JPS6291848A - Supporting medium for electrophoresis for determining base sequence of nucleic acid and supporting substrate thereof - Google Patents

Abstract

PURPOSE:To improve reliability of the result of reading and analysis of an autoradiograph by providing partition walls for partitioning gel layers to plural lanes for electrophoresis. CONSTITUTION:A supporting medium 1 is interposed with the partition walls 4 between a pair of transparent plates 2 facing each other and is partitioned to the plural lanes 6 for electrophoresis by the walls 4. The gel layers 3 are interposed in the respective lanes 6. A pair of the transparent plates 2 are united by adhesion, etc. via the walls 4 and spacers 5. Specimens migrate along the respective lanes 6 if the gel layers 3 are partitioned to the plural lanes 6 in the above-mentioned manner; therefore, migration rows 10 do not generate biasing and meandering. The reliability of the reading and analyzing of the autoradiograph is thus improved.

Description

DETAILED DESCRIPTION OF THE INVENTION [Technical Field] The present invention relates to the method of producing nucleic acids using autoradiography.
i! The present invention relates to a support medium used for base sequencing and electrophoretic development of a mixture of IIA group-specific fragments of nucleic acids, and a support substrate used for the support medium.

<Prior art and its problems> In molecular biology, which has developed rapidly in recent years, it is essential to clarify the genetic information possessed by living organisms in order to elucidate the mechanisms of n71 and replication in living organisms. It has become a thing. In particular, DNA (which carries specific genetic information)
It has become essential to determine the base sequence of nucleic acids such as DNA fragments or DNA fragments.

Maxam-Gilber uses autoradiography as a typical method for determining the base sequence of nucleic acids such as DNA and RNA.
t) method and Sanger-C
oulson) ilJ, is known. The former (7)?
The Kissam-Kilpert method first involves adding a radioactive isotope, such as F, to the end of the chain molecule of the DNA or DNA fragment whose sequence is to be determined. (By combining 0 to 1', the target is made into a radioactive labeling substance, and then the bonds between each constituent unit of the chain molecule are specifically cleaved by the 1'!! group using chemical means.
Next, the mixture of base-specific DNA cleavage products tlled by this operation was subjected to gel electrophoresis to divide the mixture into fractions #j3 (regarding the fraction 1llll, which was formed by forcefully opening a large number of Glue J cleavage products into layers). 11 the unfolded pattern (however, it cannot be seen visually).

For example, the S development pattern for this amount is uf
Visualize to obtain an autoradiograph, and from the obtained autoradiograph and each tl group-specific cleavage stage F, bases in a certain positional relationship from the end of the chain molecule to which the radioactive isotope is bound are determined. are sequentially determined, thereby making it possible to determine the base sequence of the entire target object.

In addition, the latter Sanger-Cournon method is complementary to chain molecules of DNA or DNA fragments, and is radioactively labeled! A base-specific DNA compound was synthesized using a chemical L-stage, and a mixture of base-specific DNA compounds was used to extract 114 from the autoradiograph in the same manner as J:. ), I; is a method for determining sequences.

The author of this paper intends to carry out the I'i1 group sequencing of the nucleic acids described in 2.1 as a club activity and with high precision. A patent application has already been filed for method (1) of using a radiation image conversion method using a stimulable phosphor sheet instead of the conventional radiographic method using a photosensitive material (Japanese Patent Laid-Open No. 59-83057, Patent Application (Sho 58-201231). Here, the stimulable phosphor sheet is made of stimulable phosphor, and after radiation energy is absorbed by the stimulable phosphor of the phosphor sheet, electromagnetic waves (excitation light) in the visible to infrared region are emitted. By exciting it with , radiation energy can be emitted as fluorescence. According to this method υ, the exposure time can be significantly shortened, and chemical fog, which has been a problem in the past, does not occur.

In addition, autoradiographs of radiolabeled substances are
11 It is irradiated on a phosphor sheet as radiation energy and then read out in time series as photostimulated light, so in addition to images, it can be displayed and recorded in arbitrary forms such as symbols and numbers.

