JPS63210653A - Medium membrane for electrophoresis - Google Patents

Abstract

PURPOSE:To provide a satisfactory resolving power in a wide range of molecular weight of a nucleic base fragments, by giving a gradient in the layer thickness to a gel medium layer containing a polyacrylic amide water gel and a carbamoyl group as the modifying agent. CONSTITUTION:A spacer plate 1 with a gradually varying thickness of a section along the direction of electrophoresis is fixed at both ends of a polyethylene terephthalate (PET) support with the surface thereof subjected to an ultraviolet irradiation treatment. Moreover, the work is covered with a PET cover sheet along the spacer plate 1 and the outside of the cover sheet is fixed with an aluminum plate so as to respond to changes in the thickness of the spacer plate 1, whereby a mold is formed. An aqueous solution for forming acrylic amide gels containing, for instance, urea as the modifying agent is run into the mold and irradiated with light from a high-pressure mercury lamp to cause a cross-linked polymerization. This provides a gel medium membrane having a satisfactory resolving power in a wide range of molecular weight of nucleic base fragments.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to an electrophoretic gel medium made of polyacrylamide-based aqueous gel used for nucleic acid (DNA, RNA) base sequencing. is a predetermined and controlled thickness gradient of the gel medium (
The present invention relates to an electrophoretic medium membrane having a thickness gradient).

[Conventional technology] Nucleic acid (DNA) by chemical decomposition method, dideoxy method, etc.

Slab electrophoresis using a polyacrylamide aqueous gel electrophoresis medium membrane (hereinafter sometimes referred to as polyacrylamide gel membrane or simply gel membrane) has become an essential operation in the base sequencing method of RNA). There is. In recent years, electrophoretic analysis has become frequently used.

With the development of the dideoxy method, there has been an increasing demand for polyacrylamide gel membranes that can accurately separate even high-molecular-weight portions of nucleic acid fragments.

On the other hand, in order to determine the base sequence of nucleic acids, when nucleobase fragments are electrophoretically separated depending on their molecular weight9
In a normal polyacrylamide gel membrane having a constant thickness, the interval between bands of nine separated fragments is wide in the low molecular weight part and narrow in the high molecular weight part. As a result, separation of high molecular weight portions of nucleic acid fragments becomes poor. Therefore, in order to obtain uniformly good separation performance over a wide molecular weight range from low molecular weight parts to high molecular weight parts, a polyacrylamide gel membrane (gradient gel membrane) is used that has a gradient in polyacrylamide concentration and buffer concentration in the electrophoresis direction. is used. For example, Japanese Patent Application Laid-Open No. 6O-235819 (EP 0
159694A), a polyacrylamide gel electrophoresis medium membrane having a polyacrylamide concentration (gel concentration or pore size) gradient is used, and a thin layer of an aqueous solution containing acrylamide and a crosslinking agent is applied to the surface of the support by ionizing radiation such as an electron beam. A method and apparatus for crosslinking polymerization using the method and apparatus are described.

The equipment used in this method and the method of controlling electron beams and the like to create a concentration gradient in the gel film are extremely complicated. In general, gradient gel membranes are time-consuming to manufacture, and the reproducibility of 11 degree gradients is poor. ! However, this method has the drawback that it is difficult to produce a large number of gel films with a uniform gradient. In addition, the swelling ratio differs depending on the polyacrylamide gel concentration.

There was also a drawback that the gel membrane was easily deformed during the operation of peeling the gel membrane containing the separated nucleic acid fragment image from the support after electrophoresis.

On the other hand, when producing a 2-regular gel film, it is difficult to create the desired gradual change gradient because the gel film is formed between two flat glasses.

Thickness gradient gel membranes have not previously been used.

[Object of the invention] The object of the present invention is to reduce the amount of nucleic acid fragments in a polyacrylamide-based aqueous gel electrophoresis medium (hereinafter sometimes referred to as gel medium) used for base sequencing of nucleic acids (DNA, RNA). It is an object of the present invention to provide a gel medium membrane having a thickness gradient so as to have approximately the same good high separation performance from a molecular weight portion to a high molecular weight portion.

