EP1015116A1 - Procede et dispositif destines a la production de cycles thermiques et a l'automatisation de la preparation d'echantillons au moyen de ces cycles thermiques - Google Patents

Procede et dispositif destines a la production de cycles thermiques et a l'automatisation de la preparation d'echantillons au moyen de ces cycles thermiques

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Publication number
EP1015116A1
EP1015116A1 EP97919230A EP97919230A EP1015116A1 EP 1015116 A1 EP1015116 A1 EP 1015116A1 EP 97919230 A EP97919230 A EP 97919230A EP 97919230 A EP97919230 A EP 97919230A EP 1015116 A1 EP1015116 A1 EP 1015116A1
Authority
EP
European Patent Office
Prior art keywords
reaction
reaction mixture
reaction vessel
vessels
temperature
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Withdrawn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
EP97919230A
Other languages
German (de)
English (en)
Inventor
James M. Dunn
James Leushner
John A. Renfrew
Paul Waterhouse
Alexandre M. Izmailov
Henryk Zaleski
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.)
Visible Genetics Inc
Original Assignee
Visible Genetics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/640,672 external-priority patent/US5789168A/en
Priority claimed from US08/684,498 external-priority patent/US5830657A/en
Application filed by Visible Genetics Inc filed Critical Visible Genetics Inc
Publication of EP1015116A1 publication Critical patent/EP1015116A1/fr
Withdrawn legal-status Critical Current

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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
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • B01L7/52—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • B01L7/525—Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples with physical movement of samples between temperature zones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N2035/00178—Special arrangements of analysers
    • G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks

Definitions

  • the present invention relates to a method and apparatus for automating nucleic acid sample preparation.
  • Sequencing of nucleic acids is generally performed today using the chain termination sequencing method first described by Sanger et al. This process generally involves combining a sample to be analyzed with reagents necessary for synthesis of nucleotide fragments indicative of the sequence of DNA in the sample, i.e. a polymerase enzyme, nucleoside feedstocks and a dideoxynucleoside for chain termination, followed by analysis of the fragments produced by gel electrophoresis.
  • the sample may be initially amplified, i.e., by polymerase chain reaction (PCR) amplification, to increase the amount of a selected part ofthe DNA in an original sample prior to performing the sequencing reaction.
  • PCR polymerase chain reaction
  • Thermally stable polymerases such as Taq Polymerase (Hoffman-La Roche, Inc.) and improved thermal stable chain-termination sequencing enzymes such as Thermo-SequenaseTM (Amersham Life Science, Cleveland) are used to enhance these procedures. These enzymes are utilized in conjunction with multiple successive thermal cycles for amplification or sequencing.
  • Beckman Instruments. Inc. provides the Biomek® 2000 automated pipetting apparatus, that can automate the sample preparation steps for PCR or DNA sequencing reactions in a 96 well microtiter plate using a group of eight pipetting tips. Trays containing reagents or samples are arranged for sequential liquid transfer functions.
  • Another pipette robot the QIAGEN BioRobotTM 9600 (QIAGEN Inc., Chatsworth CA). can prepare 96 bacterial minipreps in 2 hours. These robots all use a cooling plate to keep the reagents and samples at controlled temperatures (usually 4 degrees C) during sample preparation.
  • the reagent trays prepared in an apparatus of this type are then generally transferred to a separate instrument for purposes of thermal cycling.
  • a separate instrument for purposes of thermal cycling.
  • the RoboCyclerTM Gradient 96 System (Stratagene, Inc.) has 4 different temperature blocks and a lifter that moves a tray of up to 96 tubes from block to block in sequence. In this way, the apparatus cycles reaction mixtures through a series of temperature increases and decreases as appropriate for amplification or sequencing reactions.
  • Heating of the temperature blocks in thermal cycling apparatus has been accomplished in a variety of ways, including heating and cooling using liquid flow or electronic means (e.g., resistive heating).
