JP3075602B2 - Electrophoresis device - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an electrophoresis apparatus and, more particularly, to a gel electrophoresis apparatus having an improved sample introduction portion and effective for separation and detection of DNA and the like.

[0002]

2. Description of the Related Art Conventionally, including DNA sequencing,
Plate-type gels (slab gels) have been used for separation detection of DNA. The minimum detection amount of DNA in this device depends on the DNA concentration and volume in the measurement area. Therefore, it is necessary to reduce the volume of the DNA band in order to effectively detect a small amount of sample. As a method for realizing this, a capillary electrophoresis method has been proposed and used for DNA analysis (Anal. Chem. 62, 900-903, (1990)).

[0003] The apparatus currently used for capillary electrophoresis is shown in FIG.
First and second buffer over bath filled with over liquid 101,1
And a 02, electrodes 103 and 104 from the high voltage power supply B to provide the electrophoretic field potential is attached to the two buffers over tank. The buffer over liquid in the two tanks is key <br/> through Yapirari over 105 are adapted to be communicated with, the detection unit 106 to the appropriate position of the calibration peak rally is located. In performing the separation and detection of DNA or the like using this device, to disengage the front end of the calibration peak rally from the buffer over tank filled with buffer over liquid, and insert the tip into the sample reservoir S located outside after introducing a predetermined amount of the sample to the tip of the calibration peak rally <br/>, it is immersed again tip of calibration peak rally the buffer <br/> buffer over liquid within § chromatography tank. Thereafter, an analysis was electrophoresed samples gave migration electrode between bar <br/> Ffa over bath.

In this type of capillary electrophoresis, usually about nl of a sample enters a capillary from a sample reservoir of the order of μl by electric field injection and is analyzed.

[0005]

In the above prior art, the amount of the sample injected into the capillary is small, but there is a drawback that a sample that is nearly 100 times the injected amount is required in the sample reservoir. For this reason, the required minimum amount of the actual sample required for separation and detection is not necessarily small, and there has been a difficulty in analyzing trace components.

[0006] An object of the present invention is to concentrate almost all of a sample such as DNA in a sample reservoir once and then migrate it to a separation electrophoresis section, thereby obtaining a very small amount of sample actually required for analysis and detection. By providing an approximately same amount of sample to the sample reservoir, D by electrophoresis that can perform sufficient separation and detection
An object of the present invention is to provide a sample separation / analysis device such as NA.

[0007]

In order to achieve the above object, the present invention provides a fluorescence detection type electrophoresis apparatus comprising a sample injection section, a separation and migration section, a detection section, and a high voltage power supply section for migration. sample injection portion to the first molecule capable of passing material detection object located concatenated two buffer over bath and capillary tubes part tubule portion is smaller than the detection target but not pass size to each other together and a sieve membrane, the separation lane area is open in the vicinity upstream of the molecule sieve membrane, and high-voltage power supply unit for electrophoresis is and between said two buffer <br/> § over bath discloses an electrophoresis apparatus characterized by connecting freely switch and between one and the separation electrophoresis unit between two buffer over bath.

[0008] A second molecular sieve membrane is provided on the upstream side of the capillary portion to allow the detection target to pass therethrough, but to block the passage of a substance having a size larger than the detection target. The object can be further achieved by opening the capillary portion between the one molecular sieve membranes. Separating lane area may be hollow calibration pin rally filled with buffer over liquid, or may be a gel formed within Garasukya pin rally. Further, a flat gel formed between two glass flat plates may be used.

Further, the object can be further achieved by providing a means for limiting the area of the molecular sieve membrane to an area substantially equal to or smaller than the cross-sectional area of the separation / phoresis section.

