JPS613044A - Apparatus for automatically determining base sequence of nucleic acid - Google Patents

Abstract

PURPOSE:To determine the sequence of a nucleic acid with high accuracy by eliminating the distortion of an electrophoretic pattern by uniformizing the temp. of an electrophoretic gel, by preparing the support plate of the electrophoretic gel from electric insulating ceramics with high heat conductivity. CONSTITUTION:An electrophoretic separation gel 2 is held between two support plates 3 and both ends thereof are immersed in an electrolyte tank 1 and a high voltage DC power source 5 is provided to form an electric field in the gel 2. As the material of the supports 3 interposing the gel 2 therebetween, electric insualating ceramics with high heat conductivity, for example, SiC ceramics containing Be, B, Al or a compound thereof as a sintering aid is used. At this time, heat is generated during electrophoresis because of Joule heat but, because the support plate 3 is made highly heat-conductive, the temp. gradient in the gel is reduced. As a result, the distortion of the electrophoretic pattern of the specimen is not generated and the determination of the base sequence of the nucleic acid can be performed at a high speed with high accuracy.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a nucleic acid base sequencing device, and particularly to a gel electrophoresis device suitable for increasing speed and accuracy.

[Background of the invention]

Conventionally, base sequencing of nucleic acids has been determined using, for example, the Maxam-Gilbert method (Proteins, Nucleic Acids, Enzymes, Vol.

I, pp. 182-196 (1978)). In this method, radioactive isotope-labeled nucleic acids are chemically fragmented, the fragments of different lengths are aligned in a gel according to molecular weight using electrophoresis, and the gel is peeled off from a glass plate. By taking a gram, the electrophoretic band containing radioactive fragments is detected, and the base sequence of the nucleic acid is determined. In the conventional electrophoresis method, the gel is supported by a glass plate with low thermal conductivity, so the heat generated during electrophoresis is not sufficiently dissipated, resulting in uneven temperature within the gel, and the high temperature center part , the mobility of nucleic acid fragments is large.

Conversely, the mobility of nucleic acid fragments decreases in the peripheral region where the temperature is low. As a result, as shown in Figure 1, for example, there is a difference in the mobility of nucleic acid fragments between the four lanes with different terminal bases, resulting in distortion of the migration pattern and inaccurate reading of positions between lanes. It had the disadvantage of becoming In particular, when automating an apparatus, the voltage applied to the electrophoresis gel is increased to increase the mobility of nucleic acid fragments in order to speed up the processing speed, and the above-mentioned drawbacks become even more pronounced.6 [Object of the Invention] The present invention The purpose of this is to provide a high-speed nucleic acid base sequencing device that eliminates distortion of electrophoretic patterns caused by temperature non-uniformity in gels and has high reading accuracy.

[Summary of the invention]

The present invention provides a support plate for electrophoresis gel that has high thermal conductivity.
The gel is made of ceramics or metals with excellent electrophoresis properties and an insulating film coated on them, thereby minimizing the temperature non-uniformity within the gel caused by the heat generated during electrophoresis. do.

[Embodiments of the invention]

Hereinafter, one embodiment of the present invention will be described with reference to FIG. 2. This apparatus consists of a electrophoretic separation gel 2 sandwiched between two ceramic plates 3, an electrode liquid tank 1 in which both ends of the gel are immersed, and a high voltage DC power source S. Consists of.

First, as the above-mentioned ceramic plate, about 3% by weight of BeO is used.
The characteristics of the ceramic material when using SiC containing SiC will be explained in detail.

The composition of the ceramic is as follows.

100 parts of SiC with a purity of 98% (contains 0.1% Afl, O, and impurities) with the above composition was heated to a vacuum degree of 10-'- by a vacuum hot press machine.
Under reduced pressure of 10-sTorr, pressurizing force 200 kg/, 1
1. Sinter at a temperature of 2000°C to obtain a ceramic material.

A summary of the characteristics of the above ceramic material is as follows. That is, the thermal conductivity is 0,7 ca Q/aiss
e”c, which is larger than metal aluminum or BeO ceramics, and more than three orders of magnitude larger than glass plates.

The electrical resistance is 2 x 10''Ω■, which is enough to be considered an insulator.

Radioactive isotopes (e.g. 3-p) or fluorophores (
For example, when a nucleic acid sample identified with ethidium bromide (e.g., ethidium bromide) is placed on the negative electrode side of gel 2, both ends of the gel are brought into contact with electrode solution 1, and electrophoresis is performed at a voltage of about 50 V/am per gel length, components with the same molecular weight are detected. each migrates from the negative electrode toward the positive electrode with a mobility approximately inversely proportional to one molecular weight, forming a migration band 4.

Due to the Joule heat generated during the electrophoresis, the electrophoresis pattern in conventional electrophoresis devices using glass plates is distorted.
It was sometimes difficult to read. In particular, this becomes an obstacle to achieving high accuracy when automating reading and using equipment. According to this example, by using ceramics with the above characteristics in place of the glass plate, the generated heat is quickly and uniformly diffused, which reduces the temperature gradient in the gel and reduces the distortion of the migration pattern. There is.

Next, another embodiment of the present invention will be described with reference to FIG. The configuration of this device is such that the ceramic plate 3 shown in FIG. 2 has been replaced with a metal plate 6 coated with an insulating film. According to this embodiment, by coating metal with a thin film of Teflon, for example, the same effect as that described in the embodiment of FIG. 2 can be expected.

(-5i! bright effect) According to the present invention, by supporting the gel with a substrate with high thermal conductivity, the temperature gradient in the gel is reduced, so the distortion of the electrophoretic pattern is reduced, and high accuracy is achieved. Furthermore, according to the present invention, the distortion of the electrophoretic pattern is reduced, so electrophoresis can be performed at a higher voltage than conventionally, and a high-speed base sequencing apparatus can be realized.

[Brief explanation of the drawing]

FIG. 1 is a block diagram of a conventional electrophoresis method, and FIG. ff13 is a configuration diagram showing an embodiment of the present invention. 1... Electrode liquid tank, 2... Gel, 3... Ceramic plate,
4...Migration zone, 5...High voltage DC power supply, 6...Metal plate,
7...Figure 1 Figure 2 Kasumi Figure 3

Claims (1)

[Claims] 1. In an apparatus for separating the molecular weight of nucleic acid fragments by gel electrophoresis and determining the nucleic acid base sequence, an electrophoretic gel is supported by a support plate made of ceramics having high thermal conductivity and large electric insulating power. An automatic nucleic acid base sequence determination device characterized by: 2. The above ceramic material contains several percent of Be as a sintering aid.
The automatic nucleic acid base sequence determination device according to claim 1, which is a SiC ceramic material containing one or more types of Be-containing compound, B or B-containing compound, Al or Al-containing compound. 3. The automatic nucleic acid base sequence determination device according to claim 1, wherein the support plate is a metal coated with an insulating film.