaotropic ions for example sodium thiocyanate.
  • the different DNA-chaotrope solutions are aliquoted into wells of 384-well capacity microtiter dishes for storage until ready to be spotted onto a microarray.
  • the present invention can be adapted to sample and sequence the DNA in multiple wells of the same 384-well dish used as the DNA source for the spotting pens. It will be apparent that it can also be adapted to sample from dishes with more than 384 wells. Because the DNA to be sequenced is from the same sample to be spotted, numerous processing steps associated with sequencing the DNA from different samples are obviated. o This results in substantial savings of time and material costs.
  • glass capillaries are arranged into a cassette in the same pattern and inter-capillary dimensions as that of the wells in one or more rows or columns of the dish. For maximal capacity, a total of 384 capillaries are arranged into a pattern with dimensions identical to that of the dish itself.
  • the capillary cassette Prior to spotting, the capillary cassette is filled 5 with DNA-chaotrope solution (usually sodium thiocyanate) according to the methods of the present invention. After the DNA samples are immobilized and processed, they are sequenced. If any of the templates fails to give the correct sequence, the operator of the spotting apparatus knows not to spot that DNA, or if spotted, that data associated with hybridization at the corresponding spot is to an unwanted sequence and should be removed o from the resulting data set.
  • DNA-chaotrope solution usually sodium thiocyanate
  • the present reaction mixture assembly may be used for assembly of numerous 5 types of reactions.
  • the same basic method used to assemble the PCR reaction mixture may be adapted to assembly of a cycle sequencing mixture, rolling circle amplification reaction mixture, enzymatic assays, chemical reactions, or other reaction mixtures.
  • the experimenter is not obligated to carry out a reaction with the nucleic acid immobilized inside of a capillary tube.
  • the capillary as a pipettor to dispense a predetermined approximate mass of the nucleic acid in a fixed volume of liquid, and therefore at a predetermined approximate concentration, onto a substrate of the experimenter's choosing.
  • the capillary is filled with elution fluid that elutes essentially all the reversibly immobilized nucleic acid. Thereafter, the solution of the elution fluid and nucleic acid is dispensed, usually onto or into a substrate.
  • the substrate onto which the reaction mixture is transferred may be the wells of a multiwell microtiter plate, locations on a planar substrate, or wells that lead into an analytical chip.
  • the reaction may also be dispensed into a solution for further chemical or biochemical reaction.
  • the cassette becomes a multichannel parallel pipettor, and it becomes possible to dispense a large number of normalized nucleic acid samples simultaneously.
  • the dispensing can be into microtiter wells, microchips, and other chambers for further reactions.
  • the nucleic acid can be dispensed directly into the reservoirs of a capillary array electrophoresis microchip or onto a MALDI or SELDI target, or onto or into a substrate adapted to be used in other analytical modalities.
  • Different methods may be used to expel or dispense liquid from capillary tubes.
  • One method to dispense the contents of a single capillary tube or multiple similar capillaries arranged into a cassette format uses a centrifuge to dispense the fluid by centrifugal force. The centrifugal force is applied evenly to all of the capillaries in the capillary cassette such that capillaries independently dispense their contents onto a substrate situated below the orifice to the capillary from which fluid is expelled.
  • the dispensed liquid will be drawn by centrifugal force to the bottom of the wells.
  • the design for a centrifuge and associated rotor and buckets to hold a cassette is disclosed in the copending application U.S. Serial No. 09/577,199, herein incorporated by reference in its entirety.
  • a second method of dispensing the liquid contained in a capillary tube is through the use of an air displacement device.
  • the design for an air displacement device designed 5 to dispense the liquid contents of multiple capillaries arranged into a cassette is disclosed in the copending application U.S. Serial No. 09/577,199, herein incorporated by reference in its entirety.
  • the contents of a capillary could be dispensed directly into a well, or sample port (FIG. 3E 76) of an analytical device (FIG. 3E 70), such as an electrophoresis o chip. As shown in FIG. 3E, such an analytical chip would have an array of analytical lanes
  • the capillary cassette illustrated in FIG. 3C includes capillaries 12 extending through flexible strip 11. Flexible strip 11 may be used alone or in combination with other such strips. The orientation of the capillaries in an essentially straight line may be altered by bending strip 11 to form an arc.
  • FIG. 3D illustrates strip 11 bent to allow capillaries 12 to mate with input ports that are disposed on a substrate in a circular pattern.
  • the liquid in capillaries 12 may then be electrokinetically injected or otherwise dispensed from capillaries 12 into ports 76 of an analytical chip 70 if an appropriate electrode array or other dispensing methods are used.
  • Strip 11 may be positioned in the curved orientation by pressing strip 11 against a curved form, such as a curved metal block. This may be done by an automated strip mover incorporated into an automated sample preparation system. 5 [0453]
  • the capillary cassette could be dispensed by air displacement or other dispensing means preferably selected to minimize splattering and bubble formation. Prior to dispensing the prepared reaction mixture into the wells 76 for analysis, a small amount of a diluent could be added to each analytical microchip well 76.
  • the elution fluid is preferably an aqueous solution of low ionic strength, more preferably water or a low ionic strength buffer at about a pH at which the nucleic acid material is stable and substantially intact, usually between pH 6.5 and 8.5.
  • TE Buffer at 1X concentration 10 mM Tris-HCI, 1 mM ethylenediamine- tetraacetic acid (EDTA), pH 8.0) 5 and distilled or deionized water are particularly preferred elution solutions for use in the present invention.
  • the low ionic strength of the preferred forms of the elution solution described above will tend to disrupt the salt-bridges established between the nucleic acid and the material comprising the inner surface of the capillary, ensuring that the nucleic acid is eluted into the solution.
  • Other elution solutions suitable for use in the methods of this o invention will be readily apparent to one skilled in this art.
  • nucleic acid binding to the inner surface of the glass capillary tube is saturable. Under appropriate conditions, it is possible to control, with a high degree of accuracy, the quantity of nucleic acid immobilized inside any particular capillary. Thus, if the nucleic acid is eluted into an aqueous solution and 5 dispensed, the concentration of the nucleic acid in the solution can be known, as well as the total quantity of nucleic acid in any particular volume of that solution.
  • a capillary's binding capacity is 10 ng DNA
  • this is eluted into 500 nl of elution fluid
  • the concentration of the solution is 0.02 grams per liter, with the molar concentration dependent on the molecular mass of the DNA molecules. If all 500 nl is dispensed, that droplet 0 contains 10 ng DNA.
  • nucleic acid eluted 5 into the elution fluid is an approximate quantity or mass.
  • predetermined approximate mass shall mean that between similar capillaries, or repeated use of the same capillary, all other conditions being equal, the error between the mass expected to be immobilized or dispensed and actually immobilized or dispensed is not greater than 10%, more preferably 5%, more preferably 2%, and most preferably not more o than 1 % error.
  • the dispensing function of the present invention will be utilized by immobilizing a saturating quantity of nucleic acid in a particular capillary and dispensing the entire volume.
  • the experimenter will choose a capillary with a predetermined binding capacity and volume.
