JP3309951B2 - Gel surface former - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a gel electrophoresis apparatus. More specifically, the present invention relates to a gel surface former for flattening the upper surface of a gel electrolyte layer of a gel electrophoresis plate of a gel electrophoresis apparatus.

[0002]

2. Description of the Related Art Gel electrophoresis is widely used as a method for determining a base sequence of DNA or the like.

Conventionally, when performing electrophoresis, a sample is labeled with a radioisotope and analyzed, but this method has a drawback that it requires time and effort. In addition, maximum safety and control are always required from the viewpoint of radioactivity management, and analysis cannot be performed unless in a special facility. For this reason,
Recently, a method of labeling a sample with a phosphor has been studied.

In the method using light, a fluorescently labeled DN is used.
The A fragment is allowed to migrate in the gel.
A photoexciting section and a photodetector are provided for each electrophoresis path below ２０20 cm, and DNA fragments passing therethrough are sequentially measured. For example, DNAs of various lengths whose terminal base species are known are duplicated by an enzymatic reaction (dideoxy method) using a DNA chain whose sequence is to be determined as a template, and these are labeled with a fluorescent substance. That is, adenine (A) labeled with a fluorescent substance
A fragment group, a cytosine (C) fragment group, a guanine (G) fragment group, and a thymine (T) fragment group are obtained. These fragment groups are mixed and injected into separate electrophoresis lane grooves of an electrophoresis gel,
Apply voltage. Since DNA is a chain polymer polymer having a negative charge, it moves in the gel at a speed inversely proportional to the molecular weight. Shorter (small molecular weight) DNA strands move faster and longer (larger molecular weight) DNA chains move more slowly, so that DNA can be fractionated by molecular weight.

Japanese Patent Application Laid-Open No. 63-21556 discloses that a line on a gel of an electrophoresis apparatus irradiated with a laser and an arrangement direction of a photodiode array are perpendicular to a migration direction of a DNA fragment in the electrophoresis apparatus. Is disclosed.

FIG. 6 is a schematic diagram for explaining the configuration of the apparatus. In the apparatus shown in FIG. 6, the laser light emitted from the light source 70 is reflected by the mirror 72 and irradiates a predetermined point in the gel of the electrophoresis plate 74 horizontally and horizontally with the laser light. When the fluorescently labeled DNA fragment 76 that has migrated through the gel passes through the irradiated area, fluorescence is sequentially emitted from the DNA fragment. At this time, the type of the terminal base can be determined from the horizontal position of the fluorescence emission, the length of the fragment can be determined from the difference in the migration time from the start of the migration, and the sample can be further identified by the emission wavelength. The fluorescent light from each migration path forms an image at the light receiving section 82 of the image intensifier 80 by the lens 78. This signal is amplified, converted into an electric signal by the photodiode array 84, and measured. The sequence of each DNA fragment is calculated by processing the measurement result by computer, and the base sequence of the target DNA is determined.

As shown in FIG. 7, the electrophoresis plate 74 has a gel electrolyte layer 90 made of an electrophoresis gel (for example, polyacrylamide) sandwiched between glass plates 86 and 88. Spacers 92 for regulating the thickness of the gel electrolyte layer 90 are interposed at both ends in the longitudinal direction of the two glass plates. The upper end of the glass plate 88 is cut out in the width direction at a predetermined depth except for a small portion from both ends toward the center in the width direction. This cutout portion 94 is provided for bringing the upper end of the gel electrolyte layer 90 into contact with the buffer solution. The thickness of the entire electrophoresis plate is about 10 mm, while the thickness of the gel electrolyte layer 90 itself is about 0.3 mm. At the substantially same position as the lower end surface 96 of the cutout portion 94 of the glass plate 88, there is an upper end of an uneven comb-like gel electrolyte layer. A DNA fragment labeled with a fluorescent substance is injected into the concave portion 75 of the comb tooth.

