JPH06167476A - Nucleotide sequencer - Google Patents

Abstract

(57) [Summary] [Purpose] To prevent a nucleic acid fragment sample from being deteriorated due to the influence of diffusion. [Configuration] The migration voltage or the migration power output from the migration power supply 8 is controlled by the microcomputer 31 so as to change according to a program set in the microcomputer 31. As an example, the electrophoretic power is constant at 20 W from the start of migration for 2 hours, then is changed so as to increase linearly to 29 W over 6 hours, and then is maintained constant at 29 W.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a nucleotide sequence determination device for migrating and separating a nucleic acid fragment sample by a gel electrophoresis device. There are an online type and an offline type in the nucleotide sequencer, and the electrophoresis gel to be used includes a slab gel and a capillary gel. The present invention is applicable to any type of nucleotide sequence determination device.

[0002]

2. Description of the Related Art In a nucleotide sequencer, a migration voltage is applied between both ends of an electrophoretic gel in order to separate nucleic acid fragments based on their size. The migration voltage or power is constant during the migration. Is set to.

[0003]

The nucleic acid fragment sample is separated based on its size. However, in the latter half of the electrophoresis, the nucleic acid fragment sample is affected by diffusion. There is a problem that good separation cannot be obtained. The present inventor has found that the influence of the diffusion of the nucleic acid fragment sample can be suppressed by causing the nucleic acid fragment sample to migrate while increasing the migration voltage or migration power with time. Therefore, the present invention has an object to prevent deterioration of separation of a nucleic acid fragment sample under the influence of diffusion.

[0004]

SUMMARY OF THE INVENTION In the present invention, the means for applying the electrophoretic voltage is programmable such that its voltage or power increases with time, at least in the latter half of electrophoretic migration.

[0005]

EXAMPLE FIG. 1 shows an example of a base sequence determination apparatus to which the present invention is applied. 2 is a slab-like migration gel, and polyacrylamide gel is used. Both ends of the electrophoretic gel 2 are immersed in the electrode baths 4 and 6, and the electrolytic baths are stored in the electrode baths 4 and 6. A migration voltage is applied between the electrode tanks 4 and 6 by a migration power supply 8.

A sample input slot 10 for injecting a sample is provided at one end of the electrophoretic gel 2, and a sample for each end base is input to each predetermined position of the sample input slot 10. The sample is a fluorescent substance F
Four kinds of DNA fragments labeled with ITC by a known method and treated with the Sanger method so that each of the bases A (adenine), G (guanine), T (thymine) and C (cytosine) come is there. FITC is 488 nm
It is excited by an argon laser with a wavelength of 520 nm and emits fluorescence with a wavelength of 520 nm.

When the electrophoretic power supply 8 is applied, the sample becomes the electrophoretic band 16 and migrates in the electrophoretic gel 2 in the electrophoretic direction 14 over time to be separated, and reaches the measuring section.
In the measurement section, an excitation system that irradiates the excitation light from the argon laser 18 that emits a laser beam of 488 nm with the condenser lens 20 and the mirror 21 and the migration band 16 if the excitation light beam hits the migration band 16. Fluorescence emitted from the 16 fluorescent substances is collected by the objective lens 22 to obtain 520
A nm interference filter 24, a condenser lens 26, an optical fiber bundle 27, and a detection system for detecting with a photomultiplier tube 28 are provided. In the 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, the excitation light beam irradiation position is orthogonal to the migration direction 14 (scanning direction). 29)
The scanning stage 30 that is mechanically moved so as to scan the measurement line at a constant time is provided.

Detection signal of the photomultiplier tube 28 (fluorescence signal)
Is taken into a signal processing microcomputer 31 which is a data processing device through an amplifier and an A / D converter 32. When the excitation light beam irradiates the measurement section on the electrophoretic gel 2, the microcomputer 31 also captures a signal corresponding to the position of the irradiation section as scanning data. In this way, all the fluorescence signals obtained by scanning in the scanning direction are taken into the microcomputer 31 together with the location information. The migration voltage or the migration power output from the migration power supply 8 is controlled by the microcomputer 31 so as to change according to a program set in the microcomputer 31.

FIG. 2 shows an example of migration power. (A) is a schematic representation of an electrophoretic voltage application section, and (B) shows an example of electrophoretic power. In this example, the electrophoretic power is constant at 20 W for 2 hours from the start of the electrophoretic migration, and is then changed so as to increase linearly to 29 W over 6 hours, and then is maintained constant at 29 W.

FIG. 3 shows a migration pattern when the migration power is changed so as to increase according to the present invention, and FIG. 4 shows a migration pattern when the migration power and the migration voltage are kept constant as in the conventional case. Shown in. In FIG. 3, the migration power is changed as shown in FIG. 2 (B), and in FIG. 3, the migration power is 20 W.
In all cases, measurement was performed for up to 12 hours. Figure 3,
The numerical value on the horizontal axis of 4 represents the number of scans of the scanning stage 30 of the apparatus of FIG. 1, and the numerical value corresponds to time. Figures 3 and 4 are 4 types (A, G, C, T) for each end base.
Is included, and when actually output to a recorder or the like, it is output by color-coding for each terminal base. In this example, only the broadening of the peak is important, so no distinction is made by the type of terminal base.

From FIGS. 3 and 4, in the electrophoretic pattern when the electrophoretic power and electrophoretic voltage are kept constant, the peaks are broad in the latter half of the electrophoretic migration and the separation is poor, but the electrophoretic power increases. In the electrophoretic pattern with such changes, the extent to which the peak becomes broad even in the latter half of the electrophoretic migration is small, indicating that the resolution is excellent.

Although the example shows an on-line type nucleotide sequencer using a slab-like gel, the present invention is not limited to this, and an off-line type using a slab-like gel may be used.
Capillary gel may be used instead of the slab-like gel. The pattern of the program that changes the migration voltage or migration power is not limited to that shown in FIG.

[0013]

According to the present invention, the migration voltage or the migration power is changed so as to increase with time at least in the latter half of the migration, so that the spread of the migration band, which becomes conspicuous with the elapse of the migration time, can be suppressed. The separation ability between separated nucleic acid fragment samples can be enhanced. This makes it possible to increase the readable chain length.

[Brief description of drawings]

FIG. 1 is a schematic perspective view showing a base sequence determination device of one embodiment.

FIG. 2 is a diagram showing an example of migration power, (A)
Is a schematic representation of the electrophoretic voltage applying section,
(B) shows an example of migration power.

FIG. 3 is a diagram showing a migration pattern when the migration power is changed so as to increase according to the present invention.

FIG. 4 is a diagram showing a migration pattern when electrophoresis is performed while keeping the migration power and the migration voltage constant.

[Explanation of symbols]

 2 Electrophoresis gel 4,6 Electrode tank 8 Migration power supply 31 Microcomputer

Claims (1)

[Claims] 1. A nucleic acid fragment sample is placed on one end of an electrophoretic gel, and an electrophoretic voltage is applied between one end and the other end of the electrophoretic gel to cause the nucleic acid fragment sample to migrate and separate in the electrophoretic gel. In the base sequence determination device, the means for applying the electrophoretic voltage is programmable so that the voltage or the power increases with time at least in the latter half of the electrophoretic migration.