JP2009042032A - Electrophoretic apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an electrophoretic apparatus verifying whether the extraction and recovery work of a biopolymer from a support by a cutting-off instrument is normally completed. <P>SOLUTION: The electrophoretic apparatus is equipped with: an electrophoretic tank 10 in which an electrophoretic gel 101 for moving the biopolymer by electrophoresis is arranged; an image photographing means 61 for photographing the biopolymer after subjected to electrophoresis through the gel; a cutting-off instrument 20 for cutting off the biopolymer, which is set at an indicated position, along with the gel on the basis of the photographed image to hold the cut-off biopolymer therein; and a cutting-off means 62 for relatively moving the cutting-off instrument 20 with respect to the gel. The image photographing means 61 is constituted so as to photograph the region of the gel where the cutting off of the biopolymer is performed by the cutting-off instrument 20 to check the quality of cutting-off work on the basis of the photographed image of the region of the gel. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an electrophoresis apparatus for analyzing biopolymers, particularly nucleic acids, and more specifically, nucleic acids are separated into molecular weights by an electrophoresis apparatus, and the nucleic acids are cut out from a support for electrophoresis. It relates to the technology to collect.

  2. Description of the Related Art Conventionally, a technique for performing separation and analysis based on the molecular weight of a biopolymer, particularly nucleic acid, using an electrophoresis apparatus has been widely used because it is simple and inexpensive and has high analysis accuracy. Hereinafter, electrophoresis of nucleic acids will be described.

  A conventional electrophoresis apparatus is configured such that electrodes for applying a voltage are arranged at both ends of an electrophoresis tank in which a buffer solution is placed, and a support for moving a nucleic acid sample is installed in the electrophoresis tank. Yes. As a support for this electrophoresis, a gel substance such as agarose or polyacrylamide is used. As a form of the apparatus, in order to facilitate handling of a rectangular parallelepiped support, a horizontal electrophoresis apparatus in which a support is installed so that a plane on which electrophoresis is performed is horizontal is widely used.

  In the electrophoresis operation, a nucleic acid sample is dropped into a groove formed at one end of the support, a voltage is applied to both ends of the support, and the nucleic acid sample in the support is electrically moved. At this time, the movement of the nucleic acid sample is restricted by the network structure of the support, and the higher the molecular weight, the slower the mobility, thereby allowing separation based on the molecular weight of the nucleic acid.

  Further, the nucleic acid thus separated is cut out from the support for each nucleic acid fraction, and the nucleic acid is obtained by an appropriate method such as the method of Vogelstein and Gillespie (Non-patent Document 1) or the method described in Molecular Cloning (Non-patent Document 2). By extracting and recovering the protein, it is used for separation and removal of impurities from nucleic acid samples, incorporation into vectors, sequence mapping and the like.

  As a method for extracting and recovering this nucleic acid fraction, there is a method using column chromatography represented by high performance liquid chromatography in addition to a method using an electrophoresis apparatus. Considering the simplicity, flexibility, apparatus price, maintenance cost, etc., the method using the electrophoresis apparatus is extremely efficient, and therefore the method using electrophoresis is exclusively used for extraction and recovery of the nucleic acid fraction.

  Next, regarding the method for detecting nucleic acids separated by electrophoresis, a nucleic acid-binding fluorescent reagent such as ethidium bromide is mixed in advance in the support before electrophoresis, or after electrophoresis, the support is removed from these supports. After immersing in the solution and infiltrating into the support, the nucleic acid sample in the support is bound to this fluorescent reagent, and then the fluorescent reagent is excited by ultraviolet rays to develop a color of fluorescence. The separated nucleic acid sample is visualized (this apparatus for irradiating ultraviolet rays is called a transilluminator).

  The result of the nucleic acid sample visualized by this ultraviolet irradiation has been acquired by image capturing using a film camera. However, as a result of recent advances in electronic devices, an image employing a charge coupled device (CCD) camera is used. An image can be digitized by the photographing apparatus. Compared with the film type, the CCD type has higher ease of shooting, better data storage, and better compatibility with digital devices such as computers, and contributes to improving the working efficiency of a series of electrophoresis techniques as well as shortening the electrophoresis time. is doing.