  • An air flow thermal cycling mechanism is made by Idaho Technology (Idaho Falls. Idaho).
  • capillary tubes containing the desired reaction mixtures are melt-sealed at both ends with a Bunsen burner.
  • the permanently sealed capillaries are inserted through and held in a rubber plug.
  • the plug is placed into an opening in a heat chamber, such that the sealed capillaries are suspended in the heat chamber without touching the walls of the chamber.
  • Air heated to the desired temperature is circulated through the heat chamber, thereby heating and cooling the capillaries as programmed.
  • the capillaries may be removed from the rubber plug.
  • the capillaries are then broken at both ends, and the reaction mixture is extracted for analysis or for mixing with further reagents.
  • Drawbacks of using this apparatus include that the handling of the capillaries is time consuming and that the procedure requires the use of multiple capillaries for each treatment of the same sample.
  • the Vistra DNA Labstation 625 (Molecular Dynamics, Sunnyvale CA) is a pipette robot that can prepare bacterial minipreps and PCR and DNA sequencing reactions in a 96 well microtiter plate.
  • the Labstation 625 has an integrated Peltier-block thermocycler for thermal- cycling steps. Using this apparatus, a technician can prepare a sequencing experiment in about 10-15 minutes, and then start the thermocycling procedure.
  • This apparatus uses tubes, and places a layer of oil on top of the reactions to reduce loss of sample during heating.
  • One of the drawbacks of this device is the long time required for temperature changes due to the use of a heating block.
  • thermocycler which can be combined with a sample preparation apparatus if desired. It is another object of the present invention to provide an apparatus and method for automating the preparation of nucleic acid samples for rapid throughput.
  • the present invention provides an apparatus and method for thermally cycling a reaction mixture in a reaction vessel to expose the mixture to the varying temperatures necessary to, for example, achieve PCR amplification or the preparation of sequencing fragments using a cycle sequencing operation.
  • the invention makes use of flow-through reaction vessels, such as capillary tubes, for preparation and thermal cycling of reaction mixtures.
  • the thermal cycling apparatus ofthe invention provides means for sealing the proximal and distal end of each reaction vessel. The proximal ends can be sealed by coupling to a pump which permits movement ofthe samples within the reaction vessels.
  • the reaction vessels are sealed by pressing the distal end of each vessel against a sealing element with a conformable surface for reversibly sealing the reaction vessels before thermal cycling is begun.
  • This sealing element is suitably a strip of conformable material which provides a plurality of distinct locations against which successive reaction vessels can be pressed, thus reducing the likelihood of contamination between samples.
  • the distal end of each vessel is immersed in the reservoir of liquid, preferably of an oil such as mineral oil that is not miscible with the reaction mixture. The head pressure of the liquid acts to hold the reaction mixture within the vessel.
  • the thermal cycling apparatus of the invention is suitably incorporated as part of an apparatus for performing both sample preparation and thermal cycling.
  • Such an apparatus has a sample preparation region in which sample and reagents are mixed to form one or more reaction mixtures within one or more separate reaction vessels, and a thermal cycling region.
  • a robotic transference element moves the reaction vessels between the sample preparation region and the thermal cycling region, preferably under the control of a microprocessor controller.
  • the apparatus further includes a pump which is used to draw reagents and sample into and out ofthe reaction vessel within the sample preparation region, thus forming the reaction mixtures within the reaction vessels. The reaction vessels are then transferred to the thermal cycling region for thermal cycling.
  • Fig. 1 depicts a side cross-sectional view of a first embodiment of a thermal cycling apparatus having a reversible sealing element in accordance with the invention
  • Figs. 2A and 2B shows a thermal cycling apparatus in accordance with the invention
  • Fig. 3 shows the filling of a reaction vessel for use in a liquid sealed embodiment ofthe invention
  • Fig. 4 shows a thermal cycling apparatus in accordance with the invention
  • Fig. 5 depicts a robotic apparatus having a pipetting region, a thermal cycling region and a robotic transference element
  • Fig. 6 depicts a plurality of reaction vessels connected to a pump; and Fig. 7 depicts an example ofthe placement of samples and reagents in microtiter trays in accordance with an embodiment ofthe present invention.
