JPH08211018A - Gel electrophotretic method and apparatus - Google Patents

Abstract

PURPOSE: To suppress a smiling phenomenon and to accurately determine a base sequence by preheating an electrolyte in which the end part of a migration gel on the injection side of a nucleic acid fragment sample is immersed at the start of migration. CONSTITUTION: At the start of the migration of a DNA sample, the electrolyte of an electrolytic cell 8 is preheated at about 40-60 deg.C by a heater 24. The heating of the electrolyte is started before the sample is introduced into the sample charging slot 14 provided to the upper end of a slab-shaped migration gel 2 and performed even during the introduction of the sample to be continued up to the start of migration. After the start of migration, the heating of the electrolyte may be continued or stopped. This heated electrolyte becomes a heat source and the temp. of the gel 2 at the part where the slit 14 is provided becomes uniform. By this constitution, the smiling phenomenon caused by the non-uniformity of temp. is suppressed and a base sequence of nucleic acid can be accurately determined.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a gel electrophoresis method for determining the base sequence of nucleic acid such as DNA and an apparatus used therefor, and more particularly to a gel electrophoresis method and apparatus using a slab-like electrophoresis gel. .

[0002]

2. Description of the Related Art In a gel electrophoresis apparatus using a slab-like gel, Joule heat generated during electrophoresis causes a so-called smileing phenomenon in which the moving speed of the central part of the gel is higher than the moving speed of the peripheral part. . In DNA sequencing (base sequence determination) or the like, the signals of the respective migration lanes are read in the migration order to estimate the base sequence, but if the smile phenomenon occurs, the sequence is reversed and an error occurs in the sequence determination.

Therefore, the following measures are taken to reduce the smiley phenomenon. (1) The slab-like gel is used with the front and back sandwiched by glass plates, but the outside of the front and back glass plates is sandwiched by a heat dissipation plate with good thermal conductivity such as an aluminum plate, and is running. Make sure that the temperature of the entire gel does not become non-uniform due to the Joule heat generated at. (2) The outside of the glass plate in which the slab-shaped gel is sandwiched is sandwiched by a device that circulates warm water, and the gel is forcibly heated from the outside to make the entire gel a uniform temperature.

[0004]

[Problems to be Solved by the Invention] The above (1) and (2)
Although such a measure can reduce the smiley phenomenon, it still cannot sufficiently prevent misreading of sequences. For example, the migration difference between 500 bases and 501 bases is 0.2% (= 1/500), but in order to suppress the migration difference due to smileing to 0.2% or less by controlling the temperature, it is 0.1%. In reality, it is difficult to control the temperature unevenness below ℃. It is an object of the present invention to provide a gel electrophoresis method capable of suppressing the smile phenomenon and correctly determining a base sequence, and a gel electrophoresis apparatus for realizing the method.

[0005]

In the present invention, the electrolytic solution in which the end of the electrophoretic gel on the side for injecting the nucleic acid fragment sample is immersed is heated at least at the start of electrophoresis.

[0006]

1 and 2 show an example in which the present invention is applied to an on-line type nucleotide sequencer. 1 is a schematic perspective view of the whole, and FIG. 2 is a sectional view taken along line XX ′ in FIG. The slab-like migration gel 2 is formed with a transparent glass plate 4 on the front side and a transparent plate on the rear side sandwiched between the lath plates 6, and the gel sandwiched between the glass plates 4 and 6 is a gel. It is called a plate and is indicated by the symbol 2a. The side where the measurement system is provided (the front side in FIG. 1, the right side in FIG. 2) is the rear,
The opposite side is the front. An upper electrode tank 8 is provided on the upper end portion of the gel plate 2a in the front direction thereof, and the electrode tank 8 is formed in a box shape having an opening on the surface in contact with the gel plate 2a and an upper surface. A rubber seal 10 is interposed between the glass plate 4 and the end face of the opening on the gel plate side of the electrode tank 8 to prevent liquid leakage,
The electrode tank 8 is fixed while being pressed against the gel plate 2a. At the upper end of the glass plate 4, a notch 12 is provided inside the opening of the electrode tank 8 in the gel plate direction. The gel 2 is formed by cutting the glass plate 4 from the lower end of the glass plate 4 and the cutout 12
Is formed up to the bottom position, and a sample introduction slot 14 for injecting a DNA sample for each end base is formed at the upper end of the gel 2.

