JP3691258B2 - Nucleic acid electrophoresis apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a nucleic acid electrophoresis apparatus that is used in molecular biology, genetics, medicine, physics, chemistry, analysis, clinical examination, etc., and recognizes and determines a target nucleic acid by electrophoresis.
[0002]
[Prior art]
The conventional DNA electrophoresis apparatus 10 shown in FIGS. 4 and 5 is provided with an electrophoresis gel 12 at the center position in an electrophoresis tank 11, and a positive electrode 13 and a minus electrode are provided on both sides of the electrophoresis gel 12. Each of the electrodes 14 is disposed and filled with a migration buffer solution 15. In the DNA electrophoresis apparatus 10, a sample containing DNA is injected into the electrophoresis well 16 of the electrophoresis gel 12, and the DNA is electrophoresed in the electrophoresis gel 12 toward the plus electrode 13 for a predetermined time. After the electrophoresis is completed, the electrophoresis gel 12 is taken out from the electrophoresis tank 11, and the electrophoresis gel 12 is transferred to a transilluminator 19 having an ultraviolet lamp 17 and an imaging camera 18 shown in FIG. The fluorescent dye bonded to the DNA is colored by the ultraviolet light from the ultraviolet lamp 17, and the fluorescent image is taken by the imaging camera 18 as a DNA migration image.
[0003]
FIG. 7 shows an example of a DNA migration image. A target DNA band (aggregation of DNA) is prepared as a positive marker 20, and the positions of the positive marker 20 and the DNA band 21 to be determined are examined. In the DNA electrophoresis apparatus 10, due to the difference in DNA migration speed based on the difference in DNA length, the DNA migration position at an arbitrary time point and the position of the target DNA (positive marker 20) at the same time point When the two match, the DNA is recognized as the target DNA and judged.
[0004]
In addition, the code | symbol 22 in FIG. 6 is a band pass filter as an optical filter, and the code | symbol 23 is a light shielding hood.
[0005]
[Problems to be solved by the invention]
By the way, the DNA migration speed varies depending on the ambient temperature, the state of each of the electrophoresis buffer 15 or the sample (for example, DNA), and the environment such as the concentration of the electrophoresis gel 12. Therefore, DNA does not always migrate at a constant distance for a fixed time, and therefore the end time of electrophoresis varies depending on the environment. Therefore, the experimenter must end the electrophoresis by cutting off the power supply to the plus electrode 13 and the minus electrode 14 while visually confirming the electrophoresis state of the DNA when the scheduled end time of the electrophoresis approaches. . If the electrophoresis cannot be completed within the electrophoresis end time, the DNA flows out of the electrophoresis gel 12 and the precious sample containing the DNA and the expensive electrophoresis gel 12 are wasted. Become. For this reason, an experimenter needs to be present at the end of electrophoresis.
[0006]
In addition, electrophoresis can be terminated without using an experimenter by using a timer or the like. However, in this case, since the DNA after the electrophoresis is diffused in the electrophoresis gel 12 over time, it is necessary to take a photograph immediately after the electrophoresis is completed. Therefore, also in this case, the intervention of the experimenter is required after the electrophoresis is completed.
[0007]
Usually, the experimenter is busy performing other work besides electrophoresis, and it is very painful and time consuming to be restricted in time for electrophoresis.
[0008]
An object of the present invention is to provide a nucleic acid electrophoresis apparatus capable of obtaining a good electrophoretic image without attending an experimenter or the like at the end of electrophoresis. is there.
[0009]
[Means for Solving the Problems]
The invention described in claim 1 is an electrophoresis gel for electrophoresis of nucleic acids in a sample injected at a specific position, an electrophoresis tank comprising electrodes disposed on both sides of the electrophoresis gel, and the electrode An imaging device that captures an electrophoretic image of the nucleic acid that is aspirated and migrates in the electrophoresis gel. While the nucleic acid is electrophoresed, the nucleic acid in the electrophoresis is continuously or intermittently captured by the imaging device. And while the nucleic acid in the sample injected into the specific position of the electrophoresis gel is undergoing electrophoresis, another sample containing the same or different nucleic acid is injected into the specific position. The present invention is characterized in that the nucleic acid can be electrophoresed .
[0010]
The invention according to claim 2 is characterized in that, in the invention according to claim 1, the power supply to the electrode can be cut off based on the migration image of the nucleic acid photographed by the imaging device. is there.
[0011]
The invention according to claim 3 is the invention according to claim 1 or 2, wherein the sample containing the nucleic acid, an installation part for installing a reagent necessary for electrophoresis, and the sample and reagent installed in the installation part are electrically connected. And an injection part that enables injection at a specific position of the electrophoresis gel .
[0013]
The invention according to claim 1 or 3 has the following effects.