[0010]

In a preferred embodiment of the electrophoresis apparatus according to the present invention, the sample injecting section is configured by selecting a first and a second buffer tank, a sample injecting well located at the bottom of the second buffer tank, and a molecular size. It consists of a molecular sieve membrane to be separated and a capillary tube connecting the two buffer tanks. Further, the molecular sieve membrane is preferably provided in the middle of a thin tube connecting the first buffer tank and the second buffer tank. In analyzing the sample, first, the sample is injected into a sample injection well provided at the bottom in the second buffer tank, and the first and second buffer tanks are filled with a buffer solution. As a result, the inside of the thin tube is filled with the buffer solution, and the separation electrophoresis part whose entrance is opened near the molecular sieve membrane is also filled with the buffer solution.

In this state, a potential is applied between the electrodes for electrophoresis to enable electrophoresis between the first and second buffer tanks. The molecular sieve membrane is selected so that the salt having a small molecular size in the buffer solution is allowed to pass through but the DNA molecule having a large molecular size is not allowed to pass through. Therefore, the electrophoresis between the first and second buffer tanks allows the above molecular sieve to be formed. The sample is concentrated on or near the surface of the sieving membrane.

The sample such as DNA concentrated in this manner is held in a region directly connected to the capillary or the like in the separation and migration section, and then electrophoresed in the capillary or the like in the separation and migration section. It can be used for measurement of separation and detection, and the minimum amount required for measurement can be reduced. In the present invention, the term "capillary portion" refers not only to the capillaries themselves, but also to the following examples, particularly to the second, third, and third embodiments.
As shown in the fourth embodiment, the term refers to all the pipe sections communicating between the two buffer tanks.

[0013]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, an electrophoresis apparatus according to the present invention will be described in more detail with reference to the accompanying drawings. FIG. 1 shows a first embodiment, and the apparatus will be described by way of example in which a DNA fragment is analyzed. In FIG., A is a sample injection part, a first buffer over tank 1 and the second buffer over tank 2 is positioned through the septum 3, one buffer over tank buffer over in FIG. < A sample injection well 4 is provided at the bottom of the tank 2. Bottom and the other buffer over bath sample injection well 4, is the bottom of the buffer over tank 1 have been communicated by capillary 5 in the figure, near the bottom of the buffer over tank 1 of capillary tubes 5 molecular sieve film 6 It is interposed. This molecular sieve membrane 6 allows the passage of salts having a small molecular size but DNA having a large molecular size.
It has physical properties that do not allow molecules to pass through. For example, a cellulose acetate film or a polyethersulfone film can be used.

The bottom of the sample injection well 4 and the molecular sieve film 6
And in the portion of the thin tube 5 between, and the tip of <br/> capillary -50 conventional known separation lane area b is connected, the capillary
The other end of the over 50 is released in the third buffer over bath 60. Further, the lower end near the capillary over 50 known detection means c for detecting the detected sample of DNA fragments or the like which is a fluorescent label is provided.

[0015] The first, second, third buffer over tank 1,
The electrode 60 is provided with electrodes 71, 72, and 73, respectively, and each electrode is connected to a high-voltage power supply for electrophoresis 75 via a power switch 74 so as to be selectively switched to the following two modes. ing. That is, a first switching mode in which a potential is applied between the migration electrode 72 provided in the second buffer tank 2 and the migration electrode 71 provided in the first buffer tank 1 shown by a solid line in FIG. This is a second switching mode in which a potential is applied between the migration electrode 71 provided in the first buffer tank 1 and the migration electrode 73 provided in the third buffer tank 60 as shown.

Next, a DNA is prepared using this electrophoresis apparatus.
A method for analyzing a fragment will be described. First, meet all buffers reactor with buffer over liquid. Thereafter, 1-2 μl of a sample containing the fluorescently labeled DNA fragment to be measured is collected with a microsyringe or the like, and the second buffer tank 2
Is injected into the sample injection well 4 at the bottom of the sample. The sample injection well 4 is connected to the first buffer tank 1 by a thin tube 5 having a diameter approximately the same as the inner diameter of the capillary, and the middle of the thin tube 5, that is, preferably near the bottom of the first buffer tank 1, is closed with the thin tube 5. Is provided with a molecular sieve film 6, and a capillary 5 between the bottom of the sample injection well 4 and the molecular sieve film 6.
The part, the tip end of the capillary over 50 separate lane area b is connected. Therefore, the power supply switching switch 74 is not shown in FIG.
The migration electrode 72 provided in the second buffer tank 2 and the migration electrode 7 provided in the first buffer tank 2 on the side indicated by the solid line.
When a potential is applied between the sample buffer and the sample buffer, the fluorescently labeled DNA fragment migrates from the sample injection well 4 below the second buffer tank 2 toward the molecular sieve membrane 6.