A DNA fragment to be analyzed is injected into the concave portion 75 at the upper end of the gel electrolyte layer. However, the width of the recess 75 is about 1.5 mm, and the depth is only about 5 mm. The interval between the recesses is about 2 mm. Therefore, the sample must be injected into the recess 75 using a small glass tube such as a capillary. However, since both the glass plate of the electrophoresis plate and the gel electrolyte are transparent, it is extremely difficult to confirm or discriminate the position of the concave portion 75, and the injection of the sample often fails.

In order to determine the base sequence of DNA, it is necessary to regularly detect adenine (A), guanine (G), cytosine (C) and thymine (T) constituting the DNA. Nevertheless, it tends to lead to errors in the analysis results. For this reason, the injection of the sample must be performed with great care, and this has caused a great decrease in work efficiency.

In order to solve this problem, a "shark comb" 20 as shown in FIG. 8 has recently been proposed. A plurality of comb teeth 22 are formed at one end of the long side of the shark comb 20, and the comb teeth are tapered so as to become thinner from the root toward the tip.

FIG. 9 is a partially enlarged front view showing a state where the shark comb 20 is set on the electrophoresis plate 74. The shark comb 20 is inserted into a gap between the two glass plates, for example, from the upper end side of the electrophoresis plate 74. Shark comb 20
It is preferable that the tips of the comb teeth 22 slightly enter the gel electrolyte layer 90. By doing so, the adjacent comb teeth 22 serve as walls, and the sample-filled section defined by the space 25 can be isolated from the adjacent section.

FIG. 10 is a sectional view taken along line XX in FIG. As shown in the drawing, the upper end of one glass plate 88 constituting the electrophoresis plate is partially cut out along the longitudinal direction, and the comb teeth 22 have a predetermined length. The opening 27 is formed between the notch 94 and the lower end surface 96. From the opening 27, the tip of the injection means 29 such as a capillary or a micropipette can be inserted into the sample filling space 25, and the sample 31 can be injected. When this shark comb 20 is used, the work of injecting a sample into a predetermined migration path position of the gel electrolyte layer of the migration plate is remarkably promoted, and the efficiency is greatly improved.

A shark comb 20 as shown in FIG.
Before inserting into the gel electrolyte layer 90 of the electrophoresis plate 74, the upper end surface of the gel electrolyte layer 90 must be flattened.
For this reason, conventionally, a gel surface former 33 as shown in FIG. 11 has been used. FIG. 12 shows this gel surface former 33 as the gel electrolyte layer 9 of the electrophoresis plate 74.
It is a partial outline front view showing the state inserted in 0. Such a rectangular gel surface former 33 is not only difficult to insert between the glass plates at the time of forming the gel electrolyte layer, but also has a negative pressure inside when removing the gel electrolyte layer, disturbing the upper end interface of the gel electrolyte layer, and eventually, The flattening of the upper end surface of the gel electrolyte layer is hindered. Even if the shark comb is inserted into the gel electrolyte layer in such a state, the original effect to be obtained by using the shark comb cannot be obtained, which adversely affects electrophoresis.

[0014]

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a gel surface former which does not disturb the gel electrolyte layer interface when the gel electrolyte layer is inserted into and removed from the gel electrolyte layer of the electrophoresis plate.

[0015]

The object of the present invention is to provide a gel electrophoresis device comprising a glass electrolyte layer sandwiched between two glass plates, each of which is partially cut off in the width direction at one end of one glass plate. In a gel surface former inserted from the upper end of the electrophoresis plate to flatten the upper surface of the gel electrolyte layer, the gel surface former is substantially rectangular, and one end of one long side is chamfered. Solved by gel surface former.

