JP4598989B2 - Electrophoresis device - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an electrophoresis apparatus.
[0002]
[Prior art]
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).
[0003]
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.
The outline of the IGCR method will be described below with reference to the case of searching for an oncogene.
[0004]
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.
[0005]
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.
[0006]
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.
[0007]
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.
[0008]
In this case, when electrophoresis is performed in the step 4), the bands are separated from the gel, for example, gene A> gene B> gene B ′ in the gel.
[0009]
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.
[0010]
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.
[0011]
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 band of gene B ', the ratio of DNA derived from normal cells to DNA derived from cancer cells remains 0: 100.
[0012]
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.
[0013]
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 the gene B 'is composed only of DNA derived from cancer cells, it is not adsorbed to avidin and is taken out.
[0014]
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.
[0015]
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, only the gene B ′ is obtained. Is preferably detected.
[0016]
Thus, the IGCR method is an excellent gene search method. However, the IGCR method has many drawbacks in that it requires a lot of manual processes and many time-consuming processes, so that the required time is long (for example, about one week) and the work requires time and effort.
[0017]
[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.
[0018]
[Means for Solving the Problems]
  Such purposes are as follows (1) to (7This is achieved by the present invention.
[0019]
  (1) an electrophoresis medium for electrophoresis of a sample containing nucleic acid;
  Energizing means capable of energizing the electrophoresis medium and reversing its polarity;
  Temperature adjusting means for adjusting the temperature of the electrophoresis mediumA temperature adjusting means having at least a heating means for heating the electrophoresis medium;
And a control means connected to the energization means and the temperature adjustment means,
By controlling the operation of the energizing means and the temperature adjusting means by the control means,
First, the energization means is energized to the electrophoresis medium, the sample is electrophoresed in the electrophoresis medium, thereby separating the nucleic acid in the electrophoresis medium,
Next, the electrophoresis medium is heated by the temperature adjusting means to denature the separated nucleic acid in the electrophoresis medium, and then the electrophoresis medium is cooled to 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. Is configured asAn electrophoresis apparatus characterized by that.
  (2) Electrophoresis medium for electrophoresis of a sample containing nucleic acid;
  Energizing means capable of energizing the electrophoresis medium and reversing its polarity;
  Temperature adjusting means for adjusting the temperature of the electrophoresis mediumA temperature adjusting means having a heating means for heating the electrophoresis medium and a cooling means for cooling the electrophoresis medium;
And a control means connected to the energization means and the temperature adjustment means,
By controlling the operation of the energizing means and the temperature adjusting means by the control means,
First, the energization means is energized to the electrophoresis medium, the sample is electrophoresed in the electrophoresis medium, thereby separating the nucleic acid in the electrophoresis medium,
Next, the electrophoresis medium is heated by the heating means to denature the separated nucleic acid in the electrophoresis medium, and then the electrophoresis medium is forcibly cooled by the cooling means to 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. Is configured asAn electrophoresis apparatus characterized by that.
[0020]
  (3The temperature adjusting means includes a heat transfer section that is disposed around the electrophoresis medium and that transfers heat to the electrophoresis medium.(1) or (2)The electrophoresis apparatus according to 1.
[0021]
  (4) When the total length in the longitudinal direction of the electrophoretic medium is L, the heat transfer section is configured to cover the electrophoretic medium in a range of 0.45 L from the longitudinal center to both ends. Is above (3).
[0026]
  (5The energizing means reverses the polarity before and after adjusting the temperature of the electrophoretic medium by the temperature adjusting means.4).
[0027]
  (6(1) to (1) to (9) above, wherein a plurality of the migration media are arranged with a gap.5).
[0028]
  (7The electrophoretic medium is stored in a hollow member (1) to (6).
[0029]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, an electrophoretic device of the present invention will be described in detail based on preferred embodiments shown in the accompanying drawings.
[0030]
FIG. 1 is a schematic diagram showing the overall configuration of the electrophoresis apparatus of the present invention, FIG. 2 is a longitudinal sectional view showing the configuration in the vicinity of the electrophoresis unit included in the electrophoresis apparatus of the present invention, and FIG. FIG. 4 is a side view showing a configuration of temperature adjusting means provided in the electrophoresis apparatus of the present invention, and FIG. 4 is a flowchart showing an operation procedure of the electrophoresis apparatus of the present invention. In the following description, in FIG. 2, the upper side is referred to as “upper end” and the lower side is referred to as “lower end”.
