JPH0652254B2 - Electrophoretic medium film - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of use] The present invention relates to a gel medium for electrophoresis comprising a polyacrylamide-based aqueous gel used for determining the base sequence of nucleic acids (DNA, RNA). Is a gel medium thickness gradient (thickness gradient) of the gel medium, which is predetermined and controlled so as to have good separation performance over a wide molecular weight range from the low molecular weight portion to the high molecular weight portion of the nucleobase fragment, and the acrylamide compound and the cross-linking agent. The present invention relates to a medium film for electrophoresis having a combination of concentration gradients (polyacrylamide gel concentration gradient, that is, gel concentration gradient).

[Prior Art] In a nucleic acid (DNA, RNA) base sequencing method such as a chemical decomposition method or a dideoxy method, a polyacrylamide-based aqueous gel electrophoresis medium film (hereinafter, referred to as a polyacrylamide gel film or simply a gel film) is used. Slab electrophoresis is an essential operation. In recent years, electrophoretic analysis has been frequently used. With the development of the dideoxy method, there has been an increasing demand for a polyacrylamide gel membrane capable of accurately separating even high-molecular-weight portions of nucleic acid fragments.

On the other hand, when a nucleic acid base fragment is electrophoretically separated according to its molecular weight for the purpose of determining the base sequence of a nucleic acid,
In a normal polyacrylamide gel membrane with a constant thickness, the band separation of separated fragments is wide in the low molecular weight part and narrow in the high molecular weight part. Therefore, the separation of the high molecular weight portion of the nucleic acid fragment becomes poor. Therefore, in order to obtain good separation performance evenly over a wide molecular weight range from the low molecular weight portion to the high molecular weight portion, a polyacrylamide gel membrane (gradient gel membrane) with a gradient in the polyacrylamide concentration and the buffer concentration in the electrophoresis direction is used. It is used. For example, in JP-A-60-235819 (EP 0 159 694A), polyacrylamide concentration (gel concentration or pore size)
A method and apparatus for producing a gradient polyacrylamide gel electrophoresis medium film by cross-linking and polymerizing a thin layer of an aqueous solution containing acrylamide and a cross-linking agent on the surface of a support using ionizing radiation such as an electron beam is described. ing. The apparatus used for this method and the control method such as an electron beam for expressing the concentration gradient in the gel film are extremely complicated. Generally, a gradient gel film is troublesome to manufacture, the reproducibility of the concentration gradient is poor, the manufacturing is often unsuccessful, and it is difficult to manufacture a large number of gel films having a gradient with good reproducibility.

On the other hand, when a normal gel film is produced, it is difficult to give a desired gradual change in the film thickness because the gel film is formed between two flat glass sheets. It was never used.

Thus, since it is technically difficult to provide a gradient only to either the gel concentration or the film thickness, no attention has been paid to the combined use of the concentration gradient and the film thickness gradient. In addition, it is generally known that a complex gradient curve is required in order to obtain good separation performance even in the high molecular weight portion of nucleic acid when there is a gradient only in either concentration or film thickness. It was found that, when two types of gradients of concentration and film thickness are used in combination, a large effect can be obtained by combining the respective simple gradient curves (or straight lines). further,
Since the swelling ratio of the polyacrylamide aqueous gel differs depending on the concentration, in the gradient gel membrane with a complicated concentration gradient curve, the gel membrane after the electrophoresis with the separated nucleic acid fragment image is separated from the support. Although the gel film has a drawback of being easily deformed, it has been found that the gel film having a simple concentration gradient curve (or concentration gradient straight line) is unlikely to be deformed during the peeling operation after the electrophoresis.

[Object of the Invention] An object of the present invention is to reduce the content of nucleic acid fragments in a polyacrylamide-based aqueous gel electrophoresis medium (hereinafter sometimes referred to as gel medium) used for determining the nucleotide sequence of nucleic acids (DNA, RNA). It is an object of the present invention to provide a gel medium membrane having a thickness gradient and a polyacrylamide gel concentration gradient so as to have a good high-separation performance that is almost equivalent from the molecular weight portion to the high molecular weight portion.

