EP1203240A2 - Robot de laboratoire, procede et trousse de reactifs permettant d'isoler des acides nucleiques - Google Patents

Robot de laboratoire, procede et trousse de reactifs permettant d'isoler des acides nucleiques

Info

Publication number
EP1203240A2
EP1203240A2 EP00949475A EP00949475A EP1203240A2 EP 1203240 A2 EP1203240 A2 EP 1203240A2 EP 00949475 A EP00949475 A EP 00949475A EP 00949475 A EP00949475 A EP 00949475A EP 1203240 A2 EP1203240 A2 EP 1203240A2
Authority
EP
European Patent Office
Prior art keywords
particles
nucleic acids
reagent
laboratory
robot
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Withdrawn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
EP00949475A
Other languages
German (de)
English (en)
Inventor
Clemens Bergmann
Gerhard Bienhaus
Mario Berger
Jörg BÖTTGE
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.)
Bilatec Gesellschaft Zur Entwicklung Biotechnologischer Systeme MBH
Original Assignee
Bilatec Gesellschaft Zur Entwicklung Biotechnologischer Systeme MBH
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 DE19937607A external-priority patent/DE19937607A1/de
Priority claimed from DE2000117802 external-priority patent/DE10017802A1/de
Application filed by Bilatec Gesellschaft Zur Entwicklung Biotechnologischer Systeme MBH filed Critical Bilatec Gesellschaft Zur Entwicklung Biotechnologischer Systeme MBH
Publication of EP1203240A2 publication Critical patent/EP1203240A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • G01N35/0099—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor comprising robots or similar manipulators
    • 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
    • G01N35/0098—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation

Definitions

  • the invention relates to a laboratory robot with at least one robot arm which can be moved in a predetermined working area and on which a gripping device for gripping at least one functional unit to be moved by the robot arm is arranged.
  • the invention further relates to a method for isolating nucleic acids from a biological sample containing compartments containing nucleic acids as well as a method for amplifying and / or determining nucleic acids by means of PCR, and finally to a reagent kit for isolating nucleic acids from a biological sample containing compartments containing nucleic acids.
  • PCR polymerase chain reaction
  • sample preparation which contains the nucleic acids from a wide variety of materials such as saliva, blood, liquor; Organisms such as bacteria, yeasts, fungi but also plant material, meat and other nucleic acid-containing samples are released and prepared so that the PCR process can be carried out without interference.
  • sample preparation is currently the bottleneck for widespread use because, above all, there are no automated processes.
  • US Pat. No. 5,705,628 describes magnetic particles which carry carboxyl groups on the surface as adsorbers for nucleic acids.
  • the use of such particles is described in a multi-step process, in which a lysis buffer for digestion of the sample is first used in a staggered sequence, then the magnetic particle suspension and then a special buffer that adjusts the polarity and hydrophobicity of the resulting solution. that nucleic acids bind reversibly to the magnetic particles is added.
  • It is a highly viscous buffer that contains polyethylene glycol (20%) together with 2.5 M sodium chloride (hereinafter referred to as PEG buffer).
  • the nucleic acid After washing the magnetic particles one or more times and resuspending the magnetic particles in an elution buffer, the nucleic acid is desorbed and can be removed after the magnetic particles have been separated off.
  • the robot arm must be able to be coupled with a whole range of different functional units in order to be able to treat the samples in the desired manner.
  • a microtitration plate-like composite vessel cf. US Pat. No. 4,515,795
  • the laboratory robots described above are designed in such a way that they exert forces in the vertical direction (Z direction) of 15 N to 40 N, preferably 20 N to 30 N , can exercise.
  • a laboratory robot is known from EP-A-0 557 828 and parallel US-A-5,472,669, in the working area of which a plurality of functional units are kept at a predetermined parking position.
  • a gripping device is attached to the robot arm of this laboratory robot, with the aid of which the functional units can be taken from their parking positions as required and, after the functional step to be carried out with them, can be parked again at the parking position.
  • the gripper device known from these documents it can happen that when the functional unit is picked up from the parking position, during the execution of the functional step or when the functional unit is parked again at the parking position, its orientation relative to Robot arm changes. This can lead to malfunctions or even complete inability to operate the laboratory robot.
  • Another object is to provide a way to make sample preparation more effective while avoiding the disadvantages of the prior art.
  • a laboratory robot of the type mentioned in the introduction in which an orientation device is also provided on the robot arm, which is equipped with a function if desired, the counter-orientation device provided cooperates in order to impart a predetermined fixed relative orientation to the functional unit and the robot arm, and / or in which at least two functional units are provided, at least one of the functional units having such a counter-orientation device interacting with the orientation device of the robot arm. If there is still the possibility of rotation of the functional unit in the known laboratory robot after gripping the functional unit by the gripping device of the robot arm, such rotation is prevented in the laboratory robot according to the invention by the interaction of the orienting and counter-orienting device.
  • the orientation device and the counter-orientation device interact with one another by means of forces that can be switched on and off.
  • the gripping device or the robot arm and the functional unit could be coupled to one another electromagnetically after the functional unit has been gripped.
  • the desired interaction of the orienting and counter-orienting device can, however, be accomplished in a structurally and control-technically simple manner by simple mechanical intervention of the orienting and counter-orienting device.
  • one of the devices can comprise an orientation mandrel
  • the respective other device namely the counter-orientation device or the orientation device
  • the gripping device is also to be used for concealing vessels containing the samples, it is advantageous if the orientation mandrel is arranged on the functional unit and the receptacle on the robot arm or the gripping device.
  • the construction space taken up jointly by the gripping device and the orientation device only exceeds the construction space taken up by the gripping device alone slightly, so that the possibilities of movement of the robot arm, which is preferably essentially freely movable in space in a predetermined working area of the laboratory robot, are practically not restricted by the orientation device.