[0031]
The electrophoretic device 1 shown in FIG. 1 includes an electrophoretic unit 2, an energizing unit 3, a temperature adjusting unit 4, and a control unit 5.
[0032]
Hereinafter, the electrophoresis apparatus 1 will be described for each component.
As shown in FIG. 2, the electrophoresis unit 2 mainly includes an upper buffer tank 21, a lower buffer tank 22, a base 23 that supports these, and a plurality of tubes containing gels (electrophoresis media) 100. (Tubular hollow member) 20.
[0033]
The upper buffer tank 21 is composed of a box-shaped member having an opening at the upper end (upper part). In the upper buffer tank 21, for example, a buffer solution (buffer solution) such as Tris buffer or Tris / borate buffer is accommodated, and an electrode 32a of the energizing means 3 described later is immersed in the buffer solution. is set up.
[0034]
The amount of the buffer solution is such that the upper end of the tube body 20 does not protrude from the liquid surface in a state where the tube body 20 is mounted (installed) in the upper buffer tank 21.
[0035]
A plurality (three in this embodiment) of mounting holes 212 are formed in the bottom (bottom) 211 of the upper buffer tank 21 so as to penetrate in the thickness direction (vertical direction in FIG. 2). A holding member 213 that holds the tubular body 20 is fixed (attached) to each mounting hole 212 in a liquid-tight manner.
[0036]
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. When the mounting portion 213 a is inserted and fitted into the mounting hole 212, the holding member 213 is liquid-tightly fixed to the bottom portion 212 of the upper buffer tank 21.
[0037]
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.
[0038]
Further, the upper end side of the tubular body 20 described later is inserted (inserted) into the lumen portion of the holding member 213.
[0039]
Oppositely arranged leg portions 214 and 214 are formed integrally with the upper buffer tank 21 on both sides of the upper buffer tank 21 (in FIG. 2, on the back side and the front side of the page). It is installed in the base 23 via. Thereby, the upper buffer tank 21 is fixed to the base 23.
[0040]
The base 23 is for supporting the upper buffer tank 21, the lower buffer tank 22, and a heat transfer block 42 described later, and is configured by a flat plate-like member.
A lower buffer tank 22 is installed at the upper end (upper surface) of the base 23.
[0041]
The lower buffer tank 22 is composed of a box-shaped member. Inside the lower buffer tank 22, a buffer solution (buffer solution) is accommodated, and an electrode 32b of the energizing means 3 described later is installed so as to be immersed in the buffer solution.
[0042]
As this buffer solution, the same buffer solution as the buffer solution stored in the upper buffer tank 21 can be used.
[0043]
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.
[0044]
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.
[0045]
In this state, the lower buffer tank 22 stores a buffer solution having a volume that allows the lower end of the tube body 20 to be immersed therein.
[0046]
The constituent materials of the lower buffer tank 22, the upper buffer tank 21 and the base 23 are not particularly limited. For example, polyvinyl chloride, polyethylene, polypropylene, polystyrene, poly- (4-methylpentene) are available. -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, nylon) Various resin materials such as 12) can be used.
[0047]
The lower buffer tank 22 and the upper buffer tank 21 are preferably substantially transparent, respectively, in order to ensure internal visibility.
[0048]
Between the 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 via the holding members 213. It is installed by being fixed to 21.
[0049]
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.
[0050]
Although it does not specifically limit as a dimension of the tubular body 20, For example, it is preferable that an internal diameter is about 3-8 mm and an outer diameter is about 5-10 mm.
[0051]
Each tube 20 is filled (contained) with a gel (electrophoresis medium) 100, and is disposed (with a predetermined interval) through a gap (for example, about 10 to 40 mm). In other words, the plurality of gels 100 are arranged with gaps. By disposing a plurality of gels 100 in this way, the electrophoresis apparatus 1 can increase the amount of specimen that can be processed at one time.
[0052]
In addition, a portion not filled with the gel 100 is provided inside the upper end portion of the tube body 20, and the sample is filled (applied) into the portion.
[0053]
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).
