Extraction of high-purity DNA and RNA
AssigneeAtom Sciences, Inc.
InventorRichard Hurt
Life of this patent
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Abstract
The present invention is directed to a method of extracting nucleic acids from samples containing prokaryotic cells and/or eukaryotic cells by (a) lysing the cells under conditions that minimize RNA expression or enzymatic degradation of RNA; (b) dispersing and diluting the lysed cells; (c) mixing the dispersed and diluted lysed cells with a buffered solution containing cationic and anionic detergents; (d) separating non-nucleic acid contaminants from the nucleic acids by mixing with an organic extraction solvent to generate an emulsion that separates into an organic phase, an interface containing the contaminants and an aqueous phase containing the nucleic acids; (e) separating the phases, and retaining the aqueous phase; and (f) precipitating the nucleic acids in the aqueous phase.
Claims17
A method of extracting nucleic acids from samples containing prokaryotic cells and/or eukaryotic cells, which comprises:
- (a) lysing the cells in the sample under conditions that minimize RNA expression or enzymatic degradation of RNA;
- (b) dispersing and diluting the lysed cells;
- (c) mixing the dispersed and diluted lysed cells with a buffered solution containing cationic and anionic detergents, wherein the anionic detergent facilitates binding of carboxylate containing materials and ionic binding of the nucleic acids is inhibited by the cationic detergent;
- (d) separating non-nucleic acid contaminants from the nucleic acids by mixing with an organic extraction solvent to generate an emulsion that separates into an organic phase, an interface containing the contaminants and an aqueous phase containing the nucleic acids;
- (e) separating the phases, and retaining the aqueous phase;
- and
- (f) precipitating the nucleic acids in the aqueous phase followed by dissolving the precipitate in nuclease-free solvent.
A method for simultaneously extracting RNA and DNA from samples containing prokaryotic cells and/or eukaryotic cells, which comprises:
- (a) lysing sample cells;
- (b) diluting the lysed cells;
- (c) separating cell proteins and contaminant organic matter from the cell nucleic acids by mixing with an organic extraction solvent to generate an emulsion that separates into an organic phase, an interface containing the contaminants and an aqueous phase containing the nucleic acids,
- (d) separating the phases formed in step (c) with the aqueous phase being retained;
- (e) precipitating the nucleic acids from the aqueous phase followed by dissolving the nucleic acids in a nuclease-free solvent;
- and
- (f) optionally further purifying the nucleic acids by passing the dissolved nucleic acids through a mixture of molecular exclusion resin and anion exchange resin, wherein any remaining organic contaminants are captured while the nucleic acids pass through.
A method for simultaneously extracting RNA and DNA from samples containing prokaryotic cells and/or eukaryotic cells, which comprises:
- (a) lysing the cells in the samples;
- (b) dispersing and diluting the lysed cells;
- (c) preparing for separation by mixing in a buffered solution containing a solution of cationic and anionic detergents
- (d) separating cell proteins and organic contaminats by mixing with an organic extraction solvent to generate an emulsion that separates into an organic phase, an interface containing the contaminants and an aqueous phase containing the nucleic acids,
- (e) separating the phases, and retaining the aqueous phase;
- (f) precipitating the nucleic acids followed by dissolution in a nuclease-free solvent;
- and
- (g) further purifying by passing the dissolved nucleic acids through a mixture of molecular exclusion resin and anion exchange resin, wherein organic contaminants are bind to the resin and the nucleic acids pass through.
Dependencies are inferred from the claim text.