WO2000077510A2 - Procede et dispositif pour electrophorese en gel - Google Patents

Procede et dispositif pour electrophorese en gel Download PDF

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Publication number
WO2000077510A2
WO2000077510A2 PCT/EP2000/005472 EP0005472W WO0077510A2 WO 2000077510 A2 WO2000077510 A2 WO 2000077510A2 EP 0005472 W EP0005472 W EP 0005472W WO 0077510 A2 WO0077510 A2 WO 0077510A2
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WO
WIPO (PCT)
Prior art keywords
gel
samples
electrodes
sample
separating
Prior art date
Application number
PCT/EP2000/005472
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German (de)
English (en)
Other versions
WO2000077510A3 (fr
Inventor
Holger Rauth
Richard Reinhardt
Antje Starke
Original Assignee
MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
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Application filed by MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. filed Critical MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Publication of WO2000077510A2 publication Critical patent/WO2000077510A2/fr
Publication of WO2000077510A3 publication Critical patent/WO2000077510A3/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44782—Apparatus specially adapted therefor of a plurality of samples
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • G01N27/44756—Apparatus specially adapted therefor
    • G01N27/44773—Multi-stage electrophoresis, e.g. two-dimensional electrophoresis

Definitions

  • the invention relates to a method and a device for gel electrophoresis with a gel electrophoresis system in which a large number of samples are applied simultaneously and spatially separated from one another to a flat gel matrix in a buffer chamber and pass through the gel matrix under the action of an electric field.
  • Gel electrophoresis is a well-known analytical separation method in which samples composed of various components pass through a gel under the action of an electric field, the various components with different electrical properties changing in their running speed and thus as a result of the separation in their position in the gel differentiate.
  • Preferred applications arise in biochemistry, biology and medicine, e.g. when analyzing DNA samples (primers, PCR products, plasmids, etc.).
  • the gels are loaded by removing the samples in dissolved or suspended form e.g. transferred from a sample reservoir to the gel using a pipette.
  • This transfer process poses the following problems.
  • the sample transfer even if multi-channel pipettes with 8 or 12 channels are used for loading, is a considerable time factor. This is a disadvantage both because of the delay in sample processing and because of the sample stability.
  • the smallest sample quantities are provided or subjected to gel electrophoresis, which are sensitive to solvent losses due to evaporation into the environment.
  • the sample reservoirs are formed by microtiter plates with 96, 384 or more sample chambers (so-called "wells").
  • Pipette systems are known for taking samples from microtiter plates in which a multiplicity of micropipettes are arranged in accordance with the microtiter plate format. In a 96-channel pipetting system, the distance between adjacent pipette tips in rows or columns is 9 mm in each case.
  • Such a pipetting system is also suitable for taking samples on microtiter plates with 384 or more wells, since in these cases the wells are arranged in a number of staggered 96 raster frames.
  • a conventional, commonly used electrophoresis system consists of a buffer chamber with a rectangular base. area in which a gel carrier for receiving the separating gel (possibly on a separate gel carrier) is arranged.
  • a strip-shaped electrode is located on two opposite sides of the buffer chamber. The electrodes form a pair of electrodes which, when subjected to an electrical voltage in the gel, form an electrical field, under the effect of which the sample migrates.
  • the samples move along a straight path in accordance with the direction of current between the electrodes, for example in the case of DNA samples from the cathode to the anode. Due to the dimensions of the pipetting systems for sample collection from microtiter plates, the conventional gel electrophoresis systems are unsuitable for direct sample transfer in microtiter plate format.
  • the object of the invention is to provide an improved gel electrophoresis method which overcomes the disadvantages of the conventional systems and which in particular enables direct sample transfer from common sample reservoir formats to the separating gel.
  • the new process is also intended to enable the running distances in the separating gel to be extended without increasing the buffer chamber.
  • the object of the invention is also to provide a gel electrophoresis system for realizing such a method.