Another object of the present invention is to provide a gel medium film having a thickness gradient and a polyacrylamide gel concentration gradient in which deformation due to swelling is eliminated or deformation is reduced as compared with a gel film having only a concentration gradient.

[Structure of the Invention] The present invention provides a gel for electrophoresis containing a polyacrylamide-based aqueous gel obtained by cross-linking and polymerizing an acrylamide compound and a cross-linking agent in the presence of water, and a compound containing at least one carbamoyl group as a modifier. In an electrophoretic medium film in which a layer made of a medium is provided between a flat support and a flat cover sheet, the gel medium layer has a gradual change in thickness along the electrophoretic direction and an electrophoretic direction. The electrophoretic medium film is characterized in that it also has a gradual change in polyacrylamide-based aqueous gel concentration along with it.

[Detailed Description of Configuration of the Invention] Examples of acrylamide compounds (monomers) that can be used in the gel medium include acrylamide, N-methylacrylamide, N, N-dimethylacrylamide, and N-
There are acrylamide homologs such as (hydroxymethyl) acrylamide and diacetone acrylamide. These compounds can be used alone or in combination of two or more. Of these compounds, acrylamide is preferable, and it is also preferable to use acrylamide in combination with one or more other acrylamide compounds.

As a cross-linking agent, "Electrophoresis" 2 (4), 213-219
(1981), ibid. 2 (4), 220-228 (1981) and the like, and bifunctional crosslinking agent compounds described in JP-A-61-2058 and the like can be used. . Specific examples of the bifunctional cross-linking agent include N, N'-methylenebisacrylamide (BIS); N, N'-propylenebisacrylamide (PBA); diacrylamide dimethyl ether (DAE);
1,2-Diacrylamide Ethylene glycol (DEG); Ethylene ureabisacrylamide (EUB); Ethylene diacrylate (EDA); N, N'-diallyl tartardiamide (DATD); N, N'-bisacrylyl cystamine ( BAC)
There is. Specific examples of the trifunctional crosslinking agent include 1,3,5-triacryloylhexahydro-s-triazine (TAHT);
Triallyl cyanurate (TAC); triallyl isocyanurate (TAIC) and the like. BI of these cross-linking agents
S and TAHT are preferred. Two or more kinds of cross-linking agents may be used in combination.

The amount of cross-linking agent is about 1w based on the total weight of monomer and cross-linking agent.
% To about 30 w%, preferably about 2 w% to about 10 w%.

Agarose can be added to the gel medium. Examples of agarose include low electroosmotic agarose, medium electroosmotic agarose, and high electroosmosis described in JP-A-55-5730, JP-A-55-110946, JP-A-57-502098, and JP-A-59-126236. Any of the sex agarose can be used. The amount of agarose added is about 0.2 w / v% to about 2.0 w / v%, preferably about 0.3 w / v% to about 1.2 w / v% relative to the volume of the aqueous gel containing the monomer and the cross-linking agent. It is a range.

A water-soluble polymer can be added to the gel medium.
Water-soluble polymers include those disclosed in JP-A-59-126236 and JP-A-60-
Addition-polymerization type or polycondensation-type water-soluble nonionic polymers having a molecular weight of about 10,000 to about 1,000,000 described in 60548,
Crosslinkable acrylamide-based copolymers containing vinylsulfonyl groups and the like described in JP-A-61-18852, Japanese Patent Application No. 61-214878
The water-soluble cellulose derivative described in (JP-A-63-70156) can be used. Examples of the addition polymerization type water-soluble nonionic polymer include polyacrylamide, polyvinyl alcohol, and polyvinylpyrrolidone. Examples of polycondensation type water-soluble nonionic polymers are polyethylene glycol, polypropylene glycol, and poly-N-vinylpyrrolidone. Examples of the cross-linkable acrylamide copolymer include N-[[3- (vinylsulfonyl) propanamide] methyl] acrylamide-acrylamide copolymer; N-[[3- (2-chloroethylsulfonyl) propanamide] methyl] acrylamide-acrylamide −
There is N- (1,1-dimethyl-3-oxobutyl) acrylamide copolymer. Examples of water-soluble cellulose derivatives include water-soluble cellulose ethers such as methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, hydroxybutyl methyl cellulose and the like. Among these water-soluble polymers, polyacrylamide, polyethylene glycol, N
-[[3- (Vinylsulfonyl) propanamide] methyl] acrylamide-acrylamide copolymer is preferred. In the case of addition-polymerization type or polycondensation-type water-soluble nonionic polymer, the addition amount of the water-soluble polymer is about 2w% to about 100w, preferably about 5w% with respect to the total weight of the monomer and the crosslinking agent To about 50 w%, in the case of crosslinkable acrylamide copolymers, about 1 w% to about 50 w%, preferably about 5 w% to about 40 w%, based on the weight of the acrylamide compound.
Is the range.