  • the engagement of the orienting mandrel and the receptacle can be produced when the gripping device approaches the functional unit, it being possible to facilitate engagement by providing at least one bevel on the orienting mandrel and / or the receptacle.
  • the gripping device can be designed in the manner of a capping gripping device.
  • the gripping device can be elongated and, if desired, essentially cylindrical symmetrical and comprise at least one gripping tool and an actuating device for actuating the gripping tool, the gripping tool being arranged essentially within a projection of the actuating device in the direction of the longitudinal axis of the gripping device.
  • Such a capping gripping device are known, for example, from the publications EP-A-0 734 768 already mentioned above (or DE-U-295 05 652, DE-U-295 05 707 and DE-U-295 1 5 990), EP- A-0 676 643 (or DE-A-44 1 2 286) and DE-A-1 98 01 1 78 are known.
  • an engagement point for the gripping device can be provided on the functional unit, for example an engagement pin or an attack recess.
  • the gripping device can be actuated electrically, for example electromagnetically, and / or hydraulically and / or pneumatically.
  • the actuating device in the manner of a ballpoint pen or of a mechanical pencil, ie by a first start against a resistance, the gripping device can be changed from a release state to a grip state and by a renewed start against a resistance again in the release state.
  • the resistance required for "switching" the gripping device is opposed by the functional unit placed on a base plate of the working area of the laboratory robot in a parking position even to the movement of the robot arm or the gripping device, if this or this when the functional unit is picked up in a simple manner Downward movement hits the functional unit or the functional unit hits the base plate when it is parked in its assigned parking position.
  • the laboratory robot according to the invention can comprise a whole series of different functional units.
  • At least one of these functional units can comprise, for example, a fork frame for transporting containers containing the samples, preferably a fork frame for transporting at least one microtitration plate or a microtitration plate-like composite container or composite container.
  • a fork frame for transporting containers containing the samples
  • the microtitration plate can be picked up from a first position of the work area and transported to any second position, for example a treatment position, and stored there. After this function has been carried out, the fork frame can be put down again in the parking position assigned to it.
  • the microtitration plate can be held at a predetermined distance above the base plate of the work area of the laboratory robot.
  • the gripping device can then be used to discover at least one predetermined container of the microtitration plate before the robot arm is moved to another parking position and fetches another functional unit from there.
  • the further functional unit can comprise, for example, at least the pipette tip receiving part of a pipetting device.
  • various media for example nutrient solutions or liquid reagents or reagents bound to the surface of particles, for example magnetic particles, can be introduced into the sample container.
  • the magnet device can preferably be designed as a functional unit that can be picked up by the robot arm or its gripping device according to the invention and can comprise at least one permanent magnet. If one drives along the reaction vessel with this magnet device, the magnetic particles in the immediate vicinity of the magnet device are deposited on the wall of the reaction vessel and follow their movement. It is thus possible to collect them in a tapered lower end of the reaction vessel. In this way, in particular for vessels with a volume of between approximately 20 ml and approximately 200 ml, preferably between approximately 20 ml and approximately 70 ml, a practical, simple and fully automatic tool for carrying out a magnetic separation can be provided.
  • a barcode reader can be coupled to the robot arm as a further functional unit.
  • Such a barcode reader can take on a wide variety of tasks, for example the detection of sample tubes, modules or rack systems identified by barcodes, which are located in the work area of the laboratory robot.
  • the one from the barcode reader recorded data can be supplied to a control unit, which takes this into account when controlling / regulating the movement of the robot arm and / or the actuation of the gripping device or / and the operation of the magnet device or / and the operation of the pipetting unit.
  • a reagent kit for the isolation of nucleic acids from a biological sample containing nucleic acid-containing compartments which is characterized in that it a) negatively charged particles made of a polymer material, b) a reagent I consisting of a mixture of a charged one or uncharged detergent and an aliphatic alcohol, or / and a reagent II consisting of an aqueous solution of at least one chaotropic salt and optionally an aliphatic
  • Alcohol, and c) contains a proteinase solution.
  • biological, nucleic acid-containing compartments denotes all structures in samples to be examined, from which nucleic acids have to be released by lysis.
  • cells including bacterial or yeast cells, and viruses are included in this definition.
  • the reagent kit according to the invention contains, as reagents I or / and II, compositions with surprisingly simple recipes which are necessary for an effective lysis of the samples, i.e. of the biological, nucleic acid-containing compartments can be used. Furthermore, the reagents I and II contained in the reagent kit according to the invention enable the released nucleic acids to bind to the polymer particles immediately after lysis, without the buffer having to be changed or other substances having to be added. By using neutral cationic or anionic detergents in high concentrations together with aliphatic alcohols, a particularly efficient and complete lysis can be achieved, which is particularly true for samples which contain yeast cells.
  • reagent II When using reagent II containing an aqueous solution of at least one chaotropic salt, particularly effective lysis of samples containing whole blood can be effected.
  • Reagents I and II are also suitable for all other applications, although the better suitability of one or the other reagent can easily be determined in advance by a person skilled in the art for the respective application.
  • the reagent kit according to the invention enables the lysis of cells or viruses and binding of the nucleic acids to particles as well as the separation of the particles from the sample solution in a quasi one-step process in which the sample substance is added to a mixture of particles, reagent I or II and proteinase solution. and after appropriate incubation the sample solution can be separated again, leaving behind the nucleic acids separated from the cells in a form bound to the polymer particles.
  • the reagent kit additionally contains wash buffer and / or elution buffer for separating the nucleic acids from the polymer particles.