  • the experimenter can empirically determine conditions under which a predetermined non-saturating quantity of immobilized nucleic acid is bound. 5 Accordingly, using these conditions, a non-saturating predetermined quantity of nucleic acid " can be immobilized and then eluted from a capillary, allowing the experimenter to dispense any given amount of nucleic acid at will.
  • the invention provides methods and apparatus for performing enzymatic reactions - particularly, but not limited to, isothermal reactions - in small volumes, particularly submicroliter volumes.
  • the reactions can be performed in highly o parallel fashion, and can readily be interfaced, in parallel, and without substantial loss of reactants to high resolution electrophoresis instrumentation for analysis.
  • the enzymes include any that are commonly used in larger-scale assays, including proteases, such as trypsin, chymotrypsin, proteinase K, papain, pepsin, endoproteinase Glu-C, Arg-C, Lys-C, Pro-C, V8 protease, glycosidases, such as ⁇ -galactosidase, lipases, 5 oxidases and oxygenases, such as glucose oxidase, cholesterol oxidase, and lactate monooxygenase, ligases, including DNA and RNA ligases, methylases, polymerases, such as DNA-dependent DNA polymerase enzymes, terminal transferase enzymes, RNA- dependent DNA polymerase enzymes, DNA-dependent RNA polymerase enzymes, phosphatase enzymes, kinase enzymes, DNA gyrase, topoisomerases, nucleases, o including exonucleases
  • the submicroliter protein reactions are not limited to use of enzymes, and thus catalysis of chemical reactions.
  • proteins can be used for their ability to bind other molecules, and thus capture them from solution.
  • proteins can be antibodies or antigen-binding fragments thereof, such as IgG, IgE, IgM; protein G and 5 Protein A; and streptavidin, to name a few.
  • the substrates are dictated by the chosen enzyme and are, accordingly, as varied as the enzymes, and include nucleic acids, including DNA and RNA, carbohydrates, lipids, and other biological and chemical substrates.
  • nucleic acids including DNA and RNA
  • carbohydrates including DNA and RNA
  • lipids including carbohydrates, lipids, and other biological and chemical substrates.
  • submicroliter protease assays using trypsin o protease - a sequence-specific protease commonly used in the art for mass spectral peptide mapping and sequencing - are used herein to demonstrate the usefulness of such a system in proteome research and as a drug discovery platform.
  • capillaries having submicroliter volumes are used as reaction chambers for small volume enzymatic assays, and can be usefully be used in cassettes, or arrays, to conduct such assays in highly parallel fashion without significant loss of reagent or reactants before analysis.
  • the capillary typically has an internal volume of not more than 5 ⁇ L, often no more 5 than about 2 ⁇ L, frequently no more than about 1 ⁇ L, typically no more than about 750 nL,
  • Example 26 demonstrates trypsin digestion of cytochrome C in homogeneous solution.
  • Mixtures of trypsin and cytochrome C are prepared in solution at various trypsin-protein ratios, with the concentration of cytochrome C fixed at 1 mg/mL. o Aliquots of the mixture are drawn into the capillaries of a capillary cassette by capillary action, and incubated at 37°C overnight to allow the protease reaction to complete.
  • Example 27 demonstrates homogeneous assay with Asp-N, further demonstrating the multiplexing capacity of the current methods. In addition, Example 27 demonstrates that analysis can be conducted in parallel using scanners.
  • peptide CyTM5Q-YVADAPVK-Cy3 is reconstituted in assay buffer, then mixed with endoproteinase Asp-N of various concentrations. 500nL aliquots of the mixture l o are captured by a capillary cassette system due to capillary action, and incubated at room temperature to allow the reaction to complete. Digestion mixtures were then spun down to a 384 clear scan plate of which each well contains 10 uL of buffer. The resulting mixtures were scanned on TyphoonTM (Amersham Biosciences, Piscataway, NJ) to detect Cy3 emission. The signal intensity of the Cy3 emission increases linearly as the Asp-N
  • the enzyme is immobilized on a particle, or bead, so dimensioned as removably to fit within a capillary or channel, such as those present in multi- capillary cassettes, such as that shown in FIG. 3.
  • the capillary or channel has a small internal volume, desirably from
  • the bead is dimensioned itself to fill no more than about 75%, typically no more than about 50%, often no more than about 40%, 30%, 20% and even as little as 10% of the capillary volume.
  • the bead or particle is sufficiently small as to be movable solely by entrainment in the reaction volume, and thus to be of such size as to be
  • Beads suitable for surface immobilization of enzymes are known and are available commercially from a variety of vendors, such as Dynal, Miltenyi Biotec, and others.
  • Beads can usefully be magnetic or superparamagnetic, and can usefully be derivatized to permit the ready attachment of proteins or other moieties thereto.
  • the beads can usefully include a scinfillant, permitting scintillation proximity assay (Amersham Biosciences, Inc., Piscataway, NJ). In such assays, the 5 polymer beads contain scinfillant that can be stimulated to emit light, stimulation occurring only when radiolabelled molecules of interest are bound to the surface of the bead.
  • the enzyme can be immobilized on the external surface of the bead or, if the bead is porous and the pores are of sufficient size to permit enzymatic substrate to diffuse therewithin, within the bead itself.
  • trypsin immobilized on the surface of magnetic beads are used. Introduction of small magnetic beads eliminates the need for separating the enzyme from the reaction mixture prior to analysis, minimizes contamination by the proteolytic enzyme, and provides high binding surface area per unit volume for optimal accessibility of target molecules. Beads are 5 prepared by incubating streptavidin-coated magnetic beads M280 (Dynal, Oslo, Norway) with biotin-conjugated trypsin (Sigma, St.
  • the enzyme is immobilized to an interior surface of the 5 reaction chamber, usefully a channel or capillary having submicroliter volume.
  • Nonspecific immobilization of enzyme can be achieved by simple adsorption onto a relatively hydrophobic solid phase.
  • the passive adsorption of the enzyme is through its exposed hydrophobic sites.
  • Such a process is not completely general, and the optimal conditions for binding often have to be found by trial and error.
  • Enzymes bound to o the solid phase via multiple amino acid groups risk deformation of the active site and hence reduced reactivity.
  • silanization with aminoalkylsilane reagents gives a surface that is 5 funcfionalized with amino groups to which a wide variety of affinity ligands can subsequently be attached.
  • capillaries of capillary cassettes such as those shown in FIG. 3 and described above, or other kinds of reaction chambers having small volumes
  • the pyridyldithio functional group provides a convenient way to bind proteins, such as enzymes, through specific -S-S- and -SH exchange reactions.
  • the immobilized enzyme can be released by adding an excess amount of thiopyridone, regenerating the derivatized surface for tethering fresh trypsin to ensure high enzyme reactivity.
  • Another surface immobilization approach is based on a specific streptavidin-biotin reaction. Streptavidin modification enables the surface to bind biotinylated enzymes.
  • capillary cassettes can be derivatized, e.g., with 3-aminopropyltriethoxy silane, and then reacted with a bifunctional linker, such as disuccinimidyl suberate, which in turn tethers streptavidin; the streptavidin thereafter can bind any biotinylated enzyme to the 0 reaction chamber (typically capillary) interior surface.
  • the enzyme is biotinylated at a unique site - e.g., by enzymatic biotinylation of a biotin-binding site engineered into the enzyme - the high affinity, high specificity streptavidin and biotin interaction results in uniformly oriented enzymes on the inner surfaces of the capillary.
  • These protein immobilization techniques offer high surface reactivity and minimized nonspecific binding.
  • we 5 have found that proteins immobilized by such approaches remain bound, and functional, even after completing a reaction; the capillary can thus be used serially for a plurality of reactions without requiring recharging with enzyme.
  • HPLC high performance liquid chromatography
