Electrophoresis gels and cross-linking agents for their...

Synthetic resins or natural rubbers -- part of the class 520 ser – Synthetic resins – Polymers from only ethylenic monomers or processes of...

Reexamination Certificate

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C525S291000, C525S296000, C526S288000, C526S306000

Reexamination Certificate

active

06197906

ABSTRACT:

TECHNICAL FIELD
This invention relates to the separation of molecules on polymer gels, in particular to the preparation of novel crosslinked polymer gels, the separation of molecules by techniques such as electrophoresis using these gels, novel crosslinking agents useful in the preparation of the gels and novel intermediates useful in the synthesis of the crosslinking agents. The invention is especially suitable for electrophoretic applications and accordingly, for convenience, the invention will be further described with reference to electrophoresis. It is to be understood, however, that the gels, processes and crosslinking agents of the present invention are not so limited.
BACKGROUND OF INVENTION
Polyacrylamide gel electrophoresis is an analytical technique whereby fragments of biomolecules, such as DNA, enzymes and proteins, may be separated and identified on the basis of their molecular size, weight and charge. Commercially available electrophoresis gels have conventionally been produced by copolymerisation of acrylamide with the symmetrical crosslinking agent, N,N′-methylene bisacrylamide, otherwise known as BIS. Since both double bonds of BIS are of the same type their reactivities are essentially the same. Other known crosslinking agents include ethylene glycol diacrylate, dihydroxy ethylene-bisacrylamide (DHEBA), N,N′-propylenebisacrylamide, diacrylamide dimethylether, 1,2-diacrylamide ethyleneglycol, ethyleneureabisacrylamide, N,N′-bisacrylylcystamine and bisacrylamide methylether (BAME). As for BIS, the double bonds of these crosslinking agents are of the same type.
DISCLOSURE OF INVENTION
It has now been found that electrophoresis gels having surprisingly improved separating ability can be prepared using particular asymmetrical crosslinking agents.
Accordingly the invention provides a crosslinked polymer gel comprising a crosslinking moiety of the formula:
wherein X and X′ are independently selected from the group consisting of —O—, —S— and —NR—, where R is H, alkyl or cycloalkyl,
R
2
is a C
1
-C
4
alkyl group,
Y is an optionally substituted non-aromatic divalent linking group, and
Z is O or S.
Preferably R
2
is CH
3
.
The monomer or monomers used to prepare the gel may be any suitable monomer.
The crosslinked polymer gel may be prepared from monomers having the formula H
2
C═CR
5
—CO—NR
3
R
4
where R
3
, R
4
and R
5
are each independently H or alkyl optionally monosubstituted by, for example, OH or C(O)CH
2
C(O) CH
3
. Examples of monomers include acrylamide, acrylamide derivatives or acrylamide substitutes known to the art such as N,N-dimethylacrylamide, methacrylamide, methyloylacrylamide, propylacrylamide, dipropyl acrylamide, isopropyl acrylamide, diisopropyl acrylamide, lactyl acrylamide, methoxyacrylamide and mixtures thereof. Preferably the monomer is acrylamide.
The linking group may be any suitable non-aromatic hydrocarbyl group, optionally including one or more heteroatoms selected from O, S, N and P.
Preferably X and X′ are the same. Preferably Z is oxygen.
In another aspect of the invention there is provided a method of preparing a crosslinked polymer gel, said method including the step of subjecting one or more monomers to crosslinking polymerisation with one or more crosslinking agents of the formula I:
wherein X and X′ are independently selected from the group consisting of —O—, —S— and —NR—, where R is H, alkyl or cycloalkyl,
R
2
is a C
1
-C
4
alkyl group,
Y is an optionally substituted non aromatic divalent linking group, and
Z is O or S.
R
2
is preferably CH
3
.
The polymer gels according to the present invention may be prepared using one or more crosslinking agents of formula I, optionally in the presence of one or more conventional crosslinking agents known to the art. Preferably the crosslinking agent(s) is/are selected to provide a gel which is substantially transparent to visible light. Preferably the gel is an aqueous gel.