  • the reagent set according to the invention thus contains, in a particularly preferred embodiment, all reagents for carrying out nucleic acid isolation from samples. It is possible to initially keep the reagents in the reagent set individually, i.e. in separate bottles, and to prepare a stable working solution only immediately before use. This has the advantage that, depending on the sample, very different lysis principles can be used. It was surprisingly found that a buffer with sodium dodecyl sulfate together with ethanol is particularly advantageous for the isolation of nucleic acid from yeast, while a buffer with guanidinium thiocyanate and possibly ethanol is more suitable for the digestion and isolation of nucleic acids from whole blood.
  • the reagent kit according to the invention therefore contains the reagents A) particles, B) Reagent I with detergent / alcohol mixture, C) Reagent II with high-molar salts of chaotropic compounds and, if appropriate, preferably an aliphatic alcohol, and D) a proteinase solution ,
  • the reagent kit according to the invention can also hold one or more working solutions containing mixtures of A, B and D and of A, C and D ready for use.
  • a reagent kit according to the invention which contains both reagent I and reagent II, is suitable for isolating nucleic acids from a large number of samples, the final preparation of the working solution being carried out by the user in each case based on the application.
  • a reagent kit is also included of the present invention, which contains only one of the two reagents and is therefore particularly suitable for, for example, the isolation of nucleic acids from yeast-containing samples or whole blood.
  • the use of one of the reagents in other samples is not intended to be limited by these preferred uses.
  • further components of the reagent kit according to the invention can be washing solutions for the particles, which are filled in a separate bottle.
  • an elution buffer is required, which should be characterized in particular by its low ionic strength.
  • This component can also be part of the reagent set according to the invention. Both suitable washing solutions and elution buffers are known in principle to the person skilled in the art.
  • a working solution containing components A, B, D or A, C, D is added to a sample and incubated. In individual cases, the incubation can take place at a higher temperature.
  • the working solution simultaneously causes the digestion of the sample, the release of the nucleic acids and the binding to the particles that are part of the working solution.
  • the particles are separated, the clear supernatant is suctioned off and discarded.
  • the particles are then washed in a suitable washing solution, with alcoholic solutions again proving to be particularly advantageous for reasons of simple miscibility.
  • water / ethanol or generally water / aliphatic alcohol mixtures in a ratio of 70 to 30 parts of water with 30 to 70 parts of alcohol give satisfactory results in the context of the invention.
  • the washing process is repeated one or more times and the washed and deposited particles are taken up with a buffer of low ionic strength so that the nucleic acids desorb and go into solution. Then a further processing z. B. for a PCR amplification.
  • the reagent kit contains, as negatively charged particles, those made of polystyrene or polyvinyl alcohol, the particles made of polyvinyl alcohol being a particularly preferred embodiment.
  • particles which cannot be magnetically influenced it is preferred to provide particles which can be magnetically influenced and which can be particularly easily concentrated at a point in the reaction vessel using a magnet when isolating the nucleic acids and of which the remaining sample solution is particularly simple can be separated.
  • the particles in the reagent kit preferably have an average diameter of 0.1 to 100 ⁇ m and in particular 1 to 10 ⁇ m. Particles of this diameter allow good and effective binding of nucleic acids.
  • the negative charge of the particles is based on the presence of carboxyl groups on the surface of the particles.
  • Particles to be used with particular preference in the context of the present invention are e.g. described in EP 0 843 591.
  • Reagent I of the reagent kit according to the invention preferably contains sodium dodecyl sulfate or cetylammonium bromide (CTAB) as detergent, preferably in an amount of 1 to 10% based on the reagent volume.
  • Reagent I preferably contains ethanol as the aliphatic alcohol, a concentration of at least 40% by volume leading to good results and is therefore preferred.
  • Reagent II preferably contains aqueous solutions of guanidinium hydrochloride or thiocyanate, concentrations of these salts of 2 to 8 M being preferred.
  • Reagent II can also contain an aliphatic alcohol as described for Reagent I, which can be advantageous by reducing the viscosity of the sample.
  • the reagents I and II may contain other customary and suitable buffers and / or auxiliary substances.
  • TRIS / HCI may be mentioned as an example of buffer substances; auxiliary substances can e.g. Complexing agents like EDTA.
  • the pH of the reagents is preferably adjusted to approximately the physiological pH.
  • the proteinase contained in the reagent kit according to the invention serves to avoid disturbances due to the presence of proteins after digestion of the biological, nucleic acid-containing compartments.
  • Proteinase K is preferably used for this purpose in the reagent kit according to the invention, the amount of proteinase used in nucleic acid isolation being dependent on the amount of cells present and thus on the amount of proteins. Suitable concentrations of proteinase can easily be determined by the person skilled in the art.
  • the reagent kit according to the invention enables simple and rapid isolation of nucleic acids from samples, in particular for the subsequent implementation of a PCR reaction, and is intended and suitable in particular for use with a laboratory robot according to the invention. Since only sample to z. B. a prepared working solution containing all the other necessary components must be added, after separation of the particles using a magnet, for example, the remaining sample solution can easily be removed and after washing, if necessary, can be eluted to obtain the nucleic acids, a corresponding process can be easily automated. Only about 4 pipetting steps are necessary before the final sample collection.
  • the present invention therefore furthermore relates to a method for isolating nucleic acids from a biological sample containing nucleic acid-containing compartments, which is characterized in that the sample is simultaneously treated with negatively charged particles made of a polymer material and a reagent consisting of a mixture of a charged one or uncharged detergent and an aliphatic alcohol, or consisting of an aqueous solution of at least one chaotropic salt and optionally also an aliphatic alcohol, which separates the particles from the supernatant solution in a suitable manner, optionally washes and either directly or for the nucleic acids bound to the particles other processes, e.g. PCR, used, or detaches the nucleic acids bound to the particles by means of an elution buffer from the particles.
