JP2016211963A - Electrophoretic gel preparation cassette and method for preparing electrophoretic gel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To form a groove having width tapered in a depth direction to gel by simple procedures and with high yield.SOLUTION: A gel preparation cassette 100 is formed by overlapping: a lower plate 130; an intermediate plate 120; and an upper 110. The intermediate plate 120 is provided with: a center surface 125; bank parts 124a, 124b provided on both sides of the center surface 125; hole parts 126 provided at the bank parts 124a, 124b; and a long hole 121 provided near one end of the intermediate plate 120 on the center surface 125. The upper plate 110 is provided with: a flange part 116 provided on one end; a salient 115 projecting from a principal plane 112 overlapping the center plate 120 and provided near the flange part 116; and a projection 117 projecting from the principal plane 112 and provided on both side surface sides. When the upper plate 110 is overlapped with the intermediate plate 120, the salient 115 engages into the long hole 121. A tip part 115p of the salient 115 projects in a gap 140 for gel from the long hole 121.SELECTED DRAWING: Figure 7

Description

  The present invention relates to an electrophoresis gel preparation cassette and a method for producing an electrophoresis gel.

  Conventionally, for example, as disclosed in Patent Documents 1 and 2 below, gel cassettes that perform electrophoresis using a thin plate gel are known. Moreover, according to HDGP (Hyper Detection of infectious disease based on Genome Profiling) described in Non-Patent Document 1 below, temperature gradient gel electrophoresis (TGGE) More advanced analysis can be performed by pattern analysis of the electrophoretic image (band pattern) obtained on the two-dimensional plane. The applicant of the present application has developed an electrophoresis apparatus that automates electrophoresis and imaging of a thin plate-like gel as described in Patent Document 3 below.

JP 2001-165903 A Japanese Patent No. 4424938 International Publication No. 2014/178107

Yoshinori Hatakeyama, Keiichi Hamano and Hidetoshi Iwano, (2008), An Ultimate Method for Detection of Infected Pathogenic Microorganism from Silkworm using HDGP, Journal of Insect Biotechnology and Sericology 77, 1-7

  In the above conventional technique, a groove for dispensing a sample is provided on the surface of a thin plate gel. The inventor of the present application has found that the electrophoretic image (band pattern) can be sharpened by making a groove having a tapering width in the depth direction by original research. In order to actually use this electrophoretic image sharpening technique, it is required to form a groove having a taper width in the depth direction in a thin plate gel with a simple procedure and a high yield.

  The present invention has been made in order to solve the above-described problems, and is an electrophoresis gel capable of forming a groove having a tapered width in the depth direction in a gel with a simple procedure and high yield. It is an object to provide a production cassette and a method for producing an electrophoresis gel.

  The gel preparation cassette for electrophoresis according to the present invention includes a lower plate, a middle plate to be stacked on the lower plate, an upper plate to be removably stacked with the middle plate on the middle plate, The middle plate is provided with a long hole penetrating the middle plate in the thickness direction of the middle plate and linear in the plane direction of the middle plate, and at least one of the lower plate or the middle plate Comprises a bank portion that creates a gap for gel between at least the periphery of the long hole of the intermediate plate and the lower plate, and the bank portion extends to both ends in the short direction of the long hole of the intermediate plate. The upper plate has a convex portion that protrudes from the surface overlapping the middle plate and fits into the elongated hole, and the tip of the convex portion extends from the elongated hole to the gap for the gel. It protrudes inward, and the tip portion is tapered in the height direction of the convex portion.

  According to the present invention, after the gel solution is put in the gel gap and the gel solution is solidified, the upper plate can be removed from the middle plate. Accordingly, the convex portion can be extracted from the gel in a certain direction and at a certain angle while pressing the gel around the elongated hole with the intermediate plate. As a result, it is possible to accurately form a desired tapered groove while suppressing breakage of the gel. After removing the upper plate, the laminate of the middle plate, the gel, and the lower plate may constitute an electrophoresis gel cassette. The laminate may be placed in an electrophoresis cassette holder for electrophoresis. The electrophoresis may be temperature gradient electrophoresis.

  The intermediate plate is preferably made of a transparent material, and a transparent resin material may be used. Thereby, an electrophoretic image (band pattern) can be observed and imaged through the intermediate plate.

  It is preferable that the tapered shape has a corner portion of the tip portion that is partially inclined, and the most tip surface of the tip portion is parallel to the lower plate. Thereby, it can prevent that an electrophoretic image (band pattern) becomes thin too much, and can suppress the blur of a pattern. However, the tapered shape is not limited to this, and includes various specific shapes. The tapered shape may be a shape in which the side surface of the convex portion is entirely inclined (tapered shape) from the base of the convex portion to the distal end portion, and the tapered shape has a corner portion of the distal end portion. It may be partially slanted. The “oblique” may be an inclined surface having a certain inclination (that is, a flat inclined surface), and an inclined surface whose inclination angle changes by making the corner portion of the tip portion a convex curved surface or a concave curved surface ( That is, it may be a curved slope. The corner portion of the tip portion may be a polygonal slope formed by connecting a plurality of flat slopes having different inclinations.

  The said convex part may have the 1st side surface which faces the center side of the said intermediate plate, and the 2nd side surface which faces the end surface side of the said intermediate plate. The tapered shape may be a shape in which only a corner portion on the second side surface side of the tip portion is inclined. In other words, the tapered shape may have a slope on only one corner of the tip. The convex portion may be a linear convex portion that is continuous along the elongated hole, or may be a plurality of convex portions that are spaced apart along the elongated hole.

  The gel preparation cassette for electrophoresis of the present invention may be provided in a state where the gel gap is filled with a gel and a long groove corresponding to the convex portion is formed on the surface of the gel. In this case, the “gap for the gel between the middle plate and the lower plate” is filled with gel instead of air.

