Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1996-08-26
1999-10-19
Kunz, Gary L.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435 911, 536 2541, 536 2542, 935 19, 935 20, 935 21, C12P 1934, C07H 102, C07H 2100
Patent
active
059691298
DESCRIPTION:
BRIEF SUMMARY
FIELD OF INVENTION
The present invention relates to the isolation of polynucleotides from an aqueous mixture containing the polynucleotides and polysaccharides, particularly to the purification of deoxyribonucleic acid (DNA) extracted from plants.
BACKGROUND
Co-pending patent application W092/05181 herein incorporated by reference discloses the purification of polynucleotides (particularly DNA) derived from human or animal cells by the removal of protein impurities. The proteins are removed by reaction with a cross-linked silica gel material having free CHO or CO groups, so as to remove the proteins as a solid or semi-solid material in a two-phase solvent system. The lower phase is chloroform and the upper aqueous phase contains the DNA. The protein-containing silica is present as an intermediate solid or semi-solid layer which is easily separated away. The cross-linked silica is preferably prepared by cross-linking silica gel with 3-aminopropyltriethoxysilane, followed by reaction with gluteraldehyde to introduce protein-reactive CHO groups.
However, DNA extracted from plants often contains polysaccharides as an impurity, which are not encountered with DNA derived from most other organisms. Such polysaccharides may have enzyme-inhibitory properties, which may subsequently prove troublesome should the DNA be subsequently subjected to enzyme digestion, for example restriction enzyme digestion. To date, most extraction methods have employed the expensive and time-consuming caesium chloride density gradient technique to remove these polysaccharides (Bendich A J, Anderson R S, Ward B L (1980) Plant DNA: long, pure and simple. In: Leaver Cj(ed) Genome Organisation and Expression, pp 31-33. New York: Plenum Press; Murray H G., Thompson W F.(1980) Nucleic Acids Res 8:4321-4325; Taylor B, Powell A (1982) Isolation of plant DNA and RNA. Focus 4:4-6.). Other methods have also been reported (Dellaporta S L, Wood J, Hicks J B (1983) Plant Mol Biol Rep 1:19-21. Zimmer E A, Newton K J (1982) A simple method for the isolation of high molecular weight DNA from individual maize seedlings and tissues. In: Sheridan W F (ed) Maize for Biological Research. Grand Forks, N.D.: University press, University of North Dakota.) however, such techniques are generally only applicable to a limited number of plant species and tissue types. The separation of polysaccharides from nucleic acids by the differential solubilities in the presence of cetyltrimethylammonium bromide has also been reported (Rogers S O, Bendich A J (1988) Plant Mol Biol Manu.A6:1-10.); but this procedure requires organic extraction which may cause some degradation of the DNA.
It is an object of the present invention to provide a convenient process for the isolation of polynucleotides from polysaccharide-containing mixtures.
This and other objects of the present invention will become apparent from the following description and examples.
STATEMENT OF INVENTION
In one aspect, the present invention provides a process for the isolation of polynucleotides from an aqueous mixture containing polynucleotides and polysaccharides, which comprises: --B(OH).sub.2 groups, reactive with the polysaccharides (and is non-reactive to polynucleotides) wherein the polymer gel is capable of forming a solid or semi-solid material containing the polysaccharides; and polysaccharide-containing material.
DETAILED DESCRIPTION
The polymer gel may be any suitable gel material which is insoluble in the solvents employed. It may be for example a polymerised silica gel, or polyacrylate gel, a polystyrene gel or a polyester gel. The --B(OH).sub.2 groups may be present in the monomer prior to polymerisation, or may be incorporated by reaction with existing groups, such as OH or X, on the polymer.
A preferred polyacrylate is prepared by copolymerisation of dihydroxyborylanilino-substituted methacrylic acid with 1,4-butanediol dimethacrylate and is available from Aldrich Chemical Company.
A preferred polymer gel is a polymerised silica gel. Whilst the --B(OH).sub.2 groups may be attached dire
REFERENCES:
1993 Bio-Rad Lite Science Research Products Catalog, p. 23., Hercules, CA. 94547.
N. Do and R.P. Adams, "A Simple Technique for Removing Plant Polysaccharide Contaminants from DNA" Biotechniques 10 No. 2, 162-166 (1991).
B.J.B. Johnson, Synthesis of a Nitrobenzeneboronic Acid Substituted Polyacrylamide and Its Use in Purifying Isoaccepting Transfer Ribonucleic Acids; Biochemistry, 20, 6103-6108, (1981).
R.E. Duncan and P.T. Gilham; Isolation of Transfer RNA Isoacceptors by Chromatography on Dihydroxboryl-Substituted Cellulose, Polyacrylamide, and Glass; Analytical Biochemistry 66, 532-539, (1975).
Database WPI, Derwent Publications Ltd., London, GB; AN 92-311774 & JP, A,04 216 459 (Nippon Oils), Aug. 6, 1992 see abstract.
Database WPI, Derwent Publications Ltd., London, GB; AN 80-47004C & JP, A,55 066 525 (Nippon Corn Starch), May 21, 1980 see abstract.
B. Rether et al; Isolation of Polysaccharide-Free DNA from Plants; Plant Molecular Biology Reporter 11, 333-337 (1993).
Caddy Brian
Cheng Jing
Kunz Gary L.
The University of Strathclyde
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