Another object of the present invention is to provide a gel media membrane having a thickness gradient that is less deformed by swelling than concentration gradient gel membranes.

[Structure of the Invention] The present invention provides an aqueous polyacrylamide gel obtained by crosslinking polymerization of an acrylamide compound and a crosslinking agent in the presence of water, and an electrophoresis gel containing a compound containing at least one carbamoyl group as a denaturing agent. In an electrophoretic medium membrane in which a layer of a medium is provided between a planar support and a planar cover sheet, the gel medium layer exhibits a predetermined gradual change in thickness (layer thickness gradient). This is an electrophoresis medium membrane having the following properties.

[Detailed Description of the Structure of the Invention] An example of an acrylamide compound (monomer) that can be used in the gel medium is acrylamide.

N-methylacrylamide, N,N-dimethylacrylamide, N-(hydroxymethyl)acrylamide.

There are acrylamide homologs such as diacetone acrylamide. These compounds can be used alone or in combination of two or more. Among these compounds, acrylamide is preferred.

Also, 1 of acrylamide and other acrylamide-based compounds.
It is also preferable to use it in combination with more than one species.

As a crosslinking agent, rE Iectrophoresis
J2(4), 213-2l9 (1981), same magazine 2-(
4), 220-228 (1981) and the like, and trifunctional or higher-functional crosslinking compounds as described in JP-A-61-2058 and the like can be used. Specific examples of bifunctional crosslinking agents include: N,N'-methylenebisacrylamide (BIS); N,N'-propylenebisacrylamide (PBA); diacrylamide dimethyl ether (DAE); 1,2-diacrylamide ethylene Glycol (DEG); Ethyleneureabisacrylamide (EUB); Ethylene diacrylate (EDA); N,N'-diallyltartardiamide (DATD); N,N'-bisacrylycystamine ( BAC). Specific examples of trifunctional crosslinking agents include 1,3.5-)lyacryloylhexahydro-3-triazine (TAHT);)lylylcyanurate (TAC);)lylyl isocyanurate (TA
IC) etc. Among these crosslinkers, BIS and T
AHT is preferred. Two or more types of crosslinking agents can also be used in combination.

The amount of crosslinking agent is approximately 1% based on the total weight of monomer and crosslinking agent.
w% to about 30w%, preferably about 2w% to about low
Used in the range of %.

Agarose can be added to the gel medium. As for agarose, JP-A-55-5730, JP-A-55
Any of the low electroosmotic agarose, medium electroosmotic agarose, and high electroosmotic agarose described in Japanese Patent Application Laid-Open No. 57-126236, Japanese Patent Application Laid-Open No. 59-110946, etc. can be used. The amount of agarose added is approximately 0.2 to the volume of the aqueous gel containing the monomer and crosslinking agent.
% to about 2.0 %/V, preferably about 0.0%/V. Nine/
V% to about 1.2 F/V%.

Water-soluble polymers can be added to the gel medium.

Examples of water-soluble polymers include JP-A-59-126238;
Addition polymerization or condensation polymerization type water-soluble nonionic polymers with a molecular weight ranging from about 10,000 to about 1 million as described in JP-A No. 60-60548, etc., vinyl sulfonyl groups as described in JP-A-61-18852, etc. A crosslinkable acrylamide copolymer containing the like, a water-soluble cellulose derivative described in Japanese Patent Application No. 61-214878 can be used. Examples of addition polymerizable water-soluble nonionic polymers include polyacrylamide, polyvinyl alcohol, and polyvinylpyrrolidone. Polyethylene glycol and polypropylene glycol are examples of condensation type water-soluble nonionic polymers.