  • the samples are applied to the separating gel in a matrix-like manner in straight rows and columns and at least two electrode pairs are applied to the mutually opposite edges of the separating gel, the electrodes of which extend parallel to the rows or columns, with electrical voltages such that forms an electric field in the separating gel, the direction of which deviates from the row and column directions of the sample arrangement.
  • An electric current with an effective current direction depending on the ratios of the partial currents of one pair of electrodes and thus a running direction is set in such a way that the samples pass between the adjacent samples or sample paths.
  • the electrical voltages can be direct voltages, pulsed direct voltages or high-frequency alternating voltages.
  • the gel electrophoresis system is constructed essentially like a conventional system with a rectangular buffer chamber, but not only one pair of electrodes but at least two pairs of electrodes are provided. On the opposite sides of the buffer chamber in pairs there is a straight, strip or ribbon-shaped cathode or anode.
  • An important aspect of the invention is the design of the sample pockets in the separating gel with a gel comb.
  • the sample pockets are recesses in the separating gel with a usually rectangular base area, the normal to the longer side of the pocket, which forms the starting line during the separating process, has an orientation which deviates from the row or column directions and which preferably corresponds to the field direction in the separating gel.
  • a gel comb is described which has a large number of teeth for impressing the sample pockets into the gel.
  • the teeth are arranged in microtiter plate format and are rotated by a predetermined angle with respect to the matrix orientation.
  • the invention thus also relates to an overall arrangement for gel electrophoresis, consisting of a buffer chamber with a plurality of electrodes, a gel carrier and a gel comb.
  • the invention has the following advantages. For the first time, a gel electrophoresis system is created with which the separating gel can be loaded directly with a completely 96-pipetting system. This enables, especially with large sample pay, a considerable time saving (working time per gel) and also offers an automation capability. There is no need to manually load the gel with single or multipipettes. This avoids pipetting errors and drastically reduces the material consumption in relation to the pipette tips that have to be replaced during manual loading.
  • the direct, fast sample transfer from sample reservoirs to the gel prevents uncontrolled migration of the samples in the gel and undefined sample evaporation.
  • the buffer chamber designed according to the invention has the 96-microtiter plate format, so that compatibility with the available laboratory systems is maintained. Due to the inclined migration of the sample relative to the matrix orientation, running distances of more than 25 mm are achieved.
  • the utilization of the separating gels is significantly improved.
  • the gel in the buffer chamber in the 96 microtiter plate format is smaller than the gels previously used for comparable sample quantities.
  • the separation distances are closer together. This reduces the cost of providing the separation gel.
  • the sensitivity of the separation process is increased by the gel electrophoresis according to the invention.
  • less than 0.5 ⁇ l of PCR product per sample bag is required in order to nevertheless obtain a clearly visible and evaluable, meaningful band in the separating gel.
  • a particular advantage of the invention is that with a relatively simple electrode structure, electrical field conditions in the gel can be set which, on the one hand, enable the samples to skew, and at the same time are sufficiently homogeneous to achieve reproducible results on all samples. Further advantages and details of the invention will become apparent from the following description of the accompanying drawings. Show it:
  • Fig. 1 is a schematic plan view of the
  • FIG. 2 is a perspective view of the buffer chamber of FIG. 1,
  • Fig. 5 is a plan view of a separating gel after
  • the figures 1 and 2 illustrate a gel electrophoresis device 100 according to the invention in schematic plan view and in perspective view.
  • the buffer chamber 110 with the electrodes 120 and the gel carrier 130 are shown.
  • Further details of the gel electrophoresis device, such as the provision of connections with a control device, optionally covering devices, temperature control devices and the like. are known per se from conventional gel electrophoresis and are therefore not described in detail.
  • the invention is described by way of example with reference to a 96-series matrix corresponding to the frequently used microtiter plate format, but is correspondingly with larger ones (eg 384-series matrix) or smaller (e.g. 8 or 12 matrix) formats can be implemented.