  Recently, real-time monitoring of electrophoresis has been proposed by integrating an electrophoresis apparatus and an image photographing apparatus, and it is possible to perform optimal nucleic acid separation analysis and image acquisition by electrophoresis.

Regarding extraction and recovery of nucleic acids separated by electrophoresis from a support, conventionally, a target nucleic acid fraction was cut out from a support using a sharp blade, and nucleic acids were extracted and recovered by the appropriate method described above. In recent years, devices that extract a nucleic acid fraction from a support and an extraction / recovery device (see, for example, Patent Document 1, Patent Document 2, and Patent Document 3) have been proposed, and work efficiency is improved compared to conventional manual methods. It has improved significantly.
Japanese Patent Laid-Open No. 9-15206 JP 2000-88804 A JP 7-1332079 A Vogelstein. B., Gillespie. D., “Academic Bulletin of the American Academy of Sciences (Proc. Natl. Acad. Sci. USA)”, 76, 615-619, 1979 Molecular Cloning 2nd Edition, 6.22-6.35, Sambrook. J., Fritsch. EF, Maniatis. T., Cold Spring Published by Cold Spring Harbor Laboratory Press, 1989

  However, in the case of the above-described conventional electrophoresis apparatus, in particular, an apparatus that performs manual operation by the operator to cut out the nucleic acid fraction from the support after electrophoresis, the operator uses only a sharp knife to cut out, It is necessary to operate while looking directly at the ultraviolet rays. At this time, wear protective equipment to protect the worker from exposure to ultraviolet rays. Normally, ultraviolet lamps are mixed with violet visible light in addition to ultraviolet light, so high-level violet visible light is directly viewed. This is a cause of attracting serious eye fatigue to the worker. Moreover, since it is necessary to continue to irradiate ultraviolet rays during the operation, it is not possible to avoid partial interruption of the nucleic acid molecule and formation of polymerization (thymine dimer) between thymines that are bases present in the nucleic acid molecule. For this reason, it had the subject that it was unsuitable for the extraction collection | recovery of the nucleic acid from a support body.

  Further, in the above-described conventional electrophoresis apparatus in which a series of operations from the extraction of the nucleic acid fraction from the support to the recovery is automated, the extraction tool is automatically chucked, and the target fraction is extracted from the support. After the work is performed, the cut-out fraction is transferred based on a predetermined route, and the chucking of the cutting tool is released and stored in the collection container.

  However, when cutting out from the support using a cutting tool, the target fraction may not be cut out normally, and some or all of the fraction to be collected on the support will remain. was there. In such a case, there was a problem that subsequent analysis work could not be performed accurately.

  In addition, the fraction may fall during the transfer of the cutting device to the collection container and may not be stored correctly in the collection container. Even in such a case, there is a problem that the subsequent analysis work cannot be performed accurately. It was. Most of the samples used here are small and valuable, and a reliable recovery operation is required. Therefore, when a problem occurs during the collection work, the work must be immediately interrupted and a return work by the operator must be requested.

  The present invention solves the above-mentioned conventional problems, and when extracting and recovering a nucleic acid fraction from a support after electrophoresis, whether or not the fraction remains on the support is stored in a recovery container. It is an object of the present invention to provide an electrophoresis apparatus for verifying whether or not a biopolymer has been properly collected in a cut-out instrument.

  In order to solve the above-described conventional problems, the electrophoresis apparatus according to the present invention includes an electrophoresis tank in which a support for moving a biopolymer by electrophoresis is disposed, and after electrophoresis in the support. An image photographing means for photographing the biopolymer, and a cutting device for cutting out the biopolymer at a position specified based on the image photographed by the image photographing means together with the support, and placing it in the inside thereof, A cutting means for moving the cutting tool relative to the support, and the image photographing means further photographs the region of the support that has been cut out by the cutting tool of the cutting means. It is characterized in that the quality of the clipping operation is confirmed based on the photographed image of the area.