[0054]
The gel 100 has a substantially cylindrical shape (bar shape) by being accommodated in the tubular body 20.
[0055]
Although it does not specifically limit as a full length (length L in FIG. 2) of the longitudinal direction of the gel 100, Usually, about 50-200 mm is preferable and it is more preferable that it is about 100-150 mm. By setting the total length of the gel 100 in the longitudinal direction within such a range, DNA can be suitably separated.
[0056]
Such a gel 100 is energized by the energizing means 3.
This energizing means 3 is capable of energizing the gel 100 and reversing its polarity, and has an electrophoretic power supply 31 and a pair of electrodes 32a and 32b as shown in FIG. ing. The electrophoretic power supply unit 31 is connected to the electrodes 32a and 32b through signal lines.
[0057]
The electrode 32a and the electrode 32b are each made of a conductive material such as metal (aluminum or the like), for example. As described above, the electrode 32 a is installed in the upper buffer tank 21, and the electrode 32 b is installed in the lower buffer tank 22.
The temperature of the gel 100 is adjusted by the temperature adjusting means 4.
[0058]
As shown in FIG. 3, the temperature adjusting unit 4 transfers 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.
[0059]
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.
[0060]
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.
[0061]
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.
[0062]
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.
[0063]
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.
[0064]
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.
[0065]
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.
[0066]
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.
[0067]
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).
[0068]
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.
[0069]
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 disposed so as to surround the entire circumference (periphery) of each gel 100.
[0070]
Thereby, since the gel 100 can be heated and cooled evenly (uniformly) in the circumferential direction, DNA can be denatured and re-associated more quickly and reliably.
[0071]
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.
[0072]
That is, the heat transfer block 42 is not particularly limited from the center in the longitudinal direction to both ends (upper end and lower end), where L is the total length in the longitudinal direction of the gel 100. It is preferably configured to cover a range of about .45L (a range of length M in FIG. 2), and more preferably configured to cover a range of about 0.4L.
[0073]
Thereby, since the gel 100 can be heated and cooled evenly (uniformly) in the longitudinal direction, DNA can be denatured and re-associated more quickly and reliably.
[0074]
In the electrophoresis apparatus 1 of the present embodiment, the heat transfer block 42 is disposed 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 longitudinal range of the gel 100, it is possible to denature and reassociate DNA more rapidly and reliably by these synergistic effects.
[0075]
As described above, the temperature adjusting unit 4 of the present embodiment includes the heating unit 43 and the cooling unit 44, but the cooling unit 44 may be omitted. In this case, the temperature adjusting means 4 heats the gel 100 by the heating means 43, 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.
[0076]
In addition, when the gel 100 is forcibly cooled by the cooling means 44 as in the present embodiment, the denatured DNA is more quickly 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).
[0077]
The energizing means 3 and the temperature adjusting means 4 described above are controlled by the control means 5 constituted by the computer 51, respectively. The computer 51 is connected to the power supply unit 31 for the electricity supply means 3 and the temperature controller 41 (driver 432, driver 444, A / D converter 452) of the temperature adjustment means 4 via signal lines, respectively. Yes.
[0078]
The computer 51 controls the electrophoretic power supply unit 31 to apply a voltage between the electrode 32a and the electrode 32b, thereby energizing the gel 100, and also controls the electrophoretic power supply unit 31 to control the electrode 32a. And the polarity of the electrode 32b can be reversed.
[0079]
On the other hand, 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.
[0080]
Hereinafter, the usage method (action) of the electrophoresis apparatus 1 will be described with reference to FIG. 4 as an example in which the IGCR method is performed.
[0081]
[1] First, an operator (user of the electrophoresis apparatus 1) who performs the IGCR method uses the upper buffer tank 21 and the lower buffer as shown in FIG. Installed between the 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).
[0082]
On the other hand, the worker prepares a sample (specimen) containing DNA (nucleic acid). This sample contains DNA derived from cells to be examined 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.
[0083]
[2] Next, the operator operates the computer 51 to input various conditions (various setting conditions) in steps [4] to [9] described later.
[0084]
[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).
[0085]
[4] Next, when the 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. 4).
[0086]
The electrophoretic power supply unit 31 applies a predetermined voltage (constant voltage) between the electrode 32a and the electrode 32b based on a signal from the computer 51 so that the electrode 32a becomes a cathode (negative electrode), and the gel 100 Energize to.