  • the present invention provides a novel and improved apparatus for thermal cycling of reaction mixtures such as PCR amplification mixtures or cycle sequencing mixtures.
  • the thermal cycling apparatus of the invention includes a holder for receiving one or more flow- through reaction vessels; means for reversibly sealing the ends ofthe vessels; and means for con ⁇ trolling the temperature of reaction mixtures within the vessels.
  • reversibly sealing refers to the formation of a seal at an end of the reaction vessel which can be removed nondestructively and resealed multiple times if necessary without damaging the reaction vessel.
  • heat sealing the end of a capillary tube and then breaking it open to recover the contents ofthe tube is not reversible sealing within the scope of the present invention.
  • the flow-through reaction vessel in which a sample is thermally cycled in accordance with the present invention is hollow and open at both ends.
  • the reaction vessel can be made from any non-reactive substance, including glass, non-reactive metals such as stainless steel, non-reactive plastics, teflon or composite materials.
  • the reaction vessel can also be made as a layered structure with regions of high and regions of low thermal conductivity extending along the axis ofthe reaction vessel.
  • a typical reaction vessel is a capillary tube with a diameter of preferably about 1.1 mm. Suitable capillaries are FisherbrandTM or PyrexbrandTM capillaries sold by Fisher Scientific (Ottawa, Canada). Such capillaries are preferably thin-walled to allow even heat transfer across the wall. They may also be somewhat flexible to prevent breakage when reversibly sealed at the bottom.
  • Fig. 1 shows a first embodiment of the thermal cycling apparatus of the invention.
  • the apparatus includes a thermocychng chamber 123.
  • a flow-through reaction vessel 128 is placed within the chamber 123 for thermal cycling.
  • the distal end 212 of reaction vessel 128 is pressed into contact with a sealing element 410 to reversibly seal the reaction vessel 128 having a reaction mixture 431 therein.
  • Sealing element 410 has a conformable surface 414 against which the distal end 212 of reaction vessel 128 can be placed flush in sealing engagement using a force directed along the axis ofthe reaction vessel which does do not deflect from this axis in order to limit breakage.
  • Conformable surface 414 is made of a non-reactive elastic substance which must be sufficiently deformable to match the imperfections of distal ends 212, e.g., rubber or neoprene. and is preferably sterile to prevent contamination of contaminant-sensitive samples.
  • Conformable surface 414 may be a strip which forms the surface of sealing element 410 or is the sealing element 410.
  • Sealing element 410 may be wide enough to accommodate 8 to 12 or more reaction vessels, as desired. It is also preferably of small size in order to minimize thermal mass, and is preferably located entirely within thermocychng chamber 123.
  • the proximal end 214 of the reaction vessel 128 is also sealed to prevent escape ofthe reaction mixture.
  • This sealing can be done with a conformable surface comparable to that use to seal the distal ends 212.
  • the proximal end 214 is sealed by connection to a pump 216, for example via tubing 218.
  • the pump 216 can be used to increase the pressure above the reaction mixture in the reaction vessel 128 after sealing of the distal end 212, thus reducing the amount of solvent lost during the heating steps ofthe thermal cycling process.
  • a single reaction vessel 128 may be sealed at one time against conformable surface 414. However, it is preferred that multiple reaction vessels 128 be used to increase throughput. Thus, multiple reaction vessels may be present behind reaction vessel 128 along the line of sight.
  • the apparatus of the invention may include means such as driven rollers 418 and 420 for transporting sealing element 410 within thermocychng chamber 123. Operation of rollers 418 and 420 moves sealing element 410 so that a fresh portion of conformable surface 414 is placed in position for sealing engagement with reaction vessel 128 at the start of each reaction, thus reducing the likelihood of contamination of samples.