  • the buffer chamber 110 is formed by the rectangular base 111 and the side walls 112 in the form of a flat, upwardly open shell. It preferably consists of a transparent plastic material. Electrode holders 113 (not shown in FIG. 1) are provided on the inner sides of the side walls 112. The electrode holders 113 are arranged in the corner areas of the buffer chamber 110, two electrode holders serving as supports for one electrode (121-124), which extend between the electrode holders 113 just along the side wall inside the buffer chamber 110. Electrical feedthroughs 114 are also provided in the side walls 112. Each bushing 114 contains an electrical line connection between an associated electrode (121-124) and a control device (not shown). In order to avoid field distortions, the bushings 114 are preferably arranged uniformly in the corners of the buffer chamber 110.
  • the electrodes 120 comprise two pairs of electrodes 121, 122 or 123, 124, of which one electrode forms a cathode 121 or 123 and the second electrode forms an anode 122 or 124.
  • the associated cathodes and anodes are arranged opposite one another on the opposite side walls 112 of the buffer chamber 110, extending over the entire side length of the side walls 112 or at least over the side length of the gel carrier 130 (see below).
  • Each of the electrodes is constructed as is known per se from the conventional gel electrophoresis devices and consists, for example, of an inert metal strip or wire that is just stretched between the associated electrode holders 113.
  • the gel carrier 130 for receiving the separating gel 131 is arranged in the middle of the buffer chamber 110.
  • the gel carrier has a rectangular shape and a shape which leaves the same distance from the edge of the gel carrier 130 to the adjacent side wall 112 of the buffer chamber 110.
  • Typical dimensions are, for example, an area of approx. 150 mm • 100 mm and for the base area of the buffer chamber 110 and 270 mm • 220 mm.
  • a particular advantage of this dimensioning is that the gel carrier is constructed in such a way that it usually fits pipetting systems or sample application systems into the microtiter plate holder (see FIG. 3).
  • the gel carrier 130 contains the gel matrix (separating gel), which consists of a conventional separating medium, such as agarose or polyacrylamide.
  • Sample pockets 132 are formed in the surface of the separation gel.
  • a large number of sample pockets 132 are provided (only partially shown), which are arranged in a matrix-like manner, flat in straight rows or columns. In Fig. 1 only the outer sample pockets are shown.
  • the number of sample pockets and their spacing are selected depending on the application in accordance with the sample arrangement in a sample reservoir from which the samples are to be transferred to the gel.
  • the sample reservoirs are preferably microtiter plates. The arrangement of the sample pockets is therefore adapted to the microtiter plate format used in each case. Usual formats are, for example, the 96 format (as shown in FIGS.
  • sample pockets are designed with an inventive Gel comb is embossed into the separating gel 131, as will be explained in detail below with reference to FIG. 4.
  • FIG. 3 shows, according to an important aspect of the invention, a gel electrophoresis system in which a gel electrophoresis device 100 according to FIGS. 1 or 2 is combined with a pipetting system 200 known per se.
  • the pipetting system 200 comprises a pipette matrix 210, which can be moved in all spatial directions with a carrier and drive system 220 between a microtiter plate 230 and the gel electrophoresis device 100 and is formed on the microtiter plate 230 for taking samples or on the device 100 for storing samples.
  • the pipette matrix 210 comprises e.g. B. 96 micropipettes 211, which are arranged in 12 straight rows or 8 straight columns in accordance with the 96 microtiter plate format (only partially shown).
  • the pipette matrix is actuated in a manner known per se, as is customary for example in the operation of picking-spotting robots, in such a way that all micropipettes are loaded with samples simultaneously on the microtiter plate 230.
  • the pipette matrix 210 is also adapted for sample collection to the narrower microtiter plate formats mentioned above, 4 or 16 sample collection processes then optionally taking place sequentially.
  • gel electrophoresis is carried out in accordance with the following steps.
  • a gel carrier 130 with a prepared separating gel is loaded with samples in a gel electrophoresis system according to FIG. 3 (arrow A) and inserted into the buffer chamber 110 (arrow B).
  • the gel carrier with the loaded separating gel matrix is then covered with a so-called running buffer (electrolyte).
  • electrophoresis direct voltages for generating electrical fields are applied to the first and second electrode pairs 121, 122 and 123, 124, under the effect of which a current flows through the separating gel 131.
  • the field direction of the total field composed of the individual electrical fields is set in a predetermined manner deviating from the row or column directions of the sample pockets 132 by a field angle ⁇ .
  • a particular advantage of the invention is that with the two pairs of electrodes a resulting electric field is generated in the entire separating gel with the defined field angle ⁇ , which in the embodiment shown only corresponds to the currents Ii, I 2 flowing between the electrode pairs
