JPS613046A - Electrophoretic apparatus - Google Patents

Abstract

PURPOSE:To efficiently separate and detect specimens having MWs extending over a wide range, by enabling the variation in the voltage of an electrophoresis drive power source during migration corresponding to the advance situation of electrophoresis. CONSTITUTION:An anode and a cathode 2A, 2B are provided to both ends of an electrophoretic apparatus 1 and connected to an electrophoresis drive apparatus 3 while voltage is controlled by a controller 8. Detectors 4A, 4B,...4Z are provided to the migration path of a specimen and the passing specimen is detected, and amplification, data processing and outputting are respectively performed by an amplifier 5, a data processor 6 and an output device 7. The migration speed of the specimen becomes low as the MW thereof becomes larger. Because a specimen having a small MW is detected at the beginning of electrophoresis, the phoretic voltage is made low and, because MW becomes large with the elaspse of time, voltage is made high. The control of voltage is performed according to a predetermined program or while the advanced state of electrophoresis is detected. As a result, specimens having MWs extending over a wide range can be efficiently separated by one electrophoresis and a detection speed can be increased.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to an electrophoresis apparatus, and particularly to an electrophoresis apparatus used for determining the base sequence of a nucleic acid at high speed.

[Background of the invention]

Generally, in electrophoresis using polyacrylamide gel or agarose gel, it is known that the following relationship exists between the molecular weight M of the sample and the migration distance a within a certain range. (D,)1. LBishop,
J, Mou, BioQ, 26.373 (19
67)) QogM= a Q + b (a, b are constants determined by the gel) Therefore, it is difficult to simultaneously separate and detect molecules from small to large molecular weights in one electrophoresis. That is, if the electrophoresis time is too short, substances with large molecular weights will not be separated, and if the electrophoresis time is too long, substances with small molecular weights will reach the end of the gel. In order to solve this difficulty, conventional methods have been to change the gel concentration or the electrophoresis time.
A wide range of molecular weights ranging from 1 base to about 1000 bases was covered by ~3 runs of electrophoresis. ("protein·
Nucleic acids/enzymes J Vol, 23. No.3 (1978)
However, this method required 2 to 3 times more labor for experimental operations and 2 to 3 times more samples and electrophoresis equipment, and was inefficient.

In order to improve this inefficiency, conventional methods involve reading the spatial pattern of electrophoresis after the electrophoresis is completed by staining with deoxyribonucleic acid (DNA) dyes or by autoradiography, in which DNA is labeled with a radioisotope and a photographic film is exposed to light. Rather, it detects the sample passing in front of a detector fixed on the electrophoresis path during electrophoresis,
Another possible method is to read the temporal pattern of migration.

According to this method, a wide range of molecular weights can be covered in one run, but if the voltage is kept constant during the run, the time required for the sample to reach the detector position is approximately the logarithm of the sample's molecular weight. becomes longer in proportion to Therefore, as the molecular weight increases, detection takes longer,
The problem is that efficiency decreases.

In order to solve this problem, it is conceivable to increase the voltage of the electrophoresis drive power source to increase the electrophoresis speed.

However, if electrophoresis is performed at high voltage from the beginning, molecules with small molecular weights migrate faster than molecules with large molecular weights, so the speed becomes too fast and detection becomes difficult. As shown above, in order to efficiently separate and detect samples with a wide range of molecular weights ranging from 1 base to about 1000 bases in a single run by electrophoresis in a short time, it is necessary to keep the electrophoresis speed constant if the voltage of the electrophoresis drive power supply is constant. I can't control it.

[Purpose of the invention]

An object of the present invention is to solve the above-mentioned problems and provide an electrophoresis device that can efficiently separate and detect samples over a wide range of molecular weights in a short time using one electrophoresis device.

[Summary of the invention]

In order to achieve the above object, in the electrophoresis apparatus of the present invention, the electrophoresis drive power supply is operated according to a preset program or based on information regarding the progress of electrophoresis obtained from a detector provided on the electrophoresis path. The feature is that the voltage can be changed during electrophoresis. As a result, as time passes, the molecular weight of the sample molecules to be detected increases, the migration speed of the sample molecules to be detected becomes slow, and it takes a long time to reach the detector position, so the power supply voltage is gradually reduced. It is possible to increase the electrophoresis speed by increasing the electrophoresis speed. As a result, it has become possible to separate and detect substances with large molecular weights in a short time.

Therefore, according to this electrophoresis device, approximately 100
Efficient separation and separation over a wide range of molecular weights including 0 bases
Detection is now possible.

[Embodiments of the invention]

An embodiment of the present invention will be described below with reference to FIG.

Positive and negative electrodes 2A and 2B are provided at both ends of the electrophoresis tank 1, respectively.
An electric drive device 3 is installed between 2A and 2B to apply electrophoretic force to the sample. Detectors 4A to 4Z are provided on the electrophoresis path on which the sample moves, and detect the sample passing in front of them.

The output of the detector is amplified by an amplifier 5, processed by a data processing device 6, and outputted by an output device 7. A power supply control device 8 is connected to the electrophoresis drive power source 3, and this controls the voltage of the electrophoresis drive power source 3 based on a preset program or based on electrophoresis information obtained by a detector. When controlling the power supply voltage based on migration information, the power supply control device 8 is connected to the data processing device 6. It is connected to detectors 4A to 4Z via an amplifier 5. Here, the detector is a photodetector when the sample is stained or fluorescently labeled, and a radiation detector when the sample is radioiso-1-1 labeled.