In the gel medium, glycerol was added to the gel medium in an amount of about 0.1 w / v% to about 1.0 w for the purpose of broadening the migration image band width of the high molecular weight portion of the nucleic acid fragment and preventing distortion of the separation image.
It can be added in the range of / v%. As a wetting agent, a polyol compound such as glycerol or ethylene glycol can be added in the range of about 1.0 w / v% to about 40 w / v% with respect to the volume of the gel medium.

Agarose and / or a water-soluble polymer or a polyol compound such as glycerol or ethylene glycol is usually added to the gel medium from the time when the monomer and the crosslinking agent are dissolved in water to the time when the polyacrylamide-based aqueous gel is formed. Is preferred.

Nonionic, anionic or amphoteric surfactants can be added to the gel medium. Examples of surfactants include Nonionic surfactants such as Anionic surfactants such as And other amphoteric surfactants. The amount of the surfactant added is about 1 × 10 ^-4 to about 5 × 10 ^-1 w / w in the case of the nonionic or cationic surfactant, relative to the volume of the aqueous gel containing the monomer and the crosslinking agent. v%, preferably about 1 × 10 ^-3 to about 1 × 10 ^-2 w
/ v% range, about 1 x 10 ^{-4 for} anionic surfactants
To about 5 × 10 ^-2 w / v%, preferably about 1 × 10 ^-3 to about 5
It is in the range of × 10 ^-2 w / v%.

As the modifier, a compound having at least one carbamoyl group is used. Specific examples thereof include urea and formamide. The amount of the modifier added is in the range of about 40 w / v% to about 60 w / v% based on the volume of the aqueous gel containing the monomer and the crosslinking agent. When using urea as a denaturant, about 6 mol (about 360 g) per 1000 mL of aqueous gel containing monomer and crosslinking agent
To a saturated dissolution amount (about 420 g), preferably about 7 mol to a saturated dissolution amount. Since the modifier is added in a large amount, it is usually preferable to add the modifier when the various components including the monomer and the crosslinking agent are dissolved in water.

A known pH buffer may be contained in the gel medium to adjust the pH value during electrophoresis to a range of 8.0 to 0.9.
As a pH buffering agent that can be used, “Chemical Handbook Basic Edition” edited by The Chemical Society of Japan (Tokyo, Maruzen Co., Ltd., 1966) 1312-1320
Page; RMCDawson et al, “Data for Biochemical Res”
earch "Second Edition (Oxford at the Clarendon Press, 1969
Pp. 476-508; "Bio-chemistry" 5 , 467-477.
Page (1966); "Analytical Biochemistry" 104 , 300
There is a pH buffer system described in page 310 (1980) and the like. Specific examples of the pH buffer include a buffer containing tris (hydroxymethyl) aminomethane (Tris); N, N-bis (2-hydroxyethyl) glycine (Bicine); 4- (2-hydroxyethyl) -1- Piperazine propane sulfonic acid (HEPP
S), Na salt or K salt; β-hydroxy-4- (2-hydroxyethyl) -1-piperazinepropanesulfonic acid (HEP
PSO), Na salt or K salt; 3-[[2-hydroxy-1,1-
Bis (hydroxymethyl) ethyl] amino] -1-propanesulfonic acid (TAPS), Na salt or K salt, etc .; 3- (cyclohexylamino) -1-propanesulfonic acid (CAPS), N
a salt or K salt; and an acid, alkali or salt optionally combined with any of these. As an example of a preferable buffering agent, Tris-boric acid-EDTA.2Na salt (composition for pH 8.2 to 8.3)
There is.

