JP3723517B2 - Electrophoresis device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrophoresis apparatus.
[0002]
[Prior art]
In the fields of molecular biology, biochemistry, medicine, pharmacy and the like, various gene analyzes using an electrophoresis apparatus are widely performed.
[0003]
For example, the IGCR method (In Gel Competitive Reassociation method) is extremely effective in searching for oncogenes and the like, and is currently attracting much attention. This method is a kind of genome subtraction method, and is characterized by performing genome subtraction in a gel (electrophoresis medium).
[0004]
This IGCR method is advantageous for identifying relatively large gene mutations such as deletion of bases of several kb or more. For this reason, this method is extremely useful when, for example, a large difference is found between a normal cell gene and a cancer cell gene. In fact, the IGCR method has been successful in discovering genes related to gastric cancer.
An outline of the IGCR method will be described below as an example of searching for an oncogene.
[0005]
1) First, DNA derived from normal cells and DNA derived from cancer cells (for example, those treated with restriction enzymes) are prepared.
2) Next, normal cell-derived DNA is biotinylated.
[0006]
3) Next, the cancer cell-derived DNA and the normal cell-derived DNA are mixed so that the normal cell-derived DNA has a much higher concentration than the cancer cell-derived DNA (eg, about 1:50). .
4) Next, gel electrophoresis is performed on this mixture.
[0007]
5) After electrophoresis, the gel was taken out, the gel was immersed in an alkaline solution (denaturing solution), and then the gel was immersed in a neutral solution (associating solution) to denature the DNA in the gel. Later, we will re-associate.
The major features of the IGCR method are steps 3) to 5). Through this process, it is possible to detect and isolate genes that are specifically present only in cancer cells. This principle will be schematically described below.
[0008]
For example, it is assumed that a normal cell has a gene A and a gene B. Further, it is assumed that the cancer cell has a gene A and a mutated gene B ′. This gene B ′ has a shorter chain length than the gene B. In addition, the gene A of cancer cells and the gene A of normal cells are the same (identical). Gene A is longer than gene B.
[0009]
In this case, when electrophoresis is performed in the step 4), the bands are divided into, for example, gene A, gene B, and gene B ′ in the gel.
[0010]
At this time, the band of gene A contains DNA derived from normal cells and DNA derived from cancer cells. Therefore, the ratio of DNA derived from normal cells to DNA derived from cancer cells is 50: 1.
The band of gene B contains only normal cell-derived DNA. That is, the ratio of normal cell-derived DNA to cancer cell-derived DNA is 100: 0.
On the other hand, the band of gene B ′ contains only cancer cell-derived DNA. That is, the ratio of normal cell-derived DNA to cancer cell-derived DNA is 0: 100.
[0011]
Here, when the denaturation and reassociation of DNA in the step 5) is performed, the following four genes A1 to A4 are generated at a ratio of 2500: 50: 50: 1 in the band of gene A.
A1: a gene composed only of DNA derived from normal cells,
A2: a gene (type 1) composed of DNA derived from normal cells and DNA derived from cancer cells,
A3: a gene (type 2) composed of DNA derived from normal cells and DNA derived from cancer cells,
A4: A gene composed only of DNA derived from cancer cells.
Therefore, in the band of gene A, the amount of gene (gene A4) composed only of cancer cell-derived DNA is extremely small.
[0012]
On the other hand, in the band of gene B, the ratio of DNA derived from normal cells to DNA derived from cancer cells remains 100: 0 as before.
Similarly, in the gene B ′ band, the ratio of DNA derived from normal cells to DNA derived from cancer cells remains 0: 100.
[0013]
Next, the gel is cut out, DNA is extracted (collected) from the gel, and when such DNA is brought into contact with avidin, since avidin has a property of binding to biotin, the gene containing DNA derived from normal cells is Adsorbed to avidin.
In contrast, a gene composed only of cancer cell-derived DNA is not adsorbed to avidin.
[0014]
Therefore, since the gene B is composed only of DNA derived from normal cells as described above, it is adsorbed to avidin.
On the other hand, since gene B ′ is composed only of DNA derived from cancer cells, it is not adsorbed to avidin and is extracted.
[0015]
In gene A, genes A1, A2 and A3 are adsorbed to avidin, and gene A4 is not adsorbed to avidin. By the way, as described above, in the gene A, the ratio of A1: A2: A3: A4 is 2500: 50: 50: 1. In other words, the abundance of gene A4 is extremely small.
[0016]
Therefore, for example, when the adapter DNA is bound to the gene taken out without being adsorbed by avidin, PCR is performed using a primer having a base sequence complementary to the adapter DNA, and the gene is amplified. Is preferably detected.
[0017]
Thus, the IGCR method is an excellent gene search method. However, the IGCR method has many disadvantages that it requires many manual steps and many time-consuming processes, and therefore requires a long time (for example, about one week), and requires time and labor for the work.
[0018]
[Problems to be solved by the invention]
An object of the present invention is to provide an electrophoresis apparatus capable of reducing labor and time of work in, for example, the IGCR method.
[0019]
[Means for Solving the Problems]
Such an object is achieved by the present inventions (1) to (13) below.
[0020]
(1) an electrophoresis medium for electrophoresis of a specimen containing a nucleic acid and an electrophoretic marker;
Energizing means capable of energizing the electrophoresis medium and reversing its polarity;
A temperature adjusting means for adjusting the temperature of the electrophoresis medium, the temperature adjusting means having at least a heating means for heating the electrophoresis medium;
Detecting means for detecting the position of the electrophoresed marker in the electrophoresis medium,
The electrophoretic medium is energized by the energizing means, and the sample is electrophoresed in the electrophoretic medium, whereby the nucleic acid is separated in the electrophoretic medium, and the marker is contained in the electrophoretic medium by the detecting means. After detecting the arrival at a predetermined position, the energization to the electrophoresis medium is stopped,
Next, after the electrophoresis medium is heated by the heating means, the electrophoresis medium is cooled to denature the separated nucleic acid in the electrophoresis medium, and then reassociate.
Thereafter, the polarity is reversed by the energization means, the energization medium is energized, and the nucleic acid after reassociation is electrophoresed in the electrophoresis medium in the direction opposite to the electrophoresis direction and recovered from the electrophoresis medium. An electrophoretic device configured as described above.
(2) an electrophoresis medium for electrophoresis of a specimen containing a nucleic acid and an electrophoretic marker;
Energizing means capable of energizing the electrophoresis medium and reversing its polarity;
Temperature adjusting means for adjusting the temperature of the electrophoresis medium, the temperature adjusting means having heating means for heating the electrophoresis medium, and cooling means for cooling the electrophoresis medium;
Detecting means for detecting the position of the electrophoresed marker in the electrophoresis medium,
The electrophoretic medium is energized by the energizing means, and the sample is electrophoresed in the electrophoretic medium, whereby the nucleic acid is separated in the electrophoretic medium, and the marker is contained in the electrophoretic medium by the detecting means. After detecting the arrival at a predetermined position, the energization to the electrophoresis medium is stopped,
Next, after the electrophoresis medium is heated by the heating means, the electrophoresis medium is forcibly cooled by the cooling means to denature the separated nucleic acid in the electrophoresis medium, and then reassociate.
