Rapid methods for the extraction of nucleic acids from biological samples

Patent metadata

Field	Value
Publication number	US-9976136-B2
Application number	US-201615152871-A
Country	US
Kind code	B2
Filing date	May 12, 2016
Priority date	May 14, 2015
Publication date	May 22, 2018
Grant date	May 22, 2018

Abstract

The invention is directed to compositions and methods for rapidly and efficiently extracting nucleic acids and/or targeted nucleic acids sequences from biological samples. The methods of the invention comprise combining the sample with a buffer and magnetic silicon beads and concentrating the beads with a magnet or other electrical field. Liquid may be removed, or not, and an alkaline buffer is added followed by magnetic carboxy beads in a binding buffer so that nucleic acids transfer to the carboxy beads, which can be easily and quickly isolated once again with a magnet. Total nucleic acid extraction is greatly enhanced. Extracted nucleic acids can be analyzed, for example, by PCR wherein the nucleic acids can be identified and characterized. Carboxy beads may also contain a ligand so as to target specific nucleic acid sequences. The invention is also directed to kits comprising the tools and compositions for performing the methods of the invention.

First claim

The invention claimed is:

1. A method of extracting nucleic acids from a biological sample containing cells and/or microorganisms comprising:
adding a matrix material and a buffer to the biological sample, wherein the matrix material binds to nucleic acids of the sample;
isolating matrix material bound to nucleic acids of the sample;
adding an intermediate buffer that promotes a release of nucleic acids from the matrix material to form a mixture;
adding a chemically modified magnetic matrix material and a binding buffer to the mixture wherein the chemically modified matrix material comprises a ligand that binds to a specific nucleic acid sequence;
exposing the mixture to a magnetic field to concentrate the magnetic matrix material bound to the specific nucleic acid sequence; and
adding an extraction buffer to the concentrated magnetic matrix material wherein the specific nucleic acid sequence is extracted from the magnetic matrix material.

2. The method of claim 1 , wherein the biological sample comprises human, animal, microbial or plant material.

3. The method of claim 1 , wherein the matrix material comprises magnetic beads of silicone, porcelain, ceramic, plastic, glass or polymer.

4. The method of claim 1 , wherein the matrix material disrupts the cells and/or microorganisms of the biological sample and binds to nucleic acids released from the disrupted cells and/or microorganisms.

5. The method of claim 1 , wherein the matrix material does not disrupt the cells and/or microorganisms of the biological sample and binds to nucleic acids of the biological sample other than those present within the cells and/or microorganisms.

6. The method of claim 1 , wherein the buffer comprises a chaotrope, a detergent, a reducing agent, a buffer and a chelator, together at a pH of between about 6-8.

7. The method of claim 1 , wherein the buffer is a lysis buffer that lyses cells, inactivates nucleases, stabilizes macromolecules, and sterilizes the sample at ambient temperatures.

8. The method of claim 1 , wherein the buffer is a non-cell lysis buffer that maintains the integrity of cells and/or microbes of the biological sample.

9. The method of claim 1 , wherein the intermediate buffer causes the release of nucleic acids from the matrix material and has a pH above 7.

10. The method of claim 1 , wherein the intermediate buffer comprises TE, saline, an alkaline solution, NALC or a combination thereof.

11. The method of claim 1 , wherein the chemical modification of the chemically modified magnetic matrix material is a carboxy group.

12. The method of claim 1 , wherein the specific nucleic acid comprises a cancer marker, a sequence indicating the presence of a pathogenic organism or infection, a sequence that is characteristic of a phenotypic condition, a sequence indicating a lineage, a sequence indicating an identifiable characteristic, a sequence indicating a mutation, a sequence indicating a change from a wild-type sequence, or a combination thereof.

13. The method of claim 1 , wherein the specific nucleic acid sequence is indicative of the presence of a pathogen.

14. The method of claim 13 , wherein the pathogen is a virus, a bacterium, a parasite or a fungus.

15. The method of claim 1 , wherein the binding buffer comprises PEG, a salt, a buffering agent, a chelator, a detergent, and an alcohol.