  According to the electrophoresis apparatus of the present invention, whether or not the extraction and recovery operation has been normally completed can be verified by confirming the image of the cut-out support, and the recovery is not normally performed. It is possible to stop the subsequent analysis work and perform the collection work again. In addition, since the image of the area where the image is cut out can be shared with the image shooting means for shooting the support in order to specify the cut-out position, the cost can be reduced and a small apparatus can be realized. .

  Hereinafter, embodiments of the electrophoresis apparatus of the present invention will be described in detail with reference to the drawings.

(Embodiment 1)
FIG. 1 is a diagram showing a schematic configuration of the electrophoresis apparatus of the present invention. This apparatus includes a table mechanism 40 that can be driven in the Y-axis direction and a carriage mechanism 60 that can be driven in the X-axis direction. In the upper part of the table mechanism 40, the electrophoresis tank 10 and the cutting instrument holder 30 are installed. The electrophoresis tank 10 and the cutting tool holder 30 are moved integrally with the table mechanism 40 in the Y-axis direction.

  Electrodes 106 and 107 (see FIG. 2) such as platinum wires are provided at both ends of the electrophoresis tank 10, and a voltage is applied to the support installed in the electrophoresis tank 10 through the electrodes 106 and 107. Perform electrophoresis. The cutting tool holder 30 can accommodate a plurality of cutting tools 20. The carriage mechanism 60 has an image photographing means 61 and a cutting means 62 that can be driven in the Z-axis direction perpendicular to the surface on which the support of the electrophoresis tank 10 is installed, and is directly above the table mechanism 40. To position. The excision device 62 is attached to the excision means 62 in a detachable state, and the excision means 62 is driven in the Z-axis direction so that the nucleic acid sample separated by electrophoresis can be excised together with the support. .

  The operation of the electrophoresis apparatus configured as described above will be described below. FIG. 2 is a perspective view of the table mechanism 40 of the electrophoresis apparatus according to the present embodiment.

  First, an electrophoresis gel 101 is installed as a support in the electrophoresis tank 10. After injecting a nucleic acid sample into an injection port 102 provided at one end of the electrophoresis gel 101, a prescribed voltage is applied between a pair of electrodes 106 and 107 installed at both ends of the gel. When a voltage is applied, the negatively charged nucleic acid sample starts moving from the cathode toward the anode.

  The inside of the gel has a fine network, and the nucleic acid sample that moves inside the gel has a different moving speed depending on its molecular weight. It is separated into strips of approximately the same width as the entrance.

  At this time, when the nucleic acid sample is irradiated with ultraviolet light, a nucleic acid-binding fluorescent reagent such as ethidium bromide mixed in advance with the nucleic acid sample develops fluorescence. By photographing this fluorescence with the image photographing means 61, it is possible to obtain an image of an electrophoretic pattern in which the nucleic acid sample is separated into strips based on the molecular weight. In addition, although the example which implements electrophoresis was shown here in order to obtain an electrophoresis pattern, even if the gel which performed electrophoresis previously is installed in the electrophoresis tank 10, by irradiating with ultraviolet rays and photographing, An electrophoresis pattern can be obtained.

  When the nucleic acid sample that has been photographed is used in further analysis, it is necessary to extract the nucleic acid sample by cutting out the gel. In the electrophoresis apparatus according to the present embodiment, the nucleic acid fraction containing the target nucleic acid sample is determined based on the photographed electrophoresis pattern image. As the method, automatic detection by image processing, or a method in which an operator designates by manual operation based on an electrophoretic pattern image displayed on the display means is provided.

  Next, the clipping operation will be described with reference to the flowchart shown in FIG. In the cutting operation, the carriage mechanism 60 moves to a position directly above the cutting device 20 stored in advance in the cutting device holder 30 (S1), and the cutting device 62 is gripped by the cutting means 62 (S2).

