JP2006112847A - Sample extraction device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample extraction device capable of suppressing the loss of a target component caused by the staying of a gel in a sampling part of a sample, when a target part is extracted from the gel on which the sample is developed by an electrophoretic method to be transferred to a container. <P>SOLUTION: The sample extraction device is equipped with a position selection means for selecting the position of the target part, a liquid-holding means 25 in which a liquid is housed, a sampling means 32 which is connected to an air supply and discharge part 4 and has a leading end part 32d having an almost cylindrical shape and internally holding the liquid and the target part, of which the position is selected by the leading end part 32d, and is driven so as to discharge the held liquid and the target part into the container 26, and a moving means for moving the sampling means 32 in a predetermined direction. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a sample extracting device and method for extracting a portion (target portion) containing a target sample from a gel on which the sample has been developed by electrophoresis and transferring it to a container.

  Conventionally, as a method for analyzing a biological sample, a method of separating and purifying nucleic acids and proteins contained in the sample by electrophoresis using a gel has been widely used. Electrophoresis utilizes the phenomenon that when a voltage is applied to a substance in the gel, the substance moves in the gel according to its charge. In a gel with a network structure, the substance moves depending on the molecular weight and isoelectric point. Since the degrees are different, each substance can be separated.

  In the case of protein electrophoresis, a method using an acrylamide gel is often used. By changing the acrylamide concentration, the size of the mesh can be changed to separate proteins having various molecular weights. After cleaving the SS bonds between the amino acid side chains, the SDS, which is an anionic surfactant, is bound to the protein, and the respective proteins are made almost constant, so that the SDS-poly that performs separation only depending on the molecular weight One example is the acrylamide method (SDS-PAGE method), which is used for both one-dimensional and two-dimensional electrophoresis methods. Among them, according to the two-dimensional electrophoresis method, it is possible to separate several thousand kinds of proteins at a time, and it is widely used for proteome analysis for comprehensive analysis of proteins contained in a sample.

  On the other hand, agarose gel is often used in electrophoresis of nucleic acids. Since agarose gel has a larger network structure than acrylamide, it is particularly suitable for the separation of relatively large nucleic acids.

  When performing subsequent analysis using proteins and nucleic acids separated and purified by electrophoresis, it is necessary to select a band containing the target substance from the gel on which these substances are developed, and to recover each substance. . The substance in the gel can be recovered, for example, by cutting out a gel containing a band that appears to be the target substance and eluting it in a buffer solution. The gel can be cut out manually using a pipette or a gel cutter, and an apparatus has been developed that automatically performs the process from reading the pattern of the sample developed on the gel to extracting the target band.

One example of such an automated apparatus is a sample component separation apparatus described in Patent Document 1. This apparatus specifies the position of the target portion based on the development pattern of the mixed sample that appears on the development medium on which the mixed sample is developed by the separation analysis method, moves the sampling means having the tubular portion to that position, The tubular portion is inserted into a developing medium, the target portion is accommodated inside the tube, and is extracted from the remaining developing medium.
JP 2001-41928 A

  In the above-described conventional apparatus, the inner surface at the tip of the tubular portion of the sampling means has a taper that tapers to hold the developing medium (gel), and a test tube or the like to which the sample is to be discharged is placed. After the sampling means has moved to the place, the held gel is pushed out from the tip of the tubular portion by the piston. However, it is difficult to completely match the shape of the portion where the taper and the piston come into contact, and there is a possibility that the sample may remain attached to the inner surface of the tubular portion of the sampling means. Since the target component contained in the cut-out gel is very small, it is eager to prevent a slight sample loss for subsequent analysis of the sample.

  Therefore, the present invention suppresses the loss of the target component due to the gel remaining in the sample collection part when the target part is extracted from the gel on which the sample has been developed by electrophoresis and transferred to the container. It is an object of the present invention to provide a sample extraction device that can be used.

  In order to solve the above-described problems, a sample extraction device according to the present invention extracts a target portion in a gel on which a sample is developed by electrophoresis and transfers the target portion to a container. Position selection means for selecting, liquid holding means for storing liquid, tip portion is substantially cylindrical and connected to the gas supply / exhaust part, the purpose of selecting the position by the liquid and position selection means A sample collecting means which is driven so that the portion is held inside the tip portion and the held liquid and the target portion are discharged into the container; and a moving means for moving the sample collecting means in a predetermined direction; It is characterized by providing.

