Polyacrylamide medium for electrophoresis having improved elasticity

In a medium for electrophoresis comprising a polyacrylamide gel formed by crosslinking polymerization of an acrylamide compound and a crosslinking agent in the presence of water, and a modifier, the improvement in which the medium contains a water-soluble polymer.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a medium for gel electrophoresis (hereinafter 
referred to as "gel medium") used to determine the base sequence of DNA, 
DNA fragment and DNA derivative, and in particular provides a composition 
of gel medium to improve workability of the medium. 
2. Description of Prior Arts 
In the method for determination of the base sequence of DNA, RNA, their 
fragments, and their derivatives according to the post-label method, the 
operation of slab electrophoresis using a polyacrylamide gel membrane is 
now essential. The polyacrylamide membrane used for this purpose is 
obtainable, for instance, by a crosslinking polymerization between approx. 
95 parts by weight of a monomer such as acrylamide and approx. 5 parts by 
weight of a bifunctional crosslinking agent such as 
N,N'-methylenebisacrylamide in an aqueous solution containing a mixture of 
the monomer and the crosslinking agent and a polymerization initiator 
(hereinafter referred to as "gel forming solution"). 
Recently, as the study concerning the gene has progressed, a rapid 
operation for determination of the DNA base sequence is required. The 
electrophoresis using the polyacrylamide as the electrophoresis medium 
membrane is almost essential for performing the determination of DNA base 
sequence, because the polyacrylamide medium gives prominently high 
resolution. 
However, the conventional polyacrylamide gel membrane has a serious 
disadvantage that the membrane is brittle and easily breakable. Therefore, 
a polyacrylamide gel membrane for determination of the DNA base sequence 
is generally prepared by a process in which a gel forming solution is 
poured into a cell formed with two glass plates (having a clearance of 0.3 
to 1 mm) to prepare a gel membrane in the cell. In the gel membrane, 
sample inlets must be formed so that the membrane can receive DNA samples 
(e.g., Maxam-Gilbert decomposed .sup.32 P-labelled DNA or etc.). 
Accordingly, a sample slot former is generally inserted into the cell 
after the gel forming solution is poured therein but before gelation takes 
place, so that the sample slots can be formed on the gel membrane. It is 
difficult to cut off the edge from a prepared membrane with a razor or the 
like to form the sample inlets, because the gel membrane is very brittle 
and easily breakable. For this reason, the above-stated process involving 
the formation of sample slots by insertion of the sample slot former in 
advance of the gelation is utilized. This complicated process is a serious 
obstacle in producing the polyacrylamide gel membrane in a mass scale. 
The polyacrylamide gel membrane prepared as above is then kept vertically 
together with the glass plates, and the sample slot former is removed. A 
certain amount of a sample (Maxam-Gilbert decomposed .sup.32 P-labeled DNA 
or etc.) is poured into the sample slots, and the sample is 
electrophoresed. The electrophoresis is continued for a certain period of 
time, one glass plate is removed carefully, and the autoradiographic 
process is performed on the gel membrane. Thereafter, the determination of 
DNA base sequence is carried out. Even in this process, the gel membrane 
sometimes breaks when the glass plate is removed, because the conventional 
gel membrane is very brittle. Thus, the brittleness of the conventional 
membrane is a very serious problem. 
SUMMARY OF THE INVENTION 
An object of the present invention is to provide a medium for 
electrophoresis which is improved in the unfavorable features such as 
brittleness of the conventional polyacrylamide gel membrane for 
determination of DNA base sequence, in which the unfavorable feature 
arises mainly from the composition of the gel medium. 
Another object of the invention is to provide a polyacrylamide gel medium 
which is satisfactory in electrophoretic characteristics such as 
resolution and migration velocity, being inproved in brittleness, easy to 
handle, capable of being cut into a desired shape, and usable for 
producing a gel column to be employed in the disk electrophoresis if 
required. This means that the medium for electrophoresis provided by the 
invention is remarkably improved in total characteristics including the 
function as molecular sieve, as compared with the conventional medium. 
