JPS6120849A - Medium for electrophoresis - Google Patents

Abstract

PURPOSE:To improve the resolving power and workability of a medium for electrophoresis consisting of a polyacrylamide aq. gel and to make easy the preservation and transportation thereof by adding further a water soluble polymer and agarose thereto. CONSTITUTION:The electrophoresis medium consisting of the polyacrylamide aq. gel for sepn. of DNA, DNA fragments, RNA or RNA fragments is crosslinked and polymerized with a crosslinking agent in an about 0.1-30wt% range by the total weight of an acrylamide compd. and the crosslinking agent. The water soluble polymer is further incorporated therein in an about 2-100wt% range and the agarose in an about 0.2-2wt%/v% range by the weight of the polyacrylamide aq. gel. Since the gel film is formed on a polyethylene terephthalate sheet, the film is easy to handle and has excellent workability in forming a sample injecting port, etc. The dispensing operation of the DNA fragments separated on the gel film is easy as well.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of the Invention] The present invention relates to DNA, DNA fragments, RNA, RN
The present invention relates to an electrophoretic medium for separating A fragments and the like, and more particularly to an electrophoretic medium suitable for use in DNA fractionation and base sequencing operations.

[Background of the Invention] In DNA and RNA base sequencing methods based on post-label methods, slab electrophoresis using a polyacrylamide gel membrane is an essential operation. In particular, as gene-related research progresses in recent years, there is an urgent need to speed up DNA base sequencing operations.

Conventional polyacrylamide gel membranes are made by adding a monomer such as acrylamide to N in the presence of a wax.

It is obtained by crosslinking polymerization using a difunctional crosslinking agent such as N'-methylenebisacrylamide.

Note that when forming a polyacrylamide gel membrane used as an electrophoresis medium for DNA base sequencing, a denaturing agent such as urea or formamide is usually included.

The above polymerization reaction is a radical crosslinking polymerization, and since the reaction is inhibited by the influence of oxygen, the polyacrylamide gel film must be prepared in a state where oxygen is blocked. For this reason, polyacrylamide gel membranes are now generally produced by injecting a gel-forming solution into a cell (having a certain space, e.g. about 0.3 to 1 mm) formed by two glass plates, and then introducing oxygen into the cell. A gel film is formed by cross-linking polymerization in a blocked state.

Electrophoresis using the polyacrylamide gel membrane formed as described above is carried out, for example, as follows.

The polyacrylamide gel membrane is held vertically between glass plates, and after pre-electrophoresis, a sample (for example, a 32F label digested with Maxam-Gilbert) is inserted into a sample slot provided at the top of the gel membrane. D
A fixed amount of NA) is injected - then electrophoresis is performed. After performing electrophoresis for a certain period of time (for example, about 6 hours to 12 hours), carefully remove the glass plate on one side and cover the exposed surface with a synthetic resin film such as polyvinylidene chloride film. Cover and use this to perform autoradiography.

That is, an X-ray film and an intensifying screen are sequentially placed on the film covering the polyacrylamide gel membrane, and exposure is performed at a low temperature (for example, -80°C) for a certain period of time (for example, about 10 to 20 hours). . After exposure, the X-ray film is developed and the base sequence of the DNA can be determined by autoradiography, which involves reading the separation migration pattern of the DNA.

[Object of the invention] The first object of the present invention is to obtain DNA, DNA fragments,
An object of the present invention is to provide an electrophoresis medium with improved resolution suitable for use in electrophoretic analysis of RNA, RNA fragments, etc.

A second object of the present invention is to obtain DNA, DNA fragments,
An object of the present invention is to provide an electrophoresis medium with excellent processability and suitable for electrophoretic analysis for separation of RNA, RNA fragments, etc.

A third object of the present invention is to provide a lightweight and unbreakable electrophoresis medium that is easy to store, transport, etc., and that has improved resolution and processability for DNA, DNA fragments, RNA, and RNA.
An object of the present invention is to provide an electrophoretic medium suitable for electrophoretic analysis for separation of fragments and the like.

[Summary of the Invention] The present invention is a polyacrylamide-based aqueous gel electrophoresis medium in which an acrylamide-based compound and a crosslinking agent are crosslinked and polymerized in the presence of water, further comprising a water-soluble polymer and agarose. Provided is an electrophoretic medium for separating DNA, DNA fragments, RNA, or RNA fragments.

