Method for producing endotoxin-free nucleic acids and the use thereof
AssigneeXantos Biomedicine AG
InventorsStefan Grimm, Frank Neudecker
Life of this patent
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Abstract
Method for isolating and purifying nucleic acids and/or oligonucleotides from a biological sample, the use of the isolated or purified nucleic acid and/or oligonucleotide for transfecting cells and also for the production of an agent for the treatment of genetic disorders, a composition suitable for the isolation or purification method and also the use of potassium acetate and a silica gel-like support material for isolating endotoxin-free nucleic acids and/or oligonucleotides or nucleic acids and/or oligonucleotides with reduced endotoxin content.
Claims13
A method for isolating and purifying nucleic acids and/or oligonucleotides from a biological sample, said method comprising:
- (a) disrupting the biological sample;
- (b) optionally removing protein and insoluble components from said disrupted sample;
- (c) adding an aqueous solution of potassium acetate to said disrupted sample and subsequently separating non-soluble components from the aqueous solution;
- (d) mixing said aqueous solution of potassium acetate containing said disrupted sample with an alcoholic solution containing SDS;
- (e) incubating said mixed solution;
- (f) obtaining the supernatant of said mixed solution;
- (g) contacting and incubating said supernatant with a suspension of silicon dioxide or silica gel to produce a bound fraction and a soluble fraction;
- and
- (h) isolating purified nucleic acids and/or oligonucleotides from said bound fraction.
A method of transfecting eukaryotic or prokaryotic cells with nucleic acids or oligonucleotides, said method comprising:
- (a) isolating and purifying nucleic acids and/or oligonucleotides from a biological sample by the steps of:
- (1) disrupting the biological sample;
- (2) optionally removing protein and insoluble components from said disrupted sample;
- (3) adding an aqueous solution of potassium acetate to said disrupted sample and subsequently separating non-soluble components from the aqueous solution;
- (4) mixing said aqueous solution of potassium acetate containing said disrupted sample with an alcoholic solution containing SDS;
- (5) incubating said mixed solution;
- (6) obtaining the supernatant of said mixed solution;
- (7) contacting and incubating said supernatant with a silicon dioxide support material to produce a silicon dioxide bound fraction and a soluble fraction;
- and
- (8) isolating purified nucleic acids and/or oligonucleotides from said silicon dioxide bound fraction, and
- (b) transfecting said cells with said purified nucleic acids and/or oligonucleotides.
A method of producing a purified nucleic acid and/or oligonucleotide composition suitable for use in the treatment of genetic disorders, said method comprising isolating and purifying nucleic acids and/or oligonucleotides from a biological sample by the steps of:
- (a) disrupting the biological sample;
- (b) optionally removing protein and insoluble components from said disrupted sample;
- (c) adding an aqueous solution of potassium acetate to said disrupted sample and subsequently separating non-soluble components from the aqueous solution;
- (d) mixing said aqueous solution of potassium acetate containing said disrupted sample with an alcoholic solution containing SDS;
- (e) incubating said mixed solution;
- (f) obtaining the supernatant of said mixed solution;
- (g) contacting and incubating said supernatant with a silicon dioxide support material to produce a silicon dioxide bound fraction and a soluble fraction;
- and
- (h) isolating purified nucleic acids and/or oligonucleotides from said silicon dioxide bound fraction.
A kit comprising:
- (a) at least one solution suitable for the disruption of a biological sample;
- (b) an aqueous potassium acetate solution;
- (c) an alcohol solution containing 0.5% to 10% (w/v) SDS in 100% strength isopropanol;
- and
- (d) a silicon dioxide support material.
A kit comprising:
- (a) a solution suitable for alkaline lysis and disruption of biological sample material;
- (b) an aqueous salt solution containing 1 M to 6 M potassium acetate;
- (c) an alcohol solution containing 0.5% to 10% (w/v) SDS in 100% strength isopropanol;
- and
- (d) a silicon dioxide support material.
A kit comprising:
- (a) at least one solution suitable for the disruption of a biological sample;
- (b) an aqueous potassium acetate solution;
- (c) an alcohol solution containing 0.5% to 10% (w/v) SDS in 100% strength isopropanol;
- and
- (d) a suspension of silicon dioxide or silica gel.
A kit comprising:
- (a) a solution suitable for alkaline lysis and disruption of biological sample material;
- (b) an aqueous salt solution containing 1 M to 6 M potassium acetate;
- (c) an alcohol solution containing 0.5% to 10% (w/v) SDS in 100% strength isopropanol;
- and
- (d) a suspension of silicon dioxide or silica gel.
Dependencies are inferred from the claim text.