  A central hole portion in the longitudinal direction of the long hole has a first width, and a wide hole portion having a second width wider than the first width is provided at a longitudinal end of the long hole. Also good. After the sample is dispensed by the dispenser, when the air is finally discharged, the wide hole portion can receive the air. This is particularly effective in an automatic electrophoresis apparatus. That is, when the dispenser is operated according to the control program and dispensed into a plurality of gels, air can be vented at the wide hole provided at the end of the groove when the current dispensing is finished. The wide part may have a circular outline or a polygonal outline in plan view of the intermediate plate. The longitudinal end portion of the convex portion overlaps the wide hole portion, and the wide convex portion formed wider than the central convex portion of the convex portion may be provided at the convex portion end portion. The part may have the same contour as the wide hole part. It is preferable that at least the central convex portion has a tapered shape, and both the central convex portion and the wide convex portion may have a tapered shape.

  A gap is provided between the inner peripheral surface constituting the elongated hole and the convex portion, and the convex portion has a first side surface facing the center side of the intermediate plate and a first side surface facing the end surface side of the intermediate plate. It may have two side surfaces. The gap between the first side surface and the inner peripheral surface is preferably larger than the gap between the second side surface and the inner peripheral surface. Since the sample proceeds to the center side of the middle plate during electrophoresis, the groove side surface of the gel formed by the first side surface serves as an entrance surface when the sample enters the gel. By ensuring a large gap between the first side surface and the inner peripheral surface, a sufficient amount of the gel solution stably reaches the first side surface side, so that the entrance surface is smooth without unevenness. Can be formed on the surface.

  A flange that closes the gel gap at the end of the middle plate may be provided at one end of the upper plate. By injecting the gel solution into the gel gap, the gel solution proceeds in the gel gap and fills the gel gap. If the collar is not provided, the gel protrudes from between the middle plate and the upper plate, so that the gel around the groove may be damaged during the operation of removing the protruding gel. By providing the collar portion, such a work can be eliminated and a gel cassette can be produced with a high yield.

  The method for producing an electrophoresis gel according to the present invention includes a cassette preparation step of preparing the electrophoresis gel preparation cassette according to the present invention, and a gel solution in the gel gap between the middle plate and the lower plate. And a gel step for solidifying the gel solution, and an upper plate removing step for removing the upper plate from the middle plate after the gel solution is solidified. By removing the upper plate from the middle plate after the gel solution is solidified in the gel gap and the gel solution is solidified, the convex portion can be extracted from the gel in a certain direction while the middle plate presses the periphery of the long hole. Therefore, it is possible to accurately form a groove in the long hole while suppressing breakage of the gel.

  According to the present invention, a groove having a taper width in the depth direction can be formed in a gel with a simple procedure and high yield.

1 is a perspective view illustrating an electrophoresis gel cassette, an electrophoresis cassette holder, and an entire electrophoresis apparatus according to an embodiment of the present invention. FIG. It is a perspective view which shows the gel cassette for electrophoresis concerning the embodiment of this invention, and the cassette holder for electrophoresis. It is a top view which shows the cassette holder for electrophoresis concerning embodiment of this invention. It is a perspective view which shows the gel cassette for electrophoresis concerning embodiment of this invention. It is a perspective view which extracts and shows a gel from the gel cassette for electrophoresis concerning embodiment of this invention. It is sectional drawing of the gel cassette for electrophoresis concerning embodiment of this invention. It is a disassembled perspective view which shows the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is a perspective view which shows the lower board of the preparation gel cassette for electrophoresis concerning embodiment of this invention. It is a perspective view which shows the middle board of the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is a perspective view which shows the upper plate of the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is a see-through | perspective perspective view which shows the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is a see-through | perspective perspective view which shows the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is a partial expansion perspective view which shows the upper board of the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is a figure which shows the middle board of the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is a top view for demonstrating the long hole provided in the middle plate of the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is an enlarged plan view for demonstrating the long hole provided in the intermediate plate of the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is a disassembled perspective view which shows the cassette holder for electrophoresis concerning embodiment of this invention. It is a figure which shows the gel caster for electrophoresis concerning embodiment of this invention. It is a flowchart which shows the manufacturing method of the gel for electrophoresis concerning embodiment of this invention. It is a flowchart which shows the electrophoresis analysis method concerning embodiment of this invention. It is sectional drawing for demonstrating the effect obtained by the gel for electrophoresis concerning embodiment of this invention. It is sectional drawing for demonstrating the effect obtained by the gel for electrophoresis concerning embodiment of this invention. It is sectional drawing for demonstrating the effect obtained by the gel for electrophoresis concerning embodiment of this invention. It is sectional drawing of the gel for electrophoresis concerning the comparative example with respect to embodiment of this invention. It is sectional drawing which shows the modification of the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is sectional drawing which shows the modification of the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is sectional drawing which shows the modification of the gel preparation cassette for electrophoresis concerning embodiment of this invention. It is sectional drawing which shows the modification of the gel preparation cassette for electrophoresis concerning embodiment of this invention.

[Configuration of Apparatus According to Embodiment]
(Configuration of electrophoresis device)
FIG. 1 shows an electrophoresis gel cassette 101 according to an embodiment of the present invention (hereinafter also simply referred to as “gel cassette 101”), an electrophoresis cassette holder 200 (hereinafter also referred to as “cassette holder 200”), and 1 is a perspective view showing an entire electrophoresis apparatus 10. The gel cassette 101 is obtained by preparing an electrophoresis gel 150 using an electrophoresis gel preparation cassette 100 described later, and then removing the upper plate 110 that is a component of the electrophoresis gel preparation cassette 100.