The electrophoresis medium of the present invention exhibits high resolution and has good processability, that is, cutting a polyacrylamide gel membrane with a blade such as cutting an electrophoresis medium produced in a long shape or adding sample slots. can be easily and reliably performed without destroying the gel membrane or changing the shape near the cut surface, the resulting electrophoresis medium exhibits excellent performance and can be easily manufactured in mass production. .

When mass producing the electrophoresis medium, it is advantageous to attach it to a support made of a plastic material. Large-scale annual production is facilitated, and since the support is lightweight and unbreakable, storage and transportation of the electrophoresis medium are facilitated. Therefore, polyacrylamide gel membranes (electrophoresis medium) are produced intensively, and DNA
Provided to electrophoresis operators for the analysis of , RNA, their fragments, etc. This method is extremely effective in realizing a system for producing and using gel membranes that provides

[Detailed Description of the Invention] The support for the electrophoresis medium of the present invention includes a glass plate,
Any water-resistant sheet-like support can be used, such as polymer-coated paper, sheets of plastic material, and the like. However, in order to take advantage of the electric plate movement medium of the present invention,
Preferably, a sheet of plastic material is used as the support.

Various plastic sheets can be used as the plastic sheet support for the electrophoresis medium. Preferred examples of plastic sheets include wood-philic polymers or polymers whose surfaces have been made hydrophilic by known surface treatments (e.g., polyethylene terephthalate, bisphenol A).
Sheets (including films and plates) of polycarbonate, polyvinyl chloride, vinylidene chloride-vinyl chloride copolymer, polymethyl methacrylate, polyethylene, polypropylene, cellulose acetates, cellulose acetate propionate, etc.) be able to. A particularly preferred support is polyethylene terephthalate.

Next, the electrophoresis medium (hereinafter also referred to as gel medium, polyacrylamide gel membrane, or simply gel membrane) will be explained.

As mentioned above, polyacrylamide gel membranes are produced by preparing a gel-forming liquid by dissolving or dispersing an acrylamide compound and a crosslinking agent in water as an aqueous solution or dispersion, and then crosslinking and polymerizing both in the liquid. It can be obtained by forming a cross-linked aqueous gel film. In addition,
In this specification, unless otherwise specified, "in water" simply means "dissolved" (in water) and "dispersed" (in water).
It is called dissolution, and both aqueous solutions and aqueous dispersions are simply called aqueous solutions. The solvent or dispersion medium also includes a mixture of an organic solvent and water that may be added as desired.

Examples of acrylamide compounds that can be used in the present invention include acrylamide, acrylamide homologues such as N-methylacrylamide, N,N-dimethylacrylamide, N-(hydroxymethyl)acrylamide, and diacetone acrylamide, and methacrylamide compounds. Examples include methacrylamide, and these compounds can be used alone or in combination of two or more. Among these acrylamide compounds, acrylamide is most preferred, and a combination of acrylamide and one or more other acrylamide compounds is also preferred.

Examples of crosslinking agents used in the production of the electrophoresis medium of the present invention include rElectropboresis.
Known compounds described in J19B1, 2.213-228 and the like can be used. Examples of crosslinking agents include N, N
'-Methylenebisacrylamide (BIS); N.

N-propylene bisacrylamide (PBA); di(
Acrylamide dimethyl) ether (DAE); 1,2
-Diacrylamide ethylene glyco-Jlz (DEC)
; Ethylene ureabisacrylamide (EUB); Ethylene diacrylate (EDA);
rdiaa+ide: D A T D ) ;N,
N'-bisacrylylcystamine (N,N'-hisa
Difunctional compounds such as crylylc testamine, RA It will be done. The crosslinking agents may be used alone or in combination of two or more.

The crosslinking agent is used in an amount of about 0.1 to 30% by weight, preferably about 1 to 30% by weight based on the total weight of the acrylamide compound and the crosslinking agent.
It is used in an amount in the range of 10% by weight.

The gel concentration of the polyacrylamide gel used in the production of the electrophoresis medium of the present invention is S, Hje
rten: r Arch, Biochem, Bi
ophys, Jl (5upp1.), 147 (I
n2), the amount of the acrylamide compound and the crosslinking agent is about 3 to 50% based on the volume of the gel medium consisting of the acrylamide compound, the crosslinking agent, and water.
A range of 30 wt/V% is preferably used.

A water-soluble polymer is added to the electrophoresis medium of the present invention. As the water-soluble polymer, an addition polymer type or a polycondensation type water-soluble polymer can be used. Examples of addition polymers include nonionic water-soluble polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and polyacrylamide. Examples of condensation polymers include nonionic water-soluble polyalkylene glycols such as polyethylene glycol and polypropylene glycol. Among these water-soluble polymers, polyacrylamide and polyethylene glycol are preferred.