  • the field angle ⁇ (running angle) is selected depending on the application in order to achieve long, free separation distances.
  • the field angle ⁇ can be set between 0 ° and 90 ° with respect to the row direction (or correspondingly with respect to the column direction).
  • the running direction of the sample corresponds to the field angle.
  • the specific electrical parameters are selected in a manner known per se, depending in particular on the material of the separating gel 131 and on the electrolyte solution. Typical values for separations in agarose are at voltages of approx. 200 V or currents in the mA range. In the case of separations in the PCA, voltages up to the kV range are used (high-resolution sequencing). The voltage or current relationships between the electrode pairs are entered depending on the desired separation angle ⁇ . poses. Regulated power supplies are preferably used to set the electrical parameters, which keep the electrical current constant and regulate the voltage. Since heat is generated during electrophoresis, the rate of migration would otherwise disadvantageously decrease at constant voltage.
  • the separation process After passing through the separation section, the separation process is ended, the gel carrier with the separation gel 131 is removed from the buffer chamber 110 and fed to further evaluation.
  • the gel comb 140 is used to form the sample pockets 132 in the separating gel 131 (see FIG. 2).
  • the gel comb 140 has a plurality of teeth 141 arranged in a matrix in straight rows and columns, which protrude from a common carrier plate 142 in accordance with the desired depth of the sample pockets 132.
  • Adjustment elements 143 preferably at the corners of the plate, which cooperate with corresponding adjustment elements of the gel carrier 130, are also attached to the carrier plate 142.
  • Each tooth has a rectangular base. As illustrated in the top view of FIG. 4, the teeth 141 are in relation to the rows.
  • Typical dimensions of the gel comb 140 according to FIG. 4 are a carrier plate thickness of approx. 7 mm, a tooth height of approx. 3 mm, a carrier plate area of approx. 150 mm • 100 mm and a height the adjusting elements 143 of approx. 4 mm.
  • the matrix arrangement of the teeth 141 corresponds to the sample arrangement in the sample reservoir and thus preferably a microtiter plate format, as was explained above.
  • the individual teeth 141 have a cross section that becomes smaller towards the end.
  • the angle ⁇ for the inclination of the teeth is, for example, 92 °.
  • the gel carrier 130 is filled with a gel in the liquid state.
  • the gel comb 140 is placed on the gel carrier 130 overhead and the separating gel 131 is hardened. The gel comb 140 is then removed and the gel carrier is ready for the sample loading.
  • FIG. 5 illustrates a separation result achieved with the gel electrophoresis system according to the invention.
  • the dark dots indicate the positions of the sample pockets 132 with the samples to be separated that have been placed.
  • the separation results of the reference samples 133 can be seen in the left part of the figure.
  • a gel electrophoresis system can be modified as follows.
  • four individual electrodes which form the two pairs of electrodes, it is possible to provide more individual electrodes in order to homogenize or fine-tune the field profile of the sample chamber.
  • two straight, individually controllable individual electrodes can be provided on each sample chamber side, which form an electrode pair with the respective opposite electrodes.
  • the gel comb can be designed for a 384 format or also a 1536 format, tooth-tooth spacings of 4.5 mm or 2.2 mm being formed. The reason- The area of the individual sample bag is then reduced accordingly.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
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  • Biochemistry (AREA)
  • Electrochemistry (AREA)
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  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Selon l'invention, dans un système d'électrophorèse en gel dans lequel une pluralité d'échantillons sont appliqués sous forme matricielle, en rangées et en colonnes droites, sur un support de gel (131) en nappe et parcourent le gel de séparation (130) sous l'effet d'un champ électrique, il est prévu de produire un courant électrique avec au moins deux paires d'électrodes (121,122 et 123,124) comprenant des électrodes disposées sur des bords opposés du gel de séparation, de manière que les échantillons parcourent le gel de séparation conformément à un sens de marche différant des sens des rangées et des colonnes de la configuration desdits échantillons.
PCT/EP2000/005472 1999-06-14 2000-06-14 Procede et dispositif pour electrophorese en gel WO2000077510A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19926985.8 1999-06-14
DE1999126985 DE19926985B4 (de) 1999-06-14 1999-06-14 Verfahren und Vorrichtung zur Gelelektrophorese