Previously, Nagisa, who is trying to determine the base sequence of nucleic acids, has used base-specific cleavage degradation products of radioactively labeled nucleic acids or 18-group-specific composites (hereinafter referred to as nucleic acid bases) for visualized autoradiographs. 11!, J, (referred to as a specific fragment), the base arrangement of the nucleic acid is determined by visually judging the opening position of JJeC and comparing it to the phase lJ: between the separation and development columns. Therefore,
Analysis of the obtained autoradiographs is usually done through human vision, which requires a great deal of time and effort.

In addition, because it depends on human 11), there are limits to the accuracy of the information 11), such as the fact that the base sequence of a nucleic acid determined by analyzing an autoradiograph differs depending on the analyst.

Therefore, the present applicant obtains the above autoradiograph as a digital signal and then performs signal processing suitable for this digital signal, thereby obtaining the Ii! Patent applications have already been filed for a method for dynamically determining the base sequence (Japanese Patent Laid-Open Nos. 59-126527, 1982-126278,
Japanese Patent Application No. 59-89615, Japanese Patent Application No. 59-140908
When using conventional radiographic film, digital signals corresponding to radiographs are -j-
It is obtained by converting the J-autoradiograph into a visible image on the film 1;, then optically scanning the film, and photoelectrically reading the reflected light or transmitted light.

When a stimulable phosphor sheet is used, the autoradiograph can be obtained by scanning the sheet on which the autoradiograph has been stored with excitation light and photoelectrically reading the stimulated light produced by this scanning.

1; In the base sequence determination of nucleic acids accompanied by photoelectric reading of an autoradiograph by optical scanning as described above.

Structure of a support medium used in electrophoresis for separating and developing a mixture of specific fragments of nucleic acids.
Due to the above problem, the following problems exist.

As shown in FIG. 8, the conventional support medium has a structure in which a gel layer 3 is inserted between two glass plates 2.2 connected by a site spacer 5, and a plurality of slots 7 are provided at the sides. In this case, the first stage separating each slot was not provided at all, so a mixture of base-specific fragments of radioactively labeled nucleic acids (hereinafter referred to as the sample) was injected into slot 7, and electrophoresis was performed. When this was carried out, the electrophoresis column 1O often became oblique or meandered due to various reasons, as shown in an exaggerated manner in FIG.

Because of such oblique and meandering of the electrophoresis column 10, in a reading device for photoelectrically reading an autoradiograph recorded on a radiation film or a stimulable phosphor sheet by optically scanning the electrophoresis column 10, the electrophoresis column 10 is 10 must be scanned over a wide range, which makes the device complicated, large and expensive, and also increases the reading time of the autoradiograph.The most troublesome thing is that The drawback was a lack of accuracy.

In addition, because the gel membrane used as a support medium for developing the specimen was handmade by each researcher, not only did it take a lot of time and complicated work to prepare the gel membrane, but the gel membrane that was created was It was not homogeneous, and as a result, the autoradiograph reading and analysis results of the sample lacked reliability.

[Objective 1 of the Invention] The first object of the invention is that when a specimen is separated and developed in a gel membrane by ``electrophoresis'', the electrophoresis column is obliquely arranged.

It is an object of the present invention to provide a support medium for electrophoresis that prevents meandering and allows autoradiograph reading with a simple, compact, and inexpensive reading device.

In addition, the second object of the present invention is that when the support medium for electrophoresis is assembled, there is no displacement of the partition walls, and the structure is uniform.
can form a uniform gel layer,
Another object of the present invention is to provide a support plate for an electrophoresis support medium that is easy to manufacture and suitable for large clay production.

<Detailed explanation of the invention> These objects are achieved by the following invention.

That is, the present invention provides an electrophoretic support medium for separating and developing a specimen by electrophoresis, which comprises: a pair of transparent plates facing each other with a fixed distance between them; a gel layer interposed between the transparent plates; a partition wall for partitioning the gel layer into a plurality of electrophoresis lanes and separating the transparent plate by the predetermined distance;

Injection of the specimen provided at the end of each lane]-1
1. A support medium for electrophoresis for determining the base sequence of nucleic acids, characterized by comprising:

The present invention also provides a support substrate used as a support medium for electrophoresis of a specimen, characterized in that a partition wall is formed on the inner surface to limit the electrophoresis lane and fill the gel layer. It is.