Thereafter, the polarity is reversed by the energization means, the energization medium is energized, and the nucleic acid after reassociation is electrophoresed in the electrophoresis medium in the direction opposite to the electrophoresis direction and recovered from the electrophoresis medium. An electrophoretic device configured as described above.
[0021]
(3) The migration speed of the marker is substantially the same as that of the nucleic acid having the fastest migration speed, or slightly faster than the fastest migration speed of the nucleic acids (1) or ( The electrophoresis apparatus according to 2).
[0022]
(4) The electrophoresis apparatus according to any one of (1) to (3), wherein the marker has a molecular weight of 100 to 2000.
[0023]
(5) The electrophoresis apparatus according to any one of (1) to (4), wherein the marker is any one of a charged fluorescent substance, a charged luminescent substance, and a charged dye.
[0024]
(6) The electrophoretic device according to any one of (1) to (4), wherein the marker is a fluorescent substance, a luminescent substance, or a dye imparted to a nucleic acid.
[0025]
(7) The detection means includes an irradiation means for irradiating the electrophoresis medium with light,
The electrophoretic device according to any one of (1) to (6), further including a light receiving unit that receives light from the electrophoretic medium.
[0026]
(8) The electrophoretic device according to any one of (1) to (7), wherein the temperature adjusting unit includes a heat transfer unit that is provided around the electrophoretic medium and performs heat transfer to the electrophoretic medium. .
[0027]
(9) When the total length in the longitudinal direction of the electrophoresis medium is L, the heat transfer section is configured to cover the range of 0.45 L from the center in the longitudinal direction toward both ends. The electrophoresis apparatus according to the above (8).
[0032]
(10) When the total length in the longitudinal direction of the electrophoresis medium is L, the detection means detects that the marker has reached 0.9 L or more from the electrophoresis start position. ).
[0033]
(11) The electrophoresis apparatus according to any one of (1) to (10), wherein the nucleic acid after the reassociation is recovered using a nucleic acid adsorbent.
[0034]
(12) The electrophoresis device according to any one of (1) to (11), wherein a plurality of the electrophoresis media are provided side by side.
[0035]
(13) The electrophoresis device according to any one of (1) to (12), wherein the electrophoresis medium is housed in a hollow member.
[0036]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the electrophoresis apparatus of the present invention will be described in detail based on preferred embodiments shown in the accompanying drawings.
[0037]
FIG. 1 is a schematic view showing an embodiment of the electrophoresis apparatus of the present invention, and FIG. 2 is a partial longitudinal sectional view showing a configuration in the vicinity of the electrophoresis unit included in the electrophoresis apparatus shown in FIG. FIG. 4 is a perspective view showing a configuration of an upper buffer tank provided in the electrophoresis apparatus shown in FIG. 1, and FIG. 4 is a side view showing a configuration of temperature adjusting means provided in the electrophoresis apparatus shown in FIG. These are flowcharts which show the operation | movement procedure of the electrophoresis apparatus shown in FIG. In the following description, in FIGS. 2 and 3, the upper side is referred to as “upper end” and the lower side is referred to as “lower end”.
[0038]
The electrophoretic device 1 shown in FIG. 1 includes an electrophoretic unit 2, an energizing unit 3, a temperature adjusting unit 4, a control unit 5, and a detecting unit 6.
[0039]
Hereinafter, the electrophoresis apparatus 1 will be described for each component.
As shown in FIGS. 1 and 2, the electrophoresis unit 2 mainly includes a plurality (three in this embodiment) of an upper buffer tank (first buffer tank) 21 and a lower buffer tank (second buffer tank). An apparatus main body 200 having a buffer tank 22 and a base 23 for supporting them, and a plurality (three in this embodiment) of tube bodies (tubular hollow members) 20 containing the gel (electrophoresis medium) 100. And have.
[0040]
Each upper buffer tank 21 is formed of a box-shaped member that is provided corresponding to the electrophoresis medium 100 (tube body 20) and has an opening at the upper end (upper part). In the upper buffer tanks 21 (reservation spaces), buffer solutions (buffer solutions) such as Tris buffer and Tris / borate buffer are respectively stored.
[0041]
The amount of the buffer solution is, for example, such that the upper end of each tube body 20 does not protrude from the liquid surface in a state where the tube body 20 is mounted (installed) in each upper buffer tank 21.
[0042]
In this embodiment, since the structure of each upper buffer tank 21 is the same, below, it demonstrates on behalf of one.
[0043]
The upper buffer tank 21 has a partition wall 210 that is formed at a substantially central portion in the longitudinal direction and defines a storage space for storing a buffer solution into a first space 210a and a second space 210b.
[0044]
In the first space 210a, an electrode 32a included in the energizing means 3 described later is installed so as to be immersed (contacted) in the buffer solution, while in the second space 210b, the migration medium 100 (tubular body 20). The upper end (one end) of is located.
[0045]
A mounting hole 212 is formed in the bottom portion (bottom surface) 211 of the upper buffer tank 21 on the first space 210a side so as to penetrate in the thickness direction (vertical direction in FIGS. 2 and 3). A holding member 213 that holds the tubular body 20 is fixed (attached) to the attachment hole 212 in a liquid-tight manner.
[0046]
The holding member 213 is formed of a substantially cylindrical member, and a mounting portion 213a that can be fitted into the mounting hole 212 is formed on the lower end side. The holding member 213 is fixed (fixed) to the bottom 211 of the upper buffer tank 21 in a liquid-tight manner by inserting and fitting the mounting portion 213 a into the mounting hole 212.
[0047]
Although this holding member 213 is not specifically limited, For example, it is comprised with elastic materials, such as various rubber materials and various thermoplastic elastomers.
[0048]
Further, the upper end side of the tubular body 20 described later is inserted (inserted) into the lumen portion of the holding member 213.
[0049]
The partition wall 210 has a small thickness on the second space 210b side, and a concave portion 214 having an opening opened upward (upward) is formed in this portion. That is, the recess 214 communicates with the second space 210b. In this recess 214, a nucleic acid adsorbent (DNA adsorbent) R capable of adsorbing DNA (nucleic acid) detached from the gel 100 is housed (installed). That is, in this embodiment, the nucleic acid adsorbent R is provided between the gel 100 and the electrode 32a.
[0050]
Examples of the constituent material of the nucleic acid adsorbent R include ion exchange resins, various glasses, ceramic materials such as alumina and calcium phosphate, silica compounds such as amorphous silica and alkyl silica, resin materials such as polystyrene, polyethylene, and polypropylene. Etc. Among these, the nucleic acid adsorbent R is preferably composed of a material containing an ion exchange resin, and more preferably composed of an ion exchange resin as a main material. Such a nucleic acid adsorbent R has a high ability to adsorb DNA, and the adsorbed DNA can be easily recovered from the nucleic acid adsorbent R.
