JPS6370156A - Medium for electrophoresis - Google Patents

Abstract

PURPOSE:To improve workability of a gel medium, by getting a small amount of water soluble cellulose derivative contained in a medium for electrophoresis containing a compound having a carbamoyl group as modifier and a polyacrylamide based water gel. CONSTITUTION:An aqueous solution for the formation of an acrylamide gel which contains an acrylamide based compound, a crosslinking agent, a compound having a carbamoyl as modifying agent and a small amount of water soluble cellulose derivative is cast into a glass mold made of two sheets of glass plates having a spacer plate fixed at the periphery thereof in opposition to each other. The gel forming solution is irradiated with a mercury lamp of about 100W, causing a crosslinked polymerization to form a polyacrylamide water gel medi um film for electrophoresis. This enhances the hardness of the gel film to facili tate the punching of a sample injection slot and improves the workability with a smooth cut surface of the gel film.

Description

Detailed Description of the Invention [Field of Industrial Application] The present invention relates to a polyacrylamide-based aqueous gel electrophoresis medium used for nucleic acid (DNA, RNA) base sequencing, and in particular to a gel medium for nucleic acid (DNA, RNA) base sequencing. This invention relates to the composition of a polyacrylamide-based aqueous gel electrophoresis medium that maintains high separation performance for base fragments, increases the hardness of the gel medium, and improves processability.

[Prior art] Nucleic acid (DNA) by chemical decomposition method, dideoxy method, etc.

Slab electrophoresis using a polyacrylamide-based aqueous gel electrophoresis medium membrane (hereinafter sometimes referred to as polyacrylamide gel membrane or gel membrane) is an essential operation in the base sequencing method of RNA). The polyacrylamide gel membrane used for this purpose consists of about 95 parts by weight of a monoflexure such as 1-superpacked acrylamide and about 5 parts by weight of a di- or polyfunctional crosslinker such as N,N'-methylenebisacrylamide. Approximately 3 w% ~ including mixture and polymerization initiator
It is formed by cross-linking and polymerizing a monomer and a cross-linking agent in an approximately 30 w% aqueous solution (liquid layer or liquid film state).
N conversion is being carried out.

In recent years, electrophoretic analysis has become more frequently used, and the demand for polyacrylamide gel membranes has increased dramatically. Therefore, it is desired to establish a system that can produce a large number of polyacrylamide gel membranes suitable for electrophoresis in a short time, that is, mass-produce them, and supply them to electrophoresis operators.

One method that enables mass production of polyacrylamide gel membranes is to move a long organic polymer film continuously at a constant speed, and apply a cross-linked polyacrylamide gel forming solution (hereinafter referred to as "polyacrylamide gel forming solution") onto the film. An advantageous method is to apply a gel-forming solution (referred to as a gel-forming liquid) in an inert gas atmosphere (for example, in nitrogen gas) and to subject the coated layer to radical crosslinking polymerization.

On the other hand, with the development of the dideoxy method, nucleic acids (DNA, RNA)
) There has been an increasing demand for polyacrylamide gel membranes that can accurately separate even the high molecular weight portions of fragments.

In order to make a long electrophoresis medium into a usable form, it is necessary to process the polyacrylamide gel membrane with a knife, such as cutting and adding sample slots, to avoid destruction of the gel membrane or the shape of the area near the cut surface. without any change in
It must be possible to implement it easily and reliably. However, with conventional gel membranes, when the acrylamide concentration is lowered, the gel becomes softer.

Processing with a knife was difficult.

[Object of the Invention] The object of the present invention is to improve the electrophoresis 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 an improved gel medium that increases the hardness of the gel medium while maintaining high separation performance by electrophoresis so that processing can be carried out easily and reliably.

[Structure of the Invention] The present invention includes an aqueous polyacrylamide gel formed by crosslinking polymerization of an acrylamide compound and a crosslinking agent in the presence of water, and a compound having at least one carbamoyl group as a modifier. The electrophoresis medium is a polyacrylamide-based aqueous gel electrophoresis medium that further contains a small amount of a water-soluble cellulose derivative.