16. The method of claim 1 , wherein the magnetic field is an electro-magnetic field.

17. The method of claim 1 , wherein the extraction buffer comprise water, TE, saline, alcohol or a combination thereof.

18. The method of claim 1 , wherein the extracted nucleic acids are identifiable by molecular analysis.

19. The method of claim 18 , wherein the molecular analysis comprises a PCR.

20. The method of claim 1 , which does not involve centrifugation or nucleic acid purification.

21. The method of claim 1 , which is automated for high-throughput analysis of a plurality of biological samples.

22. The method of claim 1 , wherein extraction efficiency, as measured by PCR cycle threshold, is lower as compared with extraction efficiency for a conventional extraction procedure.

23. The method of claim 22 , wherein the conventional extraction procedure is a silica spin column extraction.

24. A method of extracting nucleic acids from a biological sample containing cells and/or microorganisms comprising:
combining the biological sample with magnetic silicon dioxide beads and a lysis buffer to form a solution, wherein the beads bind to nucleic acids of the sample;
exposing the solution to a magnetic field and removing liquid to concentrate the beads;
adding an alkaline buffer to the concentrated magnetic beads to form a mixture, wherein the alkaline buffer causes the release of nucleic acids from the silicon dioxide beads;
adding carboxy-modified magnetic beads in a binding buffer to the mixture wherein the carboxy-modified magnetic beads bind to a predetermined nucleic acid sequence;
exposing the mixture to a magnetic field and removing liquid to isolate the carboxy-modified magnetic beads bound to the predetermined nucleic acid sequence; and
eluting the predetermined nucleic acid sequences from the carboxy-modified magnetic beads using purified water and/or a Tris-EDTA buffer.

25. The method of claim 24 , wherein the lysis buffer comprises a chaotrope, a detergent, a reducing agent, a buffer, and a chelator at a pH of about 6-8.

26. The method of claim 24 , wherein the binding buffer comprises PEG, a salt, a buffering agent, a chelator, a detergent, NLS and an alcohol.

27. The method of claim 24 , which does not involve centrifugation.

28. The method of claim 24 , which is automated for high-throughput analysis of a plurality of biological samples.

29. The method of claim 24 , wherein extraction efficiency, as measured by PCR cycle threshold, is lower as compared with extraction efficiency for a conventional extraction procedure.

30. The method of claim 29 , wherein the conventional extraction procedure is a silica spin column extraction.

31. The method of claim 24 , further comprising analyzing the isolated nucleic acids by a PCR.

32. A kit comprising:
a solution that comprises a matrix material and a buffer;
an intermediate buffer with a pH above 7; and
carboxy-modified magnetic beads in a binding buffer, wherein the carboxy-modified magnetic beads bind to a specific nucleic acid sequence.

33. The kit of claim 32 , wherein the buffer comprises a chaotrope, a detergent, a reducing agent, a buffer and a chelator at a pH of about 6-8.

34. The kit of claim 33 , wherein the buffer lyses cells, inactivates nucleases, stabilizes nucleic acids, and sterilizes the sample at ambient temperatures.

35. The kit of claim 32 , wherein the buffer is a non-lysis buffer that maintains the integrity of cells and/or microbes.

36. The kit of claim 32 , wherein the matrix material comprises silica dioxide beads.

37. The kit of claim 32 , wherein the intermediate buffer has a pH of 8 or more.

38. The kit of claim 32 , wherein the intermediate buffer has a pH of 9 or more.

39. The it of claim 32 , wherein the binding buffer comprises a combination of PEG, a salt, a chela

Assignees

• Longhorn Vaccines & Diagnostics Llc

Inventors

• Fischer Gerald W
• Daum Luke T

Classifications

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title

• C12N15/1013 Primary

  by using magnetic beads · CPC title