[0051]
Examples of the ion exchange resin include those in which groups having various charges (for example, sulfone group, carboxyl group, trimethylammoniomethyl group, etc.) are introduced into agarose, cellulose, nitrocellulose and the like. One or more of these can be used in combination. By constituting the nucleic acid adsorbent R using such an ion exchange resin, the DNA adsorbing ability of the nucleic acid adsorbent R can be made particularly excellent, and the recovery efficiency of DNA can be improved. it can.
[0052]
In addition, the shape of the nucleic acid adsorbent R is not particularly limited, and examples thereof include granules, meshes, sponges, etc. Among these, granules are particularly preferable. By making the nucleic acid adsorbent R granular, the surface area effective for DNA adsorption can be increased, and the efficiency of DNA adsorption can be increased.
[0053]
In this case, the average particle diameter of the granular nucleic acid adsorbent R is preferably about 20 to 2000 μm, more preferably about 35 to 1000 μm, and further preferably about 50 to 150 μm. By making the average particle diameter within the above range, the DNA adsorption efficiency can be further improved.
[0054]
Further, the partition wall 210 is formed with a communication hole 215 that allows the recess 214 and the first space 210a to communicate with each other.
[0055]
The communication hole 215 is formed so as to be inclined so that the first space 210a side is higher than the second space 210b side. Thereby, when using the granular nucleic acid adsorption agent R, it can prevent suitably that the nucleic acid adsorption agent R accommodated in the recessed part 214 flows out into the 1st space 210a side.
[0056]
In this case, the angle (θ in FIG. 3) formed by the communication hole 215 and the bottom portion 211 of the upper buffer tank 21 is not particularly limited, but is preferably about 45 to 80 °, and preferably about 60 to 80 °. Is more preferable. Thereby, the said effect improves more.
[0057]
Further, the inner diameter of the communication hole 215 is not particularly limited, but is, for example, about 1 to 5 mm.
[0058]
A plurality (three) of such upper buffer tanks 21 are integrated, and leg portions 216 and 216 arranged opposite to each other are shown on both side portions (the back side and the front side in FIG. 2). It is connected (installed) to the base 23 via a connection part that is not connected. Thereby, each upper buffer tank 21 is fixed to the base 23.
[0059]
The base 23 is for supporting each upper buffer tank 21, the lower buffer tank 22, and a heat transfer block 42 to be described later, and is constituted by a flat plate member.
[0060]
A lower buffer tank 22 is installed at the upper end (upper surface) of the base 23. The lower buffer tank 22 is composed of a box-shaped member. A buffer solution (buffer solution) is stored inside the lower buffer tank 22, and an electrode 32 b included in the energizing means 3 described later is installed so as to be immersed in (contact with) the buffer solution.
[0061]
As this buffer solution, the same buffer solution as mentioned above for the buffer solution stored in the upper buffer tank 21 can be used.
[0062]
In the vicinity of the upper edge of the lower buffer tank 22 (upper right side in FIG. 2), an inlet 221 that protrudes upward is integrally formed, and the lower buffer tank 22 is formed via the inlet 221. A buffer solution can be introduced (stored) in the inside.
[0063]
A plurality of insertion holes 222 are formed in the upper end (upper surface) of the lower buffer tank 22 so as to penetrate in the thickness direction (vertical direction in FIG. 2). The lower end side of the tubular body 20 is inserted (inserted) into each insertion hole 222.
[0064]
In this state, the lower buffer tank 22 stores a buffer solution having a volume sufficient to immerse the lower end of the tube body 20.
[0065]
The constituent materials of the lower buffer tank 22, each of the upper buffer tank 21 and the base 23 are not particularly limited. For example, polyvinyl chloride, polyethylene, polypropylene, polystyrene, poly- (4-methyl) are used. Pentene-1), polycarbonate, acrylic resin, acrylonitrile-butadiene-styrene copolymer, polyester such as polyethylene terephthalate, butadiene-styrene copolymer, polyamide (for example, nylon 6, nylon 6,6, nylon 6,10, Various resin materials such as nylon 12) can be used.
[0066]
In addition, it is preferable that the lower buffer tank 22 and each upper buffer tank 21 are substantially transparent, respectively, in order to ensure internal visibility.
[0067]
Between each upper buffer tank 21 and the lower buffer tank 22, a plurality (three in this embodiment) of tube bodies (tubular hollow members) 20 are respectively connected to the upper buffer tank 21 via the holding member 213. The other end is in contact with the buffer solution stored in the upper buffer tank 21, and the other end is in contact with the buffer solution stored in the lower buffer tank 22.
[0068]
The constituent material of the tube body 20 is not particularly limited, but various resin materials such as polycarbonate, polymethylmethacrylate, polypropylene, etc., for example, are excellent in heat transfer and sufficient strength can be obtained. Various glass materials are preferably used.
[0069]
Although it does not specifically limit as a dimension of the tubular body 20, For example, an internal diameter shall be about 3-8 mm and an outer diameter shall be about 5-10 mm.
[0070]
Each tube body 20 is filled (contained) with a gel (electrophoresis medium) 100 and is provided with a predetermined distance (for example, about 10 to 40 mm) apart from each other. In other words, the plurality of gels 100 are provided with a predetermined distance apart. In this way, by providing a plurality of gels 100, the electrophoresis apparatus 1 can increase the amount of specimen that can be processed at one time.
[0071]
Further, a portion not filled with the gel 100 is provided on the inner side of the upper end portion of each tube body 20, and the sample is filled (applied) with the portion.
[0072]
The gel 100 is composed of, for example, acrylamide, agarose, etc., and a sample containing DNA (nucleic acid) is electrophoresed, whereby the DNA is separated by a difference in chain length (difference in molecular weight).
[0073]
The gel 100 has a substantially cylindrical shape (bar shape) by being accommodated in the tubular body 20.
[0074]
The total length of the gel 100 in the longitudinal direction (length L in FIG. 2) is not particularly limited, but is usually preferably about 10 to 1000 mm, more preferably about 50 to 200 mm, and about 100 to 150 mm. More preferably. By setting the total length of the gel 100 in the longitudinal direction within such a range, DNA can be suitably separated.
[0075]
The sample includes a marker having electrophoretic properties. If such a marker is added to the sample, the DNA contained in the sample (DNA fragment) is detected by detecting the position of the electrophoresed marker in the gel 100 when the sample is electrophoresed. ) Can be accurately grasped whether or not the separation is proceeding normally.
[0076]
In particular, the marker has an electrophoresis speed (electrophoretic property) that is substantially the same as or slightly higher than that of the DNA having the fastest electrophoresis speed (high electrophoretic property). Fast is preferred. When such a marker is used, the marker moves in advance in the gel 100 (electrophoresis). Therefore, by detecting the position of the marker in the gel 100, the timing for terminating the electrophoresis ( It is possible to accurately grasp the timing at which the energization of the gel 100 is stopped, that is, the electrophoresis can be completed in a state where the DNA (DNA fragment) is separated sufficiently and sufficiently.
[0077]
In this case, the molecular weight of the marker is not particularly limited, but is preferably about 100 to 2000, and more preferably about 300 to 800. The effect can be further improved by using a marker having a molecular weight in the above range.
