JPH01195366A - Method and device for determining dna base sequence - Google Patents

Abstract

PURPOSE:To eliminate the need for an intricate optical system by coupling a metal ion to DNA fragments cut to plural fragments, then effecting electrophoresis and detecting the fragments by an ion selective electrode, thereby determining the DNA base sequence. CONSTITUTION:The DNA fragments are injected as a sample into slots 8 for migration. The terminal primers of the respective fragments are previously labeled by the metal ion. Four kinds; adenine, guanine, thymine and cytosine, of the terminal bases of the sample constitute one set and 4 sets of the slots 8 are used for one set of the sample. The DNA fragments are electrically migrated and separated successively in a direction X when a migration pressure is impressed between electrode cells 4 and 6. The ion selective electrode 9 consisting of an electrode holding plate 1 made of glass and an electrode cylinder 3 is installed in a sepn. direction. The tip of the electrode in the electrode cylinder 3 comes into contact with a gel 2. The signal from the electrode enters a signal processing part 5 and the base sequence is determined in an analysis part 7.

Description

[Detailed description of the invention] (b) Industrial application field The present invention relates to a method for determining the base sequence of DN humans.

(b) Prior art Conventionally, the base sequence of a nucleic acid was determined by electrophoresing a nucleic acid fragment such as DNA labeled with a labeling dye and optically detecting the labeling dye (Japanese Unexamined Patent Application Publication No. 1989-1999-1).
No. 242368, JP-A-60-220860).

Conventional methods for determining DNA base sequences using dye labeling methods employ fluorescent dye methods that use fluorescent substances as labeling dyes (for example, "Nature J, Vol. 321, No. 12, 674 to page 679 (1
986] 1 Proceedings of the 24th Annual Meeting of the Biophysical Society of Japan 3F
i 1130 (1986)).

The final step in determining the base sequence is separation and reading of the IIi DNA fragment by electrophoresis using Polyac+7 vamide gel/I/vf-. separated n7tj
How to read DNA fragment patterns.

Two methods are considered: an offline method in which the developed DNA fragment pattern is read in a stationary state, and an online method in which the DNA fragments are read while they are being electrophoresed. All of the methods described in the above cited documents are online methods! +
, While the DNA fragments are being migrated, signals from the DNA fragments are read by a t-combeater.

In both offline and online methods, in the fluorescent dye method, Rayleigh scattering of excitation light by polyacrylamide gel and Raman scattering (Stokes a) by water interfere with the slight fluorescent light and create a background. Increase.

Therefore.

The constant fluorescence method, in which excitation light is continuously irradiated, is said to have a sensitivity that is 2 orders of magnitude lower than the radioisotope method, and an error rate of more than 1 order of magnitude (see the cited reference in FNatureJ above). .

Therefore, in the fluorescent dye method, a time-resolved fluorescence method has been proposed in place of the steady fluorescence method (see Japanese Patent Laid-Open No. 61-2077). In the time-resolved fluorescence method, the excitation light is irradiated in a P/I/N pattern, and the background is reduced by detecting the fluorescence only after the excitation light is extinguished (for example, [Fluorescence measurement - biological “Applications to Science” (Gakkai Publishing Center, 19
(published in 1983), see page 99-%-160).

(c) Problems to be Solved by the Invention In the time-resolved fluorescence method, since the light emission time of the light emitting diode is very short, it is necessary to switch the fall time of the excitation light to less than a nanosecond. Such high-speed switching cannot be achieved with a mechanical shutter and requires the use of a mode lock laser. Therefore, the device becomes expensive. In addition, high-speed signal processing is difficult, and the two-hour resolution fluorescence method is currently only a proposal.

Furthermore, the polyacrylamide gel supports used in electrophoresis devices are made of glass or polyethylene, and some of these supports emit fluorescence, albeit weakly. Therefore, even if a time-resolved fluorescence method is employed, there is no guarantee that DNA fragments can be detected with sufficiently high sensitivity using only 7n.

An object of the present invention is to provide a method for determining a base sequence without using a labeling dye.