The polymer gels according to the present invention are useful for separating molecules, especially charged species or species capable of bearing a charge such as biomolecules.
The polymer gel may be an electrophoretic gel. The electrophoretic gel may have a porosity gradient suitable for gradient gel electrophoresis. See for example, Polyacrylamide Gel Electrophoresis across a Molecular Sieve Gradient Margolis, J., Kenrick, K. G., Nature, 214, 1967, p1334-1336; Polyacrylamide Gel Electrophoresis in a Continuous Molecular Sieve Gradient, Margolis, J., Kenrick, K. G., Analytical biochemistry, 25, 1968, p347-362; and Practical System for Polyacrylamide Gradient Gel electrophoresis, Margolis, J., Laboratory Practice, 22, p107-109, 1973, the disclosures of which are incorporated herein by reference.
In a further aspect the invention provides a method of separating molecules comprising:
providing a crosslinked polymer gel by combining one or more monomers with a crosslinking agent of the formula I:
wherein X and X′ are independently selected from the group consisting of —O—, —S— and —NR—, where R is H, alkyl or cycloalkyl,
R
2
is a C
1
-C
4
alkyl group, and
Y is an optionally substituted non aromatic divalent linking group, and
Z is O or S, optionally in the presence of an initiator, subjecting the monomer solution to polymerisation and crosslinking, placing a sample containing the molecules to be separated onto the gel, and subjecting the gel and sample to a separation technique. Preferably the separation technique is electrophoresis. The electrophoresis technique employed may be any of those known to the art, including one-, two- and multi-dimensional techniques. The electrophoresis technique may be gradient gel electrophoresis.
Preferably polymerisation is carried out on a solution of the monomer or monomers with the crosslinking agent,
The linking group is preferably selected to provide a crosslinking agent which is soluble in the monomer solution. For most applications involving acrylamide the solvent will be water, and accordingly it is preferred that the linking group is selected to provide a crosslinking agent which is soluble in water or water/acrylamide. Other solvents include DMF, THF, alcohols and other water miscible systems.
Where the solvent is water and/or the monomer is acrylamide, the hydrophilic/lipophilic balance of the linking group may be controlled so that the cross-linking agent is soluble in water or water/acrylamide. Accordingly if the linking group contains a large number of carbon atoms (eg. more than about 7) the effect on solubility can be offset by including sufficient oxygen atoms or other polar groups to provide a crosslinking agent which is soluble in the acrylamide/water solution.
Examples of divalent linking groups include alkylene, oxyalkylene. polyoxyalkylene, cycloalkylene, alkanedioyl, alkylenedisulphonyl, alkylenecarbonyl, thioalkylene, ureylene, oxalyl, aminoalkylene, alkylenedisulphonyl, heterocyclyl and groups of the formula —(R
1
)
m
—R
2
—(R
3
)
n
—, where R
1
and R
3
are selected from alkylene, cycloalkylene, heterocyclyl, oxyalkylene, polyoxyalkylene, alkylenecycloalkylene and alkyleneheterocyclyl; R
2
is selected from a direct bond, —O—, —S—, —S—S—, alkylene, alkanedioyl, alkylenedioxy, alkylenedisulphonyl, —NR—, —NRC(O)O—, —NR—C(O)—NR—, —NRC(O)—, —N═N—, —NRC(O)C(O)—NR—, —C(O)—, —C(S)— and —RNNR—, where R is H, alkyl or cycloalkyl; m and n are 0 or 1 provided that m+n≠0.
As used herein the term “non-aromatic hydrocarbyl group” means any divalent group comprising carbon and hydrogen which does not include an aromatic or heteroaromatic ring.
As used herein the term “alkylene”, used either alone or in compound words such as “oxyalkylene”, “carbonylalkylene” denotes straight chain and branched C
1-10
alkylene groups. Examples include methylene, ethylene, propylene, isopropylene, butylene, isobutylene, sec-butylene, pentylene, isopentylene, sec-pentylene, 1,2-dimethylpropylene, 1,1-dimethylpropylene, hexylene, 4-methylpentylene, 1-methylpentylene, 3-methylpentylene, 1,1-dimethylbutylene, 2,2-dimethylbutyle

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