  • Dye-primer sequencing reactions were performed within a capillary cassette comprised of 96 uncoated 2.8 cm long, 150 ⁇ m I.D., 360 ⁇ m O.D. fused-silica capillaries. o Dye-primer sequencing reactions were performed by amplifying template DNA with emission-specific primers corresponding to ddT, ddA, ddC, and ddG terminated reactions. The amplification of template was performed as single reactions in each capillary and pooled into a common well for post-reaction processing and analysis.
  • the color-specific primers were based on the M 13-40 FWD primer (5'-FAM- 5 GTTTTCCCAGT*CACGACG-3'), with 5-carboxyfluorescein (FAM) as the donor dye, and a termination-specific fluor attached to the indicated thymine (T*) as the acceptor dye.
  • C-FAM FAM for ddC-terminated reactions
  • A-REG 6-carboxyrhodamine for ddA reactions
  • G-TMR N,N,N',N'-tetramethyl-5-carboxyrhodamine for ddG reactions
  • T-ROX 5-carboxy-X-rhodamine for ddT reactions
  • dye-primer solution either 1 ⁇ M T-ROX, 1 ⁇ M G- TMR, 0.5 ⁇ M A-REG, or 0.5 ⁇ M C-FAM
  • 100 ⁇ L of the corresponding deoxy- and dideoxynucleotide mix (0.94 mM dATP,
  • This solution was aliquoted into a 96-well reagent plate prior to mixing with template DNA.
  • the general mixing scheme required the use of two capillary cassettes and a 384-well "mix plate.”
  • the first capillary cassette (transfer cassette) was dipped in a 5 solution of template DNA (20 ng/ ⁇ L M13mp18), and then inverted onto the top of a 384-well "mix plate” with the short ends of the capillaries inserted into the wells.
  • the inverted transfer cassette and mix plate were placed inside a bench top centrifuge. A balance plate was added to balance the rotor and the centrifuge brought to 3,000 x g for 5 seconds.
  • the centrifugation uniformly dispensed the contents of the transfer cassette into individual wells 0 of the 384-well plate. After the centrifuge step, the transfer cassette was transferred to the capillary cassette washer 410 for cleaning, and the mix plate was used for a subsequent centrifuge step for reagent addition.
  • a second capillary cassette (the reaction cassette), was dipped into the wells containing sequencing reagents (prepared as described in the preceding 5 paragraph) and inverted over the same wells of the same 384-well plate.
  • the reaction cassette and mix plate were placed in the centrifuge, spun at 3,000 x g for 5 seconds, and removed from the centrifuge. At this point each well contained 500 nL of template DNA and 500 nL of sequencing reagents to form the final reaction mixture.
  • the second capillary cassette (used to add reagents) was then dipped into the 1 ⁇ L mixture contained in the mix o plate, filling the capillaries of the reaction cassette with 500 nL.
  • the capillary cassette was inserted into the internal chamber of an air-based thermal cycler, as described herein in FIG. 7A-C, where the ends of the capillary segments are sealed by depressing the ends of the capillaries against deformable membranes 264a and 264b. After 30 cycles of 95°C for 2 seconds, 55°C for 2 second, and 72°C for 60 5 seconds, the thermal cycler was opened, removing the ends of the capillaries from contact with the deformable membranes. The capillary cassette was removed and placed on top of a 96-well "pooling plate" with the short ends of the capillaries inserted into the wells. The capillary cassette and mix plate were placed into a centrifuge, with a balance plate.
  • reaction products were dispensed by centrifugal force (-2500 x g) into a microtiter plate o containing 40 ⁇ L of 80% isopropyl alcohol.
  • the capillaries were washed as described herein.
  • the samples were subsequently centrifuged at 3000 x g for 30 minutes.
  • the alcohol was decanted by a gentle inverted spin, and the samples were resuspended in 5 ⁇ L of ddH ⁇ O for electrokinetic injection and analysis by MegaBACETM capillary array electrophoresis.
  • the capillaries were filled with a fresh solution of 3% linear polyacrylamide (LPA) (MegaBACETM Long Read Matrix, Amersham Life Sciences, Piscataway, NJ) which was pumped through the capillaries under high pressure from the anode chamber to individual wells of a 96-well buffer plate contained in the cathode chamber. Each well was filled with 100 ⁇ L of Tris-TAPS running buffer (30 mM Tris, 100 mM TAPS, 1 mM EDTA, pH 8.0). The matrix was equilibrated for 20 minutes followed by pre-electrophoresis for 5 minutes at 180 V/cm.
  • LPA linear polyacrylamide
  • DNA sequencing samples were electrokinetically injected at constant voltage from a 96-well microtiter plate according to the specified conditions; one preferred injection condition for 500 nL samples is 40 seconds of injection at an applied voltage of 2kV.
  • the capillary ends were rinsed with water, the buffer plate was placed in the cathode chamber, and the electrophoresis run was commenced. Separations were typically for 120 minutes at 8 kV.
  • Computer controlled automation of the instrument and data collection was performed using LabBench software (Amersham Biosciences, Sunnyvale,
  • dye-primer reactions performed in the same capillary cassette were analyzed by direct injection into a 16 channel microfabricated "chip-based" analyzer described in detail in S. Liu, H. Ren, Q. Gao, D.J. Roach, R.T. Loder Jr., T.M. Armstrong, Q. Mao, I. Blaga, D.L. Barker, and S.B. Jovanovich, Proc. Natl. Acad, Sci. USA, 5-00.
  • the 16-channel chip is formed by bonding two glass wafers, the top wafer has 50 urn deep by 100 urn wide channels etched into it by standard microfabrication methods.
  • the pattern etched has a combination of two 8-channel groups, each with a common anode reservoir. Sixteen cathode reservoirs were evenly spaced at 4.5-mm intervals in a line, as were sixteen sample and sixteen waste reservoirs. The reservoirs were formed by the drilled access holes through the top etched wafer. Sixteen 250- ⁇ m long twin-T injectors were formed by the offset of channels from the sample and waste reservoirs joining the main separation channel. The distance between adjacent channels (center-to-center) was 600 ⁇ m in the detection region. The two alignment holes were used to align the chip to the detector.
  • a dye-primer reaction terminated by ddT was performed as described and dispensed into the sample wells of a microchip containing 1.5 ⁇ L of ddH ⁇ O.
  • Sample injection was performed by applying voltages of 50 and 10 volts respectively to the waste and cathode reservoirs, typically for 60 s, while the sample and anode reservoirs were grounded. Separations were carried out immediately after sample injection by applying 2,000 volts to the anode reservoir, 140 volts to sample and waste reservoirs, while grounding the cathode reservoir. The corresponding separation field strength was ca. 227 V/cm.
  • the laser-induced fluorescence was collected, digitized, and processed into the electropherogram shown in Figure 10. The electropherogram demonstrates microchip analysis of the reactions performed in the described capillary cassette system.
  • Dye-terminator cycle sequencing was demonstrated using the capillary cassette system and alcohol precipitation for cleanup prior to capillary array electrophoresis.
  • the sequencing reaction mix was prepared by mixing 400 ⁇ L of sequencing reagents (Dynamic ET terminator kit, Amersham Pharmacia Biotech, Part 81600) with 100 ⁇ L of 5 pmol/ ⁇ L of M 13-28 FWD primer (5'-TGT AAA ACG ACG GCC AGT-3'). The reaction mix was distributed in 5 ⁇ L aliquots to a 96-well "reagent" plate.
  • the cassette was removed from the cycling chamber and the contents of the capillaries dispensed by centrifugal force (3000 x g) 5 into a 96-well plate containing 40 ⁇ L of 80% ethanol.
  • the samples were centrifuged at 3000 x g for 30 minutes.
  • the alcohol was decanted by a gentle inverted spin, and the samples were resuspended in 5 ⁇ L of ddH 2 0 for electrokinetic injection and analysis by MegaBACETM capillary array electrophoresis.
  • the cleanup of dye-terminator reactions by alcohol precipitation, the reproducibility of the technique, and the application to "real-world" o templates is represented as a histogram of percent success versus read length in Figure 11.
  • Figure 11 demonstrates excellent read lengths and success rates with M13 subclone inserts prepared from a subclone library of a mouse bacterial artificial chromosome.
  • dye-terminator reactions were performed in 500 nL capillaries as described in Example 3, and the reaction products dispensed into 15 ⁇ L of ddH 2 0 by centrifugal force.