An electrolytic solution 16 is contained in the electrode tank 8, and the electrolytic solution 16 passes through the notch 12 in the glass plate 4 and then the glass plates 4, 6 are formed.
It is in contact with the gel 2 formed between them. The lower end of the gel plate 2a is immersed in the electrolytic solution 20 contained in the lower electrode tank 18. A migration voltage is applied between the upper electrolytic solution 16 and the lower electrolytic solution 20 by a high voltage power supply unit 22.

The sample to be injected into the sample introduction slot 14 at the upper end of the gel 2 is labeled by a known method with FITC which is a fluorescent substance, and the ends thereof are labeled with bases A (adenine), G (guanine), 4 types of DNA treated so that each of T (thymine) and C (cytosine) comes
It is a fragment sample. The FITC emits a fluorescence having a wavelength of 520 nm when excited by an argon laser beam having a wavelength of 488 nm.

An insulated heater 24 is installed on the inner wall surface of the upper electrode tank 8 on the side far from the gel plate 2a, and the temperature of the electrolytic solution 16 is controlled to 40 to 60 ° C. by a power supply 26.
It can be heated to.

The glass plate 4 of the gel plate 2a is provided with a front heat radiating plate 28 in contact therewith, and the glass plate 6 is provided with a rear heat radiating plate 30 in contact therewith. Heat dissipation plate 2
Reference numerals 8 and 30 are metal plates having good heat conductivity such as aluminum. The rear heat radiating plate 30 is arranged to a position above the lower end of the gel plate 2 a, and the gel plate portion exposed from the heat radiating plate 30 is irradiated with the excitation light beam 32 from the measuring unit to generate from the gel 2. Fluorescence is detected by the measuring unit.

The measuring section collects an excitation light beam 32 from an argon laser emitting a 488 nm laser beam into a condenser lens 34.
And an excitation system for irradiating the gel 2 at the lower end of the gel plate with the mirror 36, and a migration band 38 of the sample that has migrated to the place where the excitation light beam 32 hits, the migration band 38
And a detection system for collecting the fluorescence emitted from the fluorescent substance and detecting it with the photomultiplier tube 42 from the fluorescence condensing unit 40 including the 520 nm interference filter and the condensing lens through the optical fiber 41. I'm out. The condenser lens 34, the mirror 36, and the fluorescence condenser 40 are mechanical so that the irradiation position of the gel by the excitation light beam 32 scans the measurement line in the scanning direction 46 orthogonal to the migration direction 44 at regular intervals. It is fixed on the linear stage 48 which moves to the. Reference numeral 50 is an AC servo motor that scans the linear stage 48.

The CPU 52 controls the application of the migration voltage from the high-voltage power supply unit 22, and the AC servo driver 54 controls the application of the AC voltage.
The servo motor 50 is controlled to scan the linear stage 48, and the detection signal (fluorescence signal) of the photomultiplier tube 42 is detected.
Is taken in through the A / D converter 56. In the CPU 52, when the excitation light beam 32 irradiates the electrophoretic gel 2, information corresponding to the position of the irradiation portion can be used as scanning data, and thus the irradiation position by the excitation light beam 32 is scanned and obtained. The total fluorescence signal is read together with the location information of the gel in the scanning direction (corresponding to each scanning lane of A, G, C, T).

To explain the operation of this embodiment, the upper electrolytic solution 16 is heated by the heater 24 during the pre-electrophoresis performed in the usual gel electrophoresis apparatus. This pre-electrophoresis is performed to preheat the gel 2 in order to reduce the smile phenomenon, and is usually 10 to 3
It will be held for 0 minutes. It suffices that the temperature of the electrolytic solution 16 is higher than the temperature of the gel 2, and the electrolytic solution 16 can be used at the start of the migration of the DNA sample.
May be heated to 40 to 60 ° C. The heating of the electrolytic solution 16 by the heater 24 needs to be started before the sample is introduced into the sample introduction slot 14 at the upper end of the gel 2 and heated during the sample introduction, and the heating must be continued until the migration is started. There is. The heating may be continued after the migration is started, or the heating may be stopped after the migration is started. The heated electrolytic solution 16 serves as a heat source, and the gel temperature of the portion where the sample introduction slot 14 is present becomes uniform.