2) Means for Solving the Problems The present invention provides a means for solving the problems by dividing a DNA fragment whose base sequence is to be completely determined into a plurality of DNA fragments.
The method is characterized in that after cutting the DNA fragments into DNA fragments and binding metal ions to the cut DNA fragments, electrophoresis is performed and detection is performed using an ion-selective electrode.

Examples of metal ions that bind to the DNA fragment used here include Na and K.

The type of ion-selective electrode is selected depending on the type of metal ion to be bound.

Furthermore, the apparatus for carrying out method 1 of the present invention includes an injection slot for injecting cut DNA fragments to which metal ions are bound;
It consists of a migration plate for electrophoresing the injected cut DNA fragments, a nine-ion selective electrode section disposed in the migration direction of the migration plate, and an analysis section for determining the base sequence based on the signal from the ion-selective electrode section.

(e) Effect The present invention uses nucleic acid fragments labeled with metal ions.

Since detection is performed using an ion-selective electrode, a complicated optical system is not required.

(f) Example FIG. 1 shows an apparatus for carrying out the method of the invention.

2 is a slab gel for electrophoresis made of polyacrylamide, which is held between glass plates.

One end of the slurp gear A/2 is immersed in the electrolyte in the electrode tank 4, and the other end is immersed in the electrolyte in the electrode 41!16. At one end of the slab gel 2, a plurality of electrophoresis slots 8 are formed into which a sample is injected.

A migration voltage is applied between the electrolytic solution in the electrode tank 4 and the electrolytic solution in the electrode tank 6 by the migration power source 10.

The Sanger method (F
, Sanger et al; Proc, Natl, Aca
d, Sci, USA.

Vol, 74. f) 5467 to p5463 (197
DNA on the terminal base side obtained by
The fragment is injected as a sample. Each DNA fragment is pre-primed with a metal ion (at least 2 m, e.g. Na
, K). One set of samples consists of four types of terminal bases: A (adenine), G (guanine), T (teminine), and C (cytosine), and therefore, the four electrophoresis slots 8 are used for one set of samples.

When a migration voltage is applied between the electrode tank 4 and the electrode tank 6 by the migration power supply 10, the DN injected into the migration slot 8
The A fragment sample is separated by electrophoresis in the X direction (vertical direction in the figure).

An ion selective electrode section 9 is installed in the separation direction.

The ion-selective electrode section 9 consists of an electrode holding plate 1 and an electrode tube 3 made of glass. The electrode tube 3 is an indicator electrode (Na selective electrode,
A selective electrode) and a reference electrode are housed in pairs, and the number of electrode tubes 3 corresponds to the number of migration slots.

The tip of the electrode inside the electrode tube 3 contacts the gel 2.

The signal from the electrode is taken into the signal processing section 5 and sent to the analysis section 7 where the base sequence 2 is determined.

In addition, when using an ion-selective electrode that uses a membrane that allows detection without exposing all the effects of exposure to air, it is also possible to detect metal ions without binding them.

(g) Effects According to the present invention, a complicated optical system is not required because fluorescence detection is not fully utilized. Furthermore, it is effective for online detection since it is not necessary to consider the irradiation position of the excitation light.

[Brief explanation of the drawing]

FIG. 1 shows an apparatus for carrying out the method of the present invention. 2... Slab gel for electrophoresis 3... Electrode tube 7...
Analysis Department Patent Applicant: Shimazu Corporation.

Claims (1)

[Claims] 1. A DNA fragment whose base sequence is to be determined is cut into a plurality of DNA fragments, a metal ion is bound to the cut DNA fragments, electrophoresed, and detected with an ion-selective electrode. Characteristic DNA base sequencing method. 2. A labeling method in a DNA base sequencing method characterized by labeling a cut DNA fragment with a metal ion. 3. A detection method in a DNA base sequencing method, which comprises detecting electrophoresed cut DNA fragments using an ion-selective electrode. 4. An injection slot for injecting cut DNA fragments bound with metal ions, a migration plate for migrating the injected cut DNA fragments, an ion-selective electrode section disposed in the migration direction of the migration plate, and an ion selection device. A DNA base sequence determination device comprising an analysis section that determines the base sequence based on signals from a sex electrode section.