There is poly-N-vinylpyrrolidone. Examples of crosslinkable acrylamide copolymers include N([3-(vinylsulfonyl)propanamide comethylcoacrylamide-acrylamide copolymer; N-[[3-(2-chloroethylsulfonyl)propanamide]methyl]acrylamide-acrylamide-N -(1,1-dimethyl-3
-oxobutyl)acrylamide copolymers. Examples of water-soluble cellulose derivatives include water-soluble cellulose ethers such as methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, and hydroxybutylmethylcellulose. Among these water-soluble polymers, polyacrylamide,
Polyethylene glycol, N-[[3-(vinylsulfonyl)propanamidocomethylcoacrylamide-acrylamide copolymers are preferred. In the case of an addition polymerization type or condensation polymerization type water-soluble nonionic polymer, the amount of the water-soluble polymer added is about 2v% to about 100ν%, preferably about 5% based on the total weight of the monomer and crosslinking agent. -% to about 50%, in the case of crosslinkable acrylamide-based copolymers, about 1-% to about 50% by weight of the acrylamide-based compound.
% to about 50%, preferably about 5% to about 40v%.

Glycerol is added to the gel medium in an amount of approximately 0.1%/V% based on the volume of the gel medium for the purpose of widening the electrophoretic image band width of the high molecular weight portion of the nucleic acid fragment and preventing distortion of the separated image.
It can be added in a range of about 1.0 V/V%. In addition, a polyol compound such as glycerol or ethylene glycol is added as a wetting agent at a rate of about 1.0 ton/to the volume of the gel medium.
It can be added in a range of V% to about 40 w/v%.

Agarose and/or water-soluble polymers or polyol compounds such as glycerol or ethylene glycol are added to the gel medium usually between the time the monomers and crosslinker are dissolved in water and the time the polyacrylamide-based aqueous gel is formed. is preferred.

Nonionic, anionic or amphoteric surfactants can be added to the gel medium. As an example of surfactants.

C3F17SO2N (CH2CH20)+4-HE
Nonionic surfactants such as t.

Anionic surfactant such as Na03S-CHCOOCH2CH(Et)CaH9 CH2C00CH2CH(Et)C4H9.

e C1, H23CON H-(CH2)3-N" -CH
There are amphoteric surfactants such as 2COO-e. The amount of surfactant added is based on the volume of the aqueous gel containing the monomer and crosslinking agent, in the case of nonionic or cationic surfactant.

from about I x 10-' to about 5XIO-1 ton/V%, preferably from about 1X10'-3 to about I x 10-2 ton/V
% range, for anionic surfactants approximately lXl0-
4 to about 5X10-2 affected lV%, preferably about lXl0
-3 to about 5.times.10@-2 %/V.

A compound having at least one carbamoyl group is used as the modifier. Specific examples include urea and formamide. The amount of the modifier added is about 40% dlv% to about 60w based on the volume of the aqueous gel containing the monomer and crosslinking agent.
/v% range. When using urea as a denaturing agent, the saturated dissolution amount (approximately 420 g) ranges from approximately 6 mol (approximately 3608) to 10,100 O of aqueous gel containing monomers and crosslinking agent.
It is preferably used in a range from about 7 mol to a saturated dissolution amount. Since a large amount of the modifier is added, it is usually preferable to add the modifier at a time when the various components including the monomer and the crosslinking agent are dissolved in water.

The gel medium can contain a known p)I buffer to adjust the pH value during electrophoresis to a range of 8.0 to 9.0. As a pH buffering agent that can be used, 9 Japanese Chemical Society Zen "Chemical Handbook Basic Edition" (Tokyo, Maruzen ■, published in 1966) pp. 1312-1320 r R, M, C, Daw
son et a1 rData for B ioc
chemical Re5earchJ5th
rd at the C1arendonPress,
Published in 1969) pp. 476-508, r Bioch
emistryJ5.46? Pages onwards (1966), r
Analytical Biochemistry
There is a p)l buffer system described in J. Old 11, pp. 300-310 (1980). As a specific example of pt+buffer, tris(hydroxymethyl)aminomethane (Tris
); N,N-bis(2-hydroxyethyl)glycine (Bicine); N-2-hydroxypiperazine-
, N'-2-hydroxypropane-3-sulfonic acid Na
Salt or salt, etc.; N-2-hydroxyethylpiperazine-
N'-3-sulfonic acid Na salt or salt, etc.; N-[)lis(hydroxymethyl)methyl]-3-aminopropanesulfonic acid Na salt or salt, etc.; and an acid optionally combined with any of these; There are alkalis or salts. An example of a preferred pH buffer system is Tris-boric acid-EDT.
There is A.2Na salt (pt (composition for 8.2 to 8.3)).