The molecular weight of the water-soluble polymer is preferably in the range of about 10,000 to about 1,000,000. The water-soluble polymer is used in a range of about 2 to 100% by weight, and preferably in a range of about 5 to 50% by weight, based on the total weight of monomer and crosslinker.

The addition of a water-soluble polymer makes the polyacrylamide gel film plastic, which prevents it from breaking during cutting, and the gel film also has plasticity when drying, which has the advantage of improving brittleness and making it less likely to break. be. Furthermore, the viscosity of the gel film can be controlled by selecting the molecular weight and amount of the water-soluble polymer added.

The polyacrylamide gel membrane of the present invention further contains agarose. Any agarose can be used, and any of low electroosmotic, medium electroosmotic, and high electroosmotic agaroses can be used. Examples of agarose that can be used include JP-A-55-
No. 5730, Japanese Patent Publication No. 110946/1983, Special Publication No. 1983
There are agaroses and the like disclosed in various publications such as No.-502098. The amount of agarose is approximately 0.1 to 2 wt/with respect to the volume of the aqueous gel containing the acrylamide compound and the crosslinking agent.
v%, preferably about 0.3 to 1.2 w/v%.

When the polyacrylamide gel film contains agarose, it is possible to control the viscosity of the gel forming solution to an appropriate level by changing the temperature of the gel forming solution, which makes it possible to stop its fluidity and to prevent the gel film from forming. There is an advantage that molding becomes easier during the molding operation.

The electrophoresis medium of the present invention can contain a pH buffer. As a buffer, pH 8.0 to 10.0
(preferably pH 8.0 to 9.0)
It is possible to use an appropriate buffer selected from known buffering agents capable of buffering the temperature.

As a buffering agent that can be used, Nippon Kagaku complete edition "Chemistry Handbook Basic Edition" (Tokyo, Marukubi M 1! Published in 366) 1312-
Page 1320: Aomizu and Nagai eds. “Latest electrophoresis methods” (
Tokyo, Applied Shoten, published in 1873, pages 320-322; r [1ata for Biochemical
Re5search” (R,M.

C, Daw, son et al, i, 2nd edition, 0xf
ord at theClarendon Press
, published in IH9) 47El-508 pages; r Bi
oche+1istry J ”4.4137 (1
98B), rAnalytical Biochem
istryJ 104.300-310 (1980) and the like.

Examples of buffers include tris(hydroxymethyl)aminomethane (Tris) [CAS Registry
No? ? -88-1], N, N-bis(2-hydroxyethyl)glycine 7 (Bicine), N
-2-hydroxyethylpiperazine-No-2-hydroxypropane-3-sulfonic acid Na salt or salt, etc.
Na salt or salt of N-2-hydroxyethylpiperazine-No-3-propanesulfonic acid, N-[)lis(
Examples include the Na salt or di-salt of hydroxymethyl)methylco-3-7minopropanesulfonic acid, and acids, alkalis, or salts in combination with any of these if necessary. An example of a particularly preferable buffer is Tr
is, a combination of boric acid and EDTA 2Na salt (pH
8, 3).

The electrophoresis medium of the present invention can also contain a polyol compound such as glycerin or ethylene glycol as a wetting agent. The content of the polyol compound is selected from a range of about 5 to 40 w/v% based on the volume of the electrophoresis medium. Among these compounds, glycerin is particularly preferred. By adding a wetting agent, it is possible to prevent polyacrylamide gel membranes from drying out due to extreme water evaporation during storage, and also to prevent brittleness caused by extreme dryness and prevent cracking of gel membranes. There is an advantage that physical properties are improved.

The electrophoresis medium of the present invention can contain an antioxidant, if necessary. As the antioxidant, various compounds known to be able to be incorporated into polyacrylamide gel membranes can be used. Examples of antioxidants include dithiothreitol, 2-mercaptoethanol, and the like.

As mentioned above, the electrophoresis medium (reacrylamide gel membrane) of the present invention typically contains a monomer represented by acrylamide, a polyfunctional crosslinking agent, a water-soluble polymer, agarose, etc. It is obtained by radical crosslinking polymerization of an acrylamide compound and a crosslinking agent in a substantially homogeneous aqueous solution, and the water-soluble polymer and agarose are substantially added to the three-dimensional crosslinked polymer formed from the acrylamide compound and the crosslinking agent. It is presumed that the latter two polymer chains and the three-dimensionally crosslinked polymer are intertwined with each other.