  According to such a configuration, the sample collection means is moved to a desired position by the movement means. Further, when the inside of the distal end portion of the sample collection means is exhausted by the gas supply / discharge portion, the substance is sucked and held in the distal end portion, and when the air is supplied into the distal end portion, the held substance is discharged. For example, the liquid contained in the liquid holding means is first sucked into the tip of the sample collecting means, and then the target portion of the gel is punched at the tip, and then the sample collecting means is moved to the container while holding the liquid and the gel. And the liquid and the gel can be discharged into the container. In this way, since the gel is punched out after the liquid is sucked, when the gel is discharged from the tip of the sample collecting means, the liquid passes through the portion through which the gel has passed, and the gel is in the tip. For example, even if it adheres or remains on the inner wall, it is moved into the container so that it is washed away by the liquid.

  Moreover, it is preferable that the front-end | tip part of the sample collection means of the said sample extraction apparatus is provided so that attachment or detachment is possible. In this way, when the extraction of one target portion is completed and the next target portion is extracted, contamination of the sample is prevented by exchanging the tip portion. More specifically, for example, as the sample collecting means, there can be mentioned one in which a substantially cylindrical tip is attached to the tip of a substantially cylindrical body.

  Furthermore, the sample extracting device preferably includes a chip holder on which a plurality of the chips are placed. In such a configuration, it is easy to automate a series of operations such as moving the sample collecting means to the tip holder by the moving means, attaching the tip to the tip of the sample collecting means, extracting the target portion, and replacing with a new tip. It becomes.

  Furthermore, the position selection means of the sample extraction device includes an image acquisition unit that acquires an image of the gel when light of an arbitrary wavelength is projected onto the gel on which the sample is developed, and an image display unit. It is preferable that the position of the target portion is selected based on the displayed image itself.

  With such a configuration, it is easy to visualize the distribution of the sample in the gel as an image by projecting light having a wavelength appropriately selected according to the staining method of the sample onto the gel on which the sample is developed. This makes it easy to select the target part. In addition, since the position of the target part is selected based on the original image without processing the displayed image itself, that is, the image, for example, the position of the target part is selected by the operator's visual recognition. The inconvenience of failing to select a correct part can be solved.

  The sample extraction method of the present invention is a method that is effectively carried out using the sample extraction device of the present invention. That is, a target portion in a gel on which a sample is developed by electrophoresis is collected and placed in a container. A method of transferring, the first step of selecting the position of the target portion, and the tip of the sample collecting means whose tip is substantially cylindrical is immersed in a liquid holding means containing a liquid, A second step in which the inside of the tip is evacuated to suck and hold the liquid in the tip, and the sampling means is moved to the selected position, the target portion is punched out by the tip, the inside of the tip is exhausted, and the target portion is evacuated. A third step of sucking and holding in the tip, and a fourth step of moving the sample collecting means to the position where the container is placed, supplying air into the tip and discharging the liquid and the target portion into the container. Prepare.

  Furthermore, prior to the second step, it is also preferable to perform a step of causing the sampling means to suck the gas. With this gas, the liquid held in the tip can be discharged more efficiently.

  Furthermore, it is preferable that after the second step, prior to the third step, a step of sucking the gas by the sample collecting means is performed. In this case, after the liquid is sucked, the target portion of the gel is sucked after the gas is sucked, so that the target portion is prevented from being eluted due to contact between the liquid and the target portion.

  Furthermore, it is preferable to provide a fifth step of supplying the liquid to the container in advance prior to the fourth step, that is, the step of discharging the target portion of the liquid and gel into the container. By supplying a sufficient liquid (for example, a buffer solution) to the container, drying of the discharged sample is further prevented.

  Specifically, in the fourth step, it is useful to suck and discharge the liquid discharged into the container at least once by the exhaust / intake operation in the tip to clean the inside of the tip.

  In addition, in the first step of this method, light of an arbitrary wavelength is projected onto the gel on which the sample is developed to obtain an image of the gel, and the position of the target portion is displayed based on the image itself while displaying the image. Is preferably selected.