Although small molecules such as salts in the buffer solution pass freely through the molecular sieve membrane 6, the fluorescently labeled DNA fragment molecules have a large molecular size, so that the lower surface of the molecular sieve membrane 6 or the vicinity thereof can be used. It is concentrated and accumulated and held in the capillary region 5 '. By performing the electrophoresis for 2 to 5 minutes, almost all of the fluorescently labeled DNA fragment molecules can be concentrated and transferred from the second buffer tank 2 to the above-described capillary region 5 ′ which is a sample holding region. Incidentally, in this embodiment, the molecular cross-sectional area of the bottom of the capillary region 5 'of the old film 6 Yes set to be approximately equal to the cross-sectional area of the migration path sectional i.e. capillary chromatography 11 and an inner diameter 0.1mm~
0.05 mm is preferred.

[0018] Then, the power switching switch 74 in FIG. 1 Killed towards to indicate by dotted lines, between the first focusing electrode 73 that migration electrode 71 provided in the buffer tank 1 and provided with a third buffer tank 60 Apply potential. Thereby, the capillary region 5 ′
The fluorescently labeled DNA fragments held in a concentrated state start to migrate toward the third buffer tank 60 at the same time. Thereby, the fluorescently labeled DNA fragment is separated in the gel capillary 50 according to the molecular size,
Is detected by

[0019] As apparent from the above description, in this electrophoresis apparatus, after the sample injected into the sample injection wells is once concentrated, since it is introduced into the capillary over the electrophoretic separation unit, sample injection wells All the samples can be used for measurement of separation and detection without waste, and the amount of sample required for measurement can be made extremely small. FIG. 2 shows a cross section of a sample injection part a2 of a _second embodiment according to the present invention.

The sample injection section a ₂ of this embodiment is different from the first embodiment in that a _second molecular sieve film 26 ′ is provided at the bottom of a sample injection well provided at the bottom of the second buffer tank 22. Basically different from the ones. In this embodiment, the bottom portion of the first and second buffer over tank 21 is formed with a recess, the bottom of the recessed portion is communicated with each other by the capillary tube 25.

A molecular sieve film 26 similar to that of the first embodiment is attached to the recess formed at the bottom of the first buffer tank 21 so as to cover the opening of the thin tube 25. 26 is fixed by a holding plate 28 with a through hole 27. Similarly, a second molecular sieve film 26 'is attached to a concave portion formed at the bottom of the second buffer tank 22 so as to cover the opening of the thin tube 25, and the molecular sieve film 26' is used for sample injection. Holding plate 24 ′ that also serves as well 24
It is fixed by. The second molecular sieve film 26 ′
A membrane having a mesh that can transmit DNA fragments used for sequence analysis but does not transmit DNA fragments of a larger molecular size is selected.

[0022] Also in this embodiment, although not particularly shown, a third buffer over bath, other members necessary for analysis by switching power switch and the like electrophoresis is provided in the same manner as in the first embodiment. Analysis of a DNA fragment using this embodiment is performed in the same manner as in the first embodiment. That is, sample and buffer over solution was poured respectively, to start electrophoresis defeat switching switch 74 to the side indicated by the solid line in FIG. 1. In this embodiment, since the second molecular sieve film 26 'having the mesh as described above is provided between the sample injection well 24 and the capillary 25, a DNA sequence using a DNA complementary strand synthesis reaction is provided. Larger size template DNA such as M13 phage or plasmid vector used for the production of the fragment does not permeate the second molecular sieve membrane 26 ', but only the smaller size DNA permeates. Therefore, the small DNA fragment used for sequence analysis, which is the template DNA and the generated DNA fragment, is separated by the second molecular sieve membrane 26 '.