  • the sealing element may be a cylindrical roll.
  • the cylindrical sealing element is preferably elongated to allow for a plurality of reaction vessels 128 to be sealed against its conformable surface.
  • the cylindrical roll can be rotated after thermal cycling so that a clean portion of its conformable surface is moved into place for contact with distal end 212 ofthe reaction vessel 128.
  • the cylindrical sealing element may in fact be a cylindrical polygon, have a large number of flat faces to better permit sealing engagement with its conform- able surface.
  • a means for transporting this sealing element could be gears to move a member located along the axis of the cylindrical sealing element which protrudes to engage with the gears.
  • thermocychng chamber 123 is connected to a temperature control device, for example a source of temperature-controlled fluid flow, not shown.
  • a temperature control device for example a source of temperature-controlled fluid flow
  • Various types of temperature control devices are known in the art, and any can be used in conjunction with the thermocychng chamber of the present invention. See for example, U.S. Patent No. 5,455,175 to Wittwer et al., which is incorporated herein by reference.
  • fluid is circulated by the temperature control device to quickly change the temperature in the thermocychng chamber.
  • Figs. 2A and 2B shows a further embodiment of a thermal cycling apparatus in accordance with the invention.
  • the temperature control device provides a series of temperature regions, each controlled to provide one ofthe desired temperature regimes of the thermocycle program.
  • the temperature control device provides alternating bands of a high denaturation temperature 505 and a lower annealing/extension temperature 504. Temperature regulation can be achieved with resistive heaters and Peltier devices.
  • the reaction mixture in the reaction vessel is physically moved from one temperature zone to the next. This can be accomplished by shifting the reaction vessel with respect to the temperature control elements, for example by finely adjusted raising and lowering of the reaction vessel using a robotic arm. In the preferred embodiment, shown in Figs. 2A and 2B. however, the movement of the reaction mixture is accomplished by altering the position of the reaction mixture within the reaction vessel.
  • reaction vessel 128 The proximal end of reaction vessel 128 is connected to a pump 216, and the pump is used to load the reaction vessel with a layer of reaction mixture 506 and a layer of a non- miscible liquid 507 such as an oil. As shown in Fig. 3, this can be accomplished by drawing the reaction mixture into a reaction vessel 128 from a vessel containing the reaction mixture 431 covered with the non-miscible liquid 507.
  • the reaction vessel 128 is placed within the apparatus so that the alternating temperature bands 504, 505 extend along the axis of the reaction vessel 128, and the distal end of the reaction vessel is immersed in a reservoir 508 ofthe non-miscible liquid which serves as a reversible seal for the distal end ofthe reaction vessel.
  • the reaction mixture is positioned in alignment with the first high temperature band for denaturation ofthe material in the mixture as shown in Fig. 2A.
  • the pump 216 is activated to draw the reaction mixture, together with the non- miscible liquid, to the next temperature band for annealing/extension as shown in Fig. 2B. This process is repeated through each ofthe bands.
  • the temperature control device can provide multiple bands of each type, up to the number required to perform the desired number of cycles.
  • the number of temperature control bands will be less than the number required to provide an individual band for each cycle, and indeed may be as few as one temperature control band for each temperature regime to established in the cycling program as shown in Fig. 4.
  • the use of fewer temperature control bands permits greater spacing between the bands, which facilitates maintenance of disparate temperatures in the adjacent regions, and permits a greater versatility.
  • the pump 216 is used in either a forward or a reverse direction to draw the reaction mixture back and forth through the temperature control bands.
  • the positioning of the reaction mixture relative to the temperature control bands in a thermal cycling apparatus of Figs. 2A, 2B and 4 can be accomplished through careful regulation of the pump, provided that pump has sufficient sensitivity and fine control.
  • pump 216 is preferably a piston displacement pump with linear actuators. The motor must have sufficient torque to drive the linear actuator, while having sufficient sensitivity to allow precise measurements of very small liquid samples.