There is provided by the present invention a medium for electrophoresis 
comprising a polyacrylamide gel formed by crosslinking polymerization of 
an acrylamide compound and a crosslinking agent in the presence of water, 
and a modifier, which is characterized in that said medium contains a 
water-soluble polymer. 
In the present invention, the aqueous gel medium can be prepared by 
dissolving or dispersing an acrylamide compound and a crosslinking agent 
in an aqueous medium and performing the crosslinking polymerization 
therebetween in the aqueous medium to form an aqueous gel medium. 
Hereinafter, the term "dissolving (in water)" means to include both 
"dissolving (in water)" and "dispersing (in water)", and the term "aqueous 
solution" means to include both "aqueous solution" and "aqueous 
dispersion", unless otherwise indicated. The aqueous medium is used to 
include an aqueous mixture of water and an organic solvent, the latter 
being optionally added. 
DETAILED DESCRIPTION OF THE INVENTION 
Examples of the acrylamide compound employable in the invention include 
acrylamide and its homologues such as acrylamide, N-methylacrylamide , 
N,N-dimethylacrylamide, N-(hydroxymethyl)acrylamide, and 
diacetonacrylamide, and these acrylamide homologues may be employed 
independently or in combination. Acrylamide is most preferable among these 
acrylamide compounds, and said acrylamide can be also preferably employed 
in combination with one or more of other acrylamide compounds. 
As the crosslinking agent used to obtain the polyacrylamide gel of the 
invention, a crosslinking agent described in "Electrophoresis" 1981, 
2,220-228, or known as such may be employed. Examples of the crosslinking 
agent include bifunctional compounds such as N,N'-methylenebisacrylamide 
(BIS), N,N'-propylenebisacrylamide (PBA), diacrylamide dimethylether 
(DAE), 1,2-diacrylamide ethyleneglycol (DEG), ethyleneureabisacrylamide 
(EUB), ethylene diacrylate (EDA), N,N'-diallyltartardiamide (DATD), and 
N,N'-bisacrylylcystamine (BAC). The crosslinking agent can be employed in 
an amount of approx. 1 to 30 wt.%, preferably approx. 3 to 10 wt.%, based 
on the total weight of the monomer (i.e., acrylamide compound) and the 
crosslinking agent. 
As the modifier, a compound containing at least one carbamoyl group can be 
used. Examples of the modifier include urea and formamide. Urea is most 
preferred. The modifier can be used in an amount of approx. 40 to 60 wt.% 
based on the volume of the aqueous gel containing the monomer and 
crosslinking agent. In the case that urea is used as the modifier, the 
amount generally ranges from approx. 6 moles (approx. 360 g.) per one 
liter of the aqueous gel containing the monomer and crosslinking agent to 
the saturation amount, preferably from approx. 7 moles (approx. 420 g.) to 
the saturation amount. 
As the water-soluble polymer, a water-soluble polymer of the addition 
polymerization type or condensation polymerization type can be used. 
Examples of the polymer of the addition polymerization type include 
non-ionic water-soluble polymers such as polyvinyl alcohol, polyvinyl 
pyrrolidone, and polyacrylamide. Examples of the polymer of the 
condensation polymerization type include non-ionic water-soluble 
polyalkylene glycols such as polyethylene glycol and polypropylene glycol. 
The water-soluble polymer of molecular weight ranging from approx. 10,000 
to 1,000,000 is preferably used. Among these water-soluble polymers, 
polyacrylamide and polyethylene glycol are preferable. The water-soluble 
polymer is used in a range of approx. 1 to 50 wt.%, preferably, approx. 5 
to 30 wt.%, based on the total weight of the monomer and crosslinking 
agent. 
According to the present invention, the addition of a water-soluble polymer 
serves to impart elasticity to the gel medium, and thus modified gel 
medium is still elastic even if it is dried. Thus, the gel medium is so 
improved as to be almost free from the brittleness, whereby the gel medium 
becomes hardly breakable. Further, the viscosity of the gel medium can be 
controlled by selecting the molecular weight and amount of the 
water-soluble polymer. 