  • a reagent consisting of a mixture of a charged one or uncharged detergent and an aliphatic alcohol, or consisting of an aqueous solution of at least one chaotropic salt and optionally also an aliphatic
  • This method is particularly suitable and advantageous for implementation with the laboratory robot according to the invention.
  • the method according to the invention is carried out using the components already described above for the reagent kit according to the invention, which is why reference is made to the above explanations for a more detailed description of the components. It is preferred to add a proteinase, preferably proteinase K, to avoid interference from proteins.
  • a proteinase preferably proteinase K
  • the particles and reagents used correspond to those described above, as do suitable washing and elution buffers, which are likewise used in the process according to the invention be used. At least 60% ethanol is particularly preferably used for washing.
  • reagent containing detergent and alcohol corresponding to reagent I for the isolation of nucleic acids from samples containing yeasts, whereas a reagent containing chaotropic salts corresponding to reagent II is used in particular to isolate nucleic acids from whole blood.
  • the method according to the invention can in principle be used to isolate DNA or RNA, although isolation of DNA is preferred.
  • the present invention further provides a method for the amplification and / or determination of nucleic acids by means of a polymerase chain reaction, in which the nucleic acid to be amplified is obtained from a sample containing biological, nucleic acid-containing compartments with the aid of the inventive method described above or with the aid of the reagent kit according to the invention described above is isolated.
  • a particular advantage of the invention is to be seen in the fact that the nucleic acids can still be used for a PCR reaction both on the polymer particles and after separation from the polymer particles in the elution buffer.
  • the PCR reaction and / or the identification of the nucleic acids z. B. by sequencing can be carried out according to methods known per se.
  • FIG. 1 shows a perspective view of a first embodiment of a laboratory robot according to the invention
  • FIG. 2 is a view for explaining the structure of a robot arm of a second embodiment of a laboratory robot according to the invention
  • FIG. 3 shows a side view of a gripping device
  • 5a shows a functional unit designed as a fork frame
  • FIG. 5b is a view for explaining the function of the fork frame according to FIG. 5a;
  • FIG. 6 shows a rough schematic view of a functional unit designed as a pipette tip holder
  • 7a is a rough schematic view for explaining the function of a functional unit designed as a magnetic device
  • a laboratory robot according to the invention is generally designated 10. It comprises a base plate 1 2 with a working surface 1 4 and a housing 1 6, in which two robot arms 1 8 and 20 are slidably mounted. Furthermore, a control unit 22 for controlling or regulating the movement of the robot arms 1 8, 20 is accommodated in the housing 1 6.
  • the two robot arms 1 8, 20 each have a main arm 1 8 a and 20 a, which extends substantially orthogonally to a substantially vertical front surface 1 6 a of the housing 1 6 and one end of which in the housing 1 6 horizontally along a path 24 can be moved back and forth (movement in the Y direction).
  • the laboratory robot 10 is therefore a typical representative of the Cartesian XYZ laboratory robot type.
  • a gripping device 26 At the lower end of the secondary arm 1 8b or 20b, a gripping device 26 according to the invention is attached, the operation of which can also be controlled or regulated by the control unit 22 and the structure of which will be explained in more detail below with reference to FIG. 3.
  • a number of functional units which are parked in parking areas 28 of the work surface 14 and whose structure and function will be explained in more detail with reference to FIGS. 4 to 8 can be picked up and used for the treatment of samples.
  • the microtitration plates 30 receiving the samples can be provided, for example, in storage areas 34, 36 of the work surface 1 4 and can be transported into a processing area 38 by means of the robot arm 1 8 or 20.
  • the microtitration plates 30 can then be brought back to one of the storage areas 34, 36. 2 only shows the robot arm 11 8 of another type of laboratory robot for the sake of completeness.
  • the robot arm 1 1 8 has a slide 1 1 8c, which is mounted linearly movable along a rail 1 24.
  • the main arm 1 1 8a and the secondary arm 1 1 8b of the robot arm 1 1 8 are borrowed in a similar manner to a human upper or lower arm on the shoulder or elbow joint on the slide 1 1 8c or on another.
  • a gripping device 1 26 is arranged at the free end of the forearm 1 1 8b.
  • the gripping device 26 or 1 can be designed as described in EP-A-0 734 768 (or DE-U-295 05 652, DE-U-295 05 707 or DE-U-295 1 5 990) or from DE-A-1 98 01 1 78. That it is elongated along an axis A which runs essentially vertically in use and, according to FIG. 3, comprises a gripper 40 and a housing 42. An actuating device 44 is accommodated in the housing 42, which actuates the opening and closing of the gripper by movement of an actuator 46 controls in the direction of the axis A.
  • the actuating device 44 can be designed as an active actuating device which brings about the movement of the actuator 46 by means of a power device, for example an electromagnet.
  • a power device for example an electromagnet.
  • EP-A-0 734 768 it is also possible to design the actuating device 44 according to EP-A-0 734 768 as a passive actuating device which derives the movement of the actuator 46 in the manner of a ballpoint pen or mechanical pencil from a movement of the gripping device.
  • the gripping pliers are arranged essentially within a projection P of the housing 42 in the direction of the axis A enables the gripping device 26 or 1 26 to be slender in the horizontal direction, which means that they move in the area of the working surface 1 4 of the laboratory robot 1 0 relieved.
  • All of the functional units to be explained below have an attack unit 50 (see FIG. 4) which is exclusively intended to interact with the gripping device 26, 1 26.
  • the attack unit 50 has a pin sleeve 52, as is known, for example, from EP-A-0 734 769 from the lids, which close the vessels receiving the samples.