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WO2000077510A2 true WO2000077510A2 (fr) 2000-12-21
WO2000077510A3 WO2000077510A3 (fr) 2001-03-01

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006136296A1 (fr) * 2005-06-18 2006-12-28 Ge Healthcare Bio-Sciences Ab Méthode et dispositifs pour former une multitude de puits sur un gel
US7989215B2 (en) 2005-06-18 2011-08-02 Ge Healthcare Bio-Sciences Ab Methods and systems for adding a reagent to an analyte in a gel
US9753010B2 (en) 2011-02-10 2017-09-05 Biocule (Scotland) Limited Two-dimensional gel electrophoresis apparatus and method

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2225432A (en) * 1988-02-02 1990-05-30 Inst Molekularbiologie Ak Apparatus for electrophoretic separation of high-molecular dna in gel
WO1996018891A1 (fr) * 1994-12-15 1996-06-20 University College London Electrophorese de matrice en gel
US5785835A (en) * 1996-04-12 1998-07-28 One Lambda Electrophoresis method and devices
WO1999015888A1 (fr) * 1997-09-19 1999-04-01 Aclara Biosciences, Inc. Appareil et procede d'electrofluidite capillaire

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE4330307C2 (de) * 1993-09-07 1995-11-02 Georg Dr Meyer Verwendung von Nukleinsäuregelelektrophorese zur Identifizierung von Organismenteilen und dergleichen
GB2284484B (en) * 1993-12-03 1997-09-17 Univ London Gel-matrix electrophoresis

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2225432A (en) * 1988-02-02 1990-05-30 Inst Molekularbiologie Ak Apparatus for electrophoretic separation of high-molecular dna in gel
WO1996018891A1 (fr) * 1994-12-15 1996-06-20 University College London Electrophorese de matrice en gel
US5785835A (en) * 1996-04-12 1998-07-28 One Lambda Electrophoresis method and devices
WO1999015888A1 (fr) * 1997-09-19 1999-04-01 Aclara Biosciences, Inc. Appareil et procede d'electrofluidite capillaire

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006136296A1 (fr) * 2005-06-18 2006-12-28 Ge Healthcare Bio-Sciences Ab Méthode et dispositifs pour former une multitude de puits sur un gel
US7989215B2 (en) 2005-06-18 2011-08-02 Ge Healthcare Bio-Sciences Ab Methods and systems for adding a reagent to an analyte in a gel
US9753010B2 (en) 2011-02-10 2017-09-05 Biocule (Scotland) Limited Two-dimensional gel electrophoresis apparatus and method

Also Published As

Publication number Publication date
WO2000077510A3 (fr) 2001-03-01
DE19926985A1 (de) 2001-01-11
DE19926985B4 (de) 2004-12-02

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