  • the Drummond NanojectTM pump (Cat No. 3-00-203-X, Drummond Scientific Co., Broomall, PA) is sensitive to measure nanoliters of fluid.
  • the temperature control bands are suitably arranged to permit intimate contact between the temperature control devices and each individual reaction vessel.
  • the temperature control bands in the apparatus of the invention can be arranged about one or more cylindrical openings that are just sufficient in size to receive a reaction vessel.
  • the temperature control bands may be formed around an elongated rectangular opening that is divided into individual spaces for receiving individual reaction vessels with insulating spacers.
  • the outermost temperature control bands may be spaced apart from the region where the reaction mixture is positioned and set to a substantially higher temperature than the other temperature control bands.
  • These higher temperature bands can be used differentially as an alternative to a pump to shift reaction mixture back and forth within the reaction vessel.
  • heating one of the outermost bands will cause the air pressure at that end of the tube to increase, driving the reaction mixture towards the other ends of the tube.
  • the elevated temperature of these outermost bands will tend to regulate the position ofthe reaction mixture within the desired reaction zone, even when a pump is used to control the position.
  • the temperature ofthe outermost bands in this embodiment is suitably maintained at 100-300 degrees C, preferably about 200 degrees C.
  • the thermal cycling apparatus of the invention are advantageously combined in a single instrument with a sample preparation apparatus to permit automation of both the pipetting and thermal cycling portions of amplification or sequencing reactions.
  • a sample preparation apparatus has a pipetting region in which samples are prepared and a thermocychng region in which the thermocychng apparatus is disposed.
  • robotic mechanisms handle preparation of reaction mixtures within flow-through reaction vessels, which are then transferred in an automated process to and from the thermocychng region.
  • An exemplary apparatus according to this aspect of the invention is shown in Figs. 5-7.
  • Fig. 5 depicts a robotic apparatus 100 having a pipetting region 1 10, a thermal cycling region 1 12 and a robotic transference element 1 14.
  • Robotic apparatus 100 has X, Y and Z displacement axes as shown.
  • Pipetting region 1 10 has container support members 1 16 for receiving sample containers such as microtiter plates 120 having wells 122.
  • Container support members 1 16 are preferably temperature controlled for maintaining the optimum temperature of samples and/or reagents within the sample containers.
  • Thermal cycling region 1 12 has a thermocychng chamber 123 accessible to reaction vessels through opening 124.
  • Robotic transference element 1 14 includes reaction vessel holder 126 which can pick up and hold reaction vessels such as reaction vessels 128 which are shown in detail in Figure 6.
  • Y axis attachment piece 130 suspends reaction vessel holder 126.
  • Y axis attachment piece 130 is movable along the Y axis along the underside of X axis attachment piece 132 so that reaction vessels 128 can access different sample containers located along the Y axis, e.g., either of microtiter plates 120 shown.
  • X axis attachment piece 132 is movable between tracks 134 and 136 and permits reaction vessels 128 to access different sample containers along the X axis and to move between pipetting region 1 10 and thermal cycling region 1 12. Once positioned over the desired liquid sample in pipetting region 1 10.
  • reaction vessels 128 access liquid sample or reagent by movement of the reaction vessel holder 126 downward in the Z direction, for example using a linear carriage driven by a stepper motor. Movement of robotic transference element 1 14 is determined by a controller 140 which sends signals to robotic transference element 1 14 to perform various functions in accordance with a program of sample pipetting and transference operations.
  • reaction vessels 128 are used simultaneously to increase sample throughput.
  • reaction vessels 128 are held by reaction vessel holder 126.
  • Reaction vessels 128 have distal ends 212 and proximal 214 which are open.
  • Proximal ends 214 are connected to a sensitive two way pump 216, for example by way of tubing 218. to permit withdrawal or expulsion of liquid from reaction vessels 128.