It is generally preferred for the gel medium to be substantially colorless and transparent at a predetermined thickness for the detection or reading of electrophoretic images.

The gel medium is predetermined on a substantially electrically non-conductive, water-impermeable, smooth-surfaced sheet-like (film-like or planar) support or cover sheet. It is provided as a layer or film with a controlled gradual change in thickness. Known glass plates, organic polymer sheets, and the like can be used as the substantially electrically nonconductive, water-impermeable, smooth-surfaced sheet-like support or cover sheet. Specific examples of organic polymer sheets include polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose esters (e.g., cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.), and the thickness is about 5 OLIm to about 2 nv. , preferably from about 80 um to about 500 IJ1
There is a sheet or plate having a smooth surface that is transparent, ie transparent to electromagnetic radiation in a wavelength range of at least part of the wavelength range from 200n11 to about 900nm. When an organic polymer support or cover sheet is used, ultraviolet irradiation, glow discharge treatment, corona discharge treatment, flame treatment, electron beam irradiation, and chemical etching are performed to make the surface hydrophilic and improve adhesion to the gel film.

Known surface treatment methods such as electrolytic etching can be applied. The surface of the organic polymer support or cover sheet may be coated with JP-A-59-164950° as required.
9-212753. JP-A-60-194349. JP 60-239658, JP 60-244850. An undercoat layer or an adhesive layer as described in JP-A No. 61-14557 and the like can be provided to strengthen the adhesion between the gel medium layer provided thereon and the support or cover sheet. It is also possible to use a planar support or cover sheet with a predetermined gradual thickness change, as described below.

The gel medium is prepared by casting or coating an aqueous solution (hereinafter sometimes referred to as gel-forming liquid) containing the above-mentioned components and a radical polymerization initiator composition on a planar support or cover sheet in a layer or film form. A polyacrylamide-based aqueous gel medium layer or film in which a monomer (acrylamide-based compound) and a cross-linking agent are cross-linked and polymerized by irradiation with ultraviolet rays or visible light and/or heating if necessary in the absence of molecular oxygen. Manufactured and used.

Acrylamide compound (monomer) and crosslinking agent.

It is dissolved or dispersed in water as an aqueous solution or an aqueous dispersion, and the two are crosslinked and polymerized in water to form a crosslinked and polymerized aqueous gel medium. In this specification, unless otherwise specified, the term simply "dissolved (in water)" includes both dissolution (in water) and dispersion (in water).

Both aqueous solutions and aqueous dispersions are simply referred to as aqueous solutions. Water is not the only solvent or dispersion medium.

Also included are water-organic solvent mixtures with optionally added organic solvents.

As a radical polymerization initiator composition, r E Iectr
o-phOreSiSJ2(4), 213-219(1
981), same magazine Z (4), 220-228 (1981)
, JP-A-59-126236, Aomizu and Nagai, eds., Latest Electrophoresis Method J (published in 1973), and the like can be appropriately selected from the low-temperature radical polymerization initiator compositions. As an example of the radical polymerization initiator composition, β-
(dimethylamino)propionitrile (DMDPN)-
Ammonium peroxonisulfate mixture; N, N, N'
, N'-tetramethylethylenediamine (TEMED)
-Ammonium peroxonisulfate mixture; TEMED-
Riboflavin mixture; TEMED-riboflavin-hydrogen peroxide mixture: riboflavin-beroxonisulfate mixture; riboflavin-hydrogen peroxide mixture (when used in combination with a photosensitizer such as riboflavin, irradiation with ultraviolet or visible light is used), etc. There is. The amount of the radical polymerization agent composition added is approximately 0.3ν based on the total weight of the monomer and crosslinking agent.
% to about 5.0 w%, preferably about 0.5 ν% to about 3
．． It is in the range of 0-%.