  The electrophoretic device 10 shown in FIG. 1 preferably has a control unit 12, a dispenser 14, an imaging unit 15, a temperature gradient device 16, and an actuator for controlling the dispenser 14 (not shown). ), A liquid supply device 17 composed of a liquid tank storing electrophoresis solution and the like, a pump, and the like, and a DC power supply device 18 that generates a DC voltage for electrophoresis. A holder storage (not shown), a PCR (Polymerase Chain Reaction) device (not shown), a chip rack (not shown), and a chip disposal box (not shown) are further provided. It is preferable that These configurations may be housed in a housing (not shown) and controlled from an external terminal. As the temperature gradient device 16, various known devices that can form a temperature gradient in the plane direction of the surface on which the cassette holder 200 is mounted can be applied, and may be a hot plate, for example. The imaging unit 15 is arranged on the upper part of the cassette holder 200 so that the cassette holder 200 is looked down from above. The dispenser 14 is a device for dispensing the DNA sample 160 into the gel 150. The position of the dispenser 14 is controlled by the control unit 12 via an arm (not shown) or a guide rail electric motor or other various actuators. Accordingly, the dispenser 14 can be moved in the horizontal direction and the vertical direction. The dispenser 14 is connected to a micro pump (not shown) so that the suction force and the discharge force of the dispenser 14 can be adjusted by the operation of the micro pump. Thereby, injection | pouring, discharge, etc. of the DNA sample 160 by the dispenser 14 can be performed. The imaging unit 15 includes an excitation light source and a camera. The excitation light source is a light source for irradiating the DNA after dyeing through the gel cassette 101 with excitation light. The camera can capture a migration image (band pattern) formed in the planar direction of the gel 150 through the transparent intermediate plate 120 of the gel cassette 101. The chip rack stores a plurality of parts called chips that are mounted on the dispenser 14 and used. The reagent rack is used to install reagents used for electrophoresis. The reagent is called a loading buffer. The DNA sample 160 and this reagent are mixed and dispensed into the gel 150. By doing in this way, when the DNA sample 160 is dispensed into the long groove 156 of the gel 150, it can be stabilized so that the DNA sample 160 does not diffuse.

  FIG. 2 is a perspective view showing the gel cassette 101 and the cassette holder 200. FIG. 3 is a plan view showing the cassette holder 200. FIG. 17 is an exploded perspective view of the cassette holder 200. The cassette holder 200 includes an upper surface 204 and a bottom surface 206 and is made of glass or light transmissive resin. The cassette holder 200 has a thermal conductivity capable of transferring heat received by the bottom surface 206 to the gel cassette 101. The upper surface 204 is provided with a recess 210 for storing the gel cassette 101. The recess 210 is a recess having a cross-shaped outline as a whole. The concave portion 210 has a cross-shaped outline, has a central portion 211 at the center thereof, and includes liquid input portions 212 and 214 and frame portions 216 and 218 so as to surround the central portion 211. Since the end surfaces of the gel 150 are exposed from the liquid input portions 212 and 214, the end surfaces of the gel 150 can be immersed in the electrophoretic solution by supplying the electrophoretic solution to the liquid input portions 212 and 214. Electrodes 220 and 222 are provided in the liquid input portions 212 and 214. The electrodes 220 and 222 are made of, for example, a metal material such as platinum, and are connected to the electrode terminals 221 and 223. The electrode terminals 221 and 223 are arranged on one side of the cassette holder 200 and are connected to the DC power supply device 18.

  FIG. 4 is a perspective view showing the gel cassette 101. FIG. 5 is a perspective view showing an electrophoresis gel 150 extracted from the gel cassette 101 (hereinafter also simply referred to as “gel 150”). The gel cassette 101 has a gel 150 sandwiched between two transparent plates (an intermediate plate 120 and a lower plate 130). The gel cassette 101 is made of glass or light transmissive resin, and can transfer heat to the gel 150. The gel cassette 101 is obtained by preparing the gel 150 using the gel preparation cassette 100 according to the present embodiment described later and then removing the upper plate 110. The middle plate 120 is provided with a long hole 121. A long groove 156 of the gel 150 is exposed from the long hole 121.

  As shown on the surface of the gel 150 shown in FIG. 5, the long groove 156 includes a narrow groove portion 154 and a wide groove portion 155 that is wider than the narrow groove portion 154. The elongated groove portion 154 is formed in a linear shape in the vicinity of the upper end portion on the surface of the gel 150 and extends in parallel with one side of the gel 150. A wide groove 155 is provided at one end of the elongated groove 154.

  6 is a cross-sectional view of the gel cassette 101, and shows a cross-sectional view of the gel cassette 101 taken along the plane AA of FIG. As shown in FIG. 6, the long groove 156 formed in the gel 150 includes a bottom surface 154b having a width WB, a vertical side surface 154c connected to one end of the bottom surface 154b, and an inclined side surface 154a connected to the other end of the bottom surface 154b. . The inclined side surface 154a is inclined so as to reduce the width of the long groove 156 toward the bottom surface 154b. The width WA between the upper end of the inclined side surface 154a and the vertical side surface 154c at an arbitrary depth position of the long groove 156 is equal to or larger than the width WB of the bottom surface 154b. The vertical side surface 154c serves as an entrance surface for the DNA sample 160 to enter the gel 150 during electrophoresis. The gel 150 has a thickness D <b> 1 below the long groove 156, and a thickness D <b> 2 (where D <b> 2 <D <b> 1) on the center side of the lower plate 130 than the long groove 156.

(Configuration of gel preparation cassette for electrophoresis)
The gel preparation cassette 100 according to the present embodiment is obtained by stacking a lower plate 130, an intermediate plate 120, and an upper plate 110 as shown in FIGS. FIG. 7 is an exploded perspective view showing an electrophoresis gel preparation cassette 100 (hereinafter also simply referred to as “gel preparation cassette 100”) according to an embodiment of the present invention. FIG. 8 is a perspective view showing the lower plate 130 of the gel production cassette 100. FIG. 9 is a perspective view showing the middle plate 120 of the gel production cassette 100. FIG. 10 is a perspective view showing the upper plate 110 of the gel production cassette 100. FIGS. 11 and 12 are perspective perspective views showing the gel production cassette 100. FIG. FIG. 12 shows a cross section when the gel production cassette 100 is cut along the BB plane in FIG. 11. FIG. 13 is a partially enlarged perspective view showing the upper plate 110 of the gel production cassette 100. FIG. 14 is a view showing the middle plate 120 of the gel production cassette 100.