A pH buffer agent can be contained in the gel medium of the invention. Any 
buffer agent which is able to buffer a solution to a range of pH 8.0 to 
9.0, preferably pH 8.2 to 8.3 can be used. Buffer agents employable in the 
invention are described in literatures such as "Chemistry Handbook, 
Fundamental Edition" compiled by The Chemical Society of Japan (Maruzen 
Ltd., Tokyo, 1966) pages 1312-1320; "Data for Biochemical Research" 
compiled by R. M. C. Dawson et al., second edition (Oxford at the 
Clarendon Press, 1969) pages 476-508; "Biochemistry" 5, 467 (1966); and 
"Analytical Biochemistry" 104, pages 300-310 (1966). Examples of the 
buffer agent include tris(hydroxymethyl)aminomethane (Tris), 
N,N-bis(2-hydroxyethyl)glycine (Bicine), Na or K salt of 
N-2-hydroxyethylpiperazine-N'-2-hydroxypropane-3-sulfonic acid and Na or K 
salt of N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid, and 
further acids, alkalis, or salts employable together with any of these 
buffer agents. Preferred examples of the buffer agent include Tris, and 
the combination of boric acid and EDTA.2Na salt (pH 8.2). 
In the case that the gel medium of the invention is used in the form of 
layer or membrane, a gel layer or gel membrane can be prepared by a 
process in which a gel forming solution is coated by a known method on an 
electric insulation support having a smooth hydrophilic surface, and the 
gel forming solution is crosslinked thereon to polymerization. Examples of 
the support include glass plate, hydrophilic polymers in the form of plate 
or sheet, and other polymers (e.g., polyethylene terephthalate, 
polycarbonate of bisphenol A, polyvinyl chloride, vinylidene 
chloride-vinyl chloride copolymer, polymethylmethacrylate, polyethylene, 
polypropylene, cellulose acetate, and cellulose acetate propionate) in the 
form of plate or sheet, a surface of which is made hydrophilic by a known 
surface treatment. Examples of the treatment employable to make the 
surface of these polymers hydrophilic include known methods such as 
irradiation with ultra-violet rays, glow discharge treatment, corona 
discharge treatment, flame treatment, electron beam treatment, chemical 
etching, or electrochemical etching. 
In the case that the gel forming solution is crosslinked to polymerization 
on the surface of the support, the surface of the gel forming solution can 
be covered with a cover film, sheet, or plate. The same material as 
employable for the support can be employed as the cover film, sheet, and 
plate. 
The gel medium used in the invention is formed by radical 
crosslinking-polymerization between the monomer such as acrylamide with 
the bifunctional compound (crosslinking agent) in an aqueous solution in 
which the water soluble polymer is dissolved almost homogeneously. The gel 
is assumed to have a structure in which the water-soluble polymer is 
dispersed in the three dimensional crosslinked polymer, and the 
water-soluble polymer chains are entangled with the three dimensional 
crosslinked polymer chains. This structure is one of the characteristic 
features of the gel medium of the invention. 
The crosslinking polymerization can be initiated in the presence of a 
peroxide and/or under irradiation of ultra-violet rays. The reaction can 
be further accelerated by heat and irradiation with ultra-violet rays. 
As the polymerization catalyst, a known low temperature-polymerization 
initiator can be used. Examples of the initiator include 
.beta.-dimethylaminopropionitrile (DMAPN)-ammonium peroxodisulfate, 
N,N,N',N'-tetramethylethylenediamine (TEMED)-ammonium peroxodisulfate, 
TEMED-riboflavin, and TEMED-riboflavin-hydrogen peroxide. Irradiation with 
ultraviolet rays can be employed in conjunction with the initiator. 
A polyol compound such as glycerol or ethylene glycol can be incorporated 
in the gel medium of the invention as a wetting agent. The polyol compound 
can be introduced in an amount of approx. 1 to 40 wt.% based on the volume 
of the aqueous gel medium. Glycerol is particularly preferable among 
polyol compounds. 
The present invention will be more clearly understood with reference to the 
following examples, but these examples are by no means understood to 
restrict the invention.