The partition wall may preferably have a spacer interposed between the transparent plates, or may be formed integrally with at least one of the transparent plates.

It is preferable that the partition walls are formed in + rows at equal intervals.

Further, it is preferable that the partition walls have a height gradient in the direction of the long hairs so that the thickness of the electrophoresis lane becomes gradually thinner from the slot.

<Structure of the Invention> The specimen that is developed using the support medium for electrophoresis of the present invention is DNA that has been given a radioactive label.

RNA Kasa's nucleic acids 1! ! Refers to a mixture of group-specific fragments, where a fragment of a nucleic acid refers to a portion of a long-chain molecule. For example, a base-specific DNA cleavage mixture, which is a type of base-specific DNA fragment mixture, can be used to specifically cleave and decompose radioactively labeled DNA at-tij groups according to the Maxam-Gilbert υ described above. It is obtained by

In addition, for the base-specific DNA compound mixture, DNA was prepared according to the Sanger-Courlin method described above (1)
Type 4) can be obtained by synthesis using a radioactively labeled deoxynucleoside triphosphate and a DNA synthetic enzyme.

Furthermore, a mixture of base-specific RNA fragments can also be obtained as a mixture of nine fragments or as a mixture of composites by a method similar to that of the bipod. Furthermore, DNA consists of four types of bases as its constituent units: adenine, guanine, thymine, and cytosine, while RNA consists of four types of bases: adenine, guanine, uracil, and cytosine.

The radioactive label can be labeled with ':12P in a manner appropriate to these substances.
, v4c, s, 3H1IA: Which radioactive isotope is retained?

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Preferred embodiments of the electrophoresis support medium and support substrate thereof according to the present invention will be described below in detail as shown in the accompanying drawings.

FIG. 1 is a perspective view of a support medium 1 for DNA electrophoresis of the present invention.

As shown in FIG. 1, a support medium 1 is partitioned into a plurality of electrophoresis lanes 6 by a partition wall 4 interposed between a pair of opposing transparent plates 2.2. A gel layer 3 is inserted into each electrophoresis lane 6.

Note that the transparent plate 2 is a general term for a support substrate 21 and a protection plate 22, which will be described later.

A pair of opposing transparent plates 2.2 are integrated at both sides with side spacers 5 and partition walls 4 interposed therebetween. Note that the transparent plates 2, 2 may be integrated at both sides by providing protrusions on the transparent plates instead of the side spacers 5.

Transparent plate and side spacer 5? The bonding with i= is preferably performed by adhesion or melting, and it is preferable that the bonding portion has sufficient liquid tightness 41 so that the liquid (gel liquid) before the gel layer is formed does not leak.

The pair of transparent plates 2, 2 are support plates 21 having the same shape.
or use one side as the supporting substrate 21 and the other side as the holding plate 2.
Any combination, such as 2, is 11 functions.

Examples of the constituent material of the transparent plate 2 include glasses (for example, soda glass), transparent resins (for example, polyethylene terephthalate), transparent ceramics (for example, alumina), and the like.

In particular, in order to prevent the smiling phenomenon when the specimen is spread in the support medium 1, it is preferable that the transparent plate 2 is made of an expensive but highly heat conductive material such as beryllia.

The smiling phenomenon here means that the temperature distribution of the gel layer 3 becomes uneven due to the Joule heat generated when a voltage is applied to the gel layer 3, and the migration of the sample near the center of the support medium becomes uneven.
ii! refers to the phenomenon that occurs.

A feature of the present invention is that a partition wall 4 for partitioning the gel layer 3 into a plurality of lanes 6 is provided between the pair of transparent substrates 2.2.

In other words, since conventional support media were not provided with partition walls as shown in FIG. 9, when electrophoresis of a sample was performed, the electrophoresis column 10 sometimes moved diagonally or meandered;
In the support medium 1 of No. 51, the partition wall 4 is provided and the gel layer 3 is divided into a plurality of lanes 6, so that the specimen migrates along each lane 6, so that the migration column 10 does not become oblique or meandering. . Furthermore, since the partition wall 4 limits the distance between the transparent plates 2.2, distortion of the transparent plate 2 (especially near the center) can be prevented and the gel layer 3 can be made uniform.

