Methods of isolating biological target materials using silica magnetic particles

• US 6,027,945 A
• Filed: 01/21/1997
• Issued: 02/22/2000
• Est. Priority Date: 01/21/1997
• Status: Expired due to Term

• Current Assignee: Promega Corporation
• Original Assignee: Promega Corporation
• Inventors: Smith, Craig E., York, Charles K.
• Primary Examiner(s): Housel, James C.
• Assistant Examiner(s): Phan, Quang N.
• Application Number:
  US08/785,097
• Time in Patent Office: 1,127 Days
• Field of Search: 436/501, 436/526, 436/527, 423/335, 536/25.42, 252/62.51, 435/91.1, 435/6, 435/287.2
• US Class Current: 436/526
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: Y10S 977/838 Magnetic property of nanomaterial
• CPC Class Codes: Y10S 977/904 for medical, immunological, body treatment, or diagnosis
• CPC Class Codes: Y10S 977/916 Gene therapy
• CPC Class Codes: Y10S 977/92 Detection of biochemical
• CPC Class Codes: Y10S 977/924 using nanostructure as support of dna analysis
• CPC Class Codes: Y10S 977/96 Of magnetic property

Abstract

The present invention provides methods for isolating biological target materials, particularly nucleic acids, such as DNA or RNA or hybrid molecules of DNA and RNA, from other substances in a medium using silica magnetic particles. The methods of the present invention involve forming a complex of the silica magnetic particles and the biological target material in a mixture of the medium and particles, separating the complex from the mixture using external magnetic force, and eluting the biological target material from the complex. The preferred embodiments of magnetic silica particles used in the methods and kits of the present invention are capable of forming a complex with at least 2 μg of biological target material per milligram of particle, and of releasing at least 60% of the material from the complex in the elution step of the method. The methods of the present invention produce isolated biological target material which is substantially free of contaminants, such as metals or macromolecular substances, which can interfere with further processing or analysis, if present.

Claims

1. A method for isolating a biological target material from other material in a medium by:

a. providing a medium including the biological target material;

b. providing silica magnetic particles capable of directly and reversibly binding the biological target material in the absence of an intermediary substance;

c. forming a complex of the silica magnetic particles and the biological target material by combining the silica magnetic particles and the medium, the biological target material being directly adhered to the silica magnetic particles;

d. removing the complex from the medium by application of an external magnetic field; and

e. separating the biological target material from the complex by eluting the biological target material, whereby the isolated biological target material is obtained.

2. A method of isolating a biological target material according to claim 1, wherein the biological material isolated according to the method consists of a nucleic acid.

3. A method of isolating a biological target material according to claim 1, wherein the silica magnetic particles provided in step (b) are capable of reversibly binding at least 2 micrograms of biological target material per milligram of particle.

4. A method of isolating a biological target material according to claim 3, wherein the silica magnetic particles provided in step (b) of the method are siliceous-oxide coated magnetic particles.

5. A method of isolating a biological target material according to claim 1, wherein at least 60% of the biological target material in the complex is eluted from the particles in step (e).

6. A method of isolating a biological target material according to claim 1, wherein the biological target material eluted from the complex in step e. contains no more than 50 parts per million of transition metal contaminants.

7. A method of isolating a biological target material from other materials in a medium comprising the steps of:

a) providing a medium containing the biological target material;

b) providing a silica magnetic particle with the capacity to directly and reversibly bind at least 2 micrograms of biological target material per milligram of particle;

c) forming a mixture comprising the medium and the silica magnetic particle;

d) adhering the biological target material to the silica magnetic particle in the mixture;

e) removing the silica magnetic particle with the biological target material adhered thereto from the mixture by application of an external magnetic field; and

f) eluting at least 60% of the biological target material from the silica magnetic particle by exposing the particle to an elution solution;

whereby the biological target material is isolated.

8. A method of isolating a biological target material according to claim 7, wherein the biological material isolated according to the method consists of a nucleic acid material.

9. A method of isolating a biological target material according to claim 8, wherein the nucleic acid biological target material isolated according to the method consists of a plasmid DNA material.

10. A method of isolating a biological target material according to claim 8, wherein the nucleic acid biological target material isolated consists of DNA fragment material.

11. A method of isolating a biological target material according to claim 7, wherein the silica magnetic particles provided in step (b) of the method are siliceous oxide-coated magnetic particles.

12. A method of isolating a biological target material according to claim 7, wherein the mixture formed in step (c) comprises the medium, the silica magnetic particle, and a chaotropic salt, wherein the chaotropic salt concentration is sufficiently high to cause the biological target material to adhere to the silica magnetic particle in step (d).

13. A method of isolating a biological target material according to claim 12, wherein the chaotropic salt in the mixture formed in step (c) consists of a guanidinium chaotropic salt consisting of guanidine hydrochloride or guanidine thiocyanate.

14. A method of isolating a biological target material according to claim 12, wherein the concentration of chaotropic salt in the mixture formed in step (c) is at least 2 molar.

15. A method of isolating a biological target material according to claim 7, wherein the biological target material is adhered to the silica magnetic particle in step (d) by incubating the mixture.

16. A method of isolating a biological target material according to claim 15, wherein the biological target material is adhered to the silica magnetic particle in step (d) by incubating the mixture at room temperature for at least 30 seconds.

17. A method of isolating a biological target material according to claim 7, further comprising a step of washing the silica magnetic particle after removal from the medium, before eluting the biological target material from the particle.

18. A method of isolating a biological target material of claim 17, wherein the washing step is done using a wash solution comprising an alcohol and a salt.

19. A method of isolating a biological target material according to claim 18, wherein the washing step is done using a wash solution comprising at least 30% alcohol by volume and a buffer.

20. A method of isolating a biological target material according to claim 7, wherein the biological target material is eluted from the silica magnetic particle in step (f) using water or an elution solution with a low ionic strength.

21. A method of isolating biological target material according to claim 7, wherein the biological