[0087]
The predetermined voltage is appropriately set depending on the composition of the gel and the like, and is not particularly limited, but is preferably about 25 to 100 V, for example.
[0088]
Thus, the sample 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.
[0089]
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) a constant voltage between the electrodes 32a and 32b. Also good.
[0090]
In this case, the predetermined current is not particularly limited, but is preferably about 1 to 10 mA, for example.
[0091]
[5] 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 S102 in FIG. 4).
[0092]
The electrophoretic power supply unit 31 stops the application of voltage between the electrode 32a and the electrode 32b, and stops energization of the gel 100. Thereby, the electrophoresis in the gel 100 of DNA is stopped.
[0093]
The predetermined time is appropriately set depending on the gel composition and the like, and is not particularly limited, but is preferably about 0.5 to 2 hours, for example.
[0094]
[6] Next, the computer 51 drives the temperature controller 41 of the temperature adjusting means 4 (step S103 in FIG. 4).
[0095]
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.
[0096]
As a result, the separated DNA is denatured in the gel 100 to generate single-stranded DNA.
[0097]
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.
[0098]
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.
[0099]
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).
[0100]
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.
[0101]
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.
[0102]
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.
[0103]
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.
[0104]
[7] 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 S104 in FIG. 4).
[0105]
In addition, although predetermined temperature (minimum temperature) is not specifically limited, For example, it is preferable to set it as about 40 degreeC 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).
[0106]
[8] Next, the computer 51 drives the power source 31 for migration of the energization means 3 (step S105 in FIG. 4).
[0107]
Based on the signal from the computer 51, the electrophoresis power supply unit 31 sets a predetermined voltage (constant voltage) between the electrode 32a and the electrode 32b so that the electrode 32b becomes a cathode (negative electrode) (with the polarity reversed). Is applied to energize the gel 100.
[0108]
That is, the electrophoresis power supply unit 31 energizes the gel 100 in the direction opposite to the electrophoresis direction in the step [4].
[0109]
Although it does not specifically limit as this predetermined voltage, For example, it is preferable to set it as about 25-100V.
[0110]
Thereby, the DNA after reassociation is electrophoresed upward (upper end) in the gel 100 and eluted from the gel 100 into the buffer solution in the upper buffer tank 21 (recovered).
[0111]
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) a constant voltage between the electrodes 32a and 32b. Also good.
[0112]
In this case, the predetermined current is not particularly limited, but is preferably about 1 to 10 mA, for example.
[0113]
[9] 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 S106 in FIG. 4).
[0114]
The electrophoretic power supply unit 31 stops the application of voltage between the electrode 32a and the electrode 32b, and stops energization of the gel 100.
[0115]
The predetermined time is appropriately set depending on the gel composition and the like, and is not particularly limited, but is preferably about 0.5 to 2 hours, for example.
[0116]
[10] Next, the operator extracts the DNA after reassociation from the buffer solution in the upper buffer tank 21.
[0117]
Furthermore, the worker brings such DNA into contact with avidin, and uses the DNA extracted without being adsorbed by avidin for the PCR method.
[0118]
Before the step [9], a carrier such as nitrocellulose (nucleic acid adsorbing material) is placed inside the upper end portion of the tube body 20, and the adsorbed to the carrier is used to recover the DNA after reassociation. It may be.
[0119]
In this case, the DNA extraction operation after the reassociation in this step [10] can be performed more quickly, which is advantageous for shortening the time.
[0120]
Here, in summary, in the electrophoresis device 1 of the present embodiment, the gel 100 is energized by the energizing means 3 and the sample is electrophoresed in the gel 100 to separate the DNA in the gel 100. After the gel 100 is heated by the heating means 43, the gel 100 is forcibly cooled by the cooling means 44 to denature the separated DNA in the gel 100 and then reassociate. The gel 100 is energized with the polarity reversed, and the DNA after reassociation is electrophoresed in the gel 100 in the direction opposite to the direction of the electrophoresis and collected (controlled) from the gel 100.
[0121]
That is, in the electrophoretic device 1 of the present embodiment, the energizing unit 3 is configured (controlled) so as to reverse the polarity before and after adjusting the temperature of the gel 100 by the temperature adjusting unit 4.