Next, the operation of this embodiment will be explained.

When the electrophoresis voltage is kept constant during electrophoresis as in the conventional method, the following relationship exists between the electrophoresis velocity V and the molecular weight M of sample molecules.

V■a-12ogM (a is a constant) Therefore, the time required for the molecular weight to reach the detector and be detected becomes longer as the molecular weight becomes larger.

In order to shorten this time, it is possible to increase the electrophoresis voltage, but if the electrophoresis is performed at a high voltage from the beginning, the electrophoresis speed will be too high because molecules with a small molecular weight will migrate faster than molecules with a large molecular weight. I end up. For this reason,
The time the sample molecules spend in front of the detector is shortened, and if the sample is labeled with a radioisotope, the number of radiation counts decreases. If the sample is dyed or fluorescently labeled, the amount of light is insufficient to detect it. It becomes difficult.

Therefore, in this example, the electrophoresis voltage is kept low immediately after the start of electrophoresis when a sample with a small molecular weight is detected, and as time passes and the molecular weight of the detected sample increases, the electrophoresis voltage is gradually lowered. Increase the electrophoresis speed to
Even with large molecular weight substances, it is possible to migrate them to the detector position and detect them in a shorter time than with conventional methods.

Here, the voltage of the migration drive power supply 3 is increased by the power supply control device 8 according to a preset program. Any method of raising the voltage that causes the voltage to increase in a threatening manner over time will be effective, but as long as the voltage limit allows, it will increase over time. The effect of time reduction is greater if the increase ゛i is made larger.

FIG. 2 shows the relationship between the molecular weight of a sample and the time required to detect the sample. Curve (a) is the conventional method in which the power supply voltage is kept constant during electrophoresis, curve (b) is when the power supply voltage is increased linearly with respect to time, and curve (C) is the method in which the power supply voltage is increased exponentially. This is the case when As clearly seen from the figure, the time reduction effect is greater when the electrophoresis voltage is increased exponentially than when it is increased exponentially.

In this way, we aim to shorten the time required to detect a sample with a large molecular weight without making it difficult to detect a sample with a small molecular weight. This enabled good separation and detection of

Next, another embodiment of the present invention will be described with reference to FIG.

The configuration of the electrophoresis apparatus of this example is almost the same as that of the previous example, but in order to detect the progress of electrophoresis, in addition to the electrophoresis path for unknown samples, there is also a dedicated electrophoresis path. The difference is that a detector 9 is provided in the migration path A and the data processing bag e6 is connected to the power supply control device W18.

Next, the operation of this embodiment will be explained.

In this example, a dedicated electrophoresis path is provided for detecting the progress of electrophoresis, and a sample with a known molecular weight is electrophoresed here at the same time as an unknown sample. This sample may be a molecular weight marker made by cutting known DNA into small pieces with restriction enzymes, or DNA with a known base sequence may be processed for base sequencing in the same way as unknown samples (Maxam-Gilbert method or dideoxy method). It can be anything.

By detecting these samples with known molecular weights using the dedicated detector 9, it is possible to actually know the progress of electrophoresis, and this information is sent to the power supply control device 8 to control the voltage of the electrophoresis drive power source 3. The method of capping the voltage and its effects are the same as in the previous embodiment.

In general, the speed of electrophoresis can vary slightly depending on the condition of the gel and the temperature. Therefore, it is better to actually detect the progress of electrophoresis as in this example and control the electrophoresis voltage based on that information than to not actually detect the progress of electrophoresis as in the previous example. Favorable control can be achieved more reliably.

〔Effect of the invention〕

According to the present invention, even samples with large molecular weights can be detected in a shorter time than conventional methods by increasing the electric field strength, so a single electrophoresis can efficiently separate and detect samples over a wide range of molecular weights in a short time. can. Therefore, it becomes possible to speed up the conventional electrophoresis method.

[Brief explanation of the drawing]

FIG. 1 is a diagram schematically showing an electrophoresis apparatus according to an embodiment of the present invention. The second time is a diagram showing the power supply voltage and the output signal from the output device. FIG. 3 is a diagram showing another embodiment of the present invention. ■ Electrophoresis tank, 2A, 2B Electrode, 3...Electrophoresis drive power supply, 4 A, 4 F3. -42...detector, 5...
・Amplifier, 6...Data processing device, 7 Output device, 8
...Power supply control device, 9 Detector for detecting progress of swimming a.

Claims (1)

[Scope of Claims] 1. An electrophoresis apparatus comprising an electrophoresis tank, a detection unit that detects a sample migrating in the electrophoresis tank, and a migration drive power source that applies a voltage to both ends of the electrophoresis tank. . An electrophoresis apparatus, characterized in that the voltage applied to the electrophoresis drive power source is made variable, and the migration speed of the sample being electrophoresed is made variable. 2. The electrophoresis device according to claim 1, wherein the voltage is controlled according to a predetermined program. 3. The electrophoresis apparatus according to claim 1, characterized in that the voltage is controlled by detecting the progress of electrophoresis.