JPH10104201A - Gel electrophoresis - Google Patents

Abstract

(57) [Problem] To provide a method of loading a sample on an electrophoresis gel and performing electrophoresis by a novel method that does not adversely affect the sample. SOLUTION: In the present invention, as a sample, a DNA fragment (Sanger) which is labeled with, for example, a fluorescent substance, and which is treated so that any of bases A, G, T, and C comes to the terminal according to the sample by Sanger method. These reactants are added to the membrane 10 made of nylon, nitrocellulose, polyvinylidene dibrolide, or the like by using a reactant). The film 10 is placed on the heated heat block 11 and heated at 95 ° C. for 3 to 5 minutes to denature the sample on the film 10 (FIG. 2A).
After denaturing the sample on the membrane 10, the membrane 10 is placed in close contact with the upper end of the electrophoresis gel 2 (FIG. 2 (b)).
The electrophoresis is performed for a short time, and the sample (Sanger reaction product) is pushed into the upper end of the gel (FIG. 2 (c)). Thereafter, the membrane piece is removed, and normal electrophoresis is resumed.

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 in which, for example, a nucleic acid fragment sample prepared for each terminal base is injected into an electrophoresis gel and subjected to electrophoresis.

[0002]

2. Description of the Related Art Conventionally, gel electrophoresis has been used to analyze very small amounts of proteins and nucleic acids. In the gel electrophoresis method, a gel such as agar, polyacrylamide or starch having a molecular sieving effect is used as a support to stabilize a zone. When the base sequence of a nucleic acid is determined by this gel electrophoresis, for example, the terminal bases (A, G,
C, T) Another nucleic acid fragment sample is manually loaded into the upper portion of the gel by using a syringe while maintaining the solution state, for each terminal base. At this time, when processing 7 samples in one run, the terminal bases A, G, C,
Since there are 4 lanes of T, it is necessary to load 7 × 4 = 28 lanes. It takes 15 seconds per lane to perform these tasks, no matter how quick a skilled person goes, and a total of 28 × 15 = 420 seconds = 7 minutes, and it generally takes 10 to 15 minutes for ordinary people. It is the current situation.

[0003] When the loading is completed, electrophoresis is immediately performed to push the Sanger reactant (nucleic acid fragment sample) to the upper end of the gel so that the Sanger reactant does not take a three-dimensional structure. Here, "takes a three-dimensional structure" means that the DNA is in a non-denatured state.

[0004]

However, in the conventional method, since the loaded Sanger reactant is actually in a solution state at room temperature, a part of the Sanger reactant takes a three-dimensional structure before electrophoresis starts. Was. As a result, a phenomenon called "compression (condensation)" occurs, and there is a problem that a correct DNA base sequence result cannot be obtained at that location. Accordingly, an object of the present invention is to provide a method of loading a sample on an electrophoresis gel and performing electrophoresis by a novel method that does not adversely affect the sample.

[0005]

According to the present invention, there is provided a gel wherein a sample is added to a membrane, and after drying, the membrane is loaded on an electrophoresis gel to perform electrophoresis. Provide an electrophoresis method. Here, the sample corresponds to, for example, a nucleic acid fragment sample processed by the Sanger method or the Maxam Gilbird method, as well as a sample of a nucleic acid, a protein, or the like, which has been subjected to a normal pretreatment operation. The membrane may be anything that adsorbs the sample, and for example, nylon, nitrocellulose, polyvinylidene dibrolide and the like can be used. The membrane is placed vertically, so that it adheres to the top of the electrophoresis gel.
It is preferable that both sides are made smaller than the gel cross section. The size of the film is, for example, 0.25 mm long and 15 c wide.
m and a thickness of 0.1 mm can be used. The sample can be added to the membrane by a known technique such as a microsyringe or a pipette. The amount of the sample to be added varies depending on the type of the sample. For example, in the case of a Sanger reaction product, 2.0 to 3.5 μl is added.

[0006] The membrane to which the sample is added is immediately dried.
Drying is performed when the sample is a Sanger reactant and the reactant (D
NA) in a denatured state, for example, at 95 ° C.
Heat for ~ 5 minutes. Then, the Sanger reactant is adsorbed on the membrane in a denatured state, and the Sanger reactant does not take a three-dimensional structure during electrophoresis.

The term "immediately" means that when the sample is a Sanger reactant, before the sample takes a three-dimensional structure, the temperature condition for drying is not limited to the above, and is, for example, 80%.
What is necessary is just to heat at -95 degreeC for 3-10 minutes. As the heating unit, for example, a known unit such as a heat block or a surface heater can be used. In the case of a sample other than the Sanger reactant, the sample is heated at a temperature suitable for the denaturing conditions of the sample.