The radical crosslinking polymerization reaction used in the present invention can be carried out in the absence of molecular oxygen in the presence of peroxide and/or by known methods such as ultraviolet irradiation. moreover,
This crosslinking polymerization reaction can also be accelerated by heating or ultraviolet irradiation.

As a catalyst for radical crosslinking polymerization, rElectr.

phoresisJ 11381.2L213-21J
19B1.2.220-228; The low-temperature radical polymerization initiators can be appropriately selected and used from among the known low-temperature radical polymerization initiators described in "Latest Electrophoresis Methods J" (published in 1873), edited by Aomizu and Nagai. Preferred radical polymerization Specific examples of initiators include β-dimethylaminopropionitrile (DM
A PN) 11beroxonisulfate ammonium mixture,
Examples include a N,N,N',N'-tetramethylethylenediamine (TEMED)-ammonium peroxonisulfate mixture, a TEMED/riboflavin mixture, a combination of TEMED/riboflavin or a hydrogen peroxide mixture and ultraviolet irradiation. The content of the radical polymerization initiator is approximately 0.3 to 0.3 to the total weight of the acrylamide compound and crosslinking agent.
5% by weight, and preferably ranges from about 0.5 to about 3% by weight.

The polyacrylamide gel membrane of the present invention is produced by coating a gel-forming liquid on a support having a smooth surface by a known method to form a coated layer, and then subjecting the coated layer to crosslinking polymerization. However, when applying the gel-forming liquid to the support, in order to improve the adhesion between the electrophoresis medium and the support, an adhesive layer is provided on the surface of the support in advance, and the gel-forming liquid is applied onto this. It is also possible to use a gelling method. The application of an adhesive layer is particularly advantageous when plastic carrier sheets are used.

When the gel-forming liquid is cross-linked and polymerized on the surface of the support, the gel-forming liquid can be further covered with a covering material such as a cover film, sheet, or plate before or after the cross-linking polymerization. A cover film or the like used for this purpose may be made of the same material as the support. The thickness of this coating material is less than or equal to 300 pm;
A practically preferred range is about 4 to 200 ILm.

In addition, when manufacturing the electrophoresis medium of the present invention, it is also possible to use a method in which an electrophoresis medium layer is formed on a cover film or the like, and then a support is attached thereon.

The electrophoresis medium of the present invention can be used in both horizontal and vertical plate electrophoresis methods, disk electrophoresis methods, etc. according to the known methods described in the above-mentioned documents.

An electrophoresis medium to which a cover film has been attached during the production of the electrophoresis medium can be subjected to electrophoresis and then be subjected to autoradiography by removing the cover film or without removing the cover film.
Analysis of the electrophoresis results is performed.

Next, examples of the present invention will be shown.

[Example 1] 6.46 g of acrylamide, 34 g of BISO, 1.2 g of polyacrylamide, 0.3 g of agarose, and Tris were placed on a polyethylene terephthalate (PET) sheet (support) whose surface had been made hydrophilic by ultraviolet irradiation treatment. ,(
Hydroxymethyl)aminomethane 5.04g, boric acid 0
．． 28 g of EDTA-2Na salt, and 100 ml of an aqueous solution containing 47 mg of EDTA-2Na salt, 1.3 mJl of ammonium belloxonisulfate (5% by weight aqueous solution) as a polymerization initiator and 33 JLl of N,N,N',N'-tetramethylethylenediamine (TEMED). 1mm of coating solution added with
The film was coated to a thickness of 1,000 ml, and polymerized under a nitrogen atmosphere to obtain a polyacrylamide gel membrane for separating DNA fragments. This gel film was cut using a cutter knife to form a sample injection port.

The cut was sharp and a good sample injection port was formed.

E. coli plasmid pBR-322 was transformed with restriction enzyme Asul.
When the separation pattern of the DNA degradation products was examined by ethidium staining, a normal staining pattern was observed. When each band was cut out using a cutter knife based on the obtained staining pattern, DNA fragments could be separated with high precision. A polyacrylamide gel membrane was obtained.

Separately, acrylamide 7.6g, BISO, 4g)
Lis(hydroxymethyl)aminomethane 5.04 g, boric acid 0-28 g and EDTA 2Na salt 47 mg
1.3tnQ of ammonium belloxonisulfate (5% by weight aqueous solution) and TEMED337ti as polymerization initiators were prepared,
A comparative polyacrylamide gel membrane was prepared using a conventional method by polymerization in a glass mold having a space of m.