  • the gripping tongs 40 of the gripping device 26, 1 26 engage the pin 52a of this pin sleeve 52 in order to pick up the functional unit and to move it to a specific position of the working area 14 of the laboratory robot.
  • the engagement unit 50 is further provided with a positioning rod 54 which is inserted into a receiving opening 48 on the housing when the gripping device 26, 1 26 approaches the functional unit 42 of the gripping device 26, 1 26 is formed or arranged.
  • This engagement of the positioning rod 54 and the receiving opening 48 ensures that the functional unit always has the desired relative orientation with respect to the gripping device 26, 1 26 or with respect to the movement in the working area 1 4 of the laboratory robot 10 or when carrying out its intended function of the entire laboratory robot 10.
  • inclined surfaces 54a and 48a are formed both on the positioning rod 54 and on the receiving opening 48.
  • the pin sleeve 52 is designed in exactly the same way as the lids which close the vessels receiving the samples has the advantage that the gripping device 26, 1 26 can be used both for transporting the functional units and for concealing the sample vessels.
  • the material of the two journal sleeves is different. While the lids are usually made of deformable plastic, for example polypropylene or the like, the The pin sleeve 52 of the engagement unit 50 is preferably made of metal in order to be able to meet the higher mechanical stress.
  • the receiving opening 48 need not necessarily be an opening that is enclosed on all sides. Rather, it is sufficient if, in cooperation with the positioning rod 54, it prevents the functional unit from rotating about the axis A of the gripping device 26, 1 26. For this purpose, stop surfaces that are essentially orthogonal to the circumferential direction about the axis A are required.
  • FIG. 5a shows a fork frame 60 as a first example of a functional unit which can be used in the laboratory robot 10 according to the invention and which, in the manner of the fork of a forklift, for transporting microtitration plates 30 from the storage areas 34, 36 of the work surface 14 of the laboratory Robot to the treatment area 38 and vice versa.
  • the fork frame 60 is essentially U-shaped, the engagement unit 50 being attached or formed on the base leg 60c of the U-shape, while the other two legs 60a and 60b form the prongs of the receiving fork.
  • the microtitration plates 30 are preferably placed on bearing blocks 62.
  • the distance between the work surface 1 4 and the microtitration plate 30 thereby makes it easier for the fork frame 60 to reach under the microtitration plate 30.
  • the bearing blocks 62 can be used for tempering, ie cooling or heating, the microtitration plates 30. With the help of So-called "hotels" in which a plurality of microtitration plates 30 can be arranged one above the other can finally be realized in the storage areas 34 and 36.
  • the microtitration plate 30 rests on an upper surface 60d of the legs 60a, 60b and 60c of the fork frame 60.
  • the outside of these legs are provided with a web 60e which extends in the illustrated embodiment over the entire length of the legs 60a and 60b and in the end regions of the base leg 60c.
  • the fork frame 60 is one of the most important modules for laboratory robots, since the safe movement of microtitration plates, which are used as reaction vessels, as sample vessels and other tasks, is an important task in the automation of laboratory processes.
  • a pipette tip holder 70 is shown in a rough schematic in FIG.
  • a docking unit 72 for detachably receiving pipette tips 74 is arranged below the pin sleeve 52.
  • a hose 76 coming from a pipetting unit opens into the lower surface 72a of the docking unit 72.
  • the pipette tip holder 70 which is gripped by the gripping device 26, 1 26, together with the robot arm 1 8 or 1 1 8, forms a conventional pipetting robot , Therefore, the exact structure and the exact function of the docking unit 72 need not be explained in more detail here.
  • magnétique particles coated with certain reagents are often used as the solid phase.
  • the magnetic particles generally have a diameter of between approximately 0.5 ⁇ m and approximately 50 ⁇ m and are in the sample volume, which can have a content of the order of 1 ml to 500 ml, preferably 30 ml to 100 ml , usually in suspension.
  • Magnetic separation is now about separating these magnetic particles on the walls of the sample vessel in order to then be able to remove the supernatants remaining in the sample volume, for example by means of the pipetting functional unit 70 described above.
  • the reaction vessel must be brought up to a magnet, usually a permanent magnet, for example made of neodyn. Magnet systems of this type, which are also suitable for use in composite vessels, can be obtained, for example, from the applicant. Reference is also made to WO-A-92/04961.
  • the microtitration plates 30 can be placed on such a magnetic separator without any problems. After the supernatants have been pipetted off, the microtitration plates 30 can then be transported back to a storage area 34, 36 for washing the magnetic particles.
  • FIG. 7a Another variant of the magnetic separation is shown in Fig. 7a, which serves to explain the structure and function of a magnetic separation function unit 80.
  • the magnetic separation functional unit 80 has a permanent magnet 82, which can be made of neodyn, for example.
  • This permanent magnet 82 can be approximated to the upper region 84a of a reaction vessel 84 with the aid of the robot arm 18 under the control of the control unit 22. It remains there for a certain time, which depends strongly on the diameter of the reaction vessel and is usually of the order of 10 seconds to 5 minutes.
  • the permanent magnet 82 can be moved in a plurality of steps along a travel path 86, and it keeps pausing again and again in order to reliably ensure that all magnetic particles that have been collected so far have slipped onto the wall of the reaction vessel. In this way, all magnetic particles are located in the tip 84b of the reaction vessel 84 at the end of the travel path 86.
  • the magnetic separation functional unit 1 80 can also have a plurality of permanent magnets 1 82, which are arranged in a holder 1 88 around an opening 1 88 a for the reaction vessel. With this magnetic separator 1 80, too, the magnetic particles can be collected at the bottom of the reaction vessel.
  • a barcode reading unit 90 is shown in FIG. 8, which comprises a barcode reader 92 attached to the attack unit 50.