  • the 15 ⁇ L samples were transferred to a filter plate containing 45 ⁇ L of o hydrated Sephadex G-50.
  • the samples were centrifuged through the Sephadex matrix at
  • the present technology uses the disclosed system for the PCR amplification of insert DNA (e.g. subclone inserts from a DNA library).
  • the PCR reaction mixture was prepared by mixing 5 ⁇ L of 10 ⁇ M of M13 -40 FWD primer (5' GTT TTC CCA GTC ACG AC 3') and 5 ⁇ L of 10 ⁇ M -40 REV primer (5' GGA TAA CAA TTT CAC ACA GG 3') with 25 ⁇ L of 10x GeneAmp buffer, 15 ⁇ L of 25 mM MgCb, 5 ⁇ L of AmpliTaq Gold, 2.5 ⁇ L of 1 mg/mL bovine serum albumin (BSA), and 67.5 ⁇ L of ddH 2 0.
  • BSA bovine serum albumin
  • the reaction was initiated by mixing template DNA with the PCR cocktail using the two-capillary cassette and mix-plate method described.
  • the transfer cassette was dipped into the glycerol stock solutions of a subclone library and dispensed by centrifugal force into the wells of a 384-well plate.
  • a second "reaction" cassette was used to transfer 500 nL of PCR cocktail to the same wells by centrifugal force.
  • the capillaries of the reaction cassette were subsequently dipped into the combined mixture of template DNA and PCR reagents, filling the capillaries by capillary action.
  • Amplification was effected by placing the capillaries into the cycling chamber and thermally cycling with an activation step of 95°C for 12 minutes followed by 30 cycles of 64°C for 4.5 minutes and 95°C for 5 seconds.
  • the PCR products were analyzed by agarose gel electrophoresis and compared with the same subclones amplified by full volume (25 ⁇ L) reactions performed in 0.20 mL tubes. Nanoscale capillary cassette samples were dispensed into 4.5 ⁇ L of ddH ⁇ O by centrifugal force. Equivalent volume aliquots of full volume reactions were transferred manually using a low volume pipettor.
  • a preferred mode of preparing cycle sequencing samples using the present o invention is to prepare nanoscale PCR samples in the capillary cassette and related instrumentation, perform macroscale Exol/SAP reactions, and then perform the cycle sequencing in the capillary cassette and related instrumentation.
  • Nanoscale PCR template preparation for DNA sequencing was demonstrated by performing PCR amplification from glycerol stock subclones. Glycerol stock subclones were PCR amplified in the capillary 5 cassette and related hardware as described in Example 5.
  • the contents of the capillaries were dispensed by centrifugation into the wells of a 96-well plate containing 4.5 ⁇ L of 7.5 mU of shrimp alkaline phosphatase (SAP) and 37.5 mU of exonuclease I (Exol).
  • SAP shrimp alkaline phosphatase
  • Exol exonuclease I
  • the PCR products and Exol/SAP solution were allowed to incubate at 37°C for 5 minutes to digest the unincorporated primers and to dephosphorylate the o unincorporated nucleotides. After an initial incubation, the enzymes were deactivated by heating the solution to 72°C for 15 minutes.
  • the Exol/SAP treated PCR products were aliquoted to a fresh 384-well mix plate with a transfer capillary cassette and centrifugal dispensing.
  • An equal aliquot of dye- terminator sequencing reagents were added to the 500 nL of purified PCR products using 5 another capillary cassette, the reaction cassette, and centrifugal dispensing.
  • the capillaries of the reaction cassette were then filled by dipping the capillary cassette into the 1 ⁇ L reaction mixture.
  • the template was amplified according to Example 3, dispensed into 40 ⁇ L of 80% ethanol and purified as described. Analysis of the sequencing reactions was performed by MegaBACETM using electrokinetic injection.
  • o sequencing electropherograms from subclone templates prepared by nanoscale PCR amplification from glycerol stock solutions and by nanoscale cycle sequencing are shown in Figure 13.
  • the present system allows a simplified transition from nanoscale (less than 1 ⁇ L volumes) to greater than nanoscale reaction volumes.
  • the present system also allows a simplified transition from macroscale (more than 1 ⁇ L volumes) to nanoscale reaction volumes, as shown by utilizing the Exo l/SAP reactions for cycle sequencing in the capillary cassette.
  • a stock solution of 35 ⁇ M RBG was prepared in 5 mL of buffer (100 mM Tris-HCL, 20 mM KC1 , and 2 mM MgC ) to 5 mg of RBG, vortexing vigorously, and filtering the solution through a 0.40 micron filter and then adding an equal volume of buffer. A dilution curve of RBG was then prepared from the stock solution.
  • buffer 100 mM Tris-HCL, 20 mM KC1 , and 2 mM MgC
  • the cassette was placed in air cycler and after 2 minutes at 37°C, the capillary cassette was removed and the contents centrifuged out of the capillaries into a 384-well scan plate containing 5 ⁇ L of 1 M sodium carbonate.
  • the wells of the scan plate were subsequently filled with 50 ⁇ L of ddH2 ⁇ .
  • the 0.2 mL tubes were incubated at 37°C for 2 minutes and the ful volume reactions stopped by 5 adding 1 M sodium carbonate. A control aliquot from the enzyme reactions performed in the 0.20 mL tubes was added to the scan plate.
  • Figure 14 shows the expected signal versus substrate concentration for the tube reactions, and data points of signal for the pre-mixed enzyme reaction performed in the capillary cassette, and for the capillary-binding ⁇ -galactosidase assay.
  • This example serves to illustrate the compatibility of the described system for performing a range of general enzyme activity and inhibition assays. In addition, it demonstrates that solid phase capture can be applied to proteins and enzymes as well as DNA. Finally, it shows the described system can be applied to isothermal reactions.
  • Fig. 17A shows the results of sequencing PCR products mixed with the reaction mixture prior to sequencing.
  • Fig. 17B shows the results of first mixing the PCR template with sodium thiocyanate, binding the DNA to the inner surface of the capillary, washing the DNA with 80% ethanol, followed by sequencing.
  • FIG. 18 represents the retained mass of DNA following a template capture 5 protocol. The amount of DNA bound remains constant above 40 ng starting template for M13 (0), plasmid (D), and PCR product (0).
  • Template DNA was prepared by a restriction digest of M13mp18 and PUC19 DNA to form linear single and linear double stranded DNA respectively. These templates, along with a 800 bp PCR product (standard amplification conditions) were end labeled with 32 P o using [ ⁇ -32P]ATP and T4 polynucleotide kinase. The labeled DNA was seeded into unlabeled template of the same type and a calibration curve was generated for the seeded DNA solution. Template binding was performed by mixing stock DNA with 10 M sodium thiocyanate and loading into 500 nl fused-silica capillaries. After 10 minute incubation and 80% ethanol washing, the capillaries were placed in scintillation fluid and quantified. Fig. 18 5 shows definitive normalization for three sources of template DNA.
  • FIG. 19 shows a plot of read length versus starting DNA mass for samples prepared by premixing DNA and sequencing reagents (D) compared to samples prepared by template capture (D). The normalization effect is highlighted by a nearly constant read length obtained for the template capture samples, whereas for premixed samples, template overloading and reduction in read length occurs above 20 ng starting DNA.
  • Template binding was performed by mixing stock M13mp18 DNA with 10 M sodium thiocyanate and loading into 500 nL fused-silica capillaries. After 10 minute incubation and 80% ethanol washing, the capillaries were placed filled with ET terminator premixed with M13-40FWD sequencing primer. Premixed reagents were prepared in a 10 ⁇ l volume and loaded into clean sample preparation capillaries. The air-based cycle o sequencing was performed as previously described followed by ethanol precipitation and
  • PCR reactions were performed after template binding of indicated starting amount of M13mp18.
  • Standard PCR amplification reactions with M13-100 FWD and M13-400 REV primers were performed in 500 nl capillary cassette with 10 s at 95°C, 10 s at 55°C, and 120 s at 72°C.
  • Reaction products were dispensed by centrifuge into loading buffer, and transferred to a 1.5% agarose gel. The products were stained with SYBR Green dye and imaged with a Fluorimager apparatus, as shown in FIG. 20.
  • FIG. 21 represents the relative signal intensity obtained with increasing template concentration represented by the intensity of peak 79, peak 308, and peak 604 (ddT-terminated peaks early, middle, and late in the electrophoresis chromatogram).