The gel medium has a gel concentration of S, Hjerten:
rAr-chives of Biochemis
try and B 1 physics J l, (S
uppl,),! 47-151 (1962), the total amount of monomer and crosslinking agent is from about 3 w/v % to about It is used in a range of 30 w/v%.

When cross-linking and polymerizing the gel-forming liquid on the surface of a planar support (or cover sheet), the gel-forming liquid is cast and then cross-linked in the absence of molecular oxygen, such as in a nitrogen gas atmosphere. It is preferable to carry out crosslinking polymerization by immediately covering the surface of the gel-forming solution that has been carried out or cast and coated with a covering material such as a cover film, sheet, or plate. The coating material used for this purpose may be made of the same material as the above-mentioned planar support.

If the cover film is an organic polymer film,
Its thickness is about 300Lffl+ or less, with a practical range of about 4L1111 to about 20OLIff+, preferably about 4um to about 100LJII+. When the covering material is a glass plate.

The thickness of the support may be the same as that of the flat glass plate used as the support.

Generally speaking, the thickness gradient of the gel medium membrane is created so that after electrophoresis, the gradient is thicker on the side of the high-molecular-weight fragments of nucleic acids and thinner on the side of the low-molecular-weight fragments, but other gradient arrangements may be used depending on the purpose. Needless to say, it is possible to adopt The gel film thickness gradient (curve or straight line), which is a feature of the gel medium film of the present invention, is a straight line, a gently bent straight line, or an exponential function with respect to the distance from the sample injection end.

Logarithmic function, catenary line, trace line, parabola, hyperbola, ellipse, 3
A portion of a curve of gradual change expressed by a function such as the following curve,
The gradient may be represented by any other gradual curve or combination of a curve and a straight line, but increases gradually and monotonically with distance along the direction of electrophoresis. Increase from the middle. Alternatively, it is preferable to decrease the amount once and then increase it. The range of thicknesses to be varied is from about 50 um to about 5 um, preferably from about 80 um to about 1000 um. The shape of the sample injection portion can be selected from known shapes such as a rectangular shape, a square shape, a triangular shape (sharks teeth shape), and a circular shape.

A method for providing a film thickness gradient in the gel medium was predetermined on a planar support. A spacer plate with a thickness change approximately corresponding to a controlled gradual thickness change (layer thickness gradient or film thickness gradient) was fixed and covered with a covering material (sheet-like material) along the spacer plate. A method of pouring a gel-forming liquid into a mold and cross-linking polymerizing it, a spacer plate of a constant thickness is placed on the surface of a flat support (or a cover sheet) having a thickness change corresponding to a predetermined gradual thickness change. A method of cross-linking and polymerizing a gel-forming liquid by pouring it into a mold covered with a covering material (support material) along a spacer plate, and having a thickness change corresponding to a predetermined gradual thickness change. A method in which a gel-forming solution is cast on the surface of a planar support (or cover sheet) and cross-linked and polymerized in the absence of molecular oxygen, such as in a nitrogen gas atmosphere, which corresponds to a predetermined gradual thickness change. While controlling the flow rate per unit time (lower the flow rate in areas where the gel film is thinner, and increase the flow rate in areas where the gel film is thicker), A method such as casting a gel-forming solution and crosslinking polymerization in the absence of molecular oxygen such as in a nitrogen gas atmosphere can be applied.A planar support having a predetermined gradual thickness change can be used. , mold casting method, chemical etching method, cutting method, etc. can be used.The support (or cover sheet) can be used while controlling the flow rate.
In the case of cast coating, the change in the thickness of the spacer plate does not necessarily have to correspond to a predetermined gradual change in the thickness of the gel film. Also, without providing a film thickness gradient of the gel medium across the width of the gel film.

