Method of isolating and purifying nucleic acids from biological samples

Claim 1

The method of isolating and purifying cellular or viral nucleic acids from an aqueous solution of a biological sample containing nuclein acids together with water-soluble contamination components of lysed cells or virus selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, comprising: (a) selecting an anion exchange column material which effectively binds the target nucleic acids at a lower salt molarity than the molarity at which the target nucleic acids elute therefrom; (b) applying a sample containing nucleic acids together with water-soluble contaminating components of lysed cells or virus selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, to a column packed with the selected anion exchanger material, and said nucleic acids becoming bound to said column material; (c) washing said column with an aqueous solution of a salt at chloride molarity at which the nucleic acids remain bound to said column material, said wash molarity being close to the lowest molarity at which the nucleic acids begin to elute, said washing being of sufficient volume to wash the non-binding components through said column, including carboxylated mucopolysaccharides; (d) eluting the bound nucleic acids by passing through said column an aqueous solution of a salt having an eluting molarity corresponding to the lowest molarity at which the target nucleic acids will completely elute, said eluant being passed in sufficient volume to remove said nucleic acids from the column while leaving contaminating components including sulfated mucopolysaccharides in the column; and (e) recovering the eluted nucleic acids essentially free of mucopolysaccharides and other contaminating components.

Claim 2

The method of isolating and purifying cellular or viral nucleic acids from an aqueous solution of a biological sample containing nuclein acids together with water-soluble contamination components of lysed cells or virus selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, comprising: (a) selecting an anion exchange column material which effectively binds the target nucleic acids at a lower salt molarity than the molarity at which the target nucleic acids elute therefrom; (b) applying a sample containing nucleic acids together with water-soluble contaminating components of lysed cells or virus selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, to a column packed with the selected anion exchanger material, and said nucleic acids becoming bound to said column material; (c) washing said column with an aqueous solution of a salt at chloride molarity at which the nucleic acids remain bound to said column material, said wash molarity being close to the lowest molarity at which the nucleic acids begin to elute, said washing being of sufficient volume to wash the non-binding components through said column, including carboxylated mucopolysaccharides; (d) eluting the bound nucleic acids by passing through said column an aqueous solution of a salt having an eluting molarity corresponding to the lowest molarity at which the target nucleic acids will completely elute, said eluant being passed in sufficient volume to remove said nucleic acids from the column while leaving contaminating components including sulfated mucopolysaccharides in the column; and (e) recovering the eluted nucleic acids essentially free of mucopolysaccharides and other contaminating components.

Claim 12

The method of isolating microbial double-stranded nucleic acids from an aqueous solution prepared from a body fluid or excretion requiring examination for pathogenic microorganisms, said solution containing water-soluble contaminating components of the body fluid or excretion, selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, comprising: (a) selecting an anion exchange column material which effectively binds the microbial nucleic acids at a lower sodium chloride molarity than the molarity at which the nucleic acids elute therefrom; (b) applying a sample containing nucleic acids together with water-soluble contaminating components of body fluid or excretion selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, to a column packed with the selected anion exchange material, the anionic groups of said column material being in chloride form and said nucleic acids becoming bound to said column material; (c) washing said column with aqueous NaCl at a chloride molarity at which the nucleic acids remain bound to the column material, said wash molarity being close to the lowest chloride molarity at which the nucleic acids begin to elute, said washing being of sufficient volume to wash the carboxylated mucopolysaccharides out of said column together with other contaminating components; (d) eluting the bound nucleic acids by passing through said column an aqueous NaCl solution having a chloride molarity corresponding to the lowest chloride molarity at which said nucleic acids completely elute without elution of any other mucopolysaccharides which may be present, said eluant being passed in sufficient volume to remove the nucleic acids from said column; and (e) recovering the separated nucleic acids essentially free of mucopolysaccharides and other contaminating components.

Claim 13

The method of isolating microbial double-stranded nucleic acids from an aqueous solution prepared from a body fluid or excretion requiring examination for pathogenic microorganisms, said solution containing water-soluble contaminating components of the body fluid or excretion, selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, comprising: (a) selecting an anion exchange column material which effectively binds the microbial nucleic acids at a lower sodium chloride molarity than the molarity at which the nucleic acids elute therefrom; (b) applying a sample containing nucleic acids together with water-soluble contaminating components of body fluid or excretion selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, to a column packed with the selected anion exchange material, the anionic groups of said column material being in chloride form and said nucleic acids becoming bound to said column material; (c) washing said column with aqueous NaCl at a chloride molarity at which the nucleic acids remain bound to the column material, said wash molarity being close to the lowest chloride molarity at which the nucleic acids begin to elute, said washing being of sufficient volume to wash the carboxylated mucopolysaccharides out of said column together with other contaminating components; (d) eluting the bound nucleic acids by passing through said column an aqueous NaCl solution having a chloride molarity corresponding to the lowest chloride molarity at which said nucleic acids completely elute without elution of any other mucopolysaccharides which may be present, said eluant being passed in sufficient volume to remove the nucleic acids from said column; and (e) recovering the separated nucleic acids essentially free of mucopolysaccharides and other contaminating components.