[0078]
As such a marker, either a charged (negatively charged) fluorescent substance, a charged luminescent substance, or a charged dye (such as bromophenol blue), or a fluorescent substance In addition, those obtained by imparting (for example, binding) a nucleic acid to a nucleic acid are preferably used. By using such a marker, the position (electrophoretic position) of the marker in the gel 100 can be detected relatively easily without requiring a large-scale apparatus. In addition, as a marker, various labeling substances, such as what provided the radioactive substance to the nucleic acid, can also be used. Moreover, as a nucleic acid used for a marker, any of DNA, RNA, a synthetic oligonucleotide, etc. may be sufficient, for example.
[0079]
When using a fluorescent substance, a luminescent substance or a dye imparted to a nucleic acid as a marker, since the nucleic acid itself is charged, the fluorescent substance, the luminescent substance and the dye imparted to the nucleic acid are each charged. It may be either tinged or not. Specific examples of these are not particularly limited, and examples thereof include the following.
[0080]
Examples of the fluorescent substance include fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), substituted rhodamine isothiocyanate (XRITC), rhodamine B isothiocyanate, dichlorotriazine fluorescein (DTAF), Cy3, and Cy5. .
[0081]
Examples of the luminescent substance include luminol, isoluminol, alridium ester, or derivatives thereof.
[0082]
Examples of the dye include phthalocyanine pigments, azo pigments, anthraquinone pigments, azomethine pigments, quinophthalone pigments, isoindoline pigments, nitroso pigments, perinone pigments, quinacridone pigments, perylene pigments, pyrrolo. Pyrrole pigments, pigments such as dioxazine pigments, azo dyes, anthraquinone dyes, indigoid dyes, triphenylmethane dyes, pyrazolone dyes, stilbene dyes, diphenylmethane dyes, xanthene dyes, alizarin dyes, Examples include acridine dyes, quinoneimine dyes, and dyes such as metal complex dyes.
[0083]
In addition, you may make it use the marker as mentioned above combining arbitrary 2 or more types as needed.
[0084]
The position (electrophoretic position) of the electrophoresed marker in the gel 100 is detected by the detecting means 6.
[0085]
The detection position of this marker is preferably in the range of 0.9 L or more, more preferably in the range of 0.95 or more from the electrophoresis start position, where L is the total length in the longitudinal direction of the gel 100. By detecting that the marker has reached the range and then terminating the electrophoresis, DNA (DNA fragments) can be separated more accurately.
[0086]
As shown in FIGS. 1 and 2, the detection means 6 includes a light source (irradiation means) 61 that irradiates light (for example, laser light) to the gel 100, and a light receiving element that receives (detects) light from the gel 100. (Light receiving means) 62.
[0087]
The light source 61 is installed at a predetermined position below a side portion of a heat transfer block 42 described later. The light emitted from the light source 61 is formed in the heat transfer block 42, passes through the side hole 422 (see FIG. 1) communicating with the insertion hole 421, and enters the gel 100.
[0088]
The wavelength of irradiation light is appropriately selected depending on the type of marker used and the like, and is not particularly limited.
[0089]
Moreover, when using a laser beam as irradiation light, as the light source 61, a semiconductor laser, a fixed laser, a gas laser, a dye laser, an excimer laser, a free electron laser, a color center laser etc. can be used, for example.
Further, the irradiation light may be either continuous light or pulsed light.
[0090]
In the present embodiment, the irradiating means is composed of the light source 61, but is not limited to this. For example, a filter that adjusts the amount of irradiation light (irradiation intensity) between the light source 61 and the gel 100 ( An optical filter may be used.
[0091]
Further, when using a charged luminescent substance or a luminescent substance provided with a nucleic acid as a marker, it is not necessary to receive light, and the luminescent substance itself emits light. It may be omitted.
[0092]
The light receiving element 62 receives light from the gel 100, performs photoelectric conversion, and outputs a signal corresponding to the amount of received light. Specifically, the light receiving element 62 is emitted from a marker when a charged fluorescent substance or luminescent substance or a fluorescent substance or luminescent substance added to a nucleic acid is used as the marker (1). When fluorescent light (or phosphorescent light) is used, and (2) a marker, a charged dye or a dye added to a nucleic acid is used, a part of light of a specific wavelength is absorbed by the marker. The irradiation light whose light quantity has changed is received.
[0093]
The light receiving element 62 is disposed at a position corresponding to the light source 61 in the height direction and at a position that is substantially 90 ° with the light source 61. Light from the gel 100 is formed in a heat transfer block 42 described later, passes through a side hole 423 (see FIG. 2) communicating with the insertion hole 421, and enters the light receiving element 62.
[0094]
In the present embodiment, the light receiving means is configured by the light receiving element 62, but is not limited to this. For example, a lens that collects light from the gel 100 between the gel 100 and the light receiving element 62. Alternatively, a configuration having a diaphragm or the like for adjusting the amount of the condensed light may be used.
[0095]
In addition, the light receiving element 62 only needs to be provided at a position corresponding to the light source 61 in the height direction. In the case of the present embodiment, the circumferential direction of the gel 100 is not limited to the illustrated configuration, and may be any arbitrary. It can be installed at the position.
[0096]
Moreover, the light source 61 and the light receiving element 62 may be fixed, or may be installed so as to be movable in the vertical direction. In the latter case, these may be installed so as to be movable in the vertical direction corresponding to each other. In this case, whether or not DNA separation is performed normally, for example, by adding markers having various molecular weights to the specimen, and detecting (scanning) these markers. There is an advantage that it can be grasped more accurately or not.
[0097]
Now, the gel 100 is energized by the energizing means 3.
The energization means 3 is capable of energizing the gel 100 and reversing its polarity. As shown in FIG. 1, the energization means 3 is provided corresponding to the power supply unit 31 for electrophoresis and each upper buffer tank 21. And a plurality of electrodes (individual electrodes) 32a and electrodes (common electrodes) 32b facing the electrodes. These electrophoretic power supply unit 31 and each electrode 32a and electrode 32b are connected to each other by a conducting wire.
[0098]
Each of the electrodes 32a and 32b is made of a conductive material such as metal (aluminum or the like), for example. Further, as described above, each electrode 32 a is installed in the upper buffer tank 21 (first space 210 a), and the electrode 32 b is installed in the lower buffer tank 22.
[0099]
The temperature of the gel 100 is adjusted by the temperature adjusting means 4.
As shown in FIG. 4, the temperature adjusting unit 4 is configured to transfer heat between the heating unit 43 that heats the gel 100, the cooling unit 44 that cools the gel 100, and between the heating unit 43, the cooling unit 44, and the gel 100. A heat transfer block (heat transfer section) 42 to perform and a temperature sensing means 45 for detecting the temperature of the gel 100 are provided.
[0100]
The temperature controller 41 of the temperature adjusting unit 4 is configured by a driver 432 included in the heating unit 43 described later, a driver 444 included in the cooling unit 44, and an A / D converter 452 included in the temperature sensing unit 45.