EXAMPLE 1 
A glass plate cell with clearance of 0.5 mm was formed using two glass 
plates with smooth surface and a spacer of 0.5 mm thick. The acrylamide 
gel composition solution (gel forming solution) set forth in Table 1 was 
poured into the cell and crosslinked to polymerization to form a 
polyacrylamide gel membrane. One glass plate was removed after gelation 
was complete, and sample slots were formed by cutting the polyacrylamide 
gel membrane by means of a sharp cutter. 
TABLE 1 
______________________________________ 
Gel Composition 
Sample Number 
1 2 3 4 
______________________________________ 
Gel Composition 
Acrylamide 11.87 g 11.87 g 11.87 g 
11.87 g 
N,N'--Methylenebis- 
0.63 g 0.63 g 0.63 g 
0.63 g 
acrylamide 
Urea 42 g 42 g 42 g 42 g 
Solid Polyacrylamide 
None 1.25 g 2.5 g None 
(mean M.W. 50,000) 
Solid Polyacrylamide 
None None None 1.25 g 
(mean M.W. 800,000) 
Tris(hydroxymethyl)- 
1.08 g 1.08 g 1.08 g 
1.08 g 
aminomethane 
Boric acid 0.55 g 0.55 g 0.55 g 
0.55 g 
EDTA.2Na 93 mg 93 mg 93 mg 93 mg 
Water (added to make 100 ml) 
Polymerization Initiator 
Ammonium peroxodisulfate 
1.3 ml 1.3 ml 1.3 ml 
1.3 ml 
(5 wt. % aq. sol.) 
N,N,N',N'--tetramethyl- 
33 .mu.l 
33 .mu.l 
33 .mu.l 
33 .mu.l 
ethylenediamine 
______________________________________ 
Remark: A combination of tris(hydroxymethyl)aminomethane, boric acid and 
EDTA.2Na is a buffer composition showing a buffering ability to make pH 
8.2. 
As for Sample No. 1 (reference gel membrane), the cut end was cracked and 
the membrane was broken, and accordingly sample slots of sharp edge could 
not be obtained. On the other hand, as for Samples Nos. 2 to 4 (gel 
membrane according to the invention), sample slots showing sharp edge were 
easily obtained. 
The gel membrane obtained as above was again covered with a glass plate, 
and the electrophoresis was performed by the conventional method to 
observe the electrophoresed dye pattern. 
As for Sample No. 1, serious disorder of the electrophoresed dyes was 
observed. On the other hand, as for Samples Nos. 2 to 4, no disorders were 
observed, and uniform electrophoresed patterns were formed. 
Accordingly, it has been confirmed that the problem in brittleness of the 
gel membrane is well improved by the present invention, as compared with 
the conventional gel membrane. 
EXAMPLE 2 
Gel membranes were prepared using the same gel compositions as described in 
Example 1. For reference, a membrane was prepared from the same 
composition as that of Sample No. 1, except that a sample slot former was 
employed for the preparation of the sample slots (Sample No. 1-A: 
reference gel membrane). 
The experiment for the DNA base sequence determination was performed using 
these gel membranes and a sample prepared from .sup.32 P labeled DNA 
decomposed by Maxam-Gilbert method. 
Sample No. 1-A and Sample Nos. 2 to 4 exhibited normal electrophoresed 
patterns and the DNA base sequence were determined with no difficulty, but 
Sample No. 1 exhibited a disordered electrophoresed pattern. 
Accordingly, it has been confirmed that the gel membrane according to the 
present invention exhibits satisfactory electrophoresis characteristics, 
being almost free from the brittleness. 
EXAMPLE 3 
A polyacrylamide gel membrane was prepared in the same manner as in Example 
1 except that the solid polyacrylamide (water-soluble polymer) was 
replaced with polyethylene glycol (average molecular weight: 200,000). 
Thus prepared membrane was subjected to the same electrophoresis. It was 
observed that the gel membrane exhibited satisfactory electrophoresis 
characteristics, as well as that the brittleness of membrane was 
remarkably improved and the gel membrane was freely cut for the formation 
of sample slot. 
Accordingly, the advantage of the invention has been confirmed.