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

• US 8,536,322 B2
• Filed: 10/18/2010
• Issued: 09/17/2013
• Est. Priority Date: 10/19/2009
• Status: Expired due to Fees

• Current Assignee: Zhiqiang Han
• Original Assignee: Zhiqiang Han
• Inventors: Han, Zhiqiang
• Primary Examiner(s): LEWIS, PATRICK T
• Application Number:
  US12/906,969
• Publication Number:
  US 20110172409A1
• Time in Patent Office: 1,065 Days
• Field of Search: None
• 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 nucleic acids non-specifically to a solid phase material having a surface coated with one or more functional groups with multiple amide bonds without relying on any crowding reagent, the method comprising the steps of:

a) contacting a surface of a solid phase material coated with one or more functional groups with multiple amide bonds with a solution containing nucleic acids; and

b) adjusting the salt concentration and pH of the solution until suitable for binding the nucleic acids onto the surface of the solid phase material, thereby binding the nucleic acids non-specifically to the one or more functional groups with multiple amide bonds and therefore the solid phase material without relying on any crowding agent.

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 adjusted to between 4 and 11.

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

5. The method of claim 1, wherein the nucleic acids bound to the solid phase material are subsequently released from the solid phase material by dissolving the nucleic acids 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 in the elution solution is selected from the group consisting of sodium chloride, magnesium chloride, potassium chloride, sodium sulfate, magnesium sulfate, and potassium sulfate.

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

10. The method of claim 1, wherein the nucleic acid-containing solution is the result of an enzyme reaction.

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

12. The method of claim 1, wherein the nucleic acid-containing solution is the result of lysed cells, tissue, or bodily fluid.

13. The method of claim 1, wherein the nucleic acid-containing solution is a purified solution.

23. The method of claim 1 wherein the multiple amide bonds comprises two consecutive amide bonds.

24. The method of claim 1 wherein the one or more functional groups have no charge groups attached thereto.

25. The method of claim 1 wherein the one or more functional groups is represented by a general formula as follows:

26. The method of claim 1 wherein the solid phase material is selected from the group consisting of silica, plastic, and magnetic or magnetizable material.

27. The method of claim 1 wherein the solid phase material is a microparticle comprising a magnetic metal oxide core.

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

a) combining a solid phase material surface-coated with one or more functional groups with multiple amide bonds and a solution containing nucleic acids, thereby producing a first combination;

b) adjusting the salt concentration of the first combination until suitable for binding nucleic acids to the surface of the solid phase material, thereby producing a second combination comprising nucleic acids bound non-specifically to the one or more functional groups with multiple amide bonds and therefore the solid phase material without relying on any crowding agent;

c) separating the solid phase material from the second combination;

d) contacting the solid phase material separated in c) with the bound nucleic acids in an elution solution, whereby nucleic acids separate from the solid phase material and dissolve into the elution solution; and

e) separating the solid phase material from the elution solution.

15. The method of claim 14, further comprising a step of washing the solid phase material with a wash buffer solution wherein the wash buffer solution dissolves impurities bound to the solid phase material while leaving the DNA bound to the solid phase material.

16. The method of claim 14, wherein elution solution comprises one or more crowding reagents, wherein the concentration of the one or more crowding reagents is selected such that the elution solution dissolves small nucleic acid fragments bound to the solid phase materials while leaving large nucleic acid fragments bound to the solid phase material.

17. The method of claim 14 wherein the elution solution is a solution with pH above 11.

18. The method of claim 14, wherein the elution solution is a salt solution.

19. The method of claim 18, wherein the salt concentration in the elution solution is above 100 mM.

20. The method of claim 14, wherein the nucleic acids-containing solution comprises the product of enzyme reaction.

21. The method of claim 20, wherein the enzyme is selected from the group consisting of polymerase, ligase, polynucleotide kinase, reverse transcriptase and restriction enzymes.

22. The method of claim 14, wherein the nucleic acids-containing solution is a purified solution.