[0101]
The temperature adjusting means 4 heats the gel 100 by the heating means 43 and then forcibly cools the gel 100 by the cooling means 44 to denature the DNA in the gel 100 and then reassociate it.
[0102]
The heating means 43 includes a surface heater 431 that generates heat and heats the gel 100, and a driver 432 that drives and controls the surface heater 431 based on a signal from the computer 51.
[0103]
The cooling means 44 has a cooler 441 that dissipates heat from the gel 100 or the like and cools the gel 100, and a driver 444 that drives the cooler 441.
[0104]
The cooler 441 includes a heat sink 442 that dissipates heat from the gel 100 and the like, and a fan 443 that promotes heat dissipation from the heat sink 442.
[0105]
The heat sink 442 is made of a heat conductive material such as metal (aluminum or the like), for example. The heat sink 442 includes a heat dissipating portion 446 configured such that a large number of thin plates are erected, for example. In the heat radiating portion 446, the surface area, that is, the contact area between the heat sink 442 and air is increased, and heat is efficiently released to the atmosphere.
[0106]
The fan 443 can promote such heat dissipation. The fan 443 includes a blade 448 that generates wind and a motor 447 that rotates the blade 448. When the fan 443 is driven, the motor 447 embedded in the heat sink 442 rotates, and this driving force is transmitted to the blade 448. As a result, the blade 448 rotates and wind is generated. And this heat | fever promotes the thermal radiation in the thermal radiation part 446. FIG.
[0107]
The surface heater 431 described above is provided on the surface of the heat sink 442 opposite to the heat radiating portion 446 so as to be in close contact with the heat sink 442. Furthermore, in the temperature adjusting means 4, a heat transfer block 42 is provided so as to be in close contact with the surface heater 431.
[0108]
The heat transfer block 42 can transfer the heat generated by the surface heater 431 to the gel 100, and can transfer the heat of the gel 100 to the heat sink 442. The heat transfer block 42 is made of, for example, a heat conductive material such as metal (aluminum or the like).
[0109]
Through such a heat transfer block 42, the temperature sensing means 45 can sense the temperature of the gel 100. The temperature sensing means 45 includes a temperature sensor 451 that detects the temperature of the gel 100 and an A / D converter 452 that converts an analog signal from the temperature sensor into a digital signal. In the temperature sensing means 45, the temperature sensor 451 is installed so as to be embedded in the heat transfer block 42. For this reason, the temperature sensor 451 can directly detect the temperature of the heat transfer block 42. Thereby, since the temperature of the heat transfer block 42 substantially corresponds to the temperature of the gel 100, the temperature sensor 451 can detect the temperature of the gel 100.
[0110]
Further, a plurality (three in this embodiment) of insertion holes 421 are formed in a portion of the heat transfer block 42 opposite to the surface heater 431 so as to penetrate in the longitudinal direction (vertical direction in FIG. 2). Has been. Each insertion hole 421 is set to have an inner diameter substantially equal to the outer diameter of the tube body 20, and the tube body 20 is inserted into the insertion hole 421. That is, the heat transfer block 42 is provided so as to surround the entire circumference (periphery) of each gel 100.
[0111]
Thereby, since the gel 100 can be heated and cooled uniformly (uniformly) in the circumferential direction, denaturation and reassociation between the separated DNAs can be performed uniformly.
[0112]
Moreover, it is preferable that the heat transfer block 42 is configured to cover a range in the longitudinal direction of the gel 100 substantially corresponding to a range where the DNA (band) separated in the gel 100 exists.
[0113]
That is, the length of the heat transfer block 42 in the longitudinal direction is not particularly limited. For example, when the total length in the longitudinal direction of the gel 100 is L, the gel 100 is separated from the center in the longitudinal direction at both ends (upper and lower ends). It is preferably configured to cover a range of about 0.45L (in FIG. 2, a range of length M) toward the lower end), and is configured to cover a range of about 0.4L. Is more preferable.
[0114]
Thereby, since the gel 100 can be heated and cooled uniformly (uniformly) in the longitudinal direction, it can be denatured and reassociated evenly (uniformly) between the separated DNAs (bands).
[0115]
In the present embodiment, the heat transfer block 42 is provided so as to surround the entire circumference of the gel 100, and substantially corresponds to the range where the DNA separated in the gel 100 exists, By configuring so as to cover the range, these synergistic effects allow uniform (uniform), rapid and reliable denaturation and reassociation between the separated DNAs (bands).
[0116]
As described above, in the present embodiment, the temperature adjusting unit 4 includes the heating unit 43 and the cooling unit 44. However, in the present invention, the cooling unit 44 may be omitted. In this case, the temperature adjusting means 4 heats the gel 100 by the heating means 43, then stops heating the gel 100 by the heating means 43, and allows the gel 100 to cool (natural cooling). The DNA can be denatured and then reassociated.
[0117]
In addition, when the gel 100 is forcibly cooled by the cooling means 44 as in the present embodiment, the denatured DNA is more rapidly and efficiently compared to the case where the gel 100 is allowed to cool (natural cooling). There is an advantage that reassociation can be performed (with high accuracy).
[0118]
The energizing means 3, the temperature adjusting means 4, and the detecting means 6 described above are controlled by the control means 5 constituted by the computer 51.
[0119]
The computer 51 includes an electrophoretic power supply unit 31 of the energization unit 3, a temperature controller 41 (driver 432, driver 444, A / D converter 452) of the temperature adjustment unit 4, a light source 61 and a light receiving element 62 of the detection unit 6. Are connected via a signal line.
[0120]
The computer 51 controls the electrophoretic power supply unit 31 to apply a voltage between each electrode 32a and the electrode 32b, thereby energizing the gel 100, and also controls the electrophoretic power supply unit 31 to The polarity of the electrode 32a and the electrode 32b can be reversed.
[0121]
Further, the computer 51 can control the surface heater 431 and the fan 443 via the driver 432 and the driver 444. Further, the computer 51 can acquire temperature information of the gel 100 from the temperature sensor 451 via the A / D converter 452.
[0122]
The computer 51 controls the light source 61 to irradiate the gel 100 with light. Further, the computer 51 can acquire an output signal from the light receiving element 62.
[0123]
Hereinafter, the usage method (action) of the electrophoresis apparatus 1 will be described as an example with reference to FIG. 5 in the case where the IGCR method is performed.
[0124]
[1] First, an operator who performs the IGCR method (a user of the electrophoresis apparatus 1) sets the tube body 20 filled (contained) with the gel 100 as shown in FIG. Installed between the buffer tank 22 and a power switch (not shown) is turned on. Thereby, the preparation of the electrophoresis apparatus 1 is completed in a state where the electrophoresis apparatus 1 can be used (operation state).
[0125]
In addition, the worker prepares a sample (specimen) including DNA (nucleic acid) and, for example, DNA (molecular weight: about 700) provided with a fluorescent substance as a marker. This sample contains DNA derived from cells to be tested and DNA derived from normal cells (for example, those subjected to restriction enzyme treatment) in a ratio of, for example, about 1:50. The DNA concentration at this time is slightly different depending on the amount of sample to be subjected to electrophoresis, but is preferably about 0.1 to 100 μM.