That is, a solution containing the template DNA and the generated DNA fragment is injected into the sample injection well 24, and the electrophoresis electrode 72 provided in the second buffer tank 22 and the electrophoresis electrode 71 provided in the first buffer tank 21 By applying an electric potential between the two, the generated DNA fragment having a small molecular size passes through the second molecular sieve film 26 'together with the salt and the like in the buffer solution, and migrates in the direction of the first sieve film 26. . As described above, the second molecular sieve film 26 'is selected so as not to pass a template DNA having a large molecular size, and the first sieve film 26 is selected so as not to pass a DNA fragment as in the first embodiment. DNA fragment, the first
The sample is concentrated and accumulated in a capillary region 25 'which is a sample holding region at or near the lower surface of the sieve film 26. Thereby, the ratio of the sequencing DNA fragment in the total amount of DNA concentrated and accumulated in the sample holding region can be further increased as compared with that of the first embodiment. The following operation method is the same as in the first embodiment.

The present invention can be applied not only to a capillary electrophoresis apparatus but also to an electrophoresis apparatus using a flat gel (slab gel) in a separation / phoresis section. Figure 3 shows a perspective view showing the sample injection unit a ₃ for slab gel showing a third embodiment according to the present invention together with the cross-section. Slab gel 51 sandwiched between gel holding plates 55 constituting a separation / phoresis section
Sample injection unit a ₃ through a packing material 52 of the upper are attached. This sample injection part a ₃ has two buffer tanks 31 and 32 and two molecular sieve films 36 and 36 ′, respectively, as in the above embodiment. provided over, and sample injection well 44 at the bottom of the second bus Tsu fur tank 32, gel holding plate 5 to the sample injection wells plate 48
There is only the number of the migration path provided between 5 and are present as many as the number of capillaries 35 likewise migration path for communicating the bottom of the two <br/> buffer over bath. Therefore, a capillary region 35 'which is a sample holding region also exists according to the number of migration paths. Further, in this embodiment, the cross-sectional shape of the narrow tube region 35 'on a surface orthogonal to the migration path can be formed in a rectangular shape according to the shape of the migration path.

In a slab gel type electrophoresis apparatus, the cross-sectional area (or width) of an electrophoresis path is determined by the width of a portion where a sample is held and the width spread by diffusion during electrophoresis. Therefore, in order to prevent the sample from spreading due to diffusion, a polymer ribbon or a wire may be provided in the migration path, and the adjacent migration path may be made independent. How to concentrate the sample,
Other operation methods are the same as those of the first and second embodiments, and the description is omitted. In FIG. 3, the electrophoresis electrode is not shown.

FIG. 4 is a sectional view showing a sample injection part a4 according to a _fourth embodiment of the present invention. This embodiment is different from the above-mentioned other embodiments in that the first and second buffer tanks are installed in such a manner that one of them enters the other in the vertical direction. That is, the outer buffer
Another is Buffer over bath 41 is provided, functions corresponding from the first, respectively third second bar <br/> Ffa chromatography tank and a first buffer over tank in the embodiment of the chromatography tank 42 It is configured to fulfill. Capillary chromatography of the bottom of the buffer over bath 41 another embodiment similarly to the separation electrophoresis unit b 41
Has distal end of the connection, said capillary chromatography 41 through the bottom of the outer bar <br/> Ffa over tank 42 has reached the third bar <br/> Ffa over tank (not shown). It is fixed by bottom Similarly molecular sieve membrane 46 provided at the portion through the perforated presser plate 48 of the buffer over bath 41.

[0027] In this embodiment, capillary chromatography 41
Or incised slit 45 pores at points located outside the buffer over bath 42 provided is formed in the. Through the pores or interstices 45, and the space of the spatial and capillary chromatography in the buffer over <br/> tank 42 are communicated. Further, although not particularly shown, each electrophoretic electrode is attached to each buffer over tanks as well as other embodiments described above.