As the electrophoresis gel, for example, agar gel, starch gel, polyacrylamide gel, SDS-
Polyacrylamide gel, agarose gel and the like can be used, but are not limited thereto. Loading the sample on the membrane into the electrophoresis gel can be performed by closely contacting the membrane with the upper end of the electrophoresis gel and performing electrophoresis for a short time.

[0009] "Loading" is a term commonly used in electrophoresis in the sense of placing and adding.

[0010]

Embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a schematic view of a gel electrophoresis apparatus for carrying out the method of the present invention, and 2 is an electrophoresis gel. The electrophoresis gel 2 includes, for example, a polyacrylamide gel sandwiched between a pair of glass plates.
Are immersed in electrode tanks 4 and 6, and the electrode tanks 4 and 6 contain an electrolytic solution, for example, a Tris-HCl buffer solution. A migration voltage is applied between the electrode tanks 4 and 6 by a migration power supply 8.

At one end of the electrophoresis gel 2, a membrane 10 to which a Sanger reactant is added and dried by a method described later is adhered. When the electrophoresis power supply 8 is applied, the sample becomes an electrophoresis band, migrates in the electrophoresis gel 2 with time in the electrophoresis direction 14, is separated, and reaches the measurement section.

In the measuring section, an excitation system for irradiating an excitation light from an argon laser 18 for emitting a laser light of a predetermined wavelength by a condenser lens 20 and a mirror 21 and a migration band 16 at a place where the excitation light beam is applied are provided. Migrating band 1
A detection system is provided for collecting fluorescence emitted from the fluorescent substance No. 6 with the objective lens 22 and detecting the fluorescence with the photomultiplier tube 28 from the interference filter 24 and the condenser lens 26 via the optical fiber bundle 27. Further, an excitation / detection system including the condenser lens 20, the mirror 21, the objective lens 22, the interference filter 24, the condenser lens 26, and the optical fiber bundle 27 is mounted thereon, and the excitation light beam irradiation position is perpendicular to the migration direction 14 ( Scan direction 2
A scanning stage 30 that mechanically moves so as to scan the measurement line 9) at regular intervals is provided.

A detection signal from the photomultiplier tube 28 is taken into a microcomputer 31 via an amplifier and an A / D converter 32. When the excitation light beam irradiates the measurement unit on the electrophoresis gel 2 to the microcomputer 31,
A signal corresponding to the position of the irradiation unit is captured as scanning data. In this way, the microcomputer 31 captures all the fluorescence signals obtained by scanning in the scanning direction together with the location information. With the above configuration, electrophoresis is performed as follows. The sample is labeled with a fluorescent substance, for example, and bases A, G, and
Use a DNA fragment (Sanger reaction product) treated so that either T or C comes. These samples were nylon,
It is added to the membrane 10 made of nitrocellulose, polyvinylidene dibrolide or the like. The membrane 10 is cut vertically and horizontally smaller than the gel cross section so as to be in close contact with the upper end of the gel. This film 10 is placed on a heated heat block 11 (this state is shown in FIG. 2A).

The heat block 11 is used at 95 ° C. for 3 hours.
After denaturing the sample on the membrane 10 by heating for ~ 5 minutes, the membrane 1
0 is brought into close contact with the upper end of the electrophoresis gel 2 (this state is shown in FIG. 2 (b)). When the membrane 10 is brought into close contact with the upper end of the gel, the electrode tank 4 of the gel electrophoresis apparatus in FIG. 1 is removed.

The electrode tank 4 is attached, and electrophoresis is performed for a short time.
For example, for 3 to 10 minutes, the sample (Sanger reaction product) is pushed into the upper end of the gel (this state is shown in FIG. 2 (c)). Thereafter, the membrane piece is removed, and normal electrophoresis is resumed. The sample becomes a migration band 16 and migrates in the migration gel 2 with time in the migration direction 14 and is separated, and reaches the measurement unit. An excitation light beam is applied by the measurement unit, and fluorescence emitted from the fluorescent substance in the migration band is collected by the objective lens 22, and is transmitted from the interference filter 24 and the condenser lens 26 to the optical fiber bundle 27.
, And is detected by the photomultiplier tube 28.

[0016]

According to the present invention, for example, since the Sanger reactant is electrophoresed while maintaining its denatured state, it does not take a three-dimensional structure and no compression occurs.
As a result, the correct DNA base sequence is elucidated.

[Brief description of the drawings]

FIG. 1 is a schematic diagram of a gel electrophoresis apparatus of the present invention.

FIG. 2 is an explanatory diagram when a sample is put into a gel electrophoresis apparatus.

[Explanation of symbols]

2: Electrophoresis gel 4, 6: Electrode tank 10: Membrane 18: Argon laser 28: Photomultiplier tube

Claims (1)

[Claims] 1. A gel electrophoresis method comprising: adding a sample onto a membrane; drying the membrane; and loading the membrane onto an electrophoresis gel to perform electrophoresis.