  Then, the table mechanism 40 and the carriage mechanism 60 are driven, and the cutting means 62 is moved so that the cutting tool 20 is positioned at a location specified by the automatic detection by the image processing as described above or the manual operation of the operator ( S3).

  At this position, the cutting means 62 is operated to press the grasping cutting tool 20 against the gel to cut the gel (S4). After the gel cut out from the opening at the lower end of the cutting tool 20 remains, the cutting tool 20 is moved to a position immediately above the cutting tool holder 30 (S5). By removing the chucking of the cutting device 20 in a state where the gel is left inside, the cutting device 62 is removed from the cutting means 62, and the cutting device 20 is collected by the cutting device holder 30 (S6).

  Here, there is a case where the gel extraction / recovery operation is not normally performed inside the cutting device 20. In this case, the nucleic acid sample to be recovered remains in the electrophoresis gel 101 or is transferred. The gel falls out of the cutting device 20 inside. Therefore, a step of verifying whether the extraction and collection work has been normally performed is performed (S7). If there is no problem in the collection operation, it is determined whether to cut out another portion of the gel (S8). If all the cutout operations have not been completed, the process returns to step S1 to repeat the same cutout operation.

  However, when it is detected in process S7 that a problem has occurred in the collection work, the cut-out work is immediately stopped (S9). Note that when the work is interrupted by the process S9, loss of valuable specimens can be prevented by requesting assistance from the worker.

  In step S7, the determination as to whether the gel extraction / collection operation is normally performed is performed by irradiating ultraviolet rays after the gel extraction / recovery operation, and the image capturing unit 61 performs image capturing. At this time, the gel 101 for electrophoresis is used as a subject to be photographed. FIG. 4 shows an image obtained by photographing the gel 101 for electrophoresis. When the cutting operation is performed, a rectangular gap 104 is formed on the electrophoresis gel 101 in the region after the cutting. When the operation is normally performed, all the support including the nucleic acid sample in the region where the extraction operation has been performed is collected, and therefore, what emits fluorescence in the gap 104 is not confirmed as shown in FIG.

  However, when the operation is not performed normally and the nucleic acid sample remains in the electrophoresis gel 101, a residual fraction 105 that emits fluorescence is confirmed in the gap 104 as shown in FIG. Therefore, it is possible to verify whether or not the nucleic acid sample remains by confirming the fluorescent color development in the area where the extraction and recovery operation has been performed. As a result, it is possible to detect an abnormality in the work, and it is possible to interrupt the automatic work and request assistance from the worker. At this time, by limiting the range of irradiation with ultraviolet rays to a region centering on the portion where the gel is cut out, partial disruption of nucleic acid molecules of other nucleic acid samples and formation of thymine dimer can be suppressed. Therefore, in order to capture an image, an ultraviolet light source and an image capturing unit are provided on the carriage mechanism 60, and ultraviolet rays are irradiated only in a range where the image capturing unit can capture an image. It becomes possible to irradiate the limited range on the gel 101 with ultraviolet rays. In this case, although it is difficult to photograph the entire range of the electrophoresis gel 101 at a time, the ultraviolet light source and the image photographing means can be driven by the carriage mechanism 60. Images in different ranges can be taken, and an image of the entire range of the electrophoresis gel 101 can be obtained by synthesizing the plurality of images.

  As described above, in the present embodiment, it is confirmed whether or not a necessary fraction remains in the gel by taking an image of the gel for electrophoresis after the extraction and collection of the gel. It is possible to verify whether or not the minute has been successfully extracted and recovered, and whether or not the recovery operation is good.

(Embodiment 2)
Another embodiment for confirming whether the cut out gel has been properly collected and whether the collection work is good or bad is shown below.

  FIG. 5 shows an image obtained by photographing the cutting tool holder 30 containing the cutting tool 20 after the cutting work is finished. The second embodiment provides an extraction / recovery confirmation process in a manner different from that of the first embodiment. The main configuration is the same as that shown in FIGS. 1 and 2 in the first embodiment. In addition, the cutout operation is performed in the same manner as in the flowchart shown in FIG. In the present embodiment, as a confirmation means to be performed in the confirmation step of the process S7 shown in FIG.