  • the peak intensity increases to 40 ng/ ⁇ l and levels off, confirming by peak height the normalization effect and saturation level of the template capture technique.
  • the migration time of the first peak is relatively constant across template concentrations.
  • FIG. 22 shows peak height increasing with increasing template concentration, reaching a maximum due to overloading of the sequencing sample. An excess of template DNA inhibited the electrokinetic injection, reducing the current in the sample run, consequently increasing the migration time of the sample through the capillary.
  • Sample preparation for DNA sequencing could be simplified if some of the many steps involved in preparing sequencing samples from cloned DNA in bacterial cells could be eliminated.
  • bacterial cells are grown and lysed, PCR amplification is performed, followed by Exol/SAP cleanup and then cycle sequencing.
  • the instant invention provides a method to simplify the workflow by cycle - I l l -
  • the cycling protocol was for ET terminators as described in Example 1 , above.
  • the samples were ethanol precipitated by being dispensed by o centrifugation (3220 g for 30 minutes at 4°C) into a microtiter plate containing 80% ethanol.
  • FIGS. 23 A and B show a trace obtained by this method that had a Phred 20 score of 561 bases.
  • This example demonstrates the application of the instant invention to direct sequencing from frozen glycerol stocks of bacteria. It will be apparent to the skilled artisan that this method can be applied to the sequencing of bacterial colonies grown on agar plates, or similar solid growth media, o regardless whether the plates are fresh or desiccated.
  • the instant invention can be applied to perform nanoscale genotyping reactions.
  • Single-base extension (SBE) reactions were performed in the 96 channel capillary cassette.
  • the single base extension analysis consists of the single base extension of a DNA primer that terminates immediately before the base to be interrogated.
  • PCR reactions 0 of 25 ul were prepared containing 5 ng/ul of genomic human DNA, 1 ⁇ M of forward and reverse primers, buffer, MgCfe and AmpliTaq Gold.
  • the PCR cycling was 96°C for 12 min, 35 cycles of 94°C for 20 sec, 60°C for 20 sec, and 72°C for 30 sec, followed by 72°C for 2 min.
  • the single base extension reactions were performed by 25 cycles of 96°C for 10 sec, 50°C for 5 sec, 60°C for 30 sec.
  • the thermal cycling was carried out in either MJ Research tetrads (a type of thermal cycling machine) for the full volume controls, or for the o capillary cassette samples, in the air cycler disclosed in the co-pending application U.S.
  • FIG. 24 demonstrates that the capillary-based reactions could correctly identify single nucleotide polymorphisms. Traces 1 , 3, and 4 were obtained from samples 5 homozygous at the interrogated base. Trace 2 was obtained from a sample heterozygous at the interrogated base and demonstrates that allelic polymorphism can be detected using nanoscale reactions. Signal is essentially the same as that obtained with the full volume reactions. [0527] The entire process, from PCR to SBE, was accomplished using the capillary o cassette.
  • the methods of the instant invention can be used to perform AFLPs (amplified fragment length polymorphism) in nanoliter volumes.
  • genomic DNA is digested with pairs of restriction enzymes.
  • the fragments are either ligated to a linker and amplified to amplify fragments of a certain length, in a certain orientation, as determined by the two restriction enzymes used, or alternatively, amplified by PCR directly using degenerate primers.
  • the amplified fragments are analyzed by capillary electrophoresis.
  • the AFLP analysis method is used to generate a "representation" of a genome, also called an amplicon, with variable fragments as well as constant ones.
  • the amplicon is used to assess the diversity of populations of organisms or to make genome maps in organisms where little sequence and marker information is available.
  • the methods of the present invention can be used to perform direct display analysis in nanoliter volumes.
  • complementary DNA is digested with pairs of restriction enzymes.
  • the fragments are either ligated to a linker and amplified to amplify fragments of a certain length, in a certain orientation depending on the two restriction enzymes used, or alternatively, amplified by PCR directly using degenerate primers.
  • the amplified fragments are analyzed by capillary electrophoresis.
  • the direct display analysis method is used to generate a "representation" of a transcriptosome, with variable fragments as well as constant ones.
  • Direct display analysis is used to assess the quantitative change in the level of expression between organisms, or differences due to environmental or physiological effects.
  • the methods of the present invention can be used to perform genotyping by microsatellite analysis in nanoliter volumes.
  • genomic DNA is PCR amplified with marker panels such as PE Applied Biosystems Linkage Mapping Sets.
  • marker panels such as PE Applied Biosystems Linkage Mapping Sets.
  • 96 human samples are analyzed with respect to panels of 12 genotypes in about 30 minutes using a four-color analysis. Three of the colors are used with four primer sets, while the fourth color provides internal size standards.
  • PCR set-up and thermocycling is performed as recommended by the manufacturer of the primer panel.
  • the primer mix contains both forward and reverse primers, each at a final concentration of 5 ⁇ M.
  • thermal cycler program is as follows:
  • Formamide loading solution 2.75 ul Total loading volume 5.00 ul
  • the present invention is advantageously applied to performing nanoscale enzymatic reactions with nucleic acids in nanoliter volumes.
  • the nucleic acids are immobilized in a reaction chamber, such as a glass capillary, prepared according to the methods of the instant invention.
  • the capillaries are filled with reaction mixtures that comprise one or more of different enzymes, such as a restriction enzyme.
  • a typical restriction enzyme digest is performed in a total volume of 20 ⁇ L that includes 0.2 to 1.5 ⁇ g of substrate DNA and a 2-10 fold excess of restriction enzyme over DNA.
  • Reaction buffer, enzyme, water, and DNA are mixed in a reaction tube and incubated at 37°C for 1 to 4 hours.
  • template DNA is bound to the inner surface of a capillary tube.
  • a premix of restriction enzyme e.g. Hind III
  • a 1 x KGB buffer 100 mM potassium glutamate, 25 mM Tris-acetate, pH 7.5, 10 mM magnesium sulfate, 50 ⁇ g/ml bovine serum albumin, and 1 mM ⁇ -mercaptoethanol
  • the reaction is incubated at 37°C for an allotted time, after which the contents are dispensed in gel-loading buffer for agarose gel sizing, or into a solution containing 10 mM EDTA.
  • Other reactions comprising different enzymes are also possible. These enzymes include, but are not limited to methylation enzymes, DNA-dependent DNA polymerase enzymes, terminal transferase enzymes, RNA-dependent DNA polymerase enzymes, DNA- dependent RNA polymerase enzymes, phosphatase enzymes, kinase enzymes, exonuclease enzymes, such as S1 , or mung bean nucleases, other nuclease enzymes, ribonuclease enzymes, or DNA or RNA ligase enzymes. For most of these reactions, 5 control over the ratio of nucleic acid to enzyme is crucial to the success of the reaction process.
  • Use of the present application beneficially reduces the error associated with concentration dependent enzymatic reactions with nucleic acids, as well as reducing the consumption of valuable enzymes. Furthermore, through washing, use of the methods of o the present invention is effective for eliminating residual ions, such as ammonium acetate,
  • EDTA lithium chloride
  • other contaminants such as polysaccharides that interfere with enzymatic activity.
  • thermostable polymerases introduces a certain number of erroneous base pairs per thousand as it amplifies the template. If errors have been introduced they must be detected, and the amplified product or data therefrom discarded. Usually, this requires numerous processing steps separate from those associated with spotting the PCR product. However, use of an embodiment of the present invention greatly increases the efficiency of 5 sequence confirmation.
