Use of an earlier known separation material and novel forms...

Details Use of an earlier known separation material and novel forms... Use of an earlier known separation material and novel forms...

1999-03-11

2001-11-20

Warden, Jill (Department: 1743)

Chemistry: electrical and wave energy

Processes and products

Electrophoresis or electro-osmosis processes and electrolyte...

C204S456000, C204S606000, C536S051000, C536S112000, C526S238200, C526S238220

Reexamination Certificate

active

06319380

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to the electrophoresis of single-stranded or double-strand nucleic acid (ss and ds respectively), primarily polynucleotides that consist of sequences of 5-20,000 nucleotides (alternatively base pairs).
It is known that gels based, inter alia, on agarose, polyacrylamides, bis-acrylamide, etc., can be used in the electrophoresis of nucleic acid.
The gels have normally been cast or molded in the presence of an electrophoresis buffer.
In the electrophoretic analysis of single-stranded DNA (sequencing) separation is normally effected in the presence of urea in order to keep the strands dissociated (Maniatis et al, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989) New York, U.S.A.). Consequently, gels have been cast together with urea at a pH of about 8 in direct association with their use. Efforts to incorporate buffers of a higher pH value have also been made (Smith et al, Amer. Biotechnol. 8 (1990), 48-54).
Problems Associated with Hitherto Known Techniques, and Desirable Properties of Relevant Gels
Agarose gels that possess good sieving properties for the DNA-sequencing of samples are difficult to produce. Agarose also has the drawback that the gels readily become opaque and therefore are difficult to use in conjunction with optical detection. Drawbacks with polyacrylamide in electrophoresis is that the monomers (acrylamide and bisacrylamide) are toxic, and consequently great care must be observed when handling the monomers, and also that acrylates and acrylamides are hydrolysis sensitive. In the case of prefabricated gels that contain urea, the urea is also readily hydrolyzed.
As an alternative of subjecting single-stranded nucleic acid to electrophoresis in the presence of a denaturing agent, such as urea, electrophoresis may be carried out at an elevated pH (normally about pH 11) and/or at an elevated temperature (about 80-90° C.). Both alternatives result in the hydrolysis of polyacrylamide.
There is a strong general desire to find novel gel materials that can be used in the electrophoresis of nucleic acid. Objectives with regard to novel gel materials are that they shall:
1. Enable separation of single-stranded and/or double-stranded nucleic acid, primarily DNA, with a resolution that is comparable with or superior to commercial polyacrylamide gel (=PAA).
2. Enable the fabrication of gradient gels and gels that have different zones.
3. Provide low electro-endosmosis, preferably equal to or better than PAA.
4. Be stable in the pH range relevant to electrophoresis, normally 4-13.
5. Tolerate urea 8 M and other denaturing agents and conventional staining techniques for nucleic acid (e.g. silver staining and ethidium bromide staining).
6. Possess good optical properties, i.e. high light transmission 280-800 nm and non-disturbing (low) fluorescence, particularly at 480, 590 and 630 nm.
7. Be non-toxic.
The use of dextran derivative gels for protein electrophoresis was proposed about twenty years ago (Söderberg, L., U.S. Pat, No. 4,094,832). The gels were produced by free radical polymerization of a dextran derivative containing vinyl groups chosen from
H
2
C═CR—A—
and/or
H
2
C═CR—CO—
where —A— is —CH
2
— or —O— and R is H, CH
3
, F, Cl, Br or CN. Polymerization could optionally be effected in the presence of low molecular vinyl compounds. Protein electrophoresis was the stated application. The advantages obtained were said to be that the gels were inert against biological substances, were stronger, could be cast with an incorporated electrophoresis buffer, could be cast or molded in the form of easily handled plates having tailor-made properties in accordance with the choice of vinyl substituents, charged substituents and copolymers, etc.