[0014]
Since the imaging device is configured to continuously or intermittently shoot the nucleic acid in the electrophoresis while the nucleic acid in the sample is electrophoresed on the electrophoresis gel, an experimenter or the like can be used at the end of the electrophoresis. A good migrating image of the nucleic acid can be obtained without attendance, and the target nucleic acid can be determined from this migrating image. A configuration in which another nucleic acid containing the same or different nucleic acid can be injected into the specific position and the nucleic acid can be electrophoresed while the nucleic acid in the sample injected into the specific position of the electrophoresis gel is undergoing electrophoresis As a result, electrophoresis can be performed a plurality of times using the same electrophoresis gel, the frequency of use of expensive electrophoresis gel can be reduced, and cost can be reduced.
[0015]
The invention according to claim 2 has the following effects.
[0016]
Since the power supply to the electrode can be cut off based on the migration image of the nucleic acid photographed by the imaging device, the power supply to the electrode can be cut off when the target nucleic acid reaches a specific position. Unnecessary nucleic acid can be prevented from flowing out of the gel, and valuable nucleic acids can be suitably secured.
[0019]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
[0020]
FIG. 1 is a schematic sectional side view showing a DNA electrophoresis apparatus to which an embodiment of a nucleic acid electrophoresis apparatus according to the present invention is applied. FIG. 2 is a view taken in the direction of arrow II in FIG.
[0021]
A DNA electrophoresis apparatus 30 as a nucleic acid electrophoresis apparatus shown in FIGS. 1 and 2 is a submarine type electrophoresis apparatus, and includes an electrophoresis tank 34 having an electrophoresis gel 31, a plus electrode 32, and a minus electrode 33. , An imaging device 38 including a transmission ultraviolet lamp 35, a reflection ultraviolet lamp 36, and an imaging camera 37, an installation stage (not shown) as an installation unit, and a dispensing pipette (not shown) as an injection unit And a control device (not shown).
[0022]
The electrophoresis tank 34 is filled with an electrophoresis buffer 39, and an electrophoresis gel 31 is installed at the center of the electrophoresis tank 34, and is added to both end positions of the electrophoresis gel 31. The electrode 32 and the minus electrode 33 are installed, respectively, and the gel 31 for electrophoresis, the plus electrode 32, and the minus electrode 33 are immersed in the electrophoresis buffer 39.
[0023]
In the electrophoresis gel 31, a plurality of groove-shaped electrophoresis wells 40 are formed in a column at one end on the negative electrode 33 side as a specific position. Each of these electrophoresis wells 40 is provided for Lane 1 to Lane 6 (FIG. 3). Each sample containing different DNA is injected into each of the wells 40 for electrophoresis. Each sample is obtained by mixing a fluorescent dye (such as ethidium bromide) and glycerin into a solution containing DNA. The fluorescent dye binds to a double strand of DNA and emits fluorescence by ultraviolet rays. Further, unnecessary diffusion of DNA in the electrophoresis gel 31 is suppressed by the mixing of glycerin.
[0024]
DNA is generally charged with a negative charge, and when the plus electrode 32 and the minus electrode 33 are supplied with power through a power supply device (not shown), the electrophoresis gel 31 is moved from the electrophoresis well 40 toward the plus electrode 32. Electrophoresis. The migration speed of DNA varies depending on the difference in length of DNA. Therefore, the migration position of DNA at the same time also varies depending on the type of DNA.
[0025]
Here, the solution containing the DNA, the glycerin, and the fluorescent dye are each contained in a container and placed on an installation stage. The solution is collected from each container placed on the installation stage by a pipette pipette and subjected to electrophoresis. A sample is injected into each electrophoresis well 40 of the gel 31 for electrophoresis.
[0026]
On the other hand, the transmission ultraviolet lamp 35 of the imaging device 38 is disposed at a position below the electrophoresis tank 34, and can irradiate ultraviolet rays toward the electrophoresis gel 31 in the electrophoresis tank 34. An optical filter 41 is interposed between the transmitting ultraviolet lamp 35 and the electrophoresis gel 31. The reflecting ultraviolet lamp 36 is disposed above the electrophoresis tank 34 and can irradiate ultraviolet rays toward the electrophoresis gel 31 in the electrophoresis tank 34. An optical filter 42 is interposed between the reflecting ultraviolet lamp 36 and the electrophoresis gel 31. These optical filters 41 and 42 are filters that transmit only light of 330 nm or less.
[0027]
The fluorescent dye in the electrophoretic gel 31 is colored by the ultraviolet light passing through the optical filter 41 from the transmitting ultraviolet lamp 35 and the ultraviolet light passing through the optical filter 42 from the reflecting ultraviolet lamp 36. The imaging camera 37 intermittently captures this fluorescent image as a DNA migration image at regular time intervals. The imaging camera 37 is preferably a digital camera. The imaging camera 37 may take an image of a fluorescent dye in DNA that is colored by ultraviolet rays irradiated from either the transmission ultraviolet lamp 35 or the reflection ultraviolet lamp 36.