  The recovery operation is completed when the cutting device 20 is stored in the cutting device holder 30, but if the gel does not fall off from the cutting device 20 during the transfer, it is shown in FIG. 5 (a). As described above, the fluorescence development of the extracted fraction 106 is confirmed. However, when the gel falls off, a nucleic acid sample that develops fluorescence is not included in the cutting device 20, and therefore, the fluorescent coloring in the cutting device holder 30 is not confirmed as shown in FIG. Therefore, since the presence or absence of the nucleic acid sample can be determined by confirming the fluorescent color development of the fraction in the cutting instrument holder 30, it is possible to detect the dropout during the transfer of the gel. As a result, it is possible to detect an abnormality in the work, and it is possible to interrupt the automatic work and request assistance from the worker.

  At this time, in order to take an image, an ultraviolet light source and the image taking means 61 are provided on the carriage mechanism 60, so that not only the electrophoresis gel 101 but also the cutting device holder 30 can be taken. Become.

  As described above, in the present embodiment, by taking an image of the cutting tool holder 30 after the extraction and recovery of the gel, it is possible to verify whether the gel is dropped during the transfer, and the fraction is extracted normally. It can be made possible to verify whether it has been collected.

  The electrophoresis apparatus according to the present invention is capable of automatically performing batch electrophoresis of gel electrophoresis and electrophoretic patterning, and cutting out a nucleic acid sample after electrophoresis, thereby reducing worker safety and labor. It is possible to provide an apparatus capable of confirming whether or not a necessary nucleic acid sample has been collected without any surplus, and is useful in a technical field related to electrophoresis generally used for analysis of biopolymers.

The perspective view of the electrophoresis apparatus in one embodiment of the present invention The perspective view of the table mechanism part of the same electrophoresis apparatus Flow chart of cutting operation of the electrophoresis apparatus The figure explaining the collection | recovery operation | work of the same electrophoresis apparatus The figure for demonstrating the collection | recovery operation | work of the electrophoresis apparatus in other embodiment of this invention

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 Electrophoresis tank 20 Cutting tool 30 Cutting tool holder 40 Table mechanism 60 Carriage mechanism 61 Image photographing means 62 Cutting means 101 Electrophoresis gel 102 Inlet 104 Gap 105 Residual fraction 106 Extracted fraction

Claims (4)

An electrophoresis tank in which a support for moving the biopolymer by electrophoresis is disposed;
Image photographing means for photographing a biopolymer after electrophoresis in the support;
A cutting device that cuts out a biopolymer at a specified position based on an image photographed by the image photographing means together with the support and puts the biopolymer in the inside thereof; and the cutting device is relative to the support. And a cutout means for moving it automatically,
The image photographing means is for photographing an area of the support that has been cut out by the cutting tool of the cutting means, and the quality of the cutting work is confirmed based on the shot image of this area. Electrophoresis device. The electrophoretic device according to claim 1, wherein the quality of the cutting operation is confirmed based on the presence or absence of a biopolymer in the region after the cutting operation. An electrophoresis tank in which a support for moving the biopolymer by electrophoresis is disposed;
Image photographing means for photographing a biopolymer after electrophoresis in the support;
A cutting device that cuts out the biopolymer at the position specified based on the image photographed by the image photographing means, together with the support, and puts it in the inside thereof,
The cutting device is detachably attached, and a cutting means for moving the attached cutting device relative to the support,
A cutting tool holder for storing the cutting tool removed from the cutting means,
The image photographing means is further for photographing the cutting tool stored in the cutting tool holder by the cutting means after being cut out by the cutting tool, and confirming the quality of the cutting work based on the shadow image An electrophoretic device characterized by comprising: 4. The electrophoresis apparatus according to claim 3, wherein the quality of the cutting operation is confirmed based on the presence or absence of a biopolymer in the cutting device.

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