Purification of nucleic acid

Patent metadata

Field	Value
Publication number	US-9340828-B2
Application number	US-201214352122-A
Country	US
Kind code	B2
Filing date	Oct 25, 2012
Priority date	Oct 27, 2011
Publication date	May 17, 2016
Grant date	May 17, 2016

Abstract

The present invention relates to a simple and efficient method to isolate and purify nucleic acids, preferably genomic DNA, from complex samples compared with available methods, by using a ligand which relies on hydrogen bonding to purify the nucleic acids. Preferably the ligand is bound to magnetic beads/particles. More closely the method comprises adding a sample comprising nucleic acid to a polymer having neutral charge; reversibly binding said nucleic acid to said polymer by hydrogen bonding under pH conditions <5; washing said polymer; and eluting said nucleic acid from said polymer under conditions of pH >5. The method is very suitable for sample preparation of nucleic acids, for example for PCR applications.

First claim

The invention claimed is:

1. A method for purification of nucleic acid comprising:
adding a sample comprising a nucleic acid to a polymer having neutral charge;
reversibly binding said nucleic acid to said polymer by hydrogen bonding under pH conditions <5 in the presence of a binding solution that does not contain polyoxyethylene based detergents;
washing said polymer with a washing solution; and
eluting said nucleic acid from said polymer under conditions of pH >5.

2. The method of claim 1 , wherein the polymer is polycarboxylated.

3. The method of claim 1 , wherein the polymer comprises 100-500 monomer units.

4. The method of claim 1 , wherein the polymer is polyacrylic acid, poly lactic acid or carboxymethyldextran.

5. The method of claim 1 , wherein polymers are used as ligands attached to a natural or synthetic solid phase or matrix, such as a bead, particle, membrane, filter, chip, sensor chip, for example a SPR chip, monolith, microfluidic device, pipette tip or any other surface.

6. The method of claim 5 , wherein the solid phase comprises magnetic beads.

7. The method of claim 5 , wherein the solid phase comprises a filter.

8. The method of claim 1 , wherein the binding of nucleic acid occurs at pH 1-5, preferably pH 2-4, and elution occurs at pH 5-14, preferably pH 7-10.

9. The method of claim 1 , wherein the nuclei acid is amplified, such as by PCR, directly after elution.

10. The method of claim 1 , wherein the sample comprises nucleic acids of different sizes and thus different hydrogen bonding strength which are separated from each other by adding a solution or flow of a medium affecting hydrogen bonding.

11. The method of claim 1 , wherein the eluted nucleic acids are larger than 200 bp.

12. The method of claim 1 , wherein the eluted nucleic acids are 10-40 kb and are used for PCR applications.

13. The method of claim 1 , wherein the eluted nucleic acids are directly sequenced after elution (without amplification).

14. The method of claim 1 , wherein the nucleic acid is used for vaccine applications.

15. The method of claim 1 , wherein the binding solution contains organic solvents at a concentration of less than 30%.

16. The method of claim 1 , wherein the washing solution contains organic solvents, or is a pure organic solvent.

17. The method of claim 15 , wherein the washing solution comprises an organic solvent at a concentration >30%.

18. The method of claim 17 , wherein said organic solvent is ethanol.

Assignees

• Ge Healthcare Bio Sciences Ab

Inventors

• Estmer Nilsson Camilla
• Ohman Johan

Classifications

• C12N15/1006 Primary

  by means of a solid support carrier, e.g. particles, polymers · CPC title

• C12N15/1013

  by using magnetic beads · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N15/1017

  by filtration, e.g. using filters, frits, membranes · CPC title