  In each drawing, for convenience, an XYZ orthogonal coordinate system is shown. The “longitudinal direction” is the length direction of the projections 115 provided in the upper plate 110 and the long holes 121 provided in the middle plate 120, and is referred to as the X-axis direction for convenience in the XYZ orthogonal coordinate system. The short direction is the width direction of the long hole 121, and in the XYZ orthogonal coordinate system, it is the Y-axis direction for convenience. The height of the convex portion 115 and the depth of the long hole 121 are set to dimensions in the Z-axis direction for convenience in the XYZ orthogonal coordinate system.

  As shown in FIG. 8, the lower plate 130 includes a non-contact surface 131 and a non-contact surface 132 that are provided in the center and are flat with respect to the intermediate plate 120, and non-contact surfaces 131 and 132. A step 132a to be divided, a flat contact surface 134 that is provided on both sides of the non-contact surfaces 131 and 132 and that contacts the intermediate plate 120, a protrusion 135 and an elongated recess 133 provided on the contact surface 134, It has. The non-contact surface 132 is a lower surface of the step 132a, and the non-contact surface 131 is an upper surface of the step 132a. The abutting surface 134 is in contact with the bank portions 124a and 124b of the intermediate plate 120 without a gap. The protrusion 135 protrudes in the normal direction of the contact surface 134. The elongated recess 133 partitions the non-contact surfaces 131 and 132 and the contact surface 134.

  As shown in FIG. 9, the intermediate plate 120 includes a flat central surface 125 provided in the center, flat bank portions 124a and 124b provided on both sides of the central surface 125, and the two bank portions 124a, Each of the holes 126b is provided with a hole 126 that passes through the intermediate plate 120 and a long hole 121 that is provided near one end of the intermediate plate 120 in the central surface 125. The long hole 121 passes through the intermediate plate 120 in the thickness direction of the intermediate plate 120 and is formed in a linear shape in the plane direction of the intermediate plate 120. The long hole 121 has a central hole 122 at the center in the longitudinal direction, and a wide hole 123 is provided at one end of the long hole 121 in the longitudinal direction. The bank portions 124 a and 124 b are provided at both ends of the long hole 121 in the longitudinal direction. By the bank portions 124a and 124b, a gel gap 140 for forming a thin gel can be formed between the middle plate 120 and the lower plate 130. In the gel cassette 101 shown in FIG. 4, the gel gap 140 between the middle plate 120 and the lower plate 130 is filled with the gel 150, and a long groove 156 corresponding to the convex portion 115 is formed on the surface of the gel 150. It is. The gel gap 140 extends with a uniform width from the periphery of the long hole 121 to both ends in the short direction of the long hole 121 in the intermediate plate 120. Accordingly, the bank portions 124 a and 124 b cause the gel gap 140 to reach both ends of the long hole 121 in the short direction. As a result, the end surfaces 152, 153 (see FIG. 5) of the gel 150 filled in the gel gap 140 are exposed from the two opposite end surfaces of the gel cassette 101. In this embodiment, the bank portions 124a and 124b are provided only on the middle plate 120, but the bank portions 124a and 124b may be provided only on the lower plate 130, or both the lower plate 130 and the middle plate 120 may be provided. It may be provided.

  Two corners of the middle plate 120 are provided with chamfered portions 128 for forming a gap between the corner end portion of the upper plate 110 and the corner end portion of the middle plate 120. The chamfered portion 128 is provided at the other corner of the intermediate plate 120, and a gap is also formed between the intermediate plate 120 and the lower plate 130. Since the chamfered portion 128 forms a gap, the upper plate 110, the middle plate 120, and the lower plate 130 can be easily disassembled.

  The intermediate plate 120 is preferably made of a transparent material and is formed using a transparent resin material. Thereby, the electrophoretic image (band pattern) can be observed or imaged through the intermediate plate 120. The lower plate 130 may be made of a transparent material, and the upper plate 110 may be made of the same transparent material. However, the present invention is not limited to this, and materials other than the middle plate 120 may be made of a non-transparent material.

  In the present embodiment, the plan view shapes of the lower plate 130, the middle plate 120, and the upper plate 110 are all quadrangles of the same plane dimensions, more specifically rectangular. Each of the lower plate 130, the middle plate 120, and the upper plate 110 is a thin plate-like planar body, and has a front surface, a back surface, and an end surface that connects the front surface and the back surface. The end surface includes one end surface, the other end surface provided on the opposite side of the one end surface, and a set of side end surfaces connecting these end surfaces, and each end surface is connected at a right angle or a substantially right angle. Shall.

  The long hole 121 is provided so as to be biased toward one end face of the upper plate 110. The bank portions 124 a and 124 b are provided at both ends in the longitudinal direction of the long hole 121, and the plurality of bank portions 124 a and 124 b provided at both ends in the longitudinal direction form steps that extend in parallel with each other in plan view of the lower plate 130. ing. These parallel steps extend continuously from the first end surface 120a of the intermediate plate 120 to the second end surface 120b. The width dimension (X-direction dimension) of the gel gap 140 is a constant width dimension at both ends in the short-side direction of the long hole 121 in the intermediate plate 120. An elongated protrusion 127 is provided at the inner ends of the two bank portions 124a and 124b. The elongated protrusion 127 overlaps with the elongated recess 133 of the lower plate 130, and as a result, both sides of the gel gap 140 are sealed.

  The configuration of the upper plate 110 will be described with reference to FIG. The upper plate 110 includes a flange 116 provided at one end, a protrusion 115 protruding from the main surface 112 that overlaps the intermediate plate 120, and a protrusion 115 provided in the vicinity of the flange 116, and protrudes from the main surface 112 on both side surfaces. And a projecting portion 117 provided on the surface. The upper plate 110 is integrated with the middle plate 120 by overlapping the projection 117 on the middle plate 120 while being inserted into the hole 126 of the middle plate 120. Since the integration is performed by fitting the protrusion 117 and the hole 126, the upper plate 110 can be arbitrarily removed from the middle plate 120.