It is also possible to provide only an area corresponding to the number of lanes with a width slightly wider than the area of the lane in which the sample is electrophoresed, and the remaining area to have a substantially constant thickness. This embodiment can advantageously be implemented in embodiments using planar supports or cover sheets with gradual thickness changes.

The gel media membrane of the present invention can be prepared in the same manner as known polyacrylamide-based aqueous gel media membranes. Also 9
The gel medium membrane of the present invention is prepared according to the known methods described in the above-mentioned literatures and patent specifications.

It can be used in horizontal and vertical slab m pneumophoresis methods.

Example 1 and Comparative Example 1 Surface treated with ultraviolet irradiation, thickness 180um, size 20
The three types of gradual gradients shown in the cross-sectional schematic diagrams along the electrophoresis direction in Figures 1 to 3 were placed on both edges along the long sides of a Cm x 40 cm rectangular colorless transparent polyethylene terephthalate (PET) sheet (support). A spacer plate with a width of 10 degrees with a thickness change of 10 degrees (the present invention) and a constant thickness of 200 um with the same width.
spacer plates (comparative example) are each fixed, covered with a 1100u thick PET sheet (cover sheet) along the spacer plates, and further covered with a 20cII plate on the outside of the cover sheet.
A mold for preparing a medium layer for polyacrylamide aqueous gel electrophoresis was formed by fixing the spacer with a rectangular aluminum plate of 40 cm in a manner corresponding to the change in thickness of the spacer.

Pour an aqueous solution for forming an acrylamide gel with the composition shown in Table 1 into each mold.2 At an ambient temperature of 25°C, each gel forming solution was irradiated with a 100W high-pressure mercury lamp from a distance of 10cm and left to stand for 10 minutes. Three kinds of polyacrylamide aqueous gel electrophoresis media membranes of the present invention having a thickness gradient were formed by cross-linking polymerization between PET sheets, and a comparative gel membrane having a constant thickness was formed.

■ Ultra-flat V-shaped cross section in Figure 1, maximum thickness (starting end and end in migration direction) 300Lffrl, minimum (middle part in migration direction) 50 um ■ Ultra-flat wedge-shaped cross section in Figure 2, minimum thickness (starting end in migration direction) ) The thickness increases linearly from 150 um to a maximum of 300 um (at the end in the migration direction). ■The ultra-oblate wedge-shaped cross section in Figure 3, the thickness increases exponentially from 150 um at the minimum (starting end in the migration direction) to a maximum (in the migration direction). Terminating part) 300 um ■Constant thickness 200 um Using the four types of gel membranes obtained, DNA base sequence analysis experiments were carried out according to standard methods using DNA fragment samples prepared by the dideoxy method for M13-mp8 DNA. did. As a result, the range of readable base fragments in the 9-thickness gradient gel membrane of the present invention was as follows.

Gel films 060 to 240 Gel films 060 to 215 Gel films 060 to 240 and the widths and intervals of the electrophoretic images of base fragments in each lane did not become extremely narrow within the readable range.

On the other hand, in the conventional gel membrane (2) with a constant thickness, the readable range is from 60 to 200, and the width and interval of the electrophoretic image of the base fragment in each lane changes as it goes from the low molecular weight part to the high molecular weight part. It was gradually narrowing down.

These results show that the polyacrylamide aqueous gel electrophoresis medium membrane with the thickness gradient of the present invention maintains almost uniform lane spacing over a wide molecular weight range from the low molecular weight part of the nucleobase fragment to the high molecular weight part. It has become clear that good separation can be obtained using this method, and a large number of readable base fragments can be obtained, making it possible to perform DNA base sequence analysis with high accuracy.