The normal cell-derived DNA is biotinylated.
[0126]
[2] Next, the operator operates the computer 51 to input various conditions (various setting conditions) in steps [4] to [10] described later.
[0127]
[3] Next, the operator fills (applies) the sample prepared in the step [1] on the inner side of the upper end of each tubular body 20 (the upper surface of the gel 100).
[0128]
[4] Next, when an operator presses a start button (not shown), the computer 51 drives the power supply unit 31 for migration of the energization means 3 (step S101 in FIG. 5).
[0129]
Based on the signal from the computer 51, the electrophoresis power supply unit 31 uses a predetermined voltage (between each electrode 32a and the electrode 32b) so that each electrode 32a becomes a cathode (negative electrode) and the electrode 32b becomes an anode (positive electrode). A constant voltage is applied, and the gel 100 is energized.
[0130]
The predetermined voltage is appropriately set depending on the gel composition and the like, and is not particularly limited, but is preferably about 25 to 100V.
[0131]
Thus, the sample (DNA and marker) is electrophoresed (moved) downward (lower end) in the gel 100, and the DNA is separated in the gel 100 by the difference in their chain length (difference in molecular weight). At this time, these separated DNAs (DNA fragments) each form a band (electrophoretic band) in the gel 100.
[0132]
The power supply unit 31 for electrophoresis is controlled (set) so that a predetermined current (constant current) flows through the gel 100 instead of maintaining (holding) between each electrode 32a and the electrode 32b at a constant voltage. May be.
[0133]
In this case, the predetermined current is not particularly limited, but is preferably about 1 to 10 mA.
[0134]
[5] Next, when a predetermined time elapses, the computer 51 drives the light source 61 of the detection means 6 (step S102 in FIG. 5).
[0135]
The light source 61 generates and emits laser light having a predetermined wavelength, for example. Then, the laser light passes through the side hole 422 and enters a predetermined position of the gel 100.
[0136]
Next, when the marker reaches the position where the laser beam of the gel 100 is incident (electrophoresed), the laser beam hits the marker.
[0137]
At this time, the fluorescent material included in the marker is in an excited state by absorbing the laser light. Thereafter, when the excited fluorescent substance returns to the ground state, fluorescent light (or phosphorescent light) is generated.
[0138]
A part of the fluorescent light emitted from the marker (light from the gel 100) passes through the side hole 423 and enters the light receiving element 62. The light receiving element 62 receives fluorescent light, performs photoelectric conversion, and transmits (outputs) a signal corresponding to the amount of received light to the computer 51.
[0139]
In addition, you may make it start irradiation to the gel 100 of a laser beam substantially simultaneously with the said process [4].
[0140]
[6] Next, when the computer 51 obtains the output signal from the light receiving element 62, the computer 51 stops driving the power source 31 for electrophoresis of the energizing means 3 and the light source 61 of the detecting means 6 (step S103 in FIG. 5).
[0141]
The electrophoretic power supply unit 31 stops the application of voltage between the electrodes 32a and 32b, and stops energization of the gel 100. This stops the electrophoresis of the DNA and marker in the gel 100.
[0142]
[7] Next, the computer 51 drives the temperature controller 41 of the temperature adjusting means 4 (step S104 in FIG. 5).
[0143]
First, the driver 432 drives the surface heater 431 based on a signal from the computer 51 to generate heat. The heat generated by the surface heater 431 is transferred to the gel 100 through the heat transfer block 42 and the tube body 20 to heat the gel 100.
[0144]
As a result, the separated DNA is denatured in the gel 100 to generate single-stranded DNA.
[0145]
Thereafter, when the temperature of the gel 100 approaches a predetermined temperature (set temperature), the driver 432 weakens the output of the surface heater 431 based on a signal from the computer 51.
[0146]
Further, when the temperature of the gel 100 reaches a predetermined temperature, the driver 432 stops driving the surface heater 431 based on a signal from the computer 51.
[0147]
The predetermined temperature (maximum temperature) is not particularly limited, but is preferably about 60 to 90 ° C. The time for maintaining the temperature of the gel 100 in the above temperature range is not particularly limited, but is preferably about 30 seconds to 30 minutes. Thereby, the separated DNA can be denatured efficiently (accurately).
[0148]
Next, the driver 444 drives the fan 443 based on a signal from the computer 51 to promote heat dissipation by the heat sink 442. The heat of the gel 100 is transmitted to the heat sink 442 through the tube body 20 and the surface heater 431.
[0149]
This heat is dissipated from the heat radiating portion 446. At this time, if the fan 443 is rotating, wind is sent from the blade 448 to the heat radiating portion 446. With this wind, heat dissipation is promoted in the heat radiating portion 446, and heat transfer from the gel 100 to the heat sink 442 is also promoted.
[0150]
Thereby, the temperature of the gel 100 is gradually decreased, and the denatured DNA (single-stranded DNA) is re-associated (hybridized) with the temperature decrease of the gel 100.
[0151]
Thereafter, when the temperature of the gel 100 approaches a predetermined temperature, the driver 444 stops driving the fan 443 based on a signal from the computer 51.
[0152]
[8] Next, when the temperature of the gel 100 reaches a predetermined temperature, the computer 51 stops driving the temperature controller 41 of the temperature adjusting means 4 (step S105 in FIG. 5).
[0153]
In addition, although predetermined temperature (minimum temperature) is not specifically limited, It is preferable to set it as 40 degrees C or less, and it is more preferable to set it as about 10-40 degreeC. As a result, the denatured DNA can be re-associated efficiently (accurately).
[0154]
[9] Next, the computer 51 drives the power source 31 for migration of the energization means 3 (step S106 in FIG. 5).
[0155]
Based on the signal from the computer 51, the electrophoretic power supply unit 31 sets each electrode 32a and electrode 32b so that the electrode 32b becomes a cathode (negative electrode) and each electrode 32a becomes an anode (positive electrode) (with the polarity reversed). A predetermined voltage (constant voltage) is applied between and the gel 100 is energized.
[0156]
That is, the electrophoresis power supply unit 31 energizes the gel 100 in the direction opposite to the electrophoresis direction in the step [4].
[0157]
The predetermined voltage is not particularly limited, but is preferably about 25 to 100V.
[0158]
As a result, the DNA after reassociation is electrophoresed upward (upper end) in the gel 100 and released from the gel 100 into the buffer solution of the upper buffer tank 21, respectively.
[0159]
When voltage application between each electrode 32a and electrode 32b is continued, the DNA after reassociation separated from each gel 100 is directed to each electrode 32a through the buffer solution in each upper buffer tank 21. Move (drawn). That is, the DNA after reassociation tends to move from the second space 210b to the first space 210a via the recess 214 and the communication hole 215.
[0160]
At this time, since the nucleic acid adsorbent R is accommodated (installed) in the recess 214, the DNA after reassociation is sequentially adsorbed to the nucleic acid adsorbent R when passing through the recess 214.