  • Microarray spotting samples were prepared from PCR products, average of 500 bp, from human genomic DNA template. The products were purified using standard o guanidinium hydrochloride glass-filter plate processing and mixed with an equal volume of
  • sequencing reactions were performed by dipping the ends of a 96-capillary cassette into the spotting plate and binding the DNA to the inside surface of the capillary. After a wash step with 80% ethanol, the capillaries were filled with sequencing 5 mix containing buffer, polymerase, dye-labeled dideoxynucleotides, and sequencing primer at 1 x concentration. After thermal cycling (30 cycles at 95 °C for 5 s, 55 °C for 5 s, and 60 °C for 60 s), the sequencing reactions were purified by ethanol precipitation and analyzed by MegaBACETM.
  • the methods of this invention have been used to simplify the purification of PCR products prior to sequencing.
  • an enzymatic purification of the PCR product using o exonuclease I (Exol) and arctic shrimp alkaline phosphatase (SAP) to remove primer and excess dNTPs is required prior to cycle sequencing.
  • SAP arctic shrimp alkaline phosphatase
  • template binding is size dependent, however, the unincorporated primers and remaining nucleotides can instead be removed from the template by differential binding of the template to the capillary, followed by removal of nucleotides and primer by washing. This approach obviates enzymatic 5 cleanup of the PCR product and greatly simplifies the overall workflow.
  • 96 PCR products of M13 DNA containing a mouse subclone insert were directly sequenced without enzymatic purification after PCR amplification.
  • the PCR amplification reactions were performed using M13 templates containing a subclone insert (ca. 2000 bp) of mouse genomic DNA.
  • the M13 templates had previously o been prepared by polyethylene glycol precipitation and detergent solvation (Thermomax), diluted 200 fold and rearrayed into a 96-well microtiter plate.
  • a 2 ⁇ L aliquot of this solution was transferred to a PCR amplification mix prepared with 2.5 ⁇ L 10X GeneAmp buffer, 0.2 ⁇ L of 25 mM each dNTPs, 0.5 ⁇ L of 10 ⁇ M M13 - 40FWD (GTT TTC CCA GTC ACG AC), 0.5 ⁇ L of 10 ⁇ M M13 -40REV primer (GGA TAA CAA TTT CAC ACA GG), 1.5 ⁇ L of 25 mM magnesium chloride, 0.5 ⁇ L of 5 U/ ⁇ L AmpliTaq polymerase, and 17.3 ⁇ L water.
  • the capillaries of a 96-capillary cassette were dipped into the chaotrope-PCR product mixture, thus filling the cassette. After a 5 minute incubation at 60 °C, the residual chaotrope, unbound buffer components and DNA were removed with an 80% ethanol wash o applied by pulling the ethanol through the capillaries under vacuum. After drying the inside surface with a 1 minute flow of air, the capillaries were dipped into a sequencing mixture containing a 1 x solution of ET terminator reaction mix and forward sequencing primer, M13 -21 FWD (TGT AAA ACG ACG GCC AGT).
  • Cycle sequencing was performed by sealing the ends of the capillaries in the air- 5 thermal cycle. The reaction was cycled 30 times at 95 °C for 5 s, 55 ° C for 5 s, and 60 °C for 60 s. The cycle-sequencing products were dispensed into a microtiter plate containing 40 ⁇ L of 80% ethanol using centrifugal force. After a 30 minute centrifugation at 3000 x g, the alcohol was decanted, the pelleted DNA resuspended in 5 ⁇ L of ddH20, and the samples were analyzed by MegaBACETM. o [0555] For these 96 samples, an average read length of 550 bases was achieved with
  • PCR premixture is prepared by mixing template specific primer pairs with 10x GeneAmp buffer, MgCI2, AmpliTaq Gold, bovine serum albumin (BSA), dNTPs and double- o distilled water. Fifteen microliters of the premix is then aliquoted into 24 wells of microtiter plate. To each well containing PCR premix, 10 ul of genomic DNA (5 ng/ul) is added as template for the reaction. Each of 23 wells receives genomic DNA isolated from a different individual, and one well receives no template as a negative control.
  • 10x GeneAmp buffer MgCI2, AmpliTaq Gold, bovine serum albumin (BSA), dNTPs and double- o distilled water. Fifteen microliters of the premix is then aliquoted into 24 wells of microtiter plate. To each well containing PCR premix, 10 ul of genomic DNA (5 ng/ul) is added as template for the reaction. Each of 23 wells receives genomic DNA isolated from a different
  • the capillaries of a reaction cassette are filled by capillary action with about 500 nl of reaction mixture by dipping the ends of the capillaries into wells of the microtiter plate.
  • the capillary cassette is then placed into the thermal air cycler, disclosed in co-pending application U.S. Serial No. 09/577,199, herein incorporated by reference in its entirety, and 5 the capillary ends are sealed.
  • Amplification is then effected by air driven thermal cycling using the following program: 30 cycles of 93°C for 10 sec; 60°C for 10 sec, and 72°C for 60 sec.
  • the remaining PCR reaction mixture is transferred to 0.2 ml PCR tubes and amplification effected by thermal cycling using the following program: 35 cycles of 94°C for 20 sec; 60°C for 20 sec; 72°C for 30 sec, and one cycle of 72°C for 2 min. o [0557]
  • the contents of the capillaries are expelled into 7.5 ul 1x loading dye by centrifugal force.
  • An equivalent volume from each full volume PCR reaction is manually transferred using a low volume pipettor into the same amount of loading dye.
  • PCR products are then loaded into the wells of a 1.5% agarose gel and subjected to electrophoresis for 40 minutes at 15 V/cm in 1 X Tris-acetate-EDTA buffer at pH 8.0. After 5 electrophoresis is completed, the gel is stained with the DNA dye Sybr Green II (Molecular Probes, Eugene, OR), and is imaged using a two-dimensional fluorescence scanner (Fluorlmager, Amersham Biosciences, Sunnyvale, CA).
  • Sybr Green II Molecular Probes, Eugene, OR
  • FIG. 25A yields a comparable amount of product as full volume PCR reactions (FIG. 25B).
  • SBE premix is similar to PCR premix, except that primer pairs are excluded and dNTPs are replaced with fluorescently labeled dideoxyterminators. 5 After the ingredients are mixed, the reaction mixture is transferred to 0.2 ul tubes and SBE is performed by thermal cycling, as for PCR, using the following program: 25 cycles of 96°C for 10 sec; 50°C for 5 sec; 60°C for 30 sec.
  • FIG. 26 Results from four samples analyzed by full volume SBE are shown in FIG. 26.
  • FIG. 26A and FIG. 26C show heterozygous nucleotide polymorphisms at the interrogated base, whereas FIG. 26B shows a homozygous polymorphism.
  • FIG. 26D shows that a negative control, which contained no DNA, produced no single nucleotide signal.
  • capillary tubes are dipped into the same SBE primer-premix solution reaction mixture prepared for full volume SBE, and filled by capillary action with about 500 nanoliters of the mixture.
  • reaction products are expelled from the capillary tubes by centrifugation into the wells of a microtiter dish containing 20 ul of the CIAP solution described above.
  • the reaction products are treated with CIAP by incubation at 37°C for 60 min to effect the reaction and then at 72°C for 15 min to heat inactivate the CIAP enzyme.
  • CIAP Five microliters of each CIAP-treated nanovolume SBE reaction is then mixed with
  • FIG. 27 shows the results of an experiment comparing full volume (FIG. 27A) and nanovolume SBE (FIG. 27B) of the same heterozygous sample. The results demonstrate that nanovolume SBE produces similar quality data as full volume SBE.
  • FIG. 27A full volume
  • FIG. 27B nanovolume SBE
  • 23 different samples were analyzed using two distinct primers (NCBI 422 and NCBI 425) with 100 % accuracy of detection of the polymorphic nucleotide.
  • Nanovolume PCR is performed similarly as described in Example 21 , except that 5 ul of genomic DNA template are mixed with 7.5 ul PCR premix in the wells of a microtiter plate and then drawn into the capillary tubes by capillary action. After the reaction is completed, PCR product is expelled from the capillaries by centrifugation into the wells of a microtiter plate containing 500 nanoliters of 9.7M sodium thiocyanate (NaSCN). After mixing, about 500 nanoliters of the solution is drawn into new capillaries by capillary action, and incubated at 60°C for 5 min to allow the SBE product to bind to the inner surface of the capillaries. Thereafter, the solution is expelled by centrifugation, the capillaries washed with 80% ethanol/20% double distilled water and dried with flowing nitrogen. Treatment of the nanovolume PCR product with Exol/SAP is not performed.