(Configuration of convex part of upper plate)
As shown in the perspective view of FIG. 10, the protrusion 115 is a comb-like (comb-like) elongated protrusion. The convex portion 115 includes a central convex portion 113 and a wide convex portion 114 provided at one end of the convex portion 115. The central convex portion 113 is a linear convex portion having a constant thickness continuous along the long hole 121. The wide convex portion 114 provided at the end in the longitudinal direction of the convex portion 115 is formed wider than the central convex portion 113, and specifically has the same circular outline as the wide hole portion 123. The wide convex portion 114 overlaps the wide hole portion 123. In the present embodiment, both the central convex portion 113 and the wide convex portion 114 are tapered, but the present invention is not necessarily limited thereto, and at least the central convex portion 113 may be tapered.

  In order to describe the configuration of the convex portion 115 in detail, FIG. 12 and FIG. 13 are referred to. FIG. 12 shows a cross-sectional view of a laminate (that is, the gel production cassette 100) in which the lower plate 130, the middle plate 120, and the upper plate 110 are stacked. When the upper plate 110 is overlaid on the middle plate 120, the convex portion 115 fits into the long hole 121. At this time, as shown in FIG. 12, the tip 115 p of the protrusion 115 protrudes into the gel gap 140 from the elongated hole 121. As a result, the long groove 156 having the same shape as the tip portion 115p can be formed in the gel 150. The projecting dimension of the front end portion 115p is such a length that the front end portion 115p does not contact the non-contact surface 132 of the lower plate 130, and the gel 150 has a certain thickness between the non-contact surface 132 of the lower plate 130 and the front end portion 115p. To be formed.

  FIG. 13 is a perspective view of the upper plate 110 in which the vicinity of the convex portion 115 is enlarged. The convex part 115 is comprised from the center convex part 113 and the wide convex part 114 as mentioned above. The central convex portion 113 includes a first side surface 113c and a second side surface 113d that are opposite to each other. A slope 113b is provided in the vicinity of the tip of the projection 115 on the second side surface 113d side, and a region in the vicinity of the slope 113b is referred to as a tip 115p. The wide convex portion 114 has a generally cylindrical shape as a whole, and has a rounded shape with a gentle curve on the apex side. A top surface 113a extending from the central convex portion 113 to the wide convex portion 114 is provided at the most distal end of the distal end portion 115p. The top surface 113 a is parallel to the lower plate 130.

  As is well understood from the perspective view of the upper plate 110 shown in FIG. 13, the tip end portion 115 p is tapered in the height direction of the convex portion 115. The “height direction” is a direction in which the convex portion 115 protrudes and is a normal direction of the main surface 112. In the present embodiment, the tapered shape of the convex portion 115 is specifically that the corner portion of the tip portion 115p is partially inclined. This “oblique” is assumed to be a slope 113b having a certain slope (that is, a flat slope 113b). As a preferred form, the top surface 113 a is parallel to the lower plate 130. The convex portion 115 has a first side surface 113 c that faces the center side of the intermediate plate 120 and a second side surface 113 d that faces the end surface side of the intermediate plate 120. The tapered shape of the convex portion 115 is such that only the corner portion on the second side surface 113d side of the tip portion 115p is inclined.

  The convex portion 115 according to the present embodiment has a linear central convex portion 113 that continuously extends along the long hole 121 on the upper plate 110. This is because a long groove 154 continuous with the gel 150 is formed, and a sample (specifically, a DNA sample 160) to be subjected to electrophoresis analysis is dispensed here. However, the present invention is not limited to this. Instead of the central protrusion 113, a plurality of protrusions provided along the long hole 121 may be provided. In this case, the grooves provided on the surface of the gel 150 are a plurality of small grooves shorter than the long grooves 156 arranged in a line. It is also possible to perform electrophoresis by dispensing a sample into each of the plurality of small grooves. Note that the method for forming the tapered shape is not limited, and chamfering may be performed, and the tip portion 115p may be tapered in the resin molding die for the upper plate 110.

  The flange 116 is provided at one end of the upper plate 110 and closes the gel gap 140 at the end of the intermediate plate 120. By injecting the gel solution into the gel gap 140, the gel solution proceeds in the gel gap 140 and fills the gel gap 140. The flange 116 functions to protect the end of the gel 150. That is, when the collar portion 116 is not provided, the gel 150 protrudes from between the middle plate 120 and the upper plate 110, so that the gel 150 around the long groove 156 is damaged during the operation of removing the protruding gel 150. There is a risk that. In this regard, the provision of the collar portion 116 eliminates such work, and the gel cassette 101 can be manufactured with a high yield.

(Long hole configuration)
As shown in FIGS. 6 and 12, the inner peripheral surface of the long hole 121 is chamfered at the corner on the side overlapping the upper plate 110 (in other words, the edge of the long hole 121). As a result, the inner peripheral surface of the long hole 121 is constituted by the chamfered inner peripheral slope 122a and the vertical inner peripheral side surface 122b. A gap is provided between the inner peripheral side surface 122 b constituting the long hole 121 and the convex portion 115. Specifically, as shown in FIG. 12, the convex portion 115 has a first side surface 113 c facing the center side of the intermediate plate 120 and a second side surface 113 d facing the end surface side of the intermediate plate 120. A gap between the first side surface 113c and the inner peripheral side surface 122b facing the first side surface 113c is defined as O1. A gap between the second side surface 113d and the inner peripheral side surface 122b facing the second side surface 113d is defined as O2. The gap O1 is preferably larger than the gap O2. FIG. 15 is a plan view for explaining the long hole 121 provided in the middle plate 120 of the gel production cassette 100. FIG. 16 is an enlarged plan view for explaining the long hole 121. In order to realize the above-described magnitude relationship O1> O2, the width direction of the long hole 121 is enlarged only on one side as shown in FIG. 16 (dimension W3). Note that the size of the gap O2 may be zero. The effect of this will be described with reference to the cross-sectional view of FIG. 6. Since the DNA sample 160 proceeds to the center side of the intermediate plate 120 during electrophoresis, the side surface of the long groove 156 of the gel 150 formed by the first side surface 113c. Becomes an entrance surface when the DNA sample 160 enters the gel 150. By ensuring a large gap O1 between the first side surface 113c and the inner peripheral side surface 122b, a sufficient amount of the gel 150 solution can stably reach the first side surface 113c side, so that the entrance surface is not uneven. It can be formed on a smooth surface.