  • pump 216 is preferably a piston displacement pump with linear actuators which has sufficient torque to drive the linear actuator, while having sufficient sensitivity to allow precise measurements of very small liquid samples.
  • a single pump may be used for all reaction vessels as shown, or each capillary may have its own dedicated pump.
  • Wells 122 of a microtiter plate 120 are loaded with reagents for selected thermal cycling reactions, for example: PCR reagents such as primers, nucleotides, buffers, salts and enzyme; DNA sequencing reagents such as primers, nucleotides, buffers, salts and enzyme; and/or stop reagents such as formamide and visible dye, used for inhibiting enzyme action.
  • the initial preparation of a microtiter plate 120 may be performed by the robotic transference element 1 14 of the apparatus of Figs. 5-7 or by a commercially available automated sample pipetting instru ⁇ ment. The steps involve sequential pipetting and combining of different reactants. Reagents are typically positioned in a fashion convenient for sequential utilization.
  • Fig. 7 depicts an example of the placement of samples and reagents in microtiter trays in accordance with an embodiment of the present invention.
  • Reagents for nucleic acid sample preparation are present in microtiter reagent plate 301.
  • a variety of reagents are shown in alternating columns of wells.
  • PCR reagent is present in a column of wells 302.
  • the PCR reagents in the wells generally include all the reaction components required to allow for a PCR reaction of the sample DNA. This may include the following: IX Taq polymerase Buffer (final: 10mM Tris-HCl, pH 8.3. 50 mM KCl. 1.5mM MgC12. 0.001% gelatin); 8 pMol each primer
  • Sequencing reaction mixture is present in a column of wells 303 and stop solution is present in columns of wells 304.
  • the sequencing reaction mixture will include at least a chain terminating nucleoside, and may also include additional sequencing enzyme and feedstocks.
  • Sample microtiter plate 305 is loaded with sample DNA to be tested.
  • reaction vessels 128 are moved in the X direction to above opening 124 in the thermal cycling region.
  • the necessary samples and reagents are first loaded into containers in pipetting region 1 10.
  • reaction vessels is most suitable for use with standard microtiter plates.
  • a small volume, for example 0.25 microliters, of DNA from plate 305 is drawn into each capillary 128.
  • Distal ends 212 are moved upward from the sample wells by robotic transference element 1 14 and are then positioned over reagent plate 301 , for example, over the column of PCR reagent wells 302. From this position, the DNA sample is expelled into wells 302 and fully drawn up and down two or more times to mix the DNA sample and the PCR reagent.
  • a suitable reaction volume, for example 2 microliters, of reaction mixture is drawn into each reaction vessel 128.
  • the distal ends 212 are removed from the wells and the reaction mixture is drawn up each capillary approximately 2 cm.
  • Robotic transference element 1 14 then moves each reaction vessel 128 to opening 124 of thermal cycling region 1 12.
  • Reaction vessels 128 are lowered into the thermocychng chamber 123 and ends 212 are pressed against a fresh portion of the conformable surface 414 to seal ends 212 of reaction vessels 128.
  • air pressure within the reaction vessels is advantageously increased to a level higher than the vapor pressure of the sample to prevent solution from escaping, especially during the high temperature period of the thermal cycles. This can be done using the pump 216 attached to ends 214.
  • the air pressure can be set at a fixed level and maintained throughout the temperature cycles, or it may be dynamic, varying as the temperature is increased and decreased.
  • the sealed reaction vessels 128 are then exposed to a programmed series of thermal cycles, generally including at least an annealing/extension phase at a relatively low temperature (i.e. 37 to 72°C). and a denaturation phase at a relatively high temperature (i.e., 90 to 96 °C).
  • reaction mixture is brought to room temperature or below and the air pressure inside the capillary is returned to standard.
  • the air ⁇ tight seal is broken by separating the ends 212 of the reaction vessels 128 from the conformable surface 414. This is automatically accomplished by robotic transference element 114 moving the reaction vessels away from sealing element 410.