  • Nanovolume SBE and CIAP treatment of the SBE products is then performed as described in Example 22, followed by analysis of the products using MegaBACETM, also as described.
  • FIG. 28 shows the results of an experiment comparing full volume PCR treated with Exol/SAP, followed by full volume SBE (FIG. 28A) and nanovolume PCR with template capture, followed by nanovolume SBE (FIG. 28B) of the same heterozygous sample.
  • the results demonstrate that nanovolume SBE coupled with template capture produces similar quality data as full volume SBE coupled with Exol/SAP treatment of the PCR product that serves as SBE template.
  • Nanovolume SBE is performed as described in Example 23 and different methods of treating the SBE products to remove or inactivate unincorporated ddNTPs prior to analysis using MegaBACETM are compared for efficacy. Injection into MegaBACETM is performed at 2kV for 45 sec, and running of samples is performed at 6kV for 60 min. As shown in FIG. 29A, if ddNTPs are not removed or inactivated prior to injection they produce a strong signal. FIG. 29B and FIG. 29C demonstrate the effectiveness of CIAP treatment in preventing the ddNTPs from entering the MegaBACETM gel bed. Denaturation of SBE products in deionized water at 95°C for 1 minute prior to injection results in about 4 fold greater signal intensity (FIG. 29C) as compared to denaturing the products in MegaBACETM loading solution (FIG. 29B).
  • Sephadex Most effective in removing ddNTPs and increasing signal intensity however, is purifying the SBE products using Sephadex (FIG. 29D) which results in a further 2 fold increase in signal intensity.
  • Sephadex aliquoted into the wells of a microtiter plate is prewashed four times with 150 ul deionized water. Between washes, sephadex is pelleted in the well by centrifugation at 910 g for 5 min. Nanovolume SBE reactions are expelled by centrifugation into 20 ul of water, after which the diluted reactions are transferred to wells of the microtiter plate containing the sephadex. After incubation for time sufficient for ddNTPs to enter the pores of the sephadex, the sephadex is pelleted by centrifugation. A sample from each well is then injected directly into MegaBACETM.
  • Nanovolume SBE is performed as described in Example 23 using 23 unrelated human genomic DNA samples, and 12 no-DNA negative controls. Different base positions are interrogated using 12 primers. SBE product is purified with sephadex as described in Example 24. Full volume SBE using the same samples and primers is performed as described in Example 22. [0575] FIG. 30 compares the results of nanovolume and full volume SBE and shows the results for 9 of the primers. Average accuracy of nanovolume SBE (98%) is comparable to that of full volume SBE (99%). [0576] The following examples demonstrate the usefulness and effectiveness of the methods of the present invention for performing a range of general enzyme activity and inhibition assays. In addition, they demonstrate that solid phase immobilization can be applied to proteins and enzymes as well as DNA. Finally, they show the described system can be applied to isothermal reactions. A multiplex capillary system used in the following examples contains 16, 96 or 384 capillaries.
  • FITC fluorescein-5-isothiocyanate
  • the resulting mixtures were then diluted 20 to 2000 times with tris-HCI buffer, and subjected to capillary electrophoresis (CE) separation on MegaBACETM 1000 (Amersham Biosciences, Piscataway, NJ) using the MegaBACE LPA buffer and the long read matrix. Samples were injected at 1 KV for 5 sec, and separated at 9 KV for 50 min.
  • CE capillary electrophoresis
  • Endoproteinase Asp-N digestion of polypeptides is illustrated here as an additional Example. It further demonstrates the use of the described multiplex system for processing submicroliter enzymatic reactions. An enzyme-product relationship for endoproteinase Asp- N digestion was established, as well as an optimal enzyme concentration. 5 [0581] Peptide CyTM5Q-YVADAPVK-Cy3 (Amersham Biosciences, Piscataway, NJ) was used as the reaction substrate. When the peptide is intact, Cy5Q efficiently quenches Cy3 and the excitation at Cy3 wavelength results in only a residual background signal.
  • the endoproteinase Asp-N reaction was performed in homogeneous solutions. Five micrograms of the peptide was reconstituted with 20 uL dimethyl sulfoxide, then mixed with 980 uL of assay buffer (50 mM Tris, pH 8.0, + 0.005% Tween 20TM). The 5 endoproteinase Asp-N (Amersham Biosciences, Piscataway, NJ) was reconstituted with 500 uL glass distilled water, with a final concentration of 4 ug/mL. A series of dilutions were performed on the enzyme so that the final amount of Asp-N in a 500 nL reaction was between 5 and 180 picograms.
  • FIG. 36 summarizes the result of these reactions.
  • Signal intensity of Cy3 emission increases linearly as the Asp-N concentration increases, up to -50 picogram Asp-N per 500 nL reaction. Beyond that, Cy3 signal intensity continues to increase - but at a slower pace - o with the Asp-N concentration, up to 180 picogram per 500 nL reaction.
  • the optimal amount of Asp-N in a 500 nL reaction volume is - 50 picogram.
  • Trypsin immobilized magnetic beads were prepared by incubating Streptavidin coated magnetic beads M280 (Dynal, Oslo, Norway) with biotin conjugated trypsin (Sigma, St. Louis, MO) in tris-HCI buffer at a bead-trypsin ratio of 10:1 (weight/weight). After 24 hours incubation under constant end-over-end shaking at room temperature, beads were o cleaned on a Dynal MPC-96 magnet device by washing off unbound enzymes with tris buffer. These trypsin immobilized magnetic beads were then mixed with cytochrome C (Sigma, St. Louis, MO) at a bead-protein ratio of 10:1 (weight/weight).
  • capillary cassettes (or other kind of reaction chambers) were treated by 3- aminopropyltriethoxy silane, followed by N-succinimidyl 3-(2-pyridyldithio) propionatel.
  • the pyridyldithio functional group provides a convenient way to bind trypsin through specific -S- S- and -SH exchange reactions, and, if needed, the immobilized enzymes can be released by adding an excess amount of thiopyridone (Carlsson, J.; Drevin H.; Axen, R. Biochem. J. 5 1978, 173, 723).
  • the same capillary surface can be regenerated for tethering fresh trypsin to ensure high enzyme reactivity.
  • Another surface immobilization approach is based on a specific streptavidin-biotin reaction. Streptavidin modification enables the surface to bind biotinylated enzymes.
  • capillary cassettes were derivatized with 3-aminopropyltriethoxy silane, and then o reacted with a bifuncfional linker, disuccinimidyl suberate, for tethering streptavidin that allows biotinylated trypsin to be thereafter linked to the capillary surfaces.
  • the high specificity of streptavidin and biotin interaction was utilized to give uniformly oriented enzymes on the inner surfaces of the capillary (Wilchek, M.; Bayer, E.A. Methods in Enzymology, 1990, 184)
  • Protein digestion reactions were conducted by directly introducing cytochrome C (1 mg/mL) to the streptavidin-biotin immobilized-trypsin capillary microreactors by capillary action, followed by incubation at 37 °C overnight. Protein fragments were then spun out, 5 labeled with fluorescein-5-isothiocyanate (FITC), and the labeled protein digests subsequently subjected to MegaBACETM analysis, all as described in Example 26. Two untreated capillary cassettes coated with trypsin (by simple adsorption) were used as the controls.
  • FITC fluorescein-5-isothiocyanate
  • control capillary cassette showed some protein digestion only in the first run, but no protein digestion in the second or the third run. This is as expected, since nonspecific binding via physical adsorption probably reduces enzyme activity, and the binding is not as stable as covalent o binding. As a result, such immobilized enzymes do not have sufficient capacity to carry out repeated digestion reactions.
  • enzymes may be coupled to the surface of a high throughput nanoscale reactor and used to perform repeated enzymatic reactions, e.g., a proteolytic digestion, as described hereabove.
  • HPLC high performance liquid chromatography
  • FIG. 35 A representative HPLC chromatogram is shown in FIG. 35.
  • the profile of cytochrome C digests obtained on capillary cassettes is identical to literature results (Neue, U.D.; Zoubair, M.; Fallah El, HPLC Columns: Theory, Technology, and Practice, VCH Publishing, 1997). 5