[0016]

FIG. 1 is a front view of one example of a gel surface former 1 of the present invention. Although both ends of the upper end surface of the gel surface former 1 are at right angles, both end portions 3 and 3 of the lower end surface are chamfered. The chamfered shape at both ends of the lower end surface is not particularly limited. As shown, both ends of the lower end surface can be chamfered linearly. Both ends of the lower end can be chamfered at an elevation angle (θ) in the range of 50 ° to 75 °. It is preferable to chamfer at an elevation angle (θ) within the range of 60 ° to 65 °. If the angle θ is less than 50 °, the interface of the chamfered portion adheres to the gel, and when the gel surface former is extracted, the gel interface is disturbed. On the other hand, if θ exceeds 75 °, it is impossible to quickly avoid negative pressure, which causes a great obstacle to the gel interface of the sample filling section. In this case, the distance between the distance a from the end in the lateral direction and the distance b from the end in the upward direction is not particularly limited. It can be appropriately determined in consideration of the size of the shark comb, the penetration depth into the gel, the number of effective sample lanes, and the like. If one dimension of a or b is determined from the relationship of tan θ, the other is automatically determined. As a general index, 2.5 mm ≦ a ≦ 8.4 mm, and 3.0 mm ≦
It is preferable that b ≦ 10.0 mm. The gel surface former 1 of the present invention is generally used by being inserted into the gel electrolyte layer by a distance equal to b of the chamfer. Although not shown, the chamfer is not limited to a straight line but may be a curved surface. When chamfering into a curved shape, an appropriate curved shape such as an arc shape or an elliptical shape can be adopted.

The gel surface former 1 shown in FIG.
Is preferably about the same as the thickness of the gel electrolyte layer 86 of the electrophoresis plate 74, or slightly smaller than the gel electrolyte layer. For approximately the same thickness as the gel electrolyte layer, the thickness of the gel surface former is about 0.3 mm.

The material of the gel surface former 1 is not particularly limited. For example, any material such as thermoplastic synthetic resin, thermosetting synthetic resin, glass, metal foil, ceramic, and paper can be used. Thermoplastic synthetic resins such as vinyl chloride are preferred.

In the gel surface former 1 of the present invention shown in FIG. 1, the lower end face inserted into the gel electrolyte layer is chamfered at both ends, so that the gel surface former inserted into the gel electrolyte layer is used. At the time of removal, since air escapes from the chamfered portion, the space between the gel surface former and the gel electrolyte layer is not in a negative pressure state. As a result, even when the gel surface former is removed from the gel electrolyte layer, the gel electrolyte layer is not pulled up, and the upper surface of the gel electrolyte layer can be maintained in a flat state.

FIG. 2 is a front view showing another embodiment of the gel surface former 1 of the present invention, and FIG. 3 is a left side view thereof. The gel surface former 1 has a projection 5 that is flush with the upper end surface. The projections 5 are preferably formed of the same material as the material of the gel surface former 1. The thickness of the projection piece 5 is not particularly limited. Gel surface former 1 thickness is about 0.3
mm, the thickness of the protruding piece 5 is, for example, about 3 mm to
It can be 5 mm. Gel surface former 1
The protruding piece 5 and the protruding piece 5 can be bonded and integrated by a known and commonly used means such as an adhesive. Other integration means can of course be used.

Both ends 7, 7 of the lower end surface of the projection piece 5 are chamfered. The chamfering of this portion is such that when the gel surface former 1 is inserted into the electrophoresis plate 74, both ends of the lower end portion of the protruding piece 5 collide with the inner upper end of the notch 94 of one of the eared glass plates 88 of the electrophoresis plate 74. This is to avoid things. When the gel surface former 1 is inserted into the electrophoresis plate 74, the lower end surfaces 9, 9 of the protruding pieces 5 abut on the lower end surface 96 of the notch 94. Thereby, the depth of penetration of gel surface former 1 into gel electrolyte layer 90 (that is, distance b) can be kept constant. Generally, in the gel surface former 1, the gel electrolyte layer 90 is inserted into the electrophoresis plate 74 at a non-solidified stage. Therefore, when the gel surface former 1 is inserted into the electrophoresis plate 74, the gel electrolyte layer 9
From 0, the electrolyte solvent leaks. In order to temporarily retain the leaked solvent, a part is cut out from the lower end surface of the protruding piece 5 toward the upper end surface thereof to form a liquid pool portion 11.