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

The gel medium consists of a predetermined, controlled, grading on a substantially electrically non-conductive, water-impermeable, smooth surface sheet-like (film or plate) support or cover sheet. It is provided as a layer or film with a varying thickness. A known glass plate, organic polymer sheet or the like can be used as a sheet-like support or cover sheet having a substantially electrically non-conductive, water-impermeable, smooth surface. Specific examples of the organic polymer sheet include polyethylene terephthalate, polycarbonate of bisphenol A, polystyrene, cellulose ester (eg, cellulose diacetate, cellulose triacetate, cellulose acetate propionate, etc.) and the like, and the thickness is about 50 μm to about 2 m.
m, preferably transparent in the range of about 80 μm to about 500 μm,
That is, there is a sheet or plate having a smooth surface which transmits electromagnetic radiation having a wavelength in at least a part of the range of about 200 nm to about 900 nm. When using an organic polymer support or cover sheet, in order to make the surface hydrophilic and to improve the adhesion with the gel film, ultraviolet irradiation, glow discharge treatment, corona discharge treatment, flame treatment, electron beam irradiation, chemical etching, A known surface treatment method such as electrolytic etching can be applied. If necessary, the surface of the organic polymer support or the cover sheet may be used as a light-emitting device.
59-164950, JP-A-59-212753, JP-A-60-194349, JP-A-60-239658, JP-A-60-244850, JP-A-61-14557, etc. The adhesion between the gel medium layer provided thereon and the support or cover sheet can be strengthened. Further, as will be described later, it is also possible to use a planar support or a cover sheet having a predetermined gradual thickness change.

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

The acrylamide compound (monomer) and the crosslinking agent are dissolved or dispersed in water as an aqueous solution or dispersion.
Both are cross-linked and polymerized in water to form a cross-linked and polymerized aqueous gel medium. Unless otherwise specified, in this specification, both (in water) and (in water) dispersion are simply referred to as (in water) dissolution, and both an aqueous solution and an aqueous dispersion are simply referred to as an aqueous solution. Solvents or dispersion media include water as well as water-organic solvent mixtures containing optionally added organic solvents.

The radical polymerization initiator composition is “Electro-phoresi
s ” 2 (4), 213-219 (1981), ibid 2 (4), 220-228 (1
981), JP-A-59-126236, Aoki, Nagai, "Latest Electrophoresis Method" (published in 1973) and the like, and can be appropriately selected and used from low temperature radical polymerization initiator compositions.
Examples of the radical polymerization initiator composition include β- (dimethylamino) propionitrile (DMDPN) -ammonium peroxodisulfate; N, N, N ′, N′-tetramethylethylenediamine (TEMED) -ammonium peroxodisulfate; TEMED-riboflavin mixture; TEMED-riboflavin-hydrogen peroxide mixture; riboflavin-ammonium peroxodisulfate mixture; riboflavin-hydrogen peroxide mixture (in the case of using a photosensitizer such as riboflavin, UV irradiation or visible light irradiation is also used) Etc. The amount of the radical polymerization initiator composition added is in the range of about 0.3 w% to about 5.0 w%, preferably about 0.5 w% to about 3.0 w%, based on the total weight of the monomer and the crosslinking agent.

As gel concentration, S · Hjertrn: “Archives of Biochemistr
y and Biophysics ” 1 (Suppl.), 147-151 (1962), the amount of the monomer and the cross-linking agent relative to the volume of the gel medium consisting of the monomer, the cross-linking agent and water is displayed. The total amount used is in the range of about 3 w / v% to about 30 w / v%.