  In the sample extraction device and the sample extraction method according to the present invention, when the target portion is extracted from the gel on which the sample has been developed by electrophoresis and transferred to the container, the target portion of the gel is removed from the tip of the sample collecting means. It is discharged into the container and the liquid then passes through the same part. Therefore, even if the gel adheres and remains in the tip, it is washed out into the container by the liquid, so that loss of the target component can be suppressed.

  Embodiments of the present invention will be described below in detail with reference to the drawings.

  FIG. 1 is a front view showing an outline of a preferred embodiment of a sample extracting device according to the present invention. 2 is a cross-sectional view taken along the line II-II shown in FIG.

  The sample extracting device 100 is a device that extracts a target portion from the gel 24 on which the sample has been developed by electrophoresis using the sample collecting means 32 and transfers it to the container 26. Further, the sample extracting device 100 includes a gel mounting table 23, a buffer solution reservoir 25 (liquid holding means), and a titer plate 26 (container) for transferring the extracted gel on the base 2 in the housing 1. And a chip holder 27. Further, a chip exhaust box 29 for discarding the used chip 32d is embedded under the base 2. In the figure, the size of each element is different from the actual size, and in particular, the sampling means 32 is shown larger than other elements for use in explanation.

  The gel mounting table 23 is made of a light-transmitting material on the upper surface, and includes a light 22 as a light source for imaging. The light 22 is not particularly limited as long as it can be used for a normal imaging light source, and a coloring system using a sample stained with various fluorescent dyes, Coomassie Brilliant Blue (CBB) staining, silver staining, or the like. For example, it is preferable that both a white light source and a UV light source are provided so that an image can be captured regardless of whether the dye is stained with the above dye. Further, for example, if a xenon light source and an optical filter having an appropriate bandpass function are used, excitation light having an optimum wavelength can be irradiated to various fluorescent dyes. Further, when the white light source is composed of, for example, RGB LEDs, the ratio of each light quantity may be adjusted so that a color trimmer can be set.

  The buffer reservoir 25 is a container in which a desired amount of buffer solution is stored, and its activity is maintained without affecting the sample (component) such as protein or nucleic acid contained in the target portion of the gel 24. A buffer solution having such a composition is prepared.

  As the titer plate 26, a commercially available one such as a normal standard 96-hole plate can be used, and it is not particularly limited.

  On the chip holder 27, a chip 32d which is a tip portion of a sample collection means 32 described later is placed. When the chips 32d are disposable, the number of wells in the titer plate 26 and the number of chips 32d placed on the chip holder 27 are made equal, and the arrangement (for example, 12 rows × 8 columns) is also the same. Is preferred. With such a configuration, the timing for exchanging the titer plate 26 and the chip holder 27 with a new one becomes the same, so the operation is simplified.

  The chip discard box 29 is for discarding the used chip 32d as described above. Note that a container having an upper open end may be provided on the base 2 instead of providing the chip disposal box 29 in a concave shape below the base 2.

  The sample collecting means 32 has a tip 32d (tip portion) at the tip (lower end in the drawing) of a barrel 32c having a substantially cylindrical shape and having a drive shaft disposed in a space defined by the cover 32a and the fixed plate 32b. ) Is detachably mounted. In addition, an actuator 36 is connected to a drive shaft provided at a substantially middle portion of the body portion 32c. By operating this, the chip 32d is driven up and down (in the direction of the arrow Z in the figure). Further, the sample collection means 32 has a chip discard mechanism (not shown) for automatically discarding the chip 32d.

  Here, the size, shape, material, and the like of the chip 32d are not particularly limited, but a disposable one made of an inexpensive material such as polypropylene is preferable, and a structure that can be easily attached to and detached from the body portion 32c is desirable. Further, as the tip 32d, a tip having a horizontal cross-sectional shape that is easy to punch out a target portion such as a circle or a rectangle can be appropriately selected and used.

  Further, the sample collection means 32 is connected to the pump 4 (gas supply / exhaust section), and by supplying gas into the tip portion of the sample collection means 32 by the pump 4, the substance in the tip portion can be discharged, By exhausting the inside of the tip, the substance can be sucked or held in the tip.