Claim 22

The method of isolating and purifying cellular or viral nucleic acids from an aqueous solution sample contaminating components of lysed cells or virus selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, comprising: (a) selecting a first ion exchange column material comprising a weak base anion exchanger which effectively binds the target nucleic acids at a lower aqueous chloride salt molarity than the molarity at which the target nucleic acids elute therefrom; (b) selecting a second ion exchange material which comprises a strong base anion exchanger which effectively binds the target nucleic acids at a lower aqueous chloride salt molarity than the molarity at which the target nucleic acids elute therefrom, said binding molarity of the second column material corresponding to said eluting molarity of the first column material; (c) preparing first and second columns either as separate or stacked columns respectively packed with said first and second column materials in chloride form; (d) applying a sample containing nucleic acids together with water-soluble contaminating components of lysed cells or virus selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, to the first column containing said weak base exchanger, said target nucleic acids becoming bound to the first column material; (e) washing said first column with an aqueous solution of a chloride salt at a chloride molarity at which the nucleic acids remain bound to said first column material, said was molarity being close to a chloride molarity at which said nucleic acids begin to elute, said washing being sufficient to wash the nonbinding components through said first column including any carboxylated mucopolysaccharides; (f) eluting the bound nucleic acids by passing through said first column an aqueous solution of a chloride salt having an eluting chloride molarity corresponding to the lowest chloride molarity at which the target nucleic acid beings to elute, said eluant being passed in sufficient volume to remove said nucleic acids from said first column while leaving any sulfated mucopolysaccharides therein; (g) passing the eluant from said first column through said second column, the nucleic acids eluted from said first column material becoming bound to said second column material; (h) eluting the bound nucleic acid from said second material by passing through said second column an aqueous solution of a chloride salt having an eluting chloride molarity higher than that of the first column eluant, which higher molarity corresponds to the lowest chloride molarity at which said nucleic acids will completely elute from said second material, said eluant being passed through said second column in sufficient volume to remove said nucleic acids from said second column; and (i) recovering the purified and isolated nucleic acids essentially free of mucopolysaccharides and other contaminating components.

Claim 23

The method of isolating and purifying cellular or viral nucleic acids from an aqueous solution sample contaminating components of lysed cells or virus selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, comprising: (a) selecting a first ion exchange column material comprising a weak base anion exchanger which effectively binds the target nucleic acids at a lower aqueous chloride salt molarity than the molarity at which the target nucleic acids elute therefrom; (b) selecting a second ion exchange material which comprises a strong base anion exchanger which effectively binds the target nucleic acids at a lower aqueous chloride salt molarity than the molarity at which the target nucleic acids elute therefrom, said binding molarity of the second column material corresponding to said eluting molarity of the first column material; (c) preparing first and second columns either as separate or stacked columns respectively packed with said first and second column materials in chloride form; (d) applying a sample containing nucleic acids together with water-soluble contaminating components of lysed cells or virus selected from the group consisting of proteins, pigments, carboxylated mucopolysaccharides, sulfated mucopolysaccharides, and mixtures thereof, to the first column containing said weak base exchanger, said target nucleic acids becoming bound to the first column material; (e) washing said first column with an aqueous solution of a chloride salt at a chloride molarity at which the nucleic acids remain bound to said first column material, said was molarity being close to a chloride molarity at which said nucleic acids begin to elute, said washing being sufficient to wash the nonbinding components through said first column including any carboxylated mucopolysaccharides; (f) eluting the bound nucleic acids by passing through said first column an aqueous solution of a chloride salt having an eluting chloride molarity corresponding to the lowest chloride molarity at which the target nucleic acid beings to elute, said eluant being passed in sufficient volume to remove said nucleic acids from said first column while leaving any sulfated mucopolysaccharides therein; (g) passing the eluant from said first column through said second column, the nucleic acids eluted from said first column material becoming bound to said second column material; (h) eluting the bound nucleic acid from said second material by passing through said second column an aqueous solution of a chloride salt having an eluting chloride molarity higher than that of the first column eluant, which higher molarity corresponds to the lowest chloride molarity at which said nucleic acids will completely elute from said second material, said eluant being passed through said second column in sufficient volume to remove said nucleic acids from said second column; and (i) recovering the purified and isolated nucleic acids essentially free of mucopolysaccharides and other contaminating components.