[0161]
As described above, the nucleic acid adsorbent R is moved between the gel 100 and the electrode 32a and in the middle of the movement path (the recess 214 and the communication hole 215) in which the reassociated DNA separated from the gel 100 moves to the electrode 32a. By providing in, the nucleic acid adsorbent R can adsorb the DNA after reassociation more reliably.
[0162]
The power supply unit 31 for electrophoresis is controlled (set) so that a predetermined current (constant current) flows through the gel 100 instead of maintaining (holding) between each electrode 32a and the electrode 32b at a constant voltage. May be.
[0163]
In this case, the predetermined current is not particularly limited, but is preferably about 1 to 10 mA.
[0164]
[10] Next, when a predetermined time has elapsed, the computer 51 stops driving the power supply unit 31 for electrophoresis of the energization means 3 (step S107 in FIG. 5).
[0165]
The electrophoretic power supply unit 31 stops the application of voltage between the electrodes 32a and 32b, and stops energization of the gel 100.
[0166]
The predetermined time is appropriately set depending on the composition of the gel and is not particularly limited, but is preferably about 0.5 to 2 hours.
[0167]
[11] Next, the operator performs an operation of collecting the nucleic acid adsorbent R in each upper buffer tank 21 in a separately prepared container (not shown) and extracting the DNA after reassociation.
[0168]
This can be performed by bringing the nucleic acid adsorbent R into contact with a buffer solution having a high salt concentration (for example, about 0.01 to 10 mol / L). Thereby, the DNA after the reassociation is released (detached) from the nucleic acid adsorbent R and transferred into the buffer solution.
[0169]
In addition, as this buffer solution, the thing of the composition similar to the buffer solution as mentioned above can be used.
[0170]
Thus, by using the nucleic acid adsorbent R to recover the DNA after reassociation, this recovery operation can be performed more quickly, which is advantageous in reducing the work time in the IGCR method. In addition, DNA after reassociation that has detached from the gel 100 can be recovered without waste.
[0171]
Further, the operator contacts the buffer solution containing the DNA after reassociation with avidin, and uses the DNA after reassociation taken out without being adsorbed by avidin for the PCR method.
[0172]
As described above, in the electrophoresis apparatus 1 according to the present embodiment, the gel 100 is energized by the energization means 3 and the sample is electrophoresed in the gel 100, whereby the DNA is separated in the gel 100 and detected. After detecting that the marker has reached a predetermined position in the gel 100 by the means 6, the energization to the gel 100 is stopped, then the gel 100 is heated by the heating means 43, and then the gel 100 is forced by the cooling means 44 The separated DNA is denatured in the gel 100 by cooling, and then re-associated. Then, the polarity is reversed by the current-carrying means 3, and the gel 100 is energized. Electrophoresis is performed in 100 in the direction opposite to the electrophoresis direction, and the DNA after reassociation separated from the gel 100 is adsorbed to the nucleic acid adsorbent R and recovered. It is (control).
[0173]
In particular, by detecting that the marker has reached a predetermined position in the gel 100, the electrophoresis of the DNA in the gel 100 is terminated (that is, energization of the gel 100 is stopped), so that the predetermined is simply performed. Compared with the case where such an operation is terminated when time elapses, DNA can be separated in the gel 100 with higher accuracy.
[0174]
Further, when the electrophoresis of the DNA in the gel 100 is terminated by setting the time, it takes time and effort to change the time setting as appropriate depending on the composition, concentration, etc. of the gel 100 to be used. Further, by detecting that the marker has reached a predetermined position in the gel 100 and then terminating the electrophoresis of the DNA in the gel 100, there is also an advantage that such labor can be omitted.
[0175]
In addition, by configuring the DNA after reassociation to be forcibly released from the gel 100 by electrophoresis, a separate operation for extracting the reassociated DNA from the gel 100 can be omitted. The subsequent DNA can be recovered from the gel 100 more quickly and reliably.
[0176]
The direction of electrophoresis of the DNA after reassociation may be the same as the direction of electrophoresis when separating the DNA, but the direction of electrophoresis of the DNA after reassociation may be the same as that when separating the DNA. By making the direction opposite to the direction of electrophoresis, the migration distance of the DNA having a low migration speed (low mobility) in the gel 100 can be shortened, so that the DNA after the reassociation can be further quickly collected. It can be released from inside.
[0177]
In addition, the direction of electrophoresis of the DNA after reassociation is opposite to the direction of electrophoresis when separating the DNA, so that the sample is applied to the gel 100 and the reassociation from within the gel 100 is performed. Since the release (collection) of DNA is on the same side (that is, in the buffer solution of the upper buffer tank 21) and a plurality of the upper buffer tanks 21 are provided independently to correspond to the gel 100, a plurality of different types are provided. The advantage is that multiple samples (from different subjects) can be processed simultaneously.
[0178]
In the case of processing samples of the same type (from the same subject), the upper buffer tank 21 may be a common one. In this case, there is an advantage that a large amount of sample can be processed.
[0179]
When processing the same type of sample, the direction of electrophoresis of the DNA after reassociation is the same direction as the direction of electrophoresis when separating the DNA, and the DNA after reassociation is moved from the gel 100 to the bottom. You may make it discharge | release in the buffer liquid of the buffer tank 22. FIG. In this case, the nucleic acid adsorbent R may be installed in the lower buffer tank 22 so as to be positioned between each gel (electrophoresis medium) 100 and the electrode 32b.
[0180]
Such an electrophoresis apparatus 1 is controlled by the computer 51 (control means 5) to separate and terminate DNA in the gel 100, denature and reassociate DNA after separation in the gel 100, and reassociate. Since the subsequent recovery of DNA from the gel 100 can be performed, the labor and time of the IGCR method can be reduced, which contributes to the automation of the implementation of the IGCR method.
[0181]
In addition, such an electrophoresis apparatus 1 includes a step of separating DNA in the gel 100 (the above steps [4] to [6]), a step of denaturing and reassociating the separated DNA in the gel 100 (the above step). [7] and [8]), and various conditions (various setting conditions) in the step of recovering the DNA after reassociation from the gel 100 (the above steps [9] and [10]) are the computer 51 (control means). Since the control is suitably performed according to 5), it is possible to shorten the time and improve the accuracy in each of the above steps.
[0182]
The electrophoretic device of the present invention has been described based on the illustrated embodiment. However, the present invention is not limited to this, and for example, the configuration of each unit is an arbitrary configuration that can exhibit the same function. Can be substituted.
[0183]
The electrophoresis medium can be composed of, for example, a hydrophilic polymer, a water-absorbing material impregnated with a buffer solution (buffer solution), or the like.
[0184]
Further, although three migration media are installed in the illustrated configuration, the number of migration media is not limited to this. For example, the number of migration media may be one, two, or four or more. .
[0185]
Further, the overall shape of the electrophoresis medium is not limited to a cylindrical (bar) shape, and may be, for example, a flat plate.
[0186]
In the configuration shown in the figure, the detection means is installed corresponding to only one electrophoresis medium. However, a plurality of detection means may be installed corresponding to each of the plurality of electrophoresis media.