  • the data lines connecting the barcode reader 92 to the control unit 22 are not shown in FIG. 8 for the sake of clarity.
  • Barcode readers play a major role in the automation of laboratory processes, since the identification of samples using barcodes is becoming more and more common to enable clear identification. In complex robot environments, it is therefore necessary to transmit the information provided by a barcode reader 92 to the control unit, which can be formed, for example, by a computer, for example a PC, and there to control the movements of the robot arm 18 or 20 as well as the other processes. This is the only way to ensure that the right sample is always subjected to the right treatment.
  • Carboxyl groups as adsorbers and magnetic separation.
  • the particles are then separated off with a magnet, the supernatant is removed and the particles are distilled with 150 ⁇ l 80% ethanol [5054.1, Roth, Düsseldorf]. Washed water. This process is repeated once; then the particles are resuspended in 150 ⁇ l BILATEST elution buffer consisting of 10 mM Tris / HCl pH 7.0 [see above] and 1 mM EDTA [see above], incubated at 65 ° C. for 5 minutes and separated with a magnet as before. The purified nucleic acid is removed in the supernatant. The analysis in a standard agarose flat bed gel (0.8% agarose [A9311, Sigma, Kunststoff]) gives an image as in FIG. 9.
  • a working solution is first prepared from 250 ⁇ g particles of polyvinyl alcohol (manufactured according to EP 0 843 591) in 5 ⁇ l bidist. Water, 5 ul proteinase K solution [1 373 1 96, Röche, Mannheim] and 1 30 ul BILATEST lysis buffer I (SDS) consisting of 5% sodium dodecyl sulfate [L4390, Sigma, Kunststoff], 100 mM Tris / HCI [T2584, Sigma, Kunststoff], 10 mM EDTA [E51 34, Sigma, Kunststoff], 50% ethanol [5054.1, Roth, Düsseldorf].
  • SDS BILATEST lysis buffer I
  • Example 2 was repeated, but instead of centrifuging, the particles were magnetized with a permanent magnet [Fa. Rheinmagnet, Neun Meinn] separated.
  • Example 3 was repeated, the elution being carried out not at 65 ° C. but at room temperature.
  • M marker M-Hindlll 23.1 1 9.41 6.6 i 4.41 2.3 1 2.010.56 KB 5.
  • Elution at RT results in less elution of smaller nucleic acid molecules, e.g. RNA (the bottom two bands in lanes 3 and 4). This selectivity can be used to separate different nucleic acid molecules.
  • Example 3 was repeated, lysis buffers being compared with SDS or with CTAB (cetyltrimethylammonium bromide, 91 61 .1, from Roth, Düsseldorf), in each case with 50% ethanol or without ethanol.
  • CTAB cetyltrimethylammonium bromide, 91 61 .1, from Roth, Düsseldorf
  • the eluate was analyzed with a standard agarose flat bed gel (0.8% agarose [see]) and shown in FIG. Fig. 14
  • CTAB can be used instead of SDS, but without ethanol, genomic DNA does not bind in both cases.
  • cell disruption can also be achieved without ethanol, the low yield in the samples without ethanol is therefore due to the low binding of the DNA in the absence of ethanol.
  • a working solution is first prepared from 250 ⁇ g particles of polyvinyl alcohol (produced according to EP 0 843 591) in 5 ⁇ l bidist.
  • yeast cells can be selectively lysed with the SDS lysis buffer from Examples 1 and 2, while whole blood can be selectively lysed with the guanidine buffer from Example 4.
  • Example 7 was repeated with whole human blood.
  • Example 4 The experiment from Example 4 was repeated with 1 0 8 cells of the human pathogenic yeast Candida albicans. This yeast is characterized by a particularly strong and difficult to lyse cell wall. As a result, the eluate was analyzed with a standard agarose flat bed gel (0.8% agarose [see above]) and shown in FIG. Fig. 18 lane content
  • Example 4 The experiment from Example 4 was repeated with the gram-positive bacterium Lactococcus lactis. Gram-positive bacteria also have a particularly strong and difficult to lyse cell wall. Lysis times from 1 minute to 15 minutes were used.
  • Example 4 The experiment from Example 4 was repeated with agarose particles coated with glutamic acid (G2759, Sigma, Kunststoff). The result was similar to the result of Example 4 shown in FIG. 1 2, but the yield was significantly lower.
  • KV80 5'-GCG GAT CCT TAA GTC CAA TCG TCA AAA TT-3 'KV102: 5'-GCG AAT TCG TAT CTT CTT TGC CCA AGG AA-3'
  • PCR approaches contain the following components:
  • the program includes the following steps:
  • the PCR batches are carried out as described under a). After the PCR, the samples with 1 5-20% gel loading buffer with EDTA [Fa. Sigma, Kunststoff]. The amplificates were analyzed on a standard agarose gel (1.6%) (shown in FIG. 21).
  • a working solution is first prepared from 250 ⁇ g particles of polyvinyl alcohol, prepared according to EP 0 843 591 in 5 ⁇ l bidist.
  • Water 5 ul proteinase K ([1 3731 96, Röche, Mannheim], 20mg ml "1 in 1 0mM Tris-HCl pH7 [see]) and 1 30 ul BILATEST lysis buffer II (Gu-SCN) consisting of 3 M guanidinium thiocynate [G9277, Sigma, Kunststoff], 3% sodium lauroyl sulfate [L91 50, Sigma, Kunststoff], 100 mM Tris / HCl pH 7.0 [see], 1 mM EDTA [E51 34, Sigma, Kunststoff], 50% ethanol [9065, Roth, Düsseldorf].