  Still further, the fixed plate 32b of the sample collection means 32 is connected to a guide rail 37b provided with an actuator 35 and extending horizontally and in the direction of the arrow Y, and the sample collection means 32 is operated by operating the actuator 35. Is driven in the direction indicated by the arrow Y. The guide rail 37b extends horizontally and in a direction perpendicular to the guide rail 37b (in the direction of the arrow Y in the figure), and is fixed to the guide rail 37a provided with the actuator 34 via fixtures 38a, 38b, 38c. It is connected. By operating the actuator 34, the sample collection means 32 is driven in the direction indicated by the arrow X together with the guide rail 37b.

  Thus, the guide rails 37a, 37b and the actuators 34, 35, 36 constitute the moving means in the present invention, and thereby, the sample collecting means 32 can be moved in any direction of the indicated arrow directions X, Y, Z. It is possible to move to.

  Also, above the gel mounting table 23, an input device (not shown) such as a keyboard or a mouse and a CCD camera 12 (image acquisition unit) connected to a personal computer 5 (display unit) having a display 51 are installed. ing. Furthermore, the personal computer 5 is connected to the actuators 34, 35, 36 and their control unit 6 connected to the pump 4.

A method of extracting a portion (target portion) containing the target component of the gel 24 using the sample extracting device 100 configured as described above and transferring it to the titer plate 26 will be described below.
(Selection of target part)

  First, the gel 24 after electrophoresis is mounted on the gel mounting table 23, and light having a wavelength corresponding to the sample staining method is projected from the light 22 onto the gel 24. The excited fluorescent dye and the transmitted light of white light are captured as an image by the CCD camera 12, the image data is transferred to the computer 5, and the image of the gel 24 is displayed on the display 51. FIG. 3 is a diagram showing an example of the screen of the display 51 on which the image of the gel 24 is displayed in this way.

  Next, while viewing the screen 700, the operator selects a target portion on the image 71 of the gel 24 and clicks the cursor with the target portion using, for example, a mouse. Numbers such as 001, 002,... Are added and displayed on the image 71 together with a pointer. The screen 700 is provided with a picking point display section 72 that lists the coordinates of points (picking points; that is, the positions of the pointers) for extracting the sample together with the image 71, corresponding to the pointer numbers 001, 002,. Coordinates are shown (first step). Then, after the designation of the target portion is completed, the process proceeds to the next process, and sampling of the sample is started.

In addition, it is preferable that other information is also displayed on the screen 700 at the same time and can be adjusted as necessary. Examples of other information include current operating state information (status information), imaging conditions such as the type and light quantity of the selected light 22, and supply / exhaust setting conditions for the pump 4. Further, the subsequent operations are automatically performed by various command signals from the control unit 6 based on the control information set on the screen 700.
(Chip attachment)

Next, the tip 32 d is attached to the tip of the body portion 32 c of the sample collection means 32. Here, first, the actuators 34 and 35 are operated to move the sample collection means 32 above the chip 32d to be mounted. Subsequently, the actuator 36 is operated to lower the chip 32d in the Z direction until the chip 32d is sufficiently fitted to the tip of the body portion 32c. Here, FIGS. 4A to 4E schematically show a state in which the buffer solution B and the target portion M of the gel 24 are sequentially sucked and held in the tip 32d attached to the body portion 32c as will be described later. FIG. 4A shows a state where the chip 32d is fitted to the body portion 32c. After the tip 32d is thus mounted on the body portion 32c, the actuator 36 is operated to raise the tip 32d.
(Aspiration and retention of buffer B)

Next, the actuators 34 and 35 are operated, the sample collection means 32 is moved to above the buffer solution reservoir 25, and then the actuator 36 is operated so that the tip 32d is immersed until the tip of the tip 32d is immersed in the buffer solution B. Lower in the Z direction. The operation of the sampling means 32 is stopped with the tip 32d immersed in the buffer solution, the inside of the tip 32d is evacuated by the pump 4, and the buffer solution B is sucked and held in the tip 32d (see FIG. 4B); Second step). When the suction is completed, the actuator 36 is operated again to raise the chip 32d. At this time, a certain amount of air may be sucked before the buffer B is sucked. With this air, the later-described gel and buffer solution can be discharged more efficiently.
(Suction and retention of gel)

  Then, prior to punching out the target portion M of the gel 24, the inside of the tip 32d is further evacuated by the pump 4, and a certain amount of air A is sucked (see FIG. 4C). Thereby, it can prevent that the target part M contacts the buffer solution B and elutes.