[0028]
The control device controls the operation of the dispensing pipette, the power supply control from the power supply device to the plus electrode 32 and the minus electrode 33, the lighting control of the transmitting ultraviolet lamp 35 and the reflecting ultraviolet lamp 36, and the photographing control by the imaging camera 37. To implement.
[0029]
That is, the control device operates the dispensing pipette to inject each sample containing different DNA into each electrophoresis well 40 of the electrophoresis gel 31.
[0030]
Thereafter, the control device applies a predetermined voltage (for example, 100 V) to the positive electrode 32 and the negative electrode 33 from the power supply device, and causes the DNA in each sample to migrate toward the positive electrode 32 in the electrophoresis gel 31. .
[0031]
Simultaneously with the start of electrophoresis or after a lapse of a predetermined time from the start of electrophoresis, the control device turns on the transmission ultraviolet lamp 35 and the reflection ultraviolet lamp 36 at regular time intervals while performing electrophoresis of the DNA. A DNA migration image is photographed by the imaging camera 37. Note that it is preferable to temporarily stop the electrophoresis of DNA at the time of photographing by the imaging camera 37, but it is not necessary to stop the electrophoresis.
[0032]
For example, when a 1% agarose gel is used as the gel 31 for electrophoresis, a voltage of 100 V is applied to the plus electrode 32 and the minus electrode 33, and a 500 bp DNA fragment is electrophoresed for about 40 minutes as DNA (end of electrophoresis) When the time is about 40 minutes from the start of electrophoresis), 10 minutes after the start of electrophoresis, the applied voltage to the positive electrode 32 and the negative electrode 33 is set to 0 V, and the electrophoresis is temporarily stopped. The reflection ultraviolet lamp 36 is turned on, and an electrophoretic image of DNA is photographed by the imaging camera 37. Thereafter, a voltage of 100 V was applied to the plus electrode 32 and the minus electrode 33 to cause electrophoresis of the DNA again, and then the electrophoresis of the DNA was stopped and electrophoresed by the imaging camera 37 in the same manner as described above every minute. Images are taken and this is repeated 30 times to obtain 31 electrophoretic images. From these electrophoretic images, it is possible to obtain an excellent electrophoretic image capable of recognizing and determining the target DNA.
[0033]
A part of the 30 electrophoretic images is shown in FIG. At the start of the electrophoresis in FIG. 3A, it can be confirmed that the DNA (in FIG. 3, the DNA appears as a DNA band 43 (aggregate)) is in each electrophoresis well 40 in Lane 1 to Lane 6. . After 10 minutes from the start of electrophoresis shown in FIG. 3 (B), it can be confirmed that the DNA (DNA band 43) in each lane migrates to different positions based on the difference in their migration speeds. After 30 minutes from the start of electrophoresis shown in FIG. 3 (C), the small size DNA in lane 1 (left DNA band 43) and the DNA in lane 5 (DNA band 43) are transferred to the left end (positive electrode) of electrophoresis gel 31. 32 side end), but after 40 minutes from the start of electrophoresis shown in FIG. 3 (D), both the above-mentioned DNAs (DNA band 43) have flowed out of the electrophoresis gel 31. Is confirmed. Further, in the electrophoresis image of FIG. 3C, the target DNA (this DNA (DNA band) is referred to as a positive marker 44) being electrophoresed in lane 6, and lane 2 and lane 3 are electrophoresed. In the electrophoresis image of FIG. 3D, the position of the DNA (DNA band 43) electrophoresed only in lane 3 is the lane 6 position. It is confirmed that the position coincides with the position of the positive marker 44. Therefore, from the electrophoresis image of FIG. 3D, it can be determined that the DNA that is electrophoresing in lane 2 is the same target DNA as the positive marker 44.
[0034]
Further, the control device inputs data of the electrophoretic image photographed by the imaging camera 37, and when the target DNA comes to a specific position matching the positive marker 44 based on the image data, By controlling the apparatus, the power supply to the plus electrode 32 and the minus electrode 33 is cut off, and the electrophoresis is stopped, thereby preventing the DNA from flowing out from the electrophoresis gel 31 more than necessary. For example, when the DNA (DNA band 43) that runs in lane 2 shown in FIG. 3D matches the positive marker 44, that is, after about 40 minutes from the start of electrophoresis, the positive electrode 32 and the negative electrode 33 Electrophoresis is stopped by setting the applied voltage to 0V to 0V.
[0035]
Therefore, according to the above embodiment, the following effects (1) and (2) are achieved.