On the other hand, when the upper electrolytic solution 16 is not heated as in the conventional case, the gel temperature in the portion where the sample introduction slot 14 is present is not uniform even if pre-electrophoresis is performed, and the temperature nonuniformity occurs in this portion. Smiles occur as a cause,
The generated smiley is further magnified as the sample moves through the gel 2. In addition, electrophoretic distortion also occurs as a phenomenon caused by nonuniform gel temperature in the sample insertion slot 14. This migration distortion can also be eliminated by the present invention.

FIG. 3 shows an example in which a DNA sequencing sample prepared for each terminal base of A, G, C and T was electrophoresed by the apparatus of FIG. 1 and detected online. In FIG. 3, the migration bands shown on the upper side represent the migration bands detected earlier, that is, the migration bands having a smaller number of bases. When (A) is a conventional method in which the electrolytic solution on the side where the sample is injected is not heated, (B) shows that the electrolytic solution on the sample injection side is heated to 60 ° C. until the start of migration according to the present invention, and after the start of migration. Is an example of the case where the heating is stopped and the electrophoresis is performed. 4% long ranger for gel 2 (21g
(Containing urea), 2750 V was applied as a migration voltage, and x1.2 TBE was used as an electrolytic solution. From the results of FIG. 3, in the conventional method (A), the migration velocities among the A, G, C, and T migration lanes become non-uniform and smiles occur, but as shown in FIG. Thus, it is understood that smiley is eliminated by heating the sample introduction side electrolytic solution until the sample introduction.

In the embodiment shown in FIGS. 1 and 2, a heater 24 is installed on the inner wall surface of the electrode tank 12 on the sample introduction side to heat the electrolytic solution 16 in the electrode tank 12. The position of the heater 24 is not limited to the embodiment shown in FIGS. 1 and 2, but may be on the bottom side of the electrode tank 12. Instead of installing the heater 24, the electrolytic solution 16 in the electrode tank 12 may be heated by circulating heated water on the wall surface of the electrode tank 12.

[0017]

According to the present invention, the electrolyte solution in which the end of the electrophoretic gel on the side where the nucleic acid fragment sample such as DNA is injected is immersed is heated at least at the start of the migration, so that the smile phenomenon can be suppressed. This makes it possible to correctly determine the base sequence of a nucleic acid with such a simple structure.

[Brief description of drawings]

FIG. 1 is a schematic perspective view showing an embodiment.

FIG. 2 is a cross-sectional view taken along the line XX ′ in FIG.

3A and 3B are diagrams showing migration patterns detected in Examples, where FIG. 3A shows a conventional method in which the electrolyte solution is not heated, and FIG. 3B shows a sample introduction side electrolyte solution in the present invention. is there.

[Explanation of symbols]

 2 Slab-like migration gel 2a Gel plate 4,6 Glass plate 8 Sample introduction side electrode tank 14 Sample introduction slot 16 Sample introduction side electrode tank 22 High voltage power supply section 24 Heater

Claims (2)

[Claims] 1. A slab-like electrophoretic gel is dipped in both ends of the electrophoretic solution, and at one end of the electrophoretic gel immersed in one of the electrophoretic solutions, a nucleic acid fragment sample prepared for each type of terminal base is placed in each electrophoretic lane. In a gel electrophoresis method, in which the sample is injected separately, and a migration voltage is applied between both electrolytes to simultaneously perform electrophoresis in multiple migration lanes, the electrolyte solution in which the end of the migration gel on the side where the nucleic acid fragment sample is injected is immersed. A method of gel electrophoresis, wherein the gel is heated at least at the start of electrophoresis. 2. A slab-like electrophoretic gel having both ends immersed in respective electrolyte solutions, and one end of which has a sample injection part for injecting a nucleic acid fragment sample prepared for each type of terminal base, and both electrolyte solutions. In a gel electrophoresis apparatus equipped with an electrophoretic voltage application unit that applies an electrophoretic voltage between the electrophoretic cells and simultaneously electrophores in a plurality of electrophoretic lanes, an electrolytic solution in which the end of the electrophoretic gel on which the nucleic acid fragment sample is injected is immersed. A gel electrophoresis apparatus comprising a mechanism for heating a gel.