Compositions, methods and kits for isolating nucleic acids from body fluids using anion exchange media
This invention provides compositions and methods for rapid isolation and purification of nucleopolymers from biological samples using anionic exchange media. The method of this invention can utilize commercially available anion exchanger materials with selected solutions of known ionic strength for absorption and elution. The medium/nucleoprotein bound complex may be optionally stored or transported, and is subsequently treated with eluents to remove undesirable proteins and inorganic salts. The partially purified complex may also be stored or transported. This method is particularly advantageous since it allows rapid isolation and purification of soluble nucleic acids from a large volume of biological fluids with easy handling and storage, stabilizing the samples against degradation prior to analysis. It also permits the purification and identification of shorter fragments of nucleic acids from bodily fluids which, until now, had not been identified. The method is widely useful to prepare samples formats that are convenient in diagnostic analytical methods.
1. A method of isolating cell-free nucleic acids from a sample of urine comprising:
a) obtaining a cell containing urine sample from a subject and separating said sample into a cell-free fraction, containing nucleic acids in the size range of 10 to 100 base pairs, and a cell-containing fraction by filtration or centrifugation;
b) selecting an anion exchange material comprising quaternary ammonium groups and which effectively adsorbs target nucleic acids or proteinous complexes thereof present within said cell-free fraction;
c) applying said cell-free fraction of said urine sample containing nucleic acids or their proteinous complexes to the selected anion exchange material, and said nucleic acids or their complexes adsorb to said material to form bound target nucleic acids or their complexes;
d) optionally washing said anion exchange material with an aqueous solution of a salt at which the target nucleic acids or their complexes remain bound to said anion exchange material, said washing being of sufficient volume and ionic strength to wash the non-binding or weakly binding components through said anion exchange material;
e) eluting the said bound target nucleic acids or their complexes by passing through said anion exchange material an aqueous solution of increasing ionic strength to remove in succession proteins that are not bound or are weakly bound to the anion exchange material and said nucleic acids of increasing molecular weight from said material; and
f) isolating the eluted nucleic acid fraction containing nucleic acids in the size range of 10 to 100 base pairs.
2. The method of claim 1 , wherein said nucleic acids are selected from the group consisting of double- and single-stranded DNA and RNA.
3. The method of claim 1 , further comprising the step of maintaining the bound target nucleic acids or their complexes in storage for up to 3 weeks prior to elution and isolation of said nucleic acid.
4. A method of isolating cell-free nucleic acids from a sample of blood plasma comprising:
a) obtaining a cell containing blood sample from a subject and separating said sample into a cell-free fraction, containing nucleic acids in the size range of 10 to 100 base pairs, and a cell-containing fraction by filtration or centrifugation;
b) selecting an anion exchange material which effectively adsorbs target nucleic acids or proteinous complexes thereof present within said cell-free fraction;
c) applying said cell-free fraction of said blood plasma sample containing nucleic acids or their proteinous complexes to the selected anion exchange material, and said nucleic acids or their complexes adsorb to said material to form bound target nucleic acids or their complexes;
d) optionally washing said anion exchange material with an aqueous solution of a salt at which the target nucleic acids or their complexes remain bound to said anion exchange material, said washing being of sufficient volume and ionic strength to wash the non-binding or weakly binding components through said anion exchange material;
e) eluting the said bound target nucleic acids or their complexes by passing through said anion exchange material an aqueous solution of increasing ionic strength to remove in succession proteins that are not bound or are weakly bound to the anion exchange material and said nucleic acids of increasing molecular weight from said material; and
f) isolating the eluted nucleic acid fraction containing nucleic acids in the size range of 10 to 100 base pairs.
5. The method of claim 4 , wherein said nucleic acids are selected from the group consisting of double- and single-stranded DNA and RNA.
6. The method of claim 4 , further comprising the step of maintaining the bound target nucleic acids or their complexes in storage for up to 3 weeks prior to elution and isolation of said nucleic acid.
7. The method of claim 4 , wherein said anion exchange material is selected from the group consisting of tertiary and quaternary ammonium groups.
8. A method of isolating cell-free nucleic acids from a sample of urine comprising:
a) separating a cell-free fraction, containing nucleic acids in the size range of 10 to 100 base pairs, apart from cells present in a urine sample from a subject and;
b) selecting an anion exchange material comprising quaternary ammonium groups, which material effectively adsorbs target nucleic acids or proteinous complexes thereof present within said cell-free fraction;
c) applying said cell-free fraction, containing nucleic acids or their proteinous complexes, to the selected anion exchange material, and said nucleic acids or their complexes adsorb to said material to form bound target nucleic acids or their complexes;
d) optionally washing said anion exchange material with an aqueous solution of a salt at which the target nucleic acids or their complexes remain bound to said anion exchange material, said washing being of sufficient volume and ionic strength to wash the non-binding or weakly binding components through said anion exchange material;
e) eluting the said bound target nucleic acids or their complexes by passing through said anion exchange material an aqueous solution of increasing ionic strength to remove in succession proteins that are not bound or are weakly bound to the anion exchange material and said nucleic acids of increasing molecular weight from said material; and
f) isolating the eluted nucleic acid fraction containing nucleic acids in the size range of 10 to 100 base pairs.
9. The method of claim 8 , wherein said separating a cell-free fraction is by filtration or centrifugation.
10. The method of claim 8 , wherein said nucleic acids are selected from the group consisting of double- and single-stranded DNA and RNA.
11. The method of claim 8 , further comprising the step of maintaining the bound target nucleic acids or their complexes in storage for up to 3 weeks prior to elution and isolation of said nucleic acid.