Table 1: Components of gel-forming solution and agarose: low electro-osmosis, gelation temperature 36°C Tri
s: ) Lis(hydroxymethyl)aminomethane Example 2 and Comparative Example 2 Edges along the long sides of a rectangular colorless transparent PET sheet (support) with a thickness of 1BOLIII+ and a size of 20CII+ x 4QC1I whose surface was treated with ultraviolet irradiation. A spacer plate having a constant thickness of 300 LIT+ and having a width of 10 cm and a length of 40 cm was fixed by adhesive. On this support, the first
The gel-forming liquid having the composition listed in the table is flowed without changing the thickness of the gel-forming liquid film on the support by controlling the flow rate within a range such that the thickness of the gel-forming liquid film is from 150 LIITl to 300 um. The gel-forming liquid film was then cross-linked and polymerized in a nitrogen gas atmosphere while irradiating each gel-forming liquid film with a 500W xenon discharge lamp.
Then, on top of the gel film, a film with a thickness of 631 Jm and a size of 20 cm was applied.
X 40 cm colorless transparent PET sheets are closely laminated as a cover sheet to form a polyester film having a thickness change (film thickness gradient) approximately equal to the thickness change in the cross section of the spacer plate shown in Figures 1 to 3. Three types of acrylamide aqueous gel membranes (gel membranes of the present invention) were prepared.

On the other hand, the flow rate of the gel-forming liquid was changed to 20% when the thickness of the gel-forming liquid film was 20
A gel film having a constant thickness of 200 um (Comparative Example 2) was prepared in the same manner as above except that the thickness was kept constant at 0Ijffl.

D was prepared in the same manner as in Example 1 using the four types of gel films obtained.
When an experiment was carried out to read the electrophoretic images of NA fragments, the same results as in Example 1 and Comparative Example 1 were obtained.

[Brief explanation of the drawing]

Figures 1 to 3 show gradual thickness changes (film thickness FIG. Figure 1: Ultra-flat V-shaped cross section, maximum thickness (starting end and terminal end in migration direction) 300 um, minimum (middle part in migration direction) 150 um
-9 Spacer plate with a length of 40 cm (for gel film ■ preparation) Figure 2: Ultra-oblate wedge-shaped cross section, minimum thickness (starting end in migration direction) 1
Increases linearly from 50um to maximum (at end of migration direction)
300um, length 40cn+ spacer plate (gel film■
(for preparation) Figure 3: Ultra-oblate wedge-shaped cross section, minimum thickness (starting end in migration direction)
Spacer plate increasing exponentially from 150 um to a maximum of 300 um (at the end in the migration direction) and having a length of 40c+n (for gel film preparation) Patent applicant: Fuji Photo Film Co., Ltd. Figure 1 Thickness change: Ultra-flat V-shape Figure 2: Thickness change: increases linearly Figure 3: Thickness changes: increases numerically between two exponentials Length: 4000 Procedural amendment (voluntary)

Claims (6)

[Claims] (1) A layer consisting of an aqueous polyacrylamide gel obtained by crosslinking polymerization of an acrylamide compound and a crosslinking agent in the presence of water and a gel medium for electrophoresis containing a compound containing at least one carbamoyl group as a denaturing agent. The gel medium layer has a predetermined gradual thickness change (layer thickness gradient) in the electrophoresis medium membrane provided between a shaped support and a planar cover sheet. Media membrane for electrophoresis. (2) The electrophoretic medium membrane according to claim 1, wherein the support and the cover sheet are both sheet-like materials made of organic polymers. (3) The electrophoretic medium membrane according to claim 2, wherein the support and the cover sheet are both sheet-like materials made of polyethylene terephthalate. (4) The electrophoretic medium membrane according to claim 1, wherein the compound containing at least one carbamoyl group is urea. (5) The electrophoresis medium membrane according to claim 1, wherein the gel medium further contains agarose. (6) The electrophoretic medium membrane according to claim 5, wherein the gel medium further contains a water-soluble polymer.