Method for nucleic acid isolation by solid phase reversible binding of nucleic acids

• US 20110172409A1
• Filed: 10/18/2010
• Published: 07/14/2011
• Est. Priority Date: 10/19/2009
• Status: Active Grant

• Current Assignee: Zhiqiang Han
• Original Assignee: Zhiqiang Han
• Inventors: Han, Zhiqiang
• Granted Patent: US 8,536,322 B2
• Time in Patent Office: Days
• US Class Current: 536/25.4
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers

Abstract

The invention includes a method for separating polynucleotides, such as DNA, RNA and PNA, from a solution containing polynucleotides by reversibly and non-specifically binding the polynucleotides to a solid surface having a functional amide group-coated surface. The materials containing a solid surface can be in the form of microparticles, fibers, beads, membranes, test tubes, pipette tips or microwells and can further comprise a magnetic core portion. The pH, salt and concentration of crowding reagent, such as polyethylene glycol or alcohol, of the solution is adjusted to levels which result in nucleic acid binding to the solid surface. The magnetic microparticles with bound polynucleotides are separated from the solution under mild alkaline conditions and the nucleic acids are eluted from the magnetic microparticles. Solutions having different nucleic acid concentrations can be normalized by restricting the availability of the solid phase surface.

Claims

1. A method of reversibly binding DNA non-specifically to materials having functional group with multiple-amide bond-coated surface, comprising the steps of:

a) contacting the materials having functional group with multiple-amide bond-coated surface to a solution containing polynucleotides; and

b) adjusting the salt concentration and pH of the solution suitable for binding the polynucleotides onto the surfaces of the materials, thereby binding the polynucleoteds non-specifically to the magnetic microparticles.

2. The method of claim 1, wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, potassium chloride, sodium sulfate, magnesium sulfate, potassium sulfate, sodium phosphate, potassium phosphate, sodium nitrate, and potassium nitrate.

3. The method of claim 1, wherein the pH is between 4 and 11.

4. The method of claim 1, wherein the pH is preferably between 6 and 9.

5. The method of claim 1, wherein the polynucleotides are isolated by dissolving in an elution solution.

6. The method of claim 5 wherein the elution solution is a solution with pH greater than or equal to 11.

7. The method of claim 5, wherein the elution solution is a salt solution.

8. The method of claim 7, wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, potassium chloride, sodium sulfate, magnesium sulfate, potassium sulfate.

9. The method of claim 7, wherein the salt concentration is greater than 100 mM.

10. The method of claim 1, wherein the polynucleotide solution is the result of an enzyme reaction.

11. The method of claim 10, wherein the enzyme is selected from polymerase, ligase, polynucleotide kinase, reverse transcriptase or restriction enzymes.

12. The method of claim 1, wherein the polynucleotide solution is the result of lysed cells, tissue, bodily fluid.

13. The method of claim 1, wherein the polynucleotide solution is a purified polynucleotide solution.

14. A method of separating polynucleotides from a solution containing polynucleotides, comprising the steps of:

a) combining materials having functional group with multiple-amide bond-coated surfaces and a solution containing polynucleotides, thereby producing a first combination;

b) adjusting the salt concentration and the concentration of crowding reagents of the first combination to concentrations suitable for binding polynucleotides to the solid surface of the materials, thereby producing a second combination comprising polynucleotides bound non-specifically to solid phase materials;

c) separating the solid surface materials from the second combination;

d) contacting the solid surface materials separated in c) with the bound polynucleotides in a wash buffer, whereby polynucleotides remains bound to the solid surface of the materials in the absence of crowding reagents; and

e) separating the solid surface materials from the washing buffer.

15. The method of claim 14, wherein the crowding reagents are one or more of chemicals selected from polyethylene glycol, ethanol and isopropanol.

16. The method of claim 14, wherein the wash buffer solution dissolves impurities bound to the magnetic microparticles while leaving the DNA bound to the solid phase materials.

17. The method of claim 14, wherein the polynucleotides bound to the solid phase materials are washed with a selective elution solution, wherein the selective elution solution dissolves small DNA fragments bound to the solid phase materials while leaving large DNA fragments bound to the solid phase materials.

18. The method of claim 14, wherein the polynucleotides are isolated by dissolving in an elution solution.

19. The method of claim 18 wherein the elution solution is a solution with pH above 11.

20. The method of claim 18, wherein the elution solution is a salt solution.

21. The method of claim 14, wherein the salt is selected from the group consisting of sodium chloride, magnesium chloride, potassium chloride, sodium sulfate, magnesium sulfate, potassium sulfate, sodium phosphate, potassium phosphate, sodium nitrate, potassium nitrate.

22. The method of claim 20, wherein the salt concentration is above 100 mM.

23. The method of claim 14, wherein the polynucleotide solution comprises the product of enzyme reaction.

24. The method of claim 23, wherein the enzyme is selected from polymerase, ligase, polynucleotide kinase, reverse transcriptase or restriction enzymes.

25. The method of claim 14, wherein the polynucleotide solution is a purified polynucleotide solution.