When the gel-forming liquid is cross-linked and polymerized on the surface of the planar support (or cover sheet), the cast-coating of the gel-forming liquid and the subsequent cross-linking polymerization are carried out in the absence of molecular oxygen such as in a nitrogen gas atmosphere. It is preferable to carry out crosslinking polymerization by covering the surface of the gel-forming liquid which has been carried out or is cast and coated immediately with a covering material such as a covering film, sheet or plate. As the coating material used for this purpose, the same material as the above-mentioned planar support can be used. When the cover film is an organic polymer film, its thickness is about 300 μm or less, which is about 4 as a practical range.
μm to about 200 μm, preferably about 4 μm to about 100 μm
m. When the coating material is a glass plate, the same thickness as the flat glass plate used as the support can be used.

Generally, the concentration gradient of the acrylamide compound and the cross-linking agent in the gel medium membrane (polyacrylamide aqueous gel concentration gradient) is low on the nucleic acid sample injection side and high on the sample outflow side. As a general rule, the thickness gradient of 3 is provided on the inlet side of the nucleic acid sample and thicker on the outflow side of the sample, but it goes without saying that other gradient arrangements can be adopted depending on the purpose. The gel film thickness and concentration gradient (curve or straight line) that characterize the gel medium film of the present invention are as follows with respect to the distance from the sample injection end.
A portion of a curve of gradual change represented by a function such as a straight line, a loosely bent line, an exponential function, a logarithmic function, a catenary, a tracing line, a parabola, a hyperbola, an ellipse, and a cubic pole line, and any other gradual It is possible to have a gradient represented by a curve of change or a combination of a curve and a straight line. The range of thickness to be changed is about 50 μm to about 5 mm, preferably about 80 μm to about 1000 μm.
The range is m. Further, the range of the concentration gradient (gradient of polyacrylamide type aqueous gel concentration) of the acrylamide compound and the cross-linking agent to be changed is in the range of about 3 w% to about 30 w%, preferably about 4 w% to about 25 w%. Although any combination of the above gradients is possible as the combination of the film thickness gradient and the concentration gradient, the film thickness gradually increases monotonically and the concentration gradually decreases monotonically with the distance along the electrophoresis direction. The combination that makes the film thickness substantially constant in the middle and increases from the middle and decreases the concentration in the middle to become substantially constant in the middle, gradually decreases the film thickness monotonically and gradually increases the concentration. A combination of monotonically increasing, a combination of decreasing the film thickness halfway and making it substantially constant from the middle, and making the concentration substantially constant halfway and increasing from the middle is preferable. Further, the shape of the sample injection portion can be selected and provided from known shapes such as a rectangular shape, a square shape, a triangular shape (shark's teeth shape) and a circular shape.

As a method of providing a film thickness gradient to the gel medium film, there is a thickness change substantially equivalent to a predetermined controlled gradual thickness change (layer thickness gradient or film thickness gradient) on the planar support. A method in which the spacer plate is fixed and the gel forming liquid is poured into the mold covered with the cover coating material (sheet-like material) along the spacer plate to cross-link and polymerize, and a thickness corresponding to a predetermined gradual change in thickness. A spacer plate with a certain thickness is fixed on the surface of a flat support (or cover sheet) with varying thickness, and a gel forming liquid is poured into the mold covered with the cover material (support) along the spacer plate. Method of cross-linking polymerization, casting a gel-forming liquid on the surface of a flat support (or cover sheet) having a thickness change corresponding to a predetermined gradual thickness change, No oxygen Controlling the flow rate per unit time so as to correspond to the method of cross-linking polymerization under existing conditions and the predetermined gradual thickness change (the flow rate is small in the region where the gel film thickness is thin, and the flow rate is in the region where the gel thickness is thick). While increasing)
It is possible to apply a method in which a gel-forming liquid is cast on the surface of a flat support (or a cover sheet) and cross-linked and polymerized in the absence of molecular oxygen such as in a nitrogen gas atmosphere.
A flat support having a predetermined gradual thickness change is formed by a mold casting method, a chemical etching method,
A material prepared by a known technique such as a cutting method can be used. When casting is applied on the support (or cover sheet) while controlling the flow rate, the change in thickness of the spacer plate does not necessarily correspond to the predetermined gradual change in thickness of the gel film. In addition, the thickness gradient of the gel medium is not provided over the entire width of the gel film, but is provided only in a region slightly wider than the region of the lane in which the sample is electrophoresed, corresponding to the number of lanes. It is also possible to keep the thickness constant. This embodiment can be advantageously carried out in an embodiment using a planar support or cover sheet having a gradual thickness change.