  As shown in FIG. 16, the central hole portion 122 in the longitudinal direction of the long hole 121 has a first width W1. A wide hole portion 123 having a second width W2 wider than the first width W1 is provided at the longitudinal end of the long hole 121. The wide hole portion 123 has a circular outline in a plan view of the intermediate plate 120. After the DNA sample 160 is dispensed by the dispenser 14, the air when it is finally completely discharged by the micropump can be received by the wide hole portion 123. This is particularly effective in an automatic electrophoresis apparatus. That is, when the dispenser 14 is operated in accordance with the control program and dispensing is performed on the plurality of gels 150, air is removed by the wide hole portion 123 provided at the end of the long groove 156 when the current dispensing is finished. Can do. Thereby, it is possible to smoothly shift to the next dispensing operation, and it is not necessary to take measures such as moving the dispenser to a specific location just for bleeding.

(Gel caster)
FIG. 18 is a diagram showing an electrophoresis gel caster 300 (hereinafter also simply referred to as “gel caster 300”). The gel caster 300 includes a caster body 320 having a recess 322, a lid 310, and a fixing screw 330. The recess 322 has a depth dimension D3 in which three gel production cassettes 100 can be inserted in a stacked manner. The gel caster 300 has the same structure as that of the gel caster disclosed in the above-mentioned Patent Document 2 (Japanese Patent No. 4424938) and can be used in the same manner, and therefore, further explanation is omitted here.

[Method and Operation According to Embodiment]
FIG. 19 is a flowchart showing a method for manufacturing the electrophoresis gel 150 according to the embodiment of the present invention. In the flowchart of FIG. 18, first, the gel production cassette 100 is prepared, and the upper plate 110, the middle plate 120, and the lower plate 130 are assembled (step S1). Next, three gel production cassettes 100 are put on the gel caster 300 in a stacked manner (step S2). Next, the gel 150 solution is put into the gel gap 140 between the middle plate 120 and the lower plate 130 (step S3). Thereafter, the gel 150 solution is solidified (step S4). After the gel 150 solution is solidified, the gel production cassette 100 is taken out from the gel caster 300 of FIG. 17 (step S5). Since these steps are the same as the gel injection step described in Patent Document 2 (Japanese Patent No. 4424938), detailed description thereof is omitted.

  Next, the upper plate 110 is removed from the middle plate 120 (step S6). When removing the upper plate 110, the upper plate 110 can be easily removed by inserting a pin or a finger into the gap formed by the chamfered portion 128 of the middle plate 120. According to the present embodiment, after the gel 150 solution is put into the gel gap 140 and the gel 150 solution is solidified, the upper plate 110 is removed from the middle plate 120, so that the middle plate 120 holds the periphery of the long hole 121. Since the convex portion 115 can be extracted from the gel 150 in a certain direction while being attached, the long groove 156 can be accurately formed in the long hole 121 while preventing the gel 150 from being damaged. After the upper plate 110 is removed, the laminate of the middle plate 120, the gel 150, and the lower plate 130 constitutes the gel cassette 101.

  FIG. 20 is a flowchart showing the electrophoresis analysis method according to the embodiment of the present invention. The control unit 12 stores a program for executing the following control operation in advance in an internal memory. The following electrophoresis analysis method assumes that the gel cassette 101 produced by the above method is used for the automatic electrophoresis apparatus described in Patent Document 3 (International Publication No. 2014/178107). . However, this shows an example of a method of using the gel cassette 101 according to the present embodiment, and it is of course possible to use the gel cassette 101 for an electrophoresis analysis method different from the procedure shown below.

  In the flowchart of FIG. 20, first, the gel cassette 101 is prepared (step S10), and the gel cassette 101 is set in the cassette holder 200 (step S11). The cassette holder 200 is disposed on the temperature gradient device 16 (step S12). These operations are preferably automatically performed by a transport device (not shown).

  Next, the DNA sample 160 is dispensed (step S13). The control unit 12 controls the dispenser 14 to execute a suction operation, a stirring operation, an arrangement operation, and a discharge operation. The sucking operation is an operation in which the dispenser 14 attaches the chip in the chip rack and sucks the DNA sample 160 from the PCR device. The stirring operation is an operation of repeating suction and discharge so that the DNA sample 160 sucked from the PCR device is mixed with the reagent in the reagent rack. The placement operation is an operation of moving the dispenser 14 to a predetermined position of the reagent rack for the stirring operation or a predetermined position of the cassette holder 200 on the temperature gradient device 16. The discharge operation is an operation for discharging the DNA sample 160 mixed with the reagent in the chip attached to the dispenser 14 to the dispenser 14. During the discharging operation, the DNA sample 160 is dispensed from the long hole 121. The DNA sample 160 is evenly dispensed in parallel with the long groove 156 along the direction in which the long groove 156 extends. At the end of dispensing, the dispenser 14 that has reached the wide hole portion 123 performs air discharge.

  Next, a mixed solution obtained by mixing the electrophoresis solution and the staining solution is put into the liquid input unit 212, and the electrophoresis solution is put into the liquid input unit 214 (step S14). Note that the dyeing liquid may also be put into the liquid charging unit 214. Next, the electrophoresis is performed by applying a DC voltage to the electrophoresis solution, and a temperature gradient is applied to the gel 150 during electrophoresis by controlling the temperature gradient device 16 (step S15). The staining solution and the electrophoresis solution are mixed before starting electrophoresis. A charged staining solution is used as the staining solution. If electrophoresis is performed in such a state, the staining solution as well as the electrophoresis solution permeates the gel 150 by the principle of electrophoresis. As a result, electrophoresis and staining for the DNA sample 160 can be performed collectively. Since details of such electrophoresis technology are disclosed in International Publication No. 2014/178107 (International Application No. PCT / JP2013 / 062592), further explanation is omitted here.