In conjunction with the attempt to produce electrophoresis gels that would give improved resolution of low molecular compounds, low “smiling”, and improved optical properties, there have been proposed gels of polyhydroxy polymers cross-linked with a reagent that provides ether bridges with OH groups in the polymer (Kozulic, B., U.S. Pat. No. 5,371,208; see in particular column 11). The function of the gels has been shown for electrophoresis of DNA digested with the aid of restriction enzymes. Allyl agarose (Example 1) and dextran (Examples 14 and 16) can be mentioned from among a large number of examples.
A copolymer between acrylamide and allyl substituted agarose (Nochumsson, S., EP 87,995) has been proposed as a gel for electrophoresis, since such gels adhere well to conventional carrier or substrate materials.
OBJECT OF THE INVENTION
The object of the invention is to find novel gels for electrophoresis, which are suited for the separation of nucleic acid and which have improved properties with regard to the aforesaid objectives.
The invention
It has now been found that electrophoresis gels polymerized from a dextran derivative that includes groups having an alkene structure (>C═C<) are suited for electrophoretic separation of both single-stranded and double-stranded nucleic acids. The gels have improved properties with regard to several of the features mentioned in the aforegoing, such as chemical stability at high pH levels, optical clarity, are non-toxic and have a long shelf life. The polymer may be in the form of a copolymer with low molecular vinyl monomers. The gels concerned have earlier been defined in principle (Söderberg, L., U.S. Pat. No. 4,094,832).
The main aspect of the invention resides in a method for the gel electrophoretic separation of nucleic acid. The main feature of the method resides in the use of a gel which is a free radical-polymer of the same type as that used by Söderberg (U.S. Pat. No. 4,094,832). The method includes the steps typical in the gel electrophoresis of nucleic acid, i.e. the sample is placed on the gel and a voltage applied. When wishing to carry out electrophoresis on single-stranded DNA, the process is effected in the presence of a denaturing agent which dissociates double-stranded nucleic acid. See below. Alternatively, the electrophoresis can be carried out at elevated temperatures (e.g. temperatures above 50° C., a denaturing agent may be present when necessary).
Preferred groups that include alkene structures have the formula
H
2
C═CR—B—
where R may be H, F, Cl, Br or CN or a straight, branched or cyclic hydrocarbon group which may be saturated or unsaturated or contain an aromatic structure. Among saturated hydrocarbon groups, for instance groups according to the formula C
n
H
2n+1
(n is an integer) can be mentioned those that contain ten or fewer carbon atoms (n integer≦10). The most preferred groups R are hydrogen (H) and lower alkyl groups having 1-4 carbon atoms, particular methyl (CH
3
). The hydrogen atoms in H
2
C═ may be substituted with halogen, particularly Cl or F. The hydrocarbon chain in a group R may be broken or one or more of the carbons may be substituted with one or more oxygen atoms in the same manner as the bridge B (see below).
The bridge B binds directly to an oxygen atom in a hydroxyl group of dextran. The bridge B is a straight, branched or cyclic hydrocarbon bridge which may be saturated or unsaturated and is optionally broken by ether oxygen and/or has one or more carbon atoms that are substituted with one alcoholic hydroxyl group. The bridge B may include aromatic structures, such as phenylene (as cyclic structures; —C
6
H
4
—). The bridge B may alternatively be a single bond. At most, one oxygen atom is directly bound to one and the same carbon atom. The length of the bridge is normally less than 1-15 atoms, including any oxygen atoms. Unsubstituted hydrocarbon bridges are for instance —(CH
2
)
n
—, where n is an integer chosen from 1-10, for instance 2, 3 or 4. Phenylene, cyclohexandiyl, etc., are examples of cyclic structures that can be included in the bridge.
Specific examples of the group H
2
C═CR—B— allyl, 3-allyloxy-2-hydroxy-propyl, 3-(styryl-4-oxy)-2-hydroxypropyl, 1-methylallyl, 1-chloro-allyl and non-substituted vinyl

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