As a method of providing a gel concentration gradient in a gel medium film, edited by Horio and Yamashita, “Basic Experimental Methods for Proteins and Enzymes” (Nankodo, 19
(Issued in 1981), pages 304-308, as described in JP-A-54-43881, an aqueous solution containing a monomer (acrylamide), a cross-linking agent, and a polymerization initiator composition in a relatively high concentration was added. A container and a container containing an aqueous solution containing a monomer (acrylamide), a cross-linking agent, and a polymerization initiator composition at a relatively low concentration are connected by a conduit (a high-concentration aqueous solution container is at the same height as the low-concentration aqueous solution container or There is a method of using a device which is arranged at a relatively higher position than the low-concentration aqueous solution container) and which is connected from the low-concentration aqueous solution container via a pump to a casting coating device by a conduit. When this device is used, the high-concentration aqueous solution is moved to the low-concentration aqueous solution container by gravity at the same time as the solution is sent from the low-concentration aqueous solution container while stirring the low-concentration aqueous solution, and the liquid heights of both liquids are always kept equal. The concentration of the low-concentration aqueous solution gradually increases, and the concentration gradient of acrylamide and the cross-linking agent appears in the casting apparatus, and the desired gel concentration gradient is obtained after the cross-linking polymerization.

The gel medium membrane of the present invention can be prepared in the same manner as a known polyacrylamide-based aqueous gel medium membrane. Also,
The gel medium film of the present invention can be used for horizontal and vertical slab electrophoresis and the like according to known methods described in the above-mentioned documents and patent specifications.

Example 1 and Comparative Example 1 A smooth surface having a surface made hydrophilic by ultraviolet irradiation treatment has a thickness of 18
0 μm, rectangular 20 cm x 40 cm rectangular transparent and colorless polyethylene terephthalate (PET) sheet (support) along both long sides with a constant thickness of 300 μm and width of 10 mm x length of 40 cm
The spacer plate of was fixed by adhesion. 1st on this support
The thickness of the gel forming liquid film is changed from 150 μm to 300 μm while mixing the two gel forming liquids having the compositions shown in Tables A and B with the known concentration gradient forming device described in the detailed description of the invention. While controlling the flow rate in such a range to change the thickness of the gel-forming liquid film on the support, cast coating in a nitrogen gas atmosphere,
Each gel-forming liquid film is irradiated with a 500W xenon discharge lamp in a nitrogen gas atmosphere to cross-link and polymerize, and then a rectangular colorless transparent film having a thickness of 63 μm and a size of 20 cm × 40 cm is formed on the formed gel film.
A PET sheet as a cover sheet was adhered and laminated to prepare two polyacrylamide aqueous gel membranes having the gel cross-section thickness change and gel concentration change shown in FIGS. 1 and 2.

On the other hand, the flow rate of the gel forming liquid is 200 μm when the thickness of the gel forming film is 200 μm.
A gel film having a constant thickness of 200 μm and having only a gel concentration gradient (Comparative Example 1-1), and gel formation of the composition shown in Table 1C A constant thickness of 200 μm was obtained in the same manner as in Example 1 except that the thickness of the gel forming liquid film was fixed to 200 μm using a liquid.
A gel film (Comparative Example 1-2) having a constant gel concentration was prepared.

Gel film (Fig. 1): linearly increasing from minimum thickness (starting part in migration direction) of 150μm to maximum (end part in migration direction) 300μm; maximum gel concentration (starting part in migration direction) from about 20% to almost arc It is partially reduced to the minimum (end of migration direction) of about 6.0%. Gel film (Fig. 2): The maximum thickness (starting part in the migration direction) is reduced from 300 μm to a linear shape and the minimum (end part in the migration direction) is 150 μm; The minimum gel concentration (starting part in the migration direction) is approximately 6.0% and almost linear. Up to about 20%. Gel membrane (Fig. 3): Constant at a thickness of 200 μm; minimum gel concentration (starting portion in migration direction)
Increased almost linearly from about 6.0% to the maximum (end of migration direction) about 20%. Gel film (Fig. 4): constant at a thickness of 200 μm; constant at a gel concentration of about 8.0%. Using the obtained 4 kinds of gel membranes, the DNA sequence analysis experiment was performed on M13-mp8 DNA by D prepared by the dideoxy method.
The NA fragment sample was used to carry out according to a conventional method.
As a result, the range of readable base fragments in the gel film having the combination of the thickness gradient and the gel concentration gradient of the present invention was as follows.

Gel membrane Nos. 60 to 250 Gel membranes No. 60 to 240 The width and interval of the electrophoretic image of the base fragment in each lane did not become extremely narrow within the readable range.

On the other hand, the readable range of the conventional gel film having a constant thickness and only a gel concentration gradient and the gel film having a constant thickness and a constant gel concentration were as follows.

Gel Membrane No. 60-220 Gel Membrane No. 60-200 In the gel membrane, the width and interval of the electrophoretic image of the base fragment in each lane gradually narrowed from the low molecular weight portion to the high molecular weight portion.

From this result, the polyacrylamide aqueous gel electrophoresis medium membrane having the combination of the thickness gradient and the gel concentration gradient of the present invention shows that the nucleobase fragment has a uniform lane distribution over a wide molecular weight range from a low molecular weight portion to a high molecular weight portion. It was clarified that the separation was maintained, good separation was obtained, and the number of readable base fragments was large, enabling highly accurate DNA base sequence analysis.

[Brief description of drawings]

1 and 2 show a gradual thickness change (film thickness gradient) along the electrophoretic direction and a gradual change in thickness of a gel film having a combination of the thickness gradient and the gel concentration gradient of the present invention of Example 1. It is a cross-sectional schematic diagram which shows gel concentration change (concentration gradient). FIG. 3 is a schematic cross-sectional view showing a constant thickness along the electrophoresis direction and a gradual gel concentration change (concentration gradient) of a gel film having a constant thickness and a gel concentration gradient in Comparative Example 1-1, which is a conventional technique. Is. FIG. 4 is a schematic sectional view showing a constant thickness and gel concentration along the electrophoresis direction of a gel film having a constant thickness and a constant gel concentration in Comparative Example 1-2 which is a conventional technique.

Claims (6)

[Claims] 1. A layer comprising an electrophoretic gel medium containing a polyacrylamide aqueous gel obtained by cross-linking and polymerizing an acrylamide compound and a cross-linking agent in the presence of water, and a gel medium for electrophoresis containing a compound containing at least one carbamoyl group as a modifier. In an electrophoretic medium film comprising a flat support and a flat cover sheet, the gel medium layer has a gradual change in thickness along the electrophoresis direction and a gradual change along the electrophoresis direction. An electrophoretic medium film having a polyacrylamide-based aqueous gel concentration change together. 2. The electrophoretic medium film according to claim 1, wherein both the support and the cover sheet are sheet-like materials made of an organic polymer. 3. The electrophoretic medium film according to claim 2, wherein both the support and the cover sheet are sheet-like materials made of polyethylene terephthalate. 4. The electrophoretic medium film according to claim 1, wherein the compound containing at least one carbamoyl group is urea. 5. The electrophoresis medium film according to claim 1, wherein the gel medium further contains agarose. 6. The electrophoretic medium film according to claim 1, wherein the gel medium further contains a water-soluble polymer.