Compositions, methods and kits for isolating nucleic acids from body fluids using anion exchange media
This invention provides compositions and methods for rapid separation, isolation and purification of nucleic acids from biological samples using anionic exchange media. The method can utilize commercially available strong or weak anion exchanger materials with selected solutions of known ionic strength for adsorption and elution. The instant method is particularly advantageous as it permits the purification and identification of shorter fragments of nucleic acids from bodily fluids which, until now, had not been identified.
1. A method of isolating cell-free nucleic acids from a cell-containing sample of urine, the method comprising:
a) obtaining the urine sample and separating the sample into a cell-free fraction and a cell-containing fraction by centrifugation;
b) applying the cell-free fraction to a quaternary ammonium anion exchange material so as to allow adsorption of nucleic acids less than or equal to 400 base pairs in the urine sample to the quaternary ammonium anion exchange material;
c) passing, through the quaternary ammonium anion exchange material, an aqueous solution of increasing ionic strength that selectively elutes nucleic acids less than or equal to 400 base pairs; and
d) isolating an eluted nucleic acid fraction containing the nucleic acids less than or equal to 400 base pairs.
2. The method of claim 1 wherein a support for the anion exchange material is a sepharose.
3. The method of claim 1 wherein the aqueous solution is a salt solution of between 0.55M to 5M NaCl or 2.0 M guanidine isothiocyanate.
4. The method of claim 1 wherein the nucleic acids less than or equal to 400 base pairs are selectively eluted stepwise with a salt solution from 500 mM to 650 mM of NaCl.
5. The method of claim 1 further comprising, before c),
washing the quaternary ammonium anion exchange material with a sufficient volume of an aqueous solution of a salt having an ionic strength of less than 500 mM, at which the nucleic acids less than or equal to 400 base pairs remain bound to the quaternary ammonium anion exchange material during washing, and nonbinding or weakly-binding components pass through the quaternary ammonium anion exchange material.
6. The method of claim 1 wherein the aqueous solution is selected from: 300 mM NaCl/30 mM sodium citrate (pH 7.0); 300-600 nM NaCl/30 mM sodium citrate (pH 7.0); 300 mM LiCl/10 mM NaOAc (pH 5.2); and 300-600 mM LiCl/10 mM NaOAc (pH 5.2).
7. The method of claim 1 wherein the anion exchange material is a sepharose-based quaternary ammonium anion exchange medium.
8. The method of claim 7 wherein the sepharose-based quaternary ammonium anion exchange material is a Q-Sepharose.
9. The method of claim 1 wherein the elution comprises use of a LiCl solution having a concentration of 300 mM to 2.0M.
10. The method of claim 1 wherein the anion exchange material is immobilized on a column, a cartridge or a portable filtering system.
11. The method of claim 1 , wherein an eluted nucleic acid fraction containing nucleic acids of 40-400 base pairs is isolated.
12. A method of isolating cell-free nucleic acids from a cell-containing sample of urine, the method comprising:
a) obtaining the urine sample and separating the sample into a cell-free fraction and a cell-containing fraction by centrifugation;
b) applying the cell-free fraction to a quaternary ammonium anion exchange material so as to allow adsorption of nucleic acids 40 to 400 base pairs in the urine sample to the quaternary ammonium anion exchange material;
c) passing, through the quaternary ammonium anion exchange material, an aqueous solution of increasing ionic strength that selectively elutes nucleic acids 40 to 400 base pairs; and
d) isolating the eluted nucleic acid fraction containing the nucleic acids 40 to 400 base pairs.
13. The method of claim 12 , wherein the nucleic acids 40 to 400 base pairs are selectively eluted stepwise with a salt solution from 500 mM to 650 mM of NaCl.
14. The method of claim 12 , further comprising, before c), washing the quaternary ammonium anion exchange material with a sufficient volume of an aqueous solution of a salt having an ionic strength of less than 500 mM, at which the nucleic acids 40 to 400 base pairs remain bound to the quaternary ammonium anion exchange material during washing, and nonbinding or weakly-binding components pass through the quaternary ammonium anion exchange material.