  After electrophoresis, the gel 150 is imaged by the camera of the imaging unit 15 without removing the middle plate 120 by applying excitation light vertically (or obliquely) from the lower plate 130 side (or from the upper plate 110 side) (step) S16). When the step S16 is completed, it is determined whether or not the current DNA analysis is completed (step S17). If the current schedule is a single analysis, the current routine ends. On the other hand, if the analysis is scheduled to be performed a plurality of times, the process returns to step S10, and the analysis from the next time is similarly repeated using the other gel cassette 101 until the analysis for the predetermined number of times is completed. In the electrophoresis apparatus 10, the cassette holder 200 is replaced by preferably feeding the new cassette holder 200 after the cassette holder 200 is removed manually or preferably automatically by another machine. The above steps S10 to S17 are repeated as many times as necessary. Thereafter, when the number of analyzes reaches the scheduled number, the current routine is terminated. In the process of step S10, a plurality of gel cassettes 101 may be prepared in advance and complemented by a cassette rack (not shown), and a transfer device (for example, a conveyor, an arm or other transfer actuator) (not shown) is used as the cassette. The electrophoresis apparatus 10 may be configured so that the gel cassette 101 is automatically arranged at a predetermined position from the rack for the required number of times.

  FIGS. 21 to 23 are cross-sectional views for explaining the effects obtained by the gel 150. FIG. 24 is a cross-sectional view of a gel 150 according to a comparative example. The inventor of the present application has made the electrophoretic image (band pattern) clearer than in the comparative example of FIG. 24 by making the long groove 156 into which the DNA sample 160 is dispensed into a taper width in the depth direction by original research. I found out that I can do it. The fact that the electrophoretic image becomes clear has been confirmed by an experiment conducted by the present inventor, and the results of the study by the present inventor regarding the mechanism are shown below. When a DC voltage is applied and electrophoresis is started, the charged DNA sample 160 moves inside the gel 150. In the drawing, a lump of the DNA sample 160 is schematically shown as a single molecule as a sample molecule 161, but this is for convenience in explaining the movement of the DNA sample 160. In the comparative example shown in FIG. 24, the long groove 1156 of the gel 150 has a constant width, and the delay between the sample molecule 161 that has entered the gel 150 first and the sample molecule 161 that has entered the gel 150 later. Is equal to the width of the long groove 1156. On the other hand, in the gel 150 according to the present embodiment, the long groove 156 has the inclined side surface 154a. FIGS. 21 to 23 schematically show how the DNA sample 160 in the long groove 156 decreases as the sample molecules 161 enter the gel 150 during electrophoresis. On the deep side of the long groove 156, the width of the long groove 156 is smaller than that of the long groove 1156 of the comparative example. As a result, when each sample molecule 161 travels through the gel 150, the delay between the sample molecule 161 that entered the gel 150 in advance and the sample molecule 161 that entered the gel 150 later in comparison with the case of the comparative example. It is thought that it becomes smaller. If the progress delay in the gel 150 is reduced, the spread of the sample molecules 161 is reduced (in other words, the sample molecules 161 move together), so that a clearer electrophoretic image can be obtained. According to the gel preparation cassette 100 according to the present embodiment, such a long groove 156 having a tapered cross-sectional shape can be formed in the gel 150 with a simple procedure and a high yield.

  In the present embodiment, as a preferred form, the top surface 113 a of the convex portion 115 is parallel to the lower plate 130. As a result, the bottom surface 154 b can be formed in the long groove 156 of the gel 150. If there is no bottom surface 154b and the inclined side surface 154a and the vertical side surface 154c intersect, the long groove 156 can also take a shape that tapers to the bottom (see a modification of FIG. 25 described later). In this case, according to the experiment result of the inventor of the present application, there is a problem that the electrophoretic image (band pattern) is too thin and the pattern is blurred. In order to avoid this, it is considered not preferable that the long groove 156 is too sharp in the depth direction. Therefore, it is preferable to form a flat bottom surface 154 b in the long groove 156, and for this purpose, the top surface 113 a is preferably parallel to the lower plate 130.

[Modification of Embodiment]
25 to 27 are cross-sectional views showing modifications of the gel production cassette 100. FIG. The tapered shape of the convex portion 115 according to the present embodiment is a preferred embodiment in which the corner portion of the tip portion 115 p is partially inclined, and the top surface 113 a is parallel to the lower plate 130. However, the tapered shape is not limited to this, and includes various specific shapes. Like the convex part 1151 shown in FIG. 25, it is good also considering the front-end | tip part 115p as a structure only of a slope. In this case, when the inclined side surface 154a and the vertical side surface 154c intersect with each other without the bottom surface 154b, the long groove 156 has a shape that tapers to the bottom. Also in this case, an effect that the electrophoretic image can be sharpened can be obtained as compared with the comparative example of FIG. Further, the shape of the tip portion 115p is not limited to the shape of the inclined surface 113b (that is, a flat inclined surface) having a certain inclination. Like the convex part 1152 shown in FIG. 26, it may be a slope (that is, a curved slope) whose inclination angle changes by making the corner part of the tip part 115p a convex curved surface. Or it is good also as a concave curved surface contrary to FIG. Like the convex part 1153 shown in FIG. 27, it is good also considering the corner | angular part of the front-end | tip part 115p as the polygonal slope which connected the several flat slope from which inclination differs. As in the convex portion 1154 shown in FIG. 28, the tapered shape may be a shape in which the second side surface 113d of the convex portion 115 is entirely inclined (tapered shape) from the root of the convex portion 115 to the tip portion 115p. good.