[0036]
(1) Since the imaging camera 37 is configured to intermittently shoot the DNA in the electrophoresis at regular time intervals while the DNA in the sample is electrophoresed on the electrophoresis gel 31, the electrophoresis is completed. Occasionally, an excellent electrophoretic image of DNA can be obtained without the presence of an experimenter, and the target DNA can be determined from the electrophoretic image.
[0037]
(2) Since the power supply to the plus electrode 32 and the minus electrode 33 can be cut off on the basis of the DNA migration image taken by the imaging camera 37 of the imaging device 38, the target DNA is positive marker 44. Since the power supply to the plus electrode 32 and the minus electrode 33 can be cut off at the stage where the specific position coincides with, it is possible to prevent the DNA from flowing out of the gel 31 for electrophoresis more than necessary, and preferentially secure valuable DNA. .
[0038]
As mentioned above, although this invention was demonstrated based on one Embodiment, this invention is not limited to this.
[0039]
For example, while a sample is injected into each electrophoresis well 40 of one electrophoresis gel 31 in the electrophoresis tank 34 and electrophoresis is performed, each electrophoresis well 40 of the same electrophoresis gel 31 is used. Alternatively, a sample containing the same or different DNA may be injected, the DNA may be electrophoresed, and the DNA being electrophoresed may be photographed by the imaging camera 37 of the imaging device 38. In this case, as described above, even if electrophoresis is performed a plurality of times in the same electrophoresis gel 31, an electrophoretic image during the electrophoresis can be obtained by the imaging camera 37, so that the electrophoresis gel 31 later in the same lane. Even if the DNA (DNA band) that has started electrophoresis overtakes the DNA (DNA band) that has migrated first, the confirmation can be made. Therefore, in this case, since electrophoresis can be performed a plurality of times using the same electrophoresis gel 31, the frequency of use of the expensive electrophoresis gel 31 is reduced and the cost can be reduced.
[0040]
In the above-described embodiment, the imaging camera 37 is described as being intermittently photographed by a digital camera. However, DNA during electrophoresis may be continuously photographed by a video camera.
[0041]
Furthermore, in the above embodiment, the case where the nucleic acid is DNA has been described, but RNA may be used.
[0042]
【The invention's effect】
As described above, according to the nucleic acid electrophoresis apparatus according to the present invention, while performing electrophoresis of nucleic acid in the electrophoresis gel of the electrophoresis tank, the nucleic acid in the electrophoresis is continuously or intermittently captured by the imaging device. Since it is configured to take a picture, an excellent electrophoretic image can be obtained without an experimenter present at the end of electrophoresis.
[Brief description of the drawings]
FIG. 1 is a schematic side sectional view showing a DNA electrophoresis apparatus to which an embodiment of a nucleic acid electrophoresis apparatus according to the present invention is applied.
FIG. 2 is a view taken in the direction of arrow II in FIG.
3A to 3D are electrophoretic images taken by the DNA electrophoresis apparatus of FIG.
FIG. 4 is a schematic plan view showing a conventional DNA electrophoresis apparatus.
5 is a schematic sectional side view of the DNA electrophoresis apparatus of FIG.
FIG. 6 is a schematic side sectional view showing a conventional transilluminator.
FIG. 7 is an example of an electrophoretic image taken by the transilluminator of FIG.
[Explanation of symbols]
30 DNA electrophoresis device (nucleic acid electrophoresis device)
31 Electrophoresis gel 32 Positive electrode 33 Negative electrode 34 Electrophoresis tank 37 Imaging camera 38 Imaging device 40 Electrophoresis well (specific position)

Claims (3)

An electrophoresis gel for electrophoresis of nucleic acids in a sample injected at a specific position, and an electrophoresis tank comprising electrodes disposed on both sides of the electrophoresis gel;
An imaging device that captures an electrophoretic image of the nucleic acid that is aspirated by the electrode and migrates in the electrophoresis gel;
While the nucleic acid is electrophoresed, the nucleic acid in the electrophoresis is photographed continuously or intermittently by the imaging device , and the nucleic acid in the sample injected at a specific position of the electrophoresis gel is electrophoresed. In the meantime , the nucleic acid electrophoresis apparatus is configured such that another sample containing the same or different nucleic acid can be injected into the specific position and the nucleic acid can be electrophoresed.   The nucleic acid electrophoresis apparatus according to claim 1, wherein power supply to the electrode can be cut off based on a migration image of the nucleic acid photographed by the imaging device. A sample containing the nucleic acid, an installation unit for installing a reagent necessary for electrophoresis, and a sample installed in the installation unit, and an injection unit for allowing the reagent to be injected into a specific position of the electrophoresis gel. The nucleic acid electrophoresis apparatus according to claim 1 or 2, characterized in that:

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