Such a partition wall 4 may be formed by a spacer interposed between the pair of transparent substrates 2, 2, or may be formed integrally with at least one of the transparent plates.

These examples will be explained in detail below.

(1) In the case of a partition using a spacer As shown in FIG. 3, a separate straight bar-shaped spacer 8 is arranged in parallel between the transparent plates 2 and 2 to form the partition 4. However, the shape of the spacer is not limited to that shown in FIG. 3.

(2) In the case of a partition wall integrally formed on a transparent plate (support substrate) As shown in FIGS. 4 to 6, a partition wall 4 protruding from the inner surface of the support substrate 21 is integrally formed.

In the example shown in FIG. 4, the partition walls 4 having a height that specifies the thickness of the gel layer are formed on the support substrate 21, preferably in parallel and at regular intervals, and the support plate 21 is formed with a protective plate at the top 41 of the partition wall 4. 2
It is combined with 2 and integrated.

In the example shown in FIG. 5, partition walls 4 having a height approximately half the thickness of the gel layer are formed on a pair of support substrates 21.21, respectively, and both support substrates 21. top 4 of
They are combined and integrated at 1. In the example shown in FIG. 6, a pair of support substrates 21.21 are intersected by partition walls 4 having a height that specifies the thickness of the gel layer. The supporting substrates 21 are formed on the partition wall 4.
The top portion 41 of the support substrate 21 is integrated with the inner surface of the support substrate 21 . Formation of such partition walls 4 is preferably performed by a method such as press working, grinding, injection molding, or the like.

The support substrates 21 and 21 or the support substrate 21 and the protection plate 22 are preferably bonded together by adhesion or fusion.

When the partition wall 4 is integrally formed on the support substrate 2 in this way,
Manufacture of the support medium is facilitated since the alignment required in the case of partitions 4 with spacers 8 is not required.

Note that the number of partition walls 4 provided on the support substrate 21, the formation interval, etc. are appropriately determined as necessary.

In the case of (1) and (2) above, the height of the partition wall 4 is made constant, or as shown in FIG.

It is possible to provide a height gradient in the long direction of the partition wall 4 to vary the thickness of the electrophoresis lane 6, that is, the gel layer 3.

A support medium l is used using a spacer 8 having a tapered shape on the partition wall 4 or a support substrate 21 on which the tapered partition wall 4 is integrally formed.
In this case, the pair of transparent plates 2 are inclined and face each other, and a gradient end gel film (the thickness of the gel layer 3 gradually decreases from the slot 7 portion) can be easily obtained. I can do it.

As shown in FIGS. 1 and 2, in the gap between the partition walls 4,
An electrophoresis lane 6 is formed, and a gel layer 3 is formed in the lane 6.
is filled.

The constituent material of the gel layer 3 may be polyacrylamide or the like. Further, the thickness of the gel layer 3 can be uniform or tapered (gradient gel film), and is preferably about 200 to 400 gm.

A slot 7 for injecting a specimen is formed at the tip of the gel layer 3 filled in the lane 6.

This slot 7 is preferably created by solidifying the gel solution with a cap (not shown) attached.

Hereinafter, the method for producing a support medium for electrophoresis according to the K invention will be explained in detail.

A support substrate 21 (including a protection plate 22) having a predetermined shape and having partition walls 4 is created by processing a transparent material. spacer 8
When using a transparent plate 2 and a spacer 8.

Next, the pair of support plates 21, 21 (support substrate 21 and protection plate 22) are bonded and integrated by adhesion or fusion.

The same applies when using the transparent plates 2, 2 and the spacer 8.

A gel solution is injected into each lane 6 formed in the gap between the partition walls 4, and is solidified by exposure to light to form a gel layer 3. When solidifying the gel solution, a slot 7 is formed at the same time by attaching a comb to the tip of the lane 6.

The corresponding specimen is injected into each slot 7 of such a support medium 1, and the specimen is separated and developed by electrophoresis.

<Action of the invention> The operation of the present invention will be explained below.

When a sample is injected into the slot 7 formed at the tip of each electrophoresis lane 6 and a voltage is applied, the sample starts electrophoresing.

In the conventional support medium, since there were no partition walls, the migration column 10 sometimes moved obliquely or meandered as shown in FIG.