In use, the outer second buffer tank 4
2. Inject the sample into 2. The bottom of the buffer tank 42 into which the sample is injected corresponds to a sample injection well. Each buffer
Performing electrophoresis in the same manner as other examples after filling a buffer over liquid chromatography tank. Also in this embodiment, the pores provided in the capillary or the slit portion 4 cut out of the capillary are provided.
From FIG. 5, it can be easily understood that the DNA fragment enters by electrophoresis and the sample is concentrated and led to the sample holding region 45 ′ immediately below the molecular sieve membrane 46. The subsequent operation method is the same as in the first embodiment.

In this embodiment, the gel is not necessarily formed above the pores provided in the capillary or above the slit 45 formed in the capillary, that is, in the capillary connected to the first buffer tank 41 inside. May be. In the case where a cutting portion is provided, the first buffer tank 41 needs to be held in a fixed positional relationship with the outer second buffer tank 42.

Although not particularly shown, it will be easily understood that this embodiment can be applied to an apparatus using a flat gel like the embodiment shown in FIG. In the above-described first to fourth embodiments, the case where the separation portion has a gel has been described. However, the present invention can be applied to a case where a carrier such as a gel is not provided.

[0031]

According to the present invention, a sample injected into a wide sample injection well is concentrated by electrophoresis in a narrow area and held, and then subjected to electrophoretic separation, so that most of the prepared sample can be effectively analyzed. It can be used and can be separated and detected with high sensitivity even for low concentration samples.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram of a first embodiment according to the present invention.

FIG. 2 is a sectional view of a second embodiment according to the present invention.

FIG. 3 is a cross-sectional view of a third embodiment using a flat gel according to the present invention.

FIG. 4 is a sectional view of a fourth embodiment according to the present invention.

FIG. 5 is a conceptual diagram of a conventional capillary electrophoresis apparatus.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... 1st buffer tank, 2 ... 2nd buffer tank, 60 ...
Third buffer tank, 4 ... sample injection well, 5 ... capillary,
5 ′: capillary region (sample holding region), 6: first molecular sieve film, 26 ′: second molecular sieve film, 71, 72, 7
3: electrophoresis electrode, 74: electrophoretic power supply switching switch, 75:
Electrophoresis power supply, a: sample injection section, b: electrophoresis separation section, c: detection section

Continuation of the front page (56) References JP-A-63-231247 (JP, A) JP-A-2-269935 (JP, A) JP-A-61-13148 (JP, A) JP-A-4-127049 (JP, A) JP-A-2-165047 (JP, A) JP-A-1-147355 (JP, A) JP-A-3-68860 (JP, A) JP-A-63-262554 (JP, A) JP-A-3-223667 (JP, A) JP-A-3-87648 (JP, A) JP-A-50-16677 (JP, A) JP-A-63-191051 (JP, A) JP-A-5-215716 (JP, A A) Tokuhei 4-502516 (JP, A) (58) Fields surveyed (Int. Cl. ⁷ , DB name) G01N 27/447 WPI (DIALOG)

Claims (10)