  • the conformable surface remains intact upon breaking the seal with each of the reaction vessels 128.
  • Reaction vessels 128 are then removed from the thermal cycler by means of the robotic transference element 1 14.
  • reaction vessels 128 are moved to another column of wells 304 in reagent plate 301 containing stop reagents.
  • the stop reagents may be, for example, dextran blue in formamide.
  • Reaction mixture is expelled from the reaction vessels 128 into the wells 304 and drawn up and down two or more times to mix. This sample should be heat denatured for best results.
  • Two microliters of reaction mixture is drawn into each of the reaction vessels 128, capillary ends 212 are removed from the wells 304 and the mixture is drawn up each of the reaction vessels 2 cm.
  • the robotic transference element 1 14 then moves the reaction vessels 128 back into the thermal cycling chamber 123. Reaction vessels 128 are again sealed against the conformable surface 414.
  • reaction mixtures are then subjected to circulating air at 90° C to denature the sample. More commonly, however, sample preparation will not be complete after only one set of thermal cycles, but will require the addition of further reagents and a further set of thermal cycles, for example for sequencing. In this case, after the initial reactions, rather than proceeding to the stop solution wells 304. the ends 212 of reaction vessels 128 are placed in the sequencing reaction mixture in wells 303.
  • Each sequencing reaction mixture contains one species of chain terminating dideoxynucleotide (either ddATP; ddCTP; ddGTP or ddTTP), and may contain other reaction components such as: Thermo SequenaseTM buffer (final: 26 mM Tris-HCl, pH 9.5, 6.5 mM MgCl 2 ); ⁇ 30ng/5pM Fluoresceinated sequencing primer; distilled water; Thermo SequenaseTM enzyme; plus desired amounts of dNTPs.
  • Thermo SequenaseTM buffer final: 26 mM Tris-HCl, pH 9.5, 6.5 mM MgCl 2
  • ⁇ 30ng/5pM Fluoresceinated sequencing primer distilled water
  • Thermo SequenaseTM enzyme plus desired amounts of dNTPs.
  • reaction vessels 128 The products of the initial thermal cycling reactions are thoroughly mixed by successive drawing and expulsion from the reaction vessels 128. Finally, a suitable reaction volume, for example 2 microliters, is drawn into capillary 128; the capillary ends 212 are removed from the wells and the samples are drawn about 2 cm up into the capillary. The reaction vessels 128 are transferred to thermal cycling region 1 12 and lowered into the thermal cycling chamber 123. The ends 212 are pressed into the conformable surface 414. Air pressure is again increased inside reaction vessels 128 to prevent evaporation of sample, and the second round of thermal cycling then is carried out.
  • a suitable reaction volume for example 2 microliters
  • a suitable regime for thermal cycling in a sequencing reaction is: heat to 94 °C for 2 minutes 25 cycles of 94 °C for 30 seconds; 50 °C for 10 seconds; 70°C for 30 seconds; and finish at 70°C for 2 minutes, although persons skilled in the art will understand that these temperatures and times, as well as the number of cycles, depend on the specific material being sequenced and the enzyme being employed.
  • the samples are returned to room temperature or below and the air pressure inside reaction vessels 128 is returned to ambient.
  • the reaction vessels are then removed from the thermal cycler.
  • the conformable surface 414 advances so that a fresh section is in place. At this point the sample may be mixed with stop solution, as described above, or further reactions may be performed.
  • the robotic transference element 1 14 may load the samples directly into a sample analyzing instrument, such as an automated DNA sequencing apparatus or into an electrophoresis analytical system.
  • a sample analyzing instrument such as an automated DNA sequencing apparatus or into an electrophoresis analytical system.
  • the robotic arm aligns to the loading site and expels the denatured reaction products into the loading site.
  • the method and apparatus of the present invention are well-suited to performing cycle sequencing reactions and amplification reactions, and in particular to carrying out a number of successive reactions on the same sample in which the same reaction vessel can be used throughout.