[0187]
Further, for example, a Peltier element or the like may be used as the cooling means included in the temperature adjusting means.
[0188]
In the above description, DNA has been described as a representative nucleic acid. However, in the present invention, nucleic acids other than DNA, such as RNA and synthetic oligonucleotides, can also be used.
[0189]
Needless to say, the electrophoresis apparatus of the present invention can be used not only when the IGCR method is performed, but also when various gene analyzes are performed.
[0190]
【The invention's effect】
As described above, according to the electrophoresis apparatus of the present invention, for example, the labor and time of the IGCR method can be reduced, and in particular, it contributes to the automation of the implementation.
[0191]
In particular, by detecting that the marker has reached a predetermined position in the electrophoresis medium and then stopping the energization of the electrophoresis medium, the nucleic acid in the electrophoresis medium can be more accurately separated.
[0192]
In addition, the time required for recovering the nucleic acid can be shortened by configuring the energizing means to reverse the polarity before and after adjusting the temperature of the electrophoresis medium by the temperature adjusting means.
[0193]
Further, when a plurality of electrophoresis media are installed, the number and amount of specimens that can be processed at a time can be increased.
[0194]
For this reason, by using the electrophoresis apparatus of the present invention, for example, searching for an oncogene or the like can be performed more quickly and efficiently.
[Brief description of the drawings]
FIG. 1 is a schematic view showing an embodiment of an electrophoresis apparatus of the present invention.
2 is a partial longitudinal sectional view showing a configuration in the vicinity of an electrophoresis unit provided in the electrophoresis apparatus shown in FIG.
3 is a perspective view showing a configuration of an upper buffer tank provided in the electrophoresis apparatus shown in FIG. 1. FIG.
4 is a side view showing a configuration of temperature adjusting means provided in the electrophoresis apparatus shown in FIG. 1. FIG.
FIG. 5 is a flowchart showing an operation procedure of the electrophoresis apparatus shown in FIG.
[Explanation of symbols]
1 Electrophoresis device
2 electrophoresis part
200 Device body
20 tube
21 Upper buffer tank
210 Bulkhead
210a first space
210b second space
211 bottom
212 Mounting hole
213 Holding member
213a wearing part
214 recess
215 communication hole
216 legs
22 Lower buffer tank
221 inlet
222 Insertion hole
23 base
3 Energizing means
31 Power supply for electrophoresis
32a, 32b electrode
4 Temperature adjustment means
41 Temperature controller
42 Heat transfer block
421 insertion hole
422 side hole
423 side hole
43 Heating means
431 side heater
432 Driver
44 Cooling means
441 Cooler
442 heat sink
443 fans
444 driver
446 Heat dissipation part
447 motor
448 feathers
45 Temperature sensing means
451 Temperature sensor
452 A / D converter
5 Control means
51 computer
6 detection means
61 Light source
62 Light receiving element
100 gel
Steps S101 to S107

Claims (13)

An electrophoresis medium for electrophoresis of a specimen containing a nucleic acid and an electrophoretic marker;
Energizing means capable of energizing the electrophoresis medium and reversing its polarity;
A temperature adjusting means for adjusting the temperature of the electrophoresis medium, the temperature adjusting means having at least a heating means for heating the electrophoresis medium;
Detecting means for detecting the position of the electrophoresed marker in the electrophoresis medium,
The electrophoretic medium is energized by the energizing means, and the sample is electrophoresed in the electrophoretic medium, whereby the nucleic acid is separated in the electrophoretic medium, and the marker is contained in the electrophoretic medium by the detecting means. After detecting the arrival at a predetermined position, the energization to the electrophoresis medium is stopped,
Next, after the electrophoresis medium is heated by the heating means, the electrophoresis medium is cooled to denature the separated nucleic acid in the electrophoresis medium, and then reassociate.
Thereafter, the polarity is reversed by the energization means, the energization medium is energized, and the nucleic acid after reassociation is electrophoresed in the electrophoresis medium in the direction opposite to the electrophoresis direction and recovered from the electrophoresis medium. An electrophoretic device configured as described above. An electrophoresis medium for electrophoresis of a specimen containing a nucleic acid and an electrophoretic marker;
Energizing means capable of energizing the electrophoresis medium and reversing its polarity;
Temperature adjusting means for adjusting the temperature of the electrophoresis medium, the temperature adjusting means having heating means for heating the electrophoresis medium, and cooling means for cooling the electrophoresis medium;
Detecting means for detecting the position of the electrophoresed marker in the electrophoresis medium,
The electrophoretic medium is energized by the energizing means, and the sample is electrophoresed in the electrophoretic medium, whereby the nucleic acid is separated in the electrophoretic medium, and the marker is contained in the electrophoretic medium by the detecting means. After detecting the arrival at a predetermined position, the energization to the electrophoresis medium is stopped,
Next, after the electrophoresis medium is heated by the heating means, the electrophoresis medium is forcibly cooled by the cooling means to denature the separated nucleic acid in the electrophoresis medium, and then reassociate.
Thereafter, the polarity is reversed by the energization means, the energization medium is energized, and the nucleic acid after reassociation is electrophoresed in the electrophoresis medium in the direction opposite to the electrophoresis direction and recovered from the electrophoresis medium. An electrophoretic device configured as described above.   3. The electricity according to claim 1, wherein the migration speed of the marker is substantially the same as that of the nucleic acid having the fastest migration speed, or slightly faster than that of the nucleic acid having the fastest migration speed. Electrophoresis device.   The electrophoresis apparatus according to any one of claims 1 to 3, wherein the marker has a molecular weight of 100 to 2000.   The electrophoretic device according to claim 1, wherein the marker is any one of a charged fluorescent substance, a charged luminescent substance, and a charged dye.   The electrophoresis apparatus according to any one of claims 1 to 4, wherein the marker is a fluorescent substance, a luminescent substance, or a dye imparted to a nucleic acid. The detection means includes irradiation means for irradiating light to the electrophoresis medium;
The electrophoretic device according to claim 1, further comprising a light receiving unit that receives light from the electrophoresis medium.   The electrophoretic device according to claim 1, wherein the temperature adjusting unit includes a heat transfer unit that is provided around the electrophoretic medium and transfers heat to the electrophoretic medium.   When the total length in the longitudinal direction of the electrophoresis medium is L, the heat transfer section is configured to cover the range of 0.45 L from the center in the longitudinal direction to both ends of the electrophoresis medium. The electrophoresis apparatus according to claim 8.   10. The detection unit according to claim 1, wherein the detection unit detects that the marker has reached a range of 0.9 L or more from the electrophoresis start position, where L is the total length in the longitudinal direction of the electrophoresis medium. Electrophoresis device.   The electrophoresis apparatus according to claim 1, wherein the nucleic acid after the reassociation is recovered using a nucleic acid adsorbent.   The electrophoresis device according to claim 1, wherein a plurality of the electrophoresis media are provided side by side.   The electrophoresis device according to claim 1, wherein the electrophoresis medium is housed in a hollow member.

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