Abstract

La présente invention concerne des méthodes de préparation de réactions à l'échelle nanométrique à l'aide d'acides nucléiques ou de protéines. Lesdits acides nucléiques sont capturés de manière saturable, mais réversible, sur la surface intérieure de la chambre de réaction, généralement un capillaire. L'acide nucléique en excès est éliminé et la réaction est mise en oeuvre directement dans le capillaire. Des protéines sont capturées de manière spécifique et saturable sur la surface intérieure modifiée de la chambre de réaction, généralement un capillaire. Les protéines en excès sont éliminées et la réaction est mise en oeuvre directement dans le capillaire. L'invention concerne également des dispositifs de mise en oeuvre des méthodes de l'invention et un système conçu de manière avantageuse pour utiliser lesdites méthodes pour des réactions à rendement élevé impliquant des acides nucléiques ou des protéines.
EP03713412A 2002-02-08 2003-02-07 Methode et appareil de mise en oeuvre de reactions inferieures a un microlitre a l'aide d'acides nucleiques ou de proteines Withdrawn EP1567656A2 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US35566002P 2002-02-08 2002-02-08
US35564802P 2002-02-08 2002-02-08
US355648P 2002-02-08
US355660P 2002-02-08
US10/262,476 US6927045B2 (en) 1999-08-02 2002-09-30 Methods and apparatus for template capture and normalization for submicroliter reaction
US262476 2002-09-30
PCT/US2003/003986 WO2003066667A2 (fr) 2002-02-08 2003-02-07 Methode et appareil de mise en oeuvre de reactions inferieures a un microlitre a l'aide d'acides nucleiques ou de proteines

Publications (1)

Publication Number Publication Date
EP1567656A2 true EP1567656A2 (fr) 2005-08-31

Family

ID=27739086

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03713412A Withdrawn EP1567656A2 (fr) 2002-02-08 2003-02-07 Methode et appareil de mise en oeuvre de reactions inferieures a un microlitre a l'aide d'acides nucleiques ou de proteines

Country Status (5)

Country Link
EP (1) EP1567656A2 (fr)
JP (1) JP2005525797A (fr)
AU (1) AU2003217369A1 (fr)
CA (1) CA2474429A1 (fr)
WO (1) WO2003066667A2 (fr)

Families Citing this family (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050282270A1 (en) * 2004-06-21 2005-12-22 Applera Corporation System for thermally cycling biological samples with heated lid and pneumatic actuator
CA2564310A1 (fr) * 2005-11-07 2007-05-07 Nisshinbo Industries, Inc. Pointe pour reactions biomoleculaires
CA2742598A1 (fr) * 2008-11-04 2010-05-14 Blood Cell Storage, Inc. Extraction de l'acide nucleique sur des surfaces de verre courbes
EP2848309B1 (fr) * 2013-09-13 2020-06-10 NanoTemper Technologies GmbH Support pour capillaires
US9993819B2 (en) 2014-12-30 2018-06-12 Stmicroelectronics S.R.L. Apparatus for actuating and reading a centrifugal microfluidic disk for biological and biochemical analyses, and use of the apparatus
GB201501603D0 (en) * 2015-01-30 2015-03-18 Norwegian University Of Life Sciences And Hedmark University College Method and product
CN213301769U (zh) * 2019-08-01 2021-05-28 利多(香港)有限公司 单人份质控品的保存装置
CN114441264B (zh) * 2022-01-20 2023-05-30 复旦大学 一种皮升级体积单细胞样品裂解酶解反应器

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5846727A (en) * 1996-06-06 1998-12-08 Board Of Supervisors Of Louisiana State University And Agricultural & Mechanical College Microsystem for rapid DNA sequencing
US6423536B1 (en) * 1999-08-02 2002-07-23 Molecular Dynamics, Inc. Low volume chemical and biochemical reaction system
US6290995B1 (en) * 2000-02-08 2001-09-18 Zhao Xinxian Plant drug for preventing cancer II

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03066667A3 *

Also Published As

Publication number Publication date
CA2474429A1 (fr) 2003-08-14
JP2005525797A (ja) 2005-09-02
AU2003217369A1 (en) 2003-09-02
AU2003217369A8 (en) 2003-09-02
WO2003066667A2 (fr) 2003-08-14
WO2003066667A3 (fr) 2005-06-02

Similar Documents

Publication Publication Date Title
US7138254B2 (en) Methods and apparatus for performing submicroliter reactions with nucleic acids or proteins
US6927045B2 (en) Methods and apparatus for template capture and normalization for submicroliter reaction
US7238323B2 (en) Microfluidic sequencing systems
US5716825A (en) Integrated nucleic acid analysis system for MALDI-TOF MS
US6403338B1 (en) Microfluidic systems and methods of genotyping
US8014961B2 (en) Integrated systems and methods for diversity generation and screening
US20030027352A1 (en) Multiple-site reaction apparatus and method
US20030017467A1 (en) Multiple-site sample-handling apparatus and method
AU6884198A (en) Closed-loop biochemical analyzers
US9790547B2 (en) Integrated microfluidic and solid state pyrosequencing systems
EP1567656A2 (fr) Methode et appareil de mise en oeuvre de reactions inferieures a un microlitre a l'aide d'acides nucleiques ou de proteines
US20050106596A1 (en) Nucleic acid analysis methods conducted in small reaction volumes
US7341865B1 (en) Liquid delivery devices and methods
US20100203596A1 (en) Hybridization chamber for bioassay and hybridization method using the hybridization chamber
WO2003018753A2 (fr) Appareil a sites multiples et procede de traitement d'un echantillon
US20050170361A1 (en) Method of preparing reaction regions for biochips
Eickhoff et al. Eckhard Nordhoff, Lajos Nyarsik, Günther Zehetner, Wilfried Nietfeld, and Hans Lehrach

Legal Events

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

Free format text: ORIGINAL CODE: 0009012

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

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

17P Request for examination filed

Effective date: 20040818

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO

18W Application withdrawn

Effective date: 20050811