  • 1 0 l or 25 ⁇ l human whole blood (EDTA, citrate or heparin whole blood) are mixed with 1 40 ⁇ l of the previously described working solution and incubated for 5 min at room temperature.
  • sample volumes of 50 ⁇ l and 100 ⁇ l the erythrocyte lysis described above is carried out first, after the second centrifugation the pellet is resuspended in 140 ⁇ l of the previously described working solution and incubated for 5 min at room temperature.
  • the particles are then drawn to the wall with a permanent magnet and the supernatant removed.
  • the particles are mixed with 1 50 ⁇ l
  • the magnetic particles are resuspended in 1 50 ⁇ l BILATEST elution buffer (10 mM Tris / HCl pH 7.0 [see above], 1 mM EDTA [see above]) or water, incubated at 65 ° C. for 5 minutes and separated as before , The purified nucleic acid is removed in the supernatant.
  • the whole blood samples (EDTA, citrate or heparin whole blood) are mixed in a ratio of 1: 1, 4 with erythrocyte lysis buffer (1 0mM Tris-HCl pH7.5 [T2584, Sigma, Kunststoff], 300mM sucrose) before DNA isolation [9097, from Roth, Düsseldorf], 5 mM MgCl 2 [M2670, from Sigma, Kunststoff], 1% Triton X-1 00 [3051, from Roth, Düsseldorf]) mixed and centrifuged at 1,400 g for 30 s.
  • the pellet is resuspended once in the same buffer and then centrifuged off again.
  • the steps of erythrocyte lysis are currently still carried out manually.
  • a working solution is first prepared from 250 ⁇ g particles of polyvinyl alcohol, prepared according to EP 0 843 591 in 5 ⁇ l bidist.
  • the mixing of the working solution is carried out by the AUTOSPRINT pipetting robot, which takes the reagents from the storage bottles in the reagent block (position 4) and mixes them in a special vessel in the same block
  • the particles are then drawn to the wall with a permanent magnet.
  • the plate is transferred from the AUTOSPRINT to the magnetic separator (position 6), using the special system-specific plate gripper. After the magnetic particles have been separated off (1 min), the supernatant is suctioned off. The plate is transferred back to position 5, then 1 50 ⁇ l washing buffer (1 0mM Tris-HCl pH7.5 [see], 1 mM EDTA [see], 80% ethanol [see]) are added to the particles, the DNA magnetic particle complex is not destroyed .. This process is repeated at least once; the complex is then air-dried for 5 minutes (on the magnetic separator, position 6) to remove ethanol residues.
  • the worktop is transferred to position 5.
  • the particles are mixed with 100 .mu.l elution buffer (10 mM Tris / HCl pH 7.0 [see above], 1 mM EDTA [see above]) or water and incubated for 3 minutes at room temperature.
  • the particles are then resuspended by multiple uptake and delivery and used directly as a PCR template.
  • the thermal treatment for elution and the separation of the magnetic particles are omitted in this test, which leads to a substantial saving in time.
  • the magnetic particles do not influence the PCR reaction up to a final concentration in the PCR mixture of 1 ⁇ g ⁇ l '1 .
  • An HLA-DRB-PCR was carried out with DNA isolated from 50 ⁇ l EDTA whole blood (Biotest test kit). During DNA isolation, erythrocyte lysis was carried out first, the amount of magnetic particles was reduced to 1 00 ⁇ g. 3 ⁇ l of solution with resuspended magnetic particles were used in each 10 ⁇ l PCR reaction (total volume of the resuspended magnetic particles 100 ⁇ l). The approach is such that the DNA is mixed with a finished master mix and water and the resulting mix is distributed on the PCR plate with the dried primers. This step is currently carried out manually, but should also be automated.
  • Reagent block holds one bottle of solutions 1, 2, 3 or 4 [depending on the sample] and 6 as well as two bottles of solution 5.
  • the bottle for mixing the ready-to-use lysis solution by the robot is also in the reagent block).
  • the sample volume can be up to 25 ⁇ l EDTA whole blood (with upstream erythrocyte lysis, the initial volume can be up to 100 ⁇ l EDTA whole blood; the pellet after erythrocyte lysis is either resuspended manually with water or PBS [volume 25 ⁇ l] or before the samples are distributed by the AUTOSPRINT- Robot resuspended in 50 - 100 ⁇ l elution buffer [solution 6]).
  • the magnetic particles are first resuspended by recording and dispensing the full change tip volume five times, then the required amount ([N + 2] x 5 ⁇ l) removed and transferred to the vessel for the lysis solution (position 4).
  • the solution (position 4) is first mixed by taking up and dispensing the full change tip volume five times, then the required amount ([N + 2] x 5 ⁇ l) is removed and transferred to the vessel for the lysis solution (position 4).
  • the lysis solution is mixed with a fresh interchangeable tip by taking it five times and dispensing the full interchangeable tip volume, then 140 ⁇ l each are added to the samples.
  • the change tip does not come into contact with the sample and therefore does not have to be changed.
  • the robot uses a fresh interchangeable tip for each sample to avoid cross-contamination.
  • the tip change can be omitted. Aspiration is carried out by sucking up 1 60 ⁇ l, 5s waiting time and then sucking up the remaining volume from the tip of the THERMOSPRINT vessels. 23. 5 min incubation at room temperature to dry the samples (removal of the residual ethanol from the wash buffer).
  • the magnetic particle suspension from step 26 can be used directly as a PCR template (see HLA-PCR; manual distribution or automated preparation of the PRC reactions by the AUTOSPRINT robot). 16) Isolation of nucleic acids from meat / sausage using
  • BILATEST lysis buffer X (Gu-SCN) consisting of 3 MG uanide in iumthiocyn at [G 9277, Fa. Sig ma, Kunststoff], 3% sodium lauroyisulfate [Fa.