Next, the actuators 34 and 35 are operated to move the sample collection means 32 to the coordinate position of the picking point 001 above the gel 24. Then, the actuator 36 is operated to lower the tip 32d in the Z direction until the tip of the tip 32d comes into contact with the upper wall surface of the gel mounting table 23, thereby punching out the target portion M (the portion corresponding to the picking point 001) of the gel 24. . Next, while the tip 32d is in contact with the gel mounting table 23, the inside of the tip 32d is further evacuated by the pump 4, and the punched target portion M is sucked and held in the tip 32d (FIG. 4D). Reference; third step). While maintaining this state, the actuator 36 is operated again to raise the chip 32d. In addition, if the inside of the chip | tip 32d is further exhausted with the pump 4 and the air A is suck | inhaled after attracting | sucking the target part M of the gel 24, the fall of the target part M will be prevented further (refer FIG.4 (E)).
(Discharge of gel and buffer)

  Subsequently, the actuators 34 and 35 are operated to move the sample collection means 32 above a predetermined well of the titer plate 26. Then, the actuator 36 is operated to lower the chip 32d in the Z direction until the tip of the chip 32d sufficiently approaches the well. Thereafter, the air is sucked into the chip 32d by the pump 4, and the buffer B and the target portion M of the gel 24 held therein are discharged into the well (fourth step). At that time, first, after the target portion M of the gel 24 is discharged into the well, the buffer solution B is discharged. Therefore, the gel remaining on the inner wall of the chip 32d is also washed away by the buffer solution B, Moved to. At this time, it is also preferable to supply a predetermined amount of buffer B in advance in each well of the titer plate 26 (fifth step). By doing so, drying of the target portion M of the gel 24 discharged into the well can be more effectively suppressed.

Thereafter, the actuator 36 is operated to further lower the tip 32d, so that the tip of the tip 32d is immersed in the buffer B, and the suction and exhaust by the pump 4 are repeated to suck and discharge the buffer, The tip 32d can also be washed by vibrating the tip in the buffer B or the like.
(Disposal of chip)

  Next, the actuator 36 is operated to raise the chip 32 d, and the actuators 34 and 35 are operated to move the sample collection means 32 to the upper part of the chip disposal box 29. Then, the chip 32 d is thrown into the chip disposal box 29 by the chip disposal mechanism provided in the sample collecting means 32. By disposing in this way, contamination of the sample can be prevented more easily and effectively than when the sample collection means 32 is washed and reused.

  Thus, when one cycle of the sample extraction process is completed, the operation of extracting the target portion M of the gel 24 located at the picking point 002 selected previously is automatically started. The extraction of the picking point 002 is the same as the extraction of the picking point 001 except that the sample collecting means 32 is moved to the coordinate position of the picking point 002 in the step of sucking and holding the gel 24, and thus the description thereof is omitted. The target portions M of all selected picking points are extracted, transferred to the titer plate 26, and a series of processing operations is completed.

  According to the sample extraction method using the sample extraction device 100 having such a configuration, after the target portion M of the gel 24 is discharged into the well, the buffer solution B is discharged, and thereby adheres to the inner wall of the chip 32d. Since the remaining gel is also washed away with the buffer B and transferred into the well, loss of the target component can be sufficiently suppressed. Further, since the target portion M is selected by visual recognition of the operator based on the original image 71 without performing any image processing such as pattern matching on the imaged image 24 of the gel 24, Inconveniences such as selecting unnecessary portions or failing to select necessary portions can be solved.

  In addition, this invention is not limited to embodiment mentioned above, A various deformation | transformation can be added in the limit which does not change the summary. For example, when the sample in the gel 24 is stained with a fluorescent dye, the light 22 may be provided not at the inside of the gel mounting table 23 but at a position where the gel 24 is irradiated from above. Further, the sample collection means 32 may be configured such that the body portion 32c and the chip 32d are integrated. Further, the chip 32d may be formed of a corrosion-resistant material such as stainless steel, and may be used after being cleaned for each sample without being disposable.