(57) [Claims] In a fluorescence detection type electrophoresis apparatus comprising a sample injection section, a separation and migration section, a detection section, and a high voltage power supply section for electrophoresis, the sample injection section is connected to two buffers connected to each other by a thin tube portion. A first molecular sieve membrane through which the detection object located in the vessel and the capillary portion cannot pass but a substance smaller in size than the detection object can pass; switching molecule is opened near the upstream side of the sieve film, and high-voltage power supply unit for electrophoresis and between one and the separation lane area between between the two buffer <br/> § chromatography tank and two buffers over bath An electrophoresis apparatus characterized by being freely connected. 2. A second molecular sieve membrane is provided on the upstream side of the capillary portion so as to allow a detection target to pass therethrough, but to block passage of a substance having a size larger than the detection target,
2. The apparatus according to claim 1, wherein the separation electrophoresis section is open to the capillary portion between the second and first molecular sieve membranes. 3., wherein the separation lane area is hollow calibration pin rally filled with buffer over liquid, according to claim 1 or 2 wherein. 4., wherein the separation lane area is a gel formed inside Garasukya pin rally, device according to any one of claims 1 to 3. 5. The method according to claim 1, wherein the separation electrophoresis section is a flat gel formed between two glass flat plates.
Or the apparatus according to 2. 6. The apparatus according to claim 1, further comprising means for limiting an area of the molecular sieve membrane to an area substantially equal to or smaller than a cross-sectional area of the separation / phoresis section. 7. A first and second buffer tank, a thin tube connecting the first and second buffer tanks, one end of which is opened in the middle of the thin tube, and an object to be detected is electrophoretically separated. Separation electrophoresis section, which is disposed in the middle of the thin tube between the one end and the first buffer tank, and allows a substance having a size smaller than the detection target to pass without passing the detection target. A molecular sieve membrane, and first and second electrodes respectively disposed in the first and second buffer tanks, wherein a voltage is applied between the first electrode and the second electrode. The sample containing the detection object injected into the second buffer tank is concentrated, and is held in a portion of the capillary tube between the molecular sieve membrane and the one end. Electrophoresis device. 8. A first and second buffer tank, a thin tube connecting the first and second buffer tanks, one end of which is opened in the middle of the thin tube, and an object to be detected is electrophoretically separated. A separation and migration section, and a first molecular sieve membrane that is disposed on the terminal side of the first buffer tank of the thin tube and allows a substance smaller in size than the detection target to pass without passing the detection target; A second molecular sieve membrane that is disposed on the terminal side of the second buffer tank of the thin tube and that allows the detection target to pass therethrough and blocks the passage of a substance having a size larger than the detection target; And a first electrode and a second electrode disposed in the second buffer tank, respectively. A voltage is applied between the first electrode and the second electrode, and the voltage is applied to the second buffer tank. The sample containing the detection target is concentrated, An electrophoresis apparatus, wherein the electrophoresis apparatus is held in a portion of the capillary tube between the first molecular sieve membrane and the one end. 9. The first and second buffer tanks and the second buffer tank.
A plurality of sample injection wells arranged at the bottom of the buffer tank, a plurality of thin tubes connecting the first buffer tank and each of the sample injection wells, and one end portion is in the middle of each of the plurality of thin tubes. The separation electrophoresis section having a plurality of electrophoresis paths for electrophoretically separating the detection target, and disposed at the end of the plurality of thin tubes on the side of the first buffer tank,
A first molecular sieve membrane that allows passage of a substance smaller in size than the detection target without passing through the detection target, an end of the plurality of capillaries on the side of the second buffer tank, and each of the sample injection wells; And a second molecular sieve membrane that passes through the detection target and blocks a substance having a size larger than the detection target, and is disposed in the first and second buffer tanks, respectively. A first electrode, a second electrode, a voltage is applied between the first electrode and the second electrode, and the sample containing the detection target injected into each of the sample injection wells is concentrated. The electrophoresis apparatus is characterized in that the electrophoresis apparatus is held between the first molecular sieve membrane and one end of the plurality of electrophoresis paths by the plurality of capillary tubes. 10. A second buffer tank, a first buffer tank disposed inside the second buffer tank,
One end is connected to the bottom of the first buffer tank,
An electrophoresis section having pores communicating with the second buffer tank and penetrating the bottom of the second buffer tank;
A separation electrophoresis section for electrophoretically separating an object to be detected, and a molecular sieve membrane disposed at the one end of the separation electrophoresis section and passing a substance smaller in size than the object to be detected without passing through the object to be detected And a first and a second electrode respectively disposed in the first and second buffer tanks, wherein a voltage is applied between the first and the second electrodes, The sample containing the object to be detected injected into the second buffer tank is concentrated and held in a portion of the electrophoresis path between the molecular sieve membrane and the site where the pores are formed. Characteristic electrophoresis device.