[0122]
In this way, by configuring the DNA after reassociation to be eluted from the gel 100 by electrophoresis, an operation for extracting the DNA after reassociation from the gel 100 can be omitted separately. The DNA after reassociation can be recovered from the gel 100 more quickly and reliably.
[0123]
In particular, by reversing the direction of electrophoresis of the DNA after reassociation with the direction of electrophoresis when separating the DNA, the migration distance of DNA having a low migration speed (low mobility) in the gel 100 Thus, the DNA after reassociation can be recovered from the gel 100 more rapidly.
[0124]
In such an electrophoretic device 1, separation of DNA in the gel 100, denaturation and reassociation of DNA after separation in the gel 100, and after reassociation are controlled by the computer 51 (control means 5). Since the DNA can be recovered from the gel 100, the labor and time of the IGCR method can be reduced, contributing to the automation of the implementation of the IGCR method.
[0125]
Further, in such an electrophoresis apparatus 1, a step of separating DNA in the gel 100 (the above steps [4] and [5]), a step of denaturing and reassociating the separated DNA in the gel 100 (the above step) [6] and [7]), and various conditions (various setting conditions) in the step of recovering the DNA after reassociation from the gel 100 (the above steps [8] and [9]) 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.
[0126]
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.
[0127]
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.
[0128]
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. .
[0129]
Further, the overall shape of the electrophoresis medium is not limited to a cylindrical (bar) shape, and may be, for example, a flat plate shape.
[0130]
Further, for example, a Peltier element or the like may be used as the cooling means included in the temperature adjusting means.
[0131]
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.
[0132]
【The invention's effect】
As described above, according to the electrophoresis apparatus of the present invention, for example, work and time in the IGCR method can be reduced, and in particular, it contributes to automation of its implementation.
[0133]
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.
[0134]
In addition, when a plurality of electrophoresis media are installed, the amount of nucleic acid (sample) that can be processed at a time can be increased.
[0135]
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 diagram showing the overall configuration of an electrophoresis apparatus of the present invention.
FIG. 2 is a longitudinal sectional view showing a configuration in the vicinity of an electrophoresis unit provided in the electrophoresis apparatus of the present invention.
FIG. 3 is a side view showing a configuration of temperature adjusting means provided in the electrophoresis apparatus of the present invention.
FIG. 4 is a flowchart showing an operation procedure of the electrophoresis apparatus of the present invention.
[Explanation of symbols]
1 Electrophoresis device
2 electrophoresis part
20 tube
21 Upper buffer tank
211 bottom
212 Mounting hole
213 Holding member
213a wearing part
214 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
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
100 gel

Claims (7)

An electrophoresis medium for electrophoresis of a sample containing nucleic acid;
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;
And a control means connected to the energization means and the temperature adjustment means,
By controlling the operation of the energizing means and the temperature adjusting means by the control means,
First, the energization means is energized to the electrophoresis medium, the sample is electrophoresed in the electrophoresis medium, thereby separating the nucleic acid in the electrophoresis medium,
Next, the electrophoresis medium is heated by the temperature adjusting means to denature the separated nucleic acid in the electrophoresis medium, and then the electrophoresis medium is cooled to 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 sample containing nucleic acid;
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;
And a control means connected to the energization means and the temperature adjustment means,
By controlling the operation of the energizing means and the temperature adjusting means by the control means,
First, the energization means is energized to the electrophoresis medium, the sample is electrophoresed in the electrophoresis medium, thereby separating the nucleic acid in the electrophoresis medium,
Next, the electrophoresis medium is heated by the heating means to denature the separated nucleic acid in the electrophoresis medium, and then the electrophoresis medium is forcibly cooled by the cooling means to 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 . Said temperature adjusting means, wherein disposed around the electrophoretic medium, electrophoretic device according to claim 1 or 2 having a heat transfer section for heat transfer to the electrophoresis 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 3 . It said energizing means, before and after adjusting the temperature of the loading medium by the temperature adjustment means, electrophoretic device according to any one of claims 1 to invert the polarity 4. The electrophoresis medium, electrophoretic device according to any one of claims 1 are more disposed with a gap 5. The electrophoresis medium, electrophoretic device according to any one of claims 1 housed in the hollow member 6.

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