  • the processes of amplifying and sequencing nucleic acid samples for clinical analysis may progress automatically in the same reaction chamber in accordance with the present invention after initial setup and programming.
  • the method and apparatus of the invention are also well-suited for single-tube/single-reaction preparation of DNA sequencing fragments as described in U.S. Patent Application Serial No. 08/640,672 filed May 1 , 1996, and

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Abstract

On décrit un dispositif et un procédé de cyclisation thermique d'un mélange réactionnel, dans un récipient de réaction, afin d'exposer ce mélange à des températures variables, nécessaires, par exemple, pour exécuter une amplification PCR ou pour préparer des fragments de séquençage à l'aide d'une technique de séquençage par cycles. Ce procédé de cyclisation consiste à utiliser des récipients de réaction à écoulement traversant, tels que des tubes capillaires, pour la préparation et le cyclisation thermique des mélanges réactionnels. Afin d'empêcher toute perte du mélange réactionnel à partir des récipients de réaction, lors du chauffage, l'appareil de cyclisation thermique de l'invention est pourvu de moyens destinés à sceller de manière réversible les extrémités distale et proximale de chaque récipient de réaction. On peut sceller l'extrémité proximale en la couplant à une pompe qui permet la circulation des échantillons à l'intérieur des récipients de réaction. Quant à l'extrémité distale des récipients de réaction, on peut la sceller en la comprimant contre un élément de scellement présentant une surface adaptable, ou en l'immergeant dans un réservoir de liquide, de préférence une huile, qui n'est pas miscible avec le mélange réactionnel.
EP97919230A 1996-05-01 1997-05-01 Procede et dispositif destines a la production de cycles thermiques et a l'automatisation de la preparation d'echantillons au moyen de ces cycles thermiques Withdrawn EP1015116A1 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US640672 1996-05-01
US08/640,672 US5789168A (en) 1996-05-01 1996-05-01 Method for amplification and sequencing of nucleic acid polymers
US08/684,498 US5830657A (en) 1996-05-01 1996-07-19 Method for single-tube sequencing of nucleic acid polymers
US684498 1996-07-19
US703730 1996-08-27
US08/703,730 US5897842A (en) 1996-05-01 1996-08-27 Method and apparatus for thermal cycling and for automated sample preparation with thermal cycling
PCT/CA1997/000302 WO1997040939A1 (fr) 1996-05-01 1997-05-01 Procede et dispositif destines a la production de cycles thermiques et a l'automatisation de la preparation d'echantillons au moyen de ces cycles thermiques

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DE29720432U1 (de) * 1997-11-19 1999-03-25 Heimberg, Wolfgang, Dr., 85560 Ebersberg Roboter
US6780617B2 (en) 2000-12-29 2004-08-24 Chen & Chen, Llc Sample processing device and method
JP4321738B2 (ja) 1998-06-24 2009-08-26 チェン アンド チェン エルエルシー 液体試料試験システム
US6423536B1 (en) * 1999-08-02 2002-07-23 Molecular Dynamics, Inc. Low volume chemical and biochemical reaction system
WO2003022435A2 (fr) 2001-09-11 2003-03-20 Iquum, Inc. Tubes echantillons
DE10222228A1 (de) 2002-05-16 2003-11-27 Roche Diagnostics Gmbh Mikropumpe mit Heizelementen für einen pulsierten Betrieb
EP1603674B1 (fr) 2003-02-05 2016-01-06 Iquum, Inc. Traitement d'echantillons
KR101368463B1 (ko) 2010-04-23 2014-03-03 나노바이오시스 주식회사 2개의 열 블록을 포함하는 ｐｃｒ 장치
US11241692B2 (en) * 2017-08-01 2022-02-08 Mgi Tech Co., Ltd. Gene sequencing reaction device, gene sequencing system, and gene sequencing reaction method

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CA2252571A1 (fr) 1997-11-06
AU2378097A (en) 1997-11-19

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