However, since the support medium of the present invention is provided with partition walls 4, the specimen migrates along each lane 6.

Therefore, the electrophoresis column 10 does not run diagonally or meander.

Effects of the Invention> According to the present invention, by providing the partition wall that partitions the gel layer into a plurality of electrophoresis lanes, the electrophoresis column of the specimen does not become oblique or meandering.

Therefore, the reading accuracy of autoradiographs is improved, and when reading and scanning autoradiographs using a reading device, there is no need to perform extensive scanning that takes into account diagonal and meandering of the electrophoresis column. It can be downsized and its manufacturing cost can be reduced. Furthermore, it is also possible to shorten the reading time of the autoradiograph.

Further, the support substrate of the present invention is easy to manufacture, and when creating a support medium, alignment is easier than using spacers, so a support medium with a homogeneous gel layer can be easily manufactured. . As a result, the reliability of the autoradiograph reading and analysis results is improved.

Further, since the partition walls support the transparent plates and limit the distance between the transparent plates, distortion of the transparent plates (especially near the center) can be prevented and a gel layer of uniform thickness can be obtained.

Furthermore, the support substrate of the present invention is easy to manufacture, and when creating a support medium, alignment is easier than using spacers, so a support medium with a homogeneous gel layer can be easily manufactured. can.

These results improve the reliability of the autoradiograph reading analysis results.

Furthermore, if a gradient end gel film is formed by gradually reducing the thickness of the electrophoresis lane from the slot, it is possible to increase the number of analyzed bases.

[Brief explanation of drawings]

FIG. 1 is a perspective view of a support medium for electrophoresis of the present invention. FIG. 2 is a cross-sectional view of the electrophoresis support medium of FIG. 1 taken along the line ■-■. FIG. 3 is a partial perspective view of the rod-shaped spacer. 4 to 6 are exploded front views of various configuration examples of the support substrate of the present invention. FIG. 7 is a side view of a support medium with tapered partition walls. FIG. 8 is a perspective view of a conventional electrophoresis support medium. Explanation of symbols 1...Support medium, 2...Transparent plate, 21...
Supporting substrate, 22... Protective plate, 3... Gel layer, 4
...Partition, 41...Top, 5...Side spacer, 6...Lane for electrophoresis, 7...Slot, 8
...Rod-shaped spacer, 10...Except for electrophoresis of a specimen Patent applicant: Fuji Photo Film Co., Ltd. 1. Agent: Minoru Watanabe
−・ ′、・ν＼、− Patent attorney Ishi Jl゛ Yang
-nido'τba''-8I Go[pa・4!,・FIG,1 FIG,3

Claims (7)

[Claims] (1) In an electrophoresis support medium for separating and developing a mixture of base-specific DNA fragments or base-specific RNA fragments to which a radioactive label has been added by electrophoresis, a pair of cells facing each other with a certain distance apart is used. a transparent plate, a gel layer interposed between the transparent plates, a partition wall for partitioning the gel layer into a plurality of electrophoresis lanes and separating the transparent plate by the predetermined distance, and an end of each lane. 1. A support medium for electrophoresis for determining the base sequence of nucleic acids, characterized in that the support medium has an injection port for the mixture, which is provided in the inlet for the mixture. (2) The support medium for electrophoresis for determining the base sequence of nucleic acids according to claim 1, wherein a separate spacer having a plurality of partition walls is interposed between the transparent plates. (3) The support medium for electrophoresis for determining the base sequence of nucleic acids according to claim 1, wherein the partition wall is integrally formed with at least one of the transparent plates. (4) The electricity for determining the base sequence of nucleic acids according to any one of claims 1 to 3, wherein the thickness of the electrophoresis lane gradually becomes thinner from the injection port of the mixture. Support medium for migration. (5) A support substrate used as a support medium for electrophoresis of a mixture of radioactively labeled base-specific DNA fragments or base-specific RNA fragments, which limits the electrophoresis lane and is filled with a gel layer. A support substrate characterized in that a partition wall is formed to protrude from an inner surface so that the support substrate can be formed. (6) The support substrate according to claim 5, wherein the plurality of partition walls are formed in parallel and at equal intervals. (7) The support substrate according to claim 5 or 6, wherein the partition wall has a height gradient in its longitudinal direction.