  Furthermore, the supply / exhaust gas may be an inert gas such as nitrogen gas instead of air. In this case, it is preferable that the inside of the housing 1 be the inert gas atmosphere. Furthermore, the target portion M is selected by manually specifying the position while viewing the image 71, but the target portion M may be automatically detected using image processing. Both of them may be combined so that further narrowing down is performed manually.

It is a front view showing the outline of one embodiment of the sample extraction device concerning the present invention. It is sectional drawing in the II-II line shown in FIG. It is a figure which shows an example of the screen of the display 51 on which the image of the gel 24 was displayed. (A)-(E) are the perspective views which show typically the state which attracts | sucks and hold | maintains the buffer B and the target part M of the gel 24 sequentially to the chip | tip 32d with which the trunk | drum 32c was mounted | worn.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 100 ... Sample extraction apparatus, 1 ... Housing, 2 ... Base, 22 ... Light, 23 ... Gel mounting stand, 24 ... Gel, 25 ... Buffer reservoir, 26 ... Titer plate, 27 ... Chip holder, 29 ... Chip disposal box 32 ... Sample collection means, 32a ... Cover, 32b ... Fixing plate, 32c ... Body, 32d ... Tip (tip), 34-36 ... Actuator, 37a, 37b ... Guide rail, 38a, 38b, 38c ... Fixing tool 4 ... Pump (gas supply / exhaust unit), 5 ... Computer, 51 ... Display, 6 ... Control unit, 72 ... Image, 72 ... Picking point display unit, 700 ... Screen, A ... Air, B ... Buffer, M ... the target part.

Claims (11)

A sample extraction device for extracting a target portion in a gel on which a sample is developed by electrophoresis and transferring it to a container,
Position selecting means for selecting the position of the target portion;
A liquid holding means for storing the liquid;
The tip portion is substantially cylindrical and connected to a gas supply / discharge portion, the liquid and the target portion whose position is selected by the position selection means are held inside the tip portion, and Sampling means driven so that the retained liquid and the target part are discharged into the container;
A sample extracting device comprising: a moving means for moving the sample collecting means in a predetermined direction.   The sample extraction device according to claim 1, wherein the sample collection means is provided with the tip portion detachably.   The sample extracting device according to claim 1 or 2, wherein the sample collecting means has a substantially cylindrical tip attached to a tip of a substantially cylindrical body.   The sample extracting device according to claim 3, further comprising a chip holder on which a plurality of the chips are placed.   The position selection means includes an image acquisition unit that acquires an image of the gel when light of an arbitrary wavelength is projected onto the gel on which the sample is developed, and a display unit of the image, The sample extraction device according to any one of claims 1 to 4, wherein a position of the target portion is selected based on the displayed image itself. A sample extraction method in which a target portion in a gel on which a sample is developed by electrophoresis is collected and transferred to a container,
A first step of selecting a position of the target portion;
The tip of the sample collecting means having a substantially cylindrical tip is immersed in a liquid holding means containing a liquid, and the tip is exhausted to suck and hold the liquid in the tip. Process,
A third step of moving the sampling means to the selected position, punching out the target portion by the tip, evacuating the tip and sucking / holding the target in the tip;
A sample extraction method comprising: a fourth step of moving the sample collection means to a position where the container is placed, supplying air into the tip, and discharging the liquid and the target portion into the container.   The method according to claim 6, wherein a step of causing the sampling means to suck a gas is performed prior to the second step.   The method according to claim 6 or 7, wherein after the second step, prior to the third step, a step of causing the sampling means to suck a gas is performed. Prior to the fourth step, comprising a fifth step of supplying a liquid to the container in advance.
The sample extraction method according to any one of claims 6 to 8.   10. The method according to claim 6, wherein in the fourth step, the liquid discharged into the container is sucked and discharged at least once by the exhaust / air supply operation in the tip to clean the inside of the tip. 2. The sample extraction method according to item 1. In the first step, light of an arbitrary wavelength is projected onto the gel on which the sample is developed to acquire an image of the gel, and the position of the target portion is determined based on the image itself while displaying the image. The sample extraction method according to any one of claims 6 to 10, which is selected.

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