Isolation of DNA, RNA and protein from a single sample
View Patent ↗The invention provides a method for the separation and purification of two or three cellular components selected from genomic DNA, RNA and proteins from a single biological sample. The method comprises generating an aqueous solution containing the cellular components by lysing cells with a lysis solution; contacting the aqueous solution with an ion exchanger for genomic DNA and RNA to bind to the ion exchanger; collecting the flow-through which contains unbound proteins; eluting RNA from the ion exchanger; and eluting DNA from the ion exchanger. For the purification of any two of the cellular components, one of the components is not collected. The invention also provides reagent kits for carrying out the methods.
1. A method for the separation and/or purification of cellular components selected from genomic DNA, RNA and proteins, which method comprising:
a) generating an aqueous solution containing said cellular components by lysing cells with a lysis solution;
b) contacting said aqueous solution with an anion exchanger for genomic DNA and RNA to bind to the anion exchanger;
c) collecting the flow-through which contains unbound proteins;
d) eluting RNA from said anion exchanger with an RNA elution solution; and
e) eluting DNA from said anion exchanger with an DNA elution solution;
wherein said RNA elution solution has a lower salt concentration than said DNA elution solution, and said RNA elution solution has a higher salt concentration than said lysis solution.
2. The method of claim 1 , further comprising purifying said proteins collected from step c) by precipitation, gel filtration, affinity chromatography or hydrophobic interaction chromatography (HIC).
3. The method of claim 1 , further comprising purifying said genomic DNA eluted from step e) by a desalting step.
4. The method of claim 1 , further comprising purifying said RNA eluted from step d) by a desalting step.
5. The method of claim 1 , further comprising a wash step after step d), to remove any trace amount of RNA from said anion exchanger.
6. The method of claim 1 , wherein said lysis solution has a salt concentration of about 200-500 mM.
7. The method of claim 1 , wherein said RNA elution solution has a salt concentration of about 625-750 mM.
8. The method of claim 1 , wherein said DNA elution solution has a salt concentration of above about 750 mM.
9. The method of claim 1 , wherein the pH of said DNA elution solution is between about pH 9 and about pH 13.
10. The method of claim 1 , wherein the pH of said DNA elution solution is between about pH 10 and about pH 12.
11. The method of claim 1 , wherein the pH of said DNA elution solution is between about pH 10.5 and about pH 11.6.
12. The method of claim 1 , wherein said DNA elution solution comprises potassium carbonate.
13. The method of claim 12 , wherein said potassium carbonate is present at a concentration of about 0.1M-2 Molar.
14. The method of claim 1 , wherein said anion exchanger is contained within a chromatography column and separation is accomplished by gravity flow, centrifugation, positive pressure or vacuum.