φX174RF-DNA was treated with the restriction enzyme HincII, electrophoresed on each of the above-mentioned gel membranes, and the separation pattern of the DNA degradation product was examined by ethidium staining. A normal staining pattern was observed in both gel membranes.

However, the comparative gel membrane required careful handling of the glass plate after electrophoresis. On the other hand, the former gel film (according to the present invention) was easy to handle because it was formed on a PET sheet, and was also excellent in processability such as creation of a sample injection port. Furthermore, the gel membrane of the present invention was also easier to perform the fractionation operation for DNA fragments separated on the gel membrane.

[Example 3] 5.98 g of acrylamide and 0.12 g of 1,3.5-triacryloylhexahydro-5-triazine were placed on a PET sheet whose surface had been made hydrophilic by ultraviolet irradiation treatment.
g, polyacrylamide (average molecular weight approximately 500,000) 0.6
g, agarose (low electroosmoticity, gelation temperature 36°C) 0
．． 3g, tris(hydroxymethyl)aminomethane 1.
08g, boric acid 0.55g, EDTA-2Na salt 95
m g, and 100 mf containing 20 g of glycerin.
Add 1.3 m of ammonium beroxonisulfate (5% by weight aqueous solution) as a polymerization initiator to the L aqueous solution and TEMED33.
A coating solution containing JL was applied to a thickness of 1 mm to form a polyacrylamide gel membrane for separating DNA fragments which was polymerized in a nitrogen atmosphere.

Separately, 6.86 g of acrylamide, 0.14 g of 1,3.5=triacryloylhexahydro-5-)riazine,
Tris(hydroxymethyl)aminomethane 1.08g,
Boric acid 0.55g, EDTA*2N, a salt 95mg
A 100 mJl aqueous solution containing ,
A comparative polyacrylamide gel membrane based on the conventional method was prepared by adding E D 33Pi and polymerizing in a glass mold with a 1 mm space.

E. coli plasmid pBR-322 was transformed with restriction enzyme Asul.
and electrophoresed it on each of the above gel membranes,
The separation pattern of DNA degradation products was examined by ethidium staining. A normal staining pattern was observed for both gel membranes, but the resolution of the former gel membrane according to the present invention was higher. When each band was cut out using a cutter knife based on the staining pattern obtained on the gel film according to the present invention, it was also possible to separate DNA fragments with high precision.

When the gel membrane was cut with a cutter knife to form a sample injection port, in the comparison gel membrane, the cut part of the gel membrane stretched and was difficult to process, but in the gel membrane of the present invention, the sample with the sharp cut edge was difficult to process. It was possible to form an injection port.

[Example 4] In the same manner as in Example 3, a polyacrylamide gel membrane according to the present invention and a comparative polyacrylamide gel membrane were created.

φX174RF-DNA was treated with the restriction enzyme Hincll, electrophoresed on each of the above-mentioned gel membranes, and the separation pattern of DNA degradation products was examined by ethidium staining.

A normal staining pattern was observed in both gel membranes.

However, the comparative gel membrane required careful handling of the glass plate after electrophoresis. On the other hand, the gel film according to the present invention is P
Since it was formed on an ET sheet, it was easy to handle, and it was also easy to process, such as creating a sample injection port. Furthermore, the gel membrane of the present invention was also easier to perform the fractionation operation for DNA fragments separated on the gel membrane.

Claims (1)

[Claims] 1. A polyacrylamide-based aqueous gel electrophoresis medium obtained by cross-linking polymerizing an acrylamide-based compound and a cross-linking agent in the presence of water, further comprising a water-soluble polymer and agarose. Electrophoretic media for separation of DNA, DNA fragments, RNA, or RNA fragments. 2. The crosslinking agent is 0.1% to 30% by weight based on the total weight of the acrylamide compound and the crosslinking agent.
The water-soluble polymer is contained in a range of 2 wt% to 100 wt%, and the agarose is contained in a range of 0.2 wt%/v% to 2 wt%/v% based on the volume of the polyacrylamide-based aqueous gel. An electrophoresis medium according to claim 1. 3. The electrophoresis medium according to claim 1, wherein the crosslinking agent is a trifunctional crosslinking agent. 4. The electrophoresis medium according to any one of claims 1 to 3, which is attached to a support made of plastic material.