FIG. 4 is a partial schematic front view showing a state where the gel surface former 1 of the present invention shown in FIG. 2 is inserted into the electrophoresis plate 74, and FIG. 5 is taken along the line VV in FIG. It is a partial outline sectional view. As shown, the lower end surfaces 9 and 9 of the projecting pieces 5 serve as stoppers, and the glass plate 8
8 is brought into contact with the lower end surface 96 of the notch 94, and the depth of the gel surface former 1 entering the gel electrolyte layer 90 becomes constant. Also, when the gel surface former 1 is inserted into the gel electrolyte layer 90 due to the presence of the liquid pool 11,
The liquid gel electrolyte or the electrolyte solvent is prevented from leaking to the outer surface of the electrophoresis plate 74.

[0023]

As described above, the gel surface former of the present invention has a chamfered lower end face inserted into the gel electrolyte layer, so that the gel surface former inserted into the gel electrolyte layer is chamfered. When removing the gel, air is released from the chamfered portion, so that there is no negative pressure between the gel surface former and the gel electrolyte layer. As a result, even when the gel surface former is removed from the gel electrolyte layer, the gel electrolyte layer is not pulled up, and the upper surface of the gel electrolyte layer can be maintained in a flat state.

[Brief description of the drawings]

FIG. 1 is a front view of an example of a gel surface former according to the present invention.

FIG. 2 is a front view of another example of the gel surface former of the present invention.

FIG. 3 is a left side view of the gel surface former shown in FIG. 2;

FIG. 4 is a partial schematic front view showing a state where the gel surface former of the present invention shown in FIG. 2 is inserted into an electrophoresis plate.

FIG. 5 is a partial schematic sectional view taken along line VV in FIG. 4;

FIG. 6 shows a D disclosed in JP-A-63-21556.
FIG. 2 is a schematic configuration diagram of an NA base sequencer.

FIG. 7 is a partially enlarged perspective view of an electrophoresis plate used in the apparatus shown in FIG.

FIG. 8 is a front view of an example of a shark comb.

9 is a partial schematic front view showing a state where the shark comb shown in FIG. 8 has been inserted into an electrophoresis plate.

FIG. 10 is a partial schematic sectional view taken along line XX in FIG. 9;

FIG. 11 is a front view of an example of a conventional gel surface former.

12 is a partial schematic front view showing a state where the gel surface former shown in FIG. 11 is inserted into an electrophoresis plate.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Gel surface former of this invention 3 Chamfer part 5 Projection piece 7 Chamfer part of a protrusion piece 9 Stopper 11 Liquid pool part

Claims (6)

(57) [Claims] 1. An electrophoresis plate for gel electrophoresis in which a gel electrolyte layer is sandwiched between two glass plates in which a part of an upper end portion of one glass plate is cut out in a width direction is inserted. A gel surface former for flattening the upper surface of the gel electrolyte layer, wherein the gel surface former has a substantially rectangular shape and one end of one long side is chamfered. 2. 50 ° to 75 ° with respect to the long side to be chamfered
2. The gel surface former according to claim 1, wherein both ends of said long side are chamfered linearly at an elevation angle (θ) within a range of ゜. 3. A protrusion having the same length as the gel surface former and having a width shorter than the width of the gel surface former is joined to the end surface of the long side of the gel surface former which is not chamfered in a flush manner. The gel surface former of claim 1 wherein said gel surface former is formed. 4. Both ends of the long side of the protruding piece facing the long side which is chamfered of the gel surface former are chamfered, and there is a horizontal plane part extending inward in the horizontal direction from the lower end of the chamfered part. 2. The gel surface former according to claim 1, wherein there is a liquid pool portion cut out at a predetermined width from an inner end of the horizontal surface portion to the other long side. 5. The gel surface former according to claim 1, wherein the gel surface former is formed of a material made of a synthetic resin. 6. A projection piece is formed from a material made of a synthetic resin, a gel surface former is formed from the same material as the projection piece or a different synthetic resin material, and the projection piece and the gel surface former are integrally joined with an adhesive. Claim 3
Gel surface former.