DESCRIPTION OF SYMBOLS 10 Electrophoresis apparatus 12 Control part 14 Dispenser 15 Imaging part 16 Temperature gradient apparatus 17 Liquid supply apparatus 18 DC power supply apparatus 100 Electrophoresis gel preparation cassette 101 Electrophoresis gel cassette 110 Upper plate 112 Main surface 113 Central convex part 113a Top surface 113b Slope (flat slope)
113c 1st side surface 113d 2nd side surface 114 Wide convex part 115,1151,1152,1153,1154 Convex part 115p Tip part 116 Edge part 117 Protrusion part 120 Middle plate 120a 1st end surface 120b 2nd end surface 121 Long hole 122 Central hole part 122a Inner peripheral slope 122b Inner peripheral side surface 123 Wide hole portion 124 Bank portion 124a Bank portion 125 Central surface 126 Hole portion 127 Elongated projection portion 128 Chamfered portion 130 Lower plate 131, 132 Non-contact surface 132a Step 133 Elongated recess portion 134 Contact Surface 135 Protrusion 140 Gel gap 150 Electrophoresis gels 152, 153 End face 154 Elongated groove 154a Inclined side 154b Bottom 154c Vertical side 155 Wide groove 156 Long groove 160 Sample 161 Sample molecule 200 Electrophoresis cassette holder 204 Upper surface 206 Bottom 210 Recess 211 medium Center portion 212, 214 Liquid inlet portion 216, 218 Frame portion 220, 222 Electrode 221, 223 Electrode terminal 300 Gel caster 310 for electrophoresis 310 Cover 320 Caster body 322 Recess

Claims (9)

A lower plate,
An intermediate plate to be stacked on the lower plate;
An upper plate to be detachably stacked on the middle plate;
With
The middle plate is provided with a long hole that penetrates the middle plate in the thickness direction of the middle plate and is linear in the plane direction of the middle plate,
At least one of the lower plate or the middle plate includes a bank portion that creates a gap for gel between at least the periphery of the elongated hole of the middle plate and the lower plate,
The bank portion is to reach the gel gap to both ends in the short direction of the long hole in the middle plate,
The upper plate has a convex portion that protrudes from the surface overlapping the middle plate and fits into the elongated hole,
A gel preparation cassette for electrophoresis, wherein a tip end portion of the convex portion protrudes from the elongated hole into the gel gap, and the tip portion is tapered in the height direction of the convex portion.   2. The electrophoresis according to claim 1, wherein the tapered shape has a corner portion of the tip portion that is partially inclined, and a tip end surface of the tip portion is parallel to the lower plate. Gel preparation cassette. The convex portion has a first side surface facing the center side of the intermediate plate, and a second side surface facing the end surface side of the intermediate plate,
2. The electrophoresis gel preparation cassette according to claim 1, wherein the tapered shape is formed by obliquely forming a corner portion on the second side surface side of the tip portion. The longitudinal central hole in the elongated hole has a first width;
2. The electrophoresis gel preparation cassette according to claim 1, wherein a wide hole portion having a second width wider than the first width is provided at a longitudinal end of the long hole.   A longitudinal end portion of the convex portion overlaps the wide hole portion, and a wide convex portion formed wider than a central convex portion of the convex portion is provided at a longitudinal end portion of the convex portion, and the wide convex portion is provided. 5. The electrophoresis gel preparation cassette according to claim 4, wherein the portion has the same outline as the wide hole portion, and both the central convex portion and the wide convex portion are tapered. A gap is provided between the inner peripheral surface constituting the elongated hole and the convex portion,
The convex portion has a first side surface facing the center side of the intermediate plate, and a second side surface facing the end surface side of the intermediate plate,
The gel cassette for electrophoresis according to claim 1, wherein a gap between the first side surface and the inner peripheral surface is larger than a gap between the second side surface and the inner peripheral surface.   The electrophoresis gel preparation cassette according to claim 1, wherein a collar portion that closes the gap for the gel at an end of the middle plate is provided at one end portion of the upper plate. The lower plate, the middle plate, and the upper plate are quadrilaterals having the same planar dimensions,
The lower plate, the middle plate, and the upper plate each have a front surface, a back surface, and an end surface that connects the front surface and the back surface,
The end face includes a first end face, a second end face provided on the opposite side of the first end face, and a set of third end faces connecting the first and second end faces, and these first to third end faces are Connected at right angles or almost right angles,
The long hole provided in the intermediate plate extends in parallel with the first and second end surfaces from one of the pair of third end surfaces to the other,
The long hole is provided on the first end face side from the center of the middle plate,
The bank portions are provided at both ends in the longitudinal direction of the long hole, and the plurality of bank portions form steps that are parallel to each other, and the parallel steps are continuous from the first end surface to the second end surface. 2. The electrophoresis gap according to claim 1, wherein a width dimension of the gap for the gel is equal to a width direction end part of the elongated hole in the intermediate plate. Gel production cassette. A lower plate, an intermediate plate overlaid on the lower plate, and an upper plate overlaid on the intermediate plate, wherein the intermediate plate has the intermediate plate in the thickness direction of the intermediate plate. A long hole that is linear in the plane direction of the middle plate is provided, and at least one of the lower plate or the middle plate is a gel between at least the periphery of the middle plate and the lower plate. A bank portion that creates a gap for use, and the bank portion allows the gel gap to reach both ends in the short direction of the long hole in the middle plate, and the upper plate overlaps the middle plate. A convex portion that protrudes from the surface and fits into the elongated hole, a distal end portion of the convex portion projects from the elongated hole into the gap for the gel, and the distal end portion tapers in a height direction of the convex portion. A cassette preparation step of preparing a gel preparation cassette for electrophoresis;
A gel step of putting the gel solution in the gap for the gel between the middle plate and the lower plate, and solidifying the gel solution;
After the gel solution is solidified, an upper plate removing step of removing the upper plate from the middle plate;
A method for producing an electrophoresis gel, comprising:

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