  • the samples are centrifuged off and the supernatant is treated with 1 50 ⁇ l of ethanol [9065, Roth. Düsseldorf] and 250 ⁇ g particles of polyvinyl alcohol, produced according to EP 0 843 591, in 5 ⁇ l bidist. Mixed water.
  • the samples are incubated for 5 minutes at room temperature.
  • the particles are then pulled onto the floor with a permanent magnet and the supernatant removed.
  • the particles are washed with 1 50 ⁇ l washing buffer (1 0mM Tris-HCl pH7.5 [see above], 1 mM EDTA [see above], 80% ethanol [see above]), the DNA-magnetic particle complex not being resuspended. This process becomes repeated at least once; the complex is then air dried for 5 minutes to remove residual ethanol.
  • the magnetic particles are resuspended in 100 ⁇ l water. 2 ⁇ l of these samples are used directly or after elution of the DNA and separation of the particles in a PCR reaction with MT2 and MT1 1 primers (total volume of the PCR mixture 50 ⁇ l).
  • the plant material (wheat leaf pieces) is mechanically crushed in 100 - 300 ⁇ l extraction buffer and the resulting extract is clarified by centrifugation. 75 ⁇ l of the clear supernatant are mixed with a mixture of 10 ⁇ g magnetic particles made of polyvinyl alcohol, produced according to EP 0 843 591, in 75 ⁇ l ethanol (9065, Roth, Düsseldorf) and mixed. The mixture is incubated for 5 min at room temperature for DNA binding.
  • the particles are then pulled onto the floor with a permanent magnet and the supernatant removed.
  • the particles are washed with 1 50 ⁇ l washing buffer (10mM Tris-HCl pH7.5 [see above], 1 mM EDTA [see above], 80% ethanol [see above]), the DNA-magnetic particle complex not being resuspended. This process is carried out at least repeated twice; the complex is then air-dried for 5 minutes to remove ethanol residues.
  • the magnetic particles are then resuspended in 10 ⁇ l BILATEST elution buffer (10 mM Tris / HCl pH 7.0 [see above], 1 mM EDTA [see above]) or water and used directly in the PCR. Analysis of the isolated DNA in a standard agarose flat-bed gel (0.8% agarose [from Sigma, Kunststoff] gives an image like FIG. 27. Trace content

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Abstract

La présente invention concerne un robot de laboratoire présentant au moins un bras de robot pouvant se déplacer dans une zone de travail prédéfinie. Un dispositif de prise (26), placé sur ledit bras, permet de saisir au moins une unité fonctionnelle devant être déplacée par le bras de robot. Selon cette invention, un dispositif d'orientation (48) est également pourvu sur le bras de robot et sur le dispositif de prise (26). Ce dispositif d'orientation coopère avec un dispositif d'orientation contraire qui est éventuellement pourvu sur l'unité fonctionnelle, afin de conférer à l'unité fonctionnelle et au dispositif de prise (26) une orientation relative fixe prédéfinie. Le procédé permettant d'isoler des acides nucléiques à partir d'un compartiment biologique contenant des acides nucléiques, notamment par utilisation d'un robot de laboratoire selon l'invention, consiste à mélanger l'échantillon avec des particules chargées négativement, formées d'un matériau polymère et d'un réactif composé soit d'un mélange d'un détergent chargé ou non et d'un alcool soit d'une solution aqueuse d'au moins un sel chaotrope et d'éventuellement un alcool aliphatique, à séparer, de façon appropriée, les particules de la solution en saillie, à éventuellement laver et à dissoudre les acides nucléiques liés aux particules au moyen d'un tampon d'élution des particules, ou à utiliser lesdits acides nucléiques liés aux particules directement pour un procédé ultérieur, tel qu'une réaction enchaîne de la polymérase (PCR).
EP00949475A 1999-08-09 2000-08-08 Robot de laboratoire, procede et trousse de reactifs permettant d'isoler des acides nucleiques Withdrawn EP1203240A2 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
DE19937607A DE19937607A1 (de) 1999-08-09 1999-08-09 Reagenzienkit zur Isolierung von Nukleinsäuren
DE19937607 1999-08-09
DE2000117802 DE10017802A1 (de) 2000-04-10 2000-04-10 Labor-Roboter mit Vielzweckgreifer
DE10017802 2000-04-10
PCT/EP2000/007711 WO2001010554A2 (fr) 1999-08-09 2000-08-08 Robot de laboratoire, procede et trousse de reactifs permettant d'isoler des acides nucleiques

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US7001724B1 (en) * 2000-11-28 2006-02-21 Applera Corporation Compositions, methods, and kits for isolating nucleic acids using surfactants and proteases
KR20050008720A (ko) * 2002-05-17 2005-01-21 벡톤 디킨슨 앤드 컴퍼니 표적 핵산 서열의 분리, 증폭 및 검출을 위한 자동화 시스템
US20120122099A1 (en) 2003-09-11 2012-05-17 Rangarajan Sampath Compositions for use in identification of bacteria
DE102005047736B4 (de) 2005-09-29 2008-08-14 Aj Innuscreen Gmbh Verfahren und System zur Isolierung von Nukleinsäuren aus beliebigen komplexen Ausgangsmaterialien
US8148163B2 (en) 2008-09-16 2012-04-03 Ibis Biosciences, Inc. Sample processing units, systems, and related methods
CN110208063A (zh) * 2019-06-27 2019-09-06 中国农业科学院农业质量标准与检测技术研究所 一种在线自动化磁性纳米材料震荡-解吸装置

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NL8900725A (nl) * 1989-03-23 1990-10-16 Az Univ Amsterdam Werkwijze en combinatie van middelen voor het isoleren van nucleinezuur.
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