[Detailed explanation of the structure of the invention] In the present invention, an acrylamide compound (monomer) and a crosslinker (the three agents are an aqueous solution or a water dispersion) are dissolved or dispersed in water overnight, and both are crosslinked and polymerized in water. to form a cross-linked aqueous gel medium. In this specification, unless otherwise specified, the term ``dissolved in water'' includes both ``dissolution'' and ``dispersion'', and the term ``aqueous solution'' includes both an aqueous solution and an aqueous dispersion. The solvent or dispersion medium includes not only water but also a water-organic solvent mixture containing an optionally added organic solvent.

Acrylamide compounds that can be used in the present invention (
Examples of monomers include acrylamide.

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

There are acrylamide 1ζ 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, r E I electrophores
i 5J2(4), 213-219 (1981),
Bifunctional crosslinking agent compounds described in the same magazine 2(4), 220-228 (+981) etc., JP-A-Sho [3! -2058 and the like can be used. Specific examples of bifunctional crosslinking agents include N,N'-methylenebisacrylamide (BIS); N,N''
-Propylene bisacrylamide (PBA); diacrylamide dimethyl ether (DAE); 1,2-diacrylamide ethylene glycol (D E C); ethylene ureabis acrylamide (E U B); ethylene diacrylate (EDA); N, N' -Diallyltartardiamide (D A T D ); N,N'-bisacrylylcystamine (B A C). Specific examples of the trifunctional crosslinking agent include 1,3,5-triacryloylhexahythro S-triazine (TAHT); ) realyl cyanurate (TAC); ) realyl isocyanurate (TA, TC). Among these crosslinking agents, BIS and TAHT are preferred. Two or more crosslinking agents may 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 10w
Used in the range of %.

Agarose can be added to the gel medium of the invention. As for agarose, JP-A-55-5730, JP-A-55-110946, JP-A-57-502098
Any of low electroosmotic agarose, medium electroosmotic agarose, and high electroosmotic agarose described in JP-A-59-126236 and the like can be used. The amount of agarose added is about 0.2 v/v% to about 2.0 v/v%, preferably about 0.3 v/v% to about 1.2 v/v%, based on the volume of the aqueous gel containing the monomer and crosslinking agent. /v% range.

Water-soluble polymers other than water-soluble cellulose derivatives may be added to the gel media of the present invention.

Examples of water-soluble polymers include JP-A-59-126236;
Addition polymerization or condensation polymerization type water-soluble nonionic polymers with a molecular weight ranging from about 10,000 to about 1,000,000 as described in JP-A-60-60548, vinyl sulfonyl groups as described in JP-A-61-18852, etc. A crosslinkable acrylamide-based polymer can be used. Examples of addition polymerizable water-soluble nonionic polymers include polyacrylamide, polyvinyl alcohol, and polyvinylpyrrolidone. Examples of condensation type water-soluble nonionic polymers include polyethylene glycol and polypropylene glycol.

As an example of a crosslinkable acrylamide copolymer, N-
[[3-(vinylsulfonyl)propanamidocomethylcoacrylamide-acrylamide copolymer; N-
[[3-(2-chloroethylsulfonyl)propanamidocomethylcoacrylamide-acrylamide-N-(
1,1-dimethyl-3-oxobutyl)acrylamide copolymer. Among these water-soluble polymers, polyacrylamide, polyethylene glycol, N-
[[3-(vinylsulfonyl)propanamidocomethylcoacrylamide-acrylamide copolymer is preferred. In the case of addition polymerization type or condensation polymerization type water-soluble nonionic polymer, the amount of water-soluble polymer added is about 2ν% to about 109% based on the total weight of monomer and crosslinking agent, preferably about 5 w% to about 50 w% for crosslinkable acrylamide-based copolymers.

The amount ranges from about 1 w% to about 50 w%, preferably from about 5 v% to about 40 w%, based on the weight of the acrylamide compound.

The timing of adding agarose and/or water-soluble polymer to the gel medium is usually preferably between the time when the monomer and crosslinking agent are dissolved in water and the time when the polyacrylamide-based aqueous gel is formed.

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

C8F, 7S O2N - (CtoI2CH2O)1
．． Nonionic W surface active agents such as IH.

Na03S -CHCOOCH2CH(E t)C4H
Anionic surfactants such as 9CH2C00CH2CH(Et)C4H9.

e C1l H23CON H-(CH2)3-N" -C
There are amphoteric surfactants such as H2COO-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.

In the range of from about 1X10-4 to about 5XIO" w/v%, preferably from about 1X10-3 to about IX10"w/v%, for anionic surfactants from about 1X10-4 to about 5X]0 -2% v/v, preferably in the range from about 1X10-3 to about 5X to-2% v/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 40w/v% to about 60v/v% based on the volume of the aqueous gel containing the monomer and crosslinking agent.
V% range. When using urea as a denaturing agent, from about 6 mol (about 360 g) to saturated solution (about 420 g), preferably from about 7 mol to saturated solution IW, It is used in the range up to fl. 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 water-soluble cellulose derivative which characterizes the component of the gel medium of the present invention has some or substantially all of its hydroxyl groups formed by an alkyl group having 1 to 3 carbon atoms or a hydroxyalkyl group having 1 to 4 carbon atoms. It is an etherified water-soluble cellulose ether. Examples of cellulose ethers include methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, and hydroxybutylmethylcellulose.
Among these, methylcellulose is preferred. Water-soluble cellulose ether generally has a molecular weight of about 8,000 to about 100.
10,000, preferably in the range of about 10,000 to about 300,000. The amount of water-soluble cellulose derivative added is about 1.
% Ow/v or less, preferably in the range from about 0.05% to about 5%. The water-soluble cellulose derivative is preferably added between the time when the monomer and the crosslinking agent are dissolved in water and the time when the polyacrylamide-based aqueous gel is formed.

The gel TF of the present invention and the medium for pneumophoresis can contain a known ρ)1 buffer to adjust the PAL value during electrophoresis to a range of 8.0 to 9.0. As buffering agents that can be used, 9. Chemical Handbook, Basic Edition, edited by the Chemical Society of Japan, Tokyo,
Maruzen ■, published in 1966) pp. 1312-1320,
RoM, C, Dawson et al. Dat
a for B iochemical research
hJ 2nd edition (Oxford at the C1are
ndonPress, published in 1969) 476-5
p. 08, r B iochemistry J5, 467
pages onwards (1966), rAnalytical
Biochem-istryJ 104.300-31
There is a plim shock agent system described in, for example, p. 0 (1980).

Specific examples of p)l buffers include 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, etc.
Salt or I (salt etc., N-[)lis(hydroxymethyl)
Methyl]-3-aminopropanesulfonic acid Na salt or salt, etc., and an acid that is optionally combined with any of these.

There are alkalis or salts. An example of a preferred p++ softening agent system is Tris-M-EDTA-2Na salt (pl+8
．． 2 to 8.3).

It is generally preferred that the gel medium of the present invention be substantially colorless and transparent for the detection or reading of electrophoretic images.

When the gel medium of the present invention is used as a gel membrane, it is preferably provided on a substantially electrically nonconductive, water-impermeable, smooth-surfaced sheet-like (film-like or flat-like) support. . As the substantially electrically non-conductive, water-impermeable, smooth surface sheet-like support, known glass plates, organic polymer sheets, etc. can be used. Specific examples of organic polymer sheets include polyethylene terephthalate, bisphenol polycarbonate, and polystyrene.

Cellulose esters (e.g. cellulose diacetate).

(e.g. cellulose triacetate, cellulose acetate propionate, etc.) with a thickness in the range of about 50 to about 2 mm, preferably in the range of about 80 to about 500 um, i.e., with a wavelength of about 200 nm to about 90 nm.
There is a sheet-like or flat-like support having a smooth surface that is transparent to electromagnetic radiation in at least a part of the range of wavelengths within the +1 range. When using an organic polymer support, in order to make the surface hydrophilic and improve adhesion with the gel film, it may be subjected to ultraviolet irradiation, glow discharge treatment, corona discharge treatment, flame treatment, electron beam irradiation, chemical etching, electrolytic etching, etc. Any known surface treatment method can be applied.

The surface of the polymer support may be coated with JP-A-59
-IG4950. JP-A-59-212752, JP-A-60-194349.

JP-A-60-239658, JP-A-60-24485
0, it is possible to provide a subbing layer or an adhesive layer as described in JP-A No. 61-14557 and the like to strengthen the bond between the gel film provided on the support and the support.

The gel medium of the present invention is produced by casting or coating an aqueous solution (hereinafter sometimes referred to as gel forming solution) containing the above-mentioned components and a radical polymerization initiator composition onto a planar support in a layered manner.
In the absence of molecular oxygen, if necessary, ultraviolet or visible light! It is produced and used as a polyacrylamide-based aqueous gel medium membrane in which a monomer (acrylamide-based compound) and a crosslinking agent are cross-linked and polymerized by Ig and/or heating.

As a radical polymerization initiator composition, r E 1ectr
o-phores 1sJ2(4), 2+3-21
9 (1981), same magazine 2 (4), 220-228 (1
981), JP-A-59-12G236, Aomizu and Nagai (eds.), "Latest Electrophoresis Methods J" (published in 1973), etc., low-temperature radical polymerization initiator compositions can be appropriately selected and used. As an example of the polymerization initiator composition, β-(dimethylamino)propionitrile (DMD
PN) - ammonium peroxonisulfate mixture; N,
N,N',N'-tetramethylethylenediamine (TE
MED) - ammonium peroxonisulfate mixture; TE
MED-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 rays or visible light is used) ) etc. The amount of the radical polymerization agent NH component added is about 0.3v% to about 5.0w%, preferably about 0.5% to about 5.0w%, based on the total weight of the monomer and crosslinking agent.
% to about 3.0v%.

The gel concentration of the electrophoresis medium of the present invention is S.

Archives of
B 1oche*1try andB 1o
physicsJ 11 (5upp1.), 147-1
51 (1962) and expressed in terms of the volume of the gel medium consisting of monomer, crosslinker and water,
The total amount of monomer and crosslinking agent is about 3 w/v% to about 30 w/v%.
It is used in the range of v%.

When the gel medium of the present invention is used in the form of a film, the thickness of the gel film is about 50 mm to about 5 mm, preferably 8'180 mm.
The range is approximately 500 um. The gel membrane of the present invention has good separation ability even when made into a thin layer, so the thickness of the gel membrane is approximately 50μ.
The resolution of the electrophoretic image can be improved by setting mfM to about 300 um.

When cross-linking the gel-forming solution on the surface of a planar support, the casting application of the gel-forming solution and the subsequent cross-linking polymerization are carried out in the absence of molecular oxygen, such as in a nitrogen gas atmosphere. or immediately cover the surface of the gel-forming solution by casting.

It is preferable to cover with a covering material such as a sheet or plate and carry out crosslinking polymerization. The covering material ↑:I used for this purpose may be made of the same material as the above-mentioned planar support. When the cover film is an organic polymeric film, its thickness is less than or equal to about 300 μm, with a practical range of from about 41+ m to about 200 μm, preferably from about 4 IJrn to about 100 μm. When the coating material is a glass plate, its thickness may be the same as that of a flat glass plate.

The gel medium of the present invention can be prepared in the same manner as known polyacrylamide-based aqueous gel media. Further, the gel medium of the present invention can be formed into a gel film and used in accordance with known methods described in the above-mentioned literatures and patent specifications, such as horizontal and vertical slab electrophoresis.

Neko I Wataru Example 1 and Comparative Example 1 Smooth surface thickness 2 mm, size 20 cm x 40 cm
Two long flat glass plates with a thickness of 400u at the edges.
A glass mold was formed by fixing and opposing spacer plates having a width of m+10 mm, and the acrylamide gel-forming aqueous solution listed in Table 1 was poured into the glass mold. At an ambient temperature of 25°C, each gel-forming solution was irradiated with a 100-V high-pressure mercury lamp from a distance of locm and allowed to stand for 10 minutes. Cross-linking polymerized between two glass plates to form a 4001 Jm thick polyacrylamide aqueous gel for electrophoresis. A media film was formed.

When the elastic modulus of the four types of gel films obtained was measured, the first
It was as described in the table.

Using these gel medium membranes for electrophoresis, an experiment of NA base sequence analysis was carried out according to a conventional method using a DNA fragment sample prepared by the dideoxy method for M+3-mp8DN, □~.

It showed a normal electrophoresis pattern and was able to perform highly accurate DNA sequence analysis.

As described above, it is clear that the gel film of the present invention has improved workability in that the hardness is increased, the punching process of the Zamble injection slot is easy, and the cut surface of the gel film is smooth, and it is also possible to perform DNA base sequence analysis. It is clear that the electrophoretic properties suitable for use are maintained.

Part 1 Table 1! I BiS: NIN'-methylenebisacrylamide Tris: ) Lis(hydroxymethyl)aminomethane TEMED: N, N, N', N'-tetramethylethylenediamine Example 2 and Comparative Example 2 Colorless and transparent polyethylene terephthalate (PET) with a thickness of 1801 ffn, with a diacetyl cellulose undercoat layer of about 0.5 um after the surface was irradiated with ultraviolet rays by the method described in Example 1 of JP-A-239658. A sheet was prepared as a support.This PET sheet was cut into a rectangle of 20 cm x 40 cm, and a thickness of 5 mm was placed on the center side of the PET sheet.
001J] Tl.

A spacer plate with a width of 10 mm was fixed. 1,3 crosslinking agent
゜Four types of acrylamide gel-forming aqueous solutions having the same compositions as in Example 1 and Comparative Example 1, except that 5-triacryloylhexahydro-5-)riazine was used, were applied to this PET.
Casting onto a support in a nitrogen atmosphere at an ambient temperature of 25°C,
Apply a 100W high-pressure mercury lamp to each gel-forming solution from a distance of 10cm! '1. ! The film was allowed to stand still for 10 minutes to undergo cross-linking polymerization, and then a film with a thickness of 631mm was formed on top of the film. lIr1 colorless transparent PET
Closely apply the film as a cover film for 1ffF' and 4
A gel medium membrane for seed electrophoresis was formed.

When the obtained gel medium membrane for electrophoresis was subjected to a DNA base sequence analysis experiment in the same manner as in Example 1, the same results as in Example 1 and Comparative Example 1 were obtained.

Example 3 Thickness: 180 μm, surface made hydrophilic by ultraviolet irradiation treatment,
Colorless and transparent P with a smooth surface, size 20cm x 40cm
A support was prepared in which a spacer plate with a thickness of 300 μm was fixed to the edge of the ET film. A gel-forming solution having the composition shown in Table 2 was cast onto this PET support, and cross-linked and polymerized in a nitrogen atmosphere under irradiation with a 500W xenon discharge lamp at an ambient temperature of 25°C. 631 sheets of colorless transparent PET film are closely laminated as a cover film to make polyacrylamide!・An aqueous gel film was formed.

When the resulting gel film was cut with a knife to provide a shark-teeth-shaped sample injection slot, the cut section was smooth and no breakage of the gel film was observed.

Using this gel membrane, an experiment for DNA base sequence analysis was conducted in the same manner as in Example 1, and the same results as in Example 1 were obtained11).

The polyacrylamide aqueous gel membrane of the present invention has increased hardness while maintaining high resolution, and it is clear that the processability has been improved in that the sample injection slot can be easily punched out and the cut surface of the gel membrane is smooth. .

mouth] wa [mouth Table 2 Components of gel forming solution

Claims (4)

[Claims] (1) An aqueous polyacrylamide gel formed by crosslinking polymerization of an acrylamide compound and a crosslinking agent in the presence of water, and an electrophoresis medium containing a compound having at least one carbamoyl group as a denaturing agent, further soluble in water. An electrophoresis medium characterized by containing a small amount of cellulose derivative. (2) The electrophoresis medium according to claim 1, wherein the content of the water-soluble cellulose derivative is 1.0 w/v% or less based on the volume of the gel medium. (3) The electrophoresis medium according to claim 2, wherein the water-soluble cellulose derivative is methylcellulose. (4) The electrophoresis medium according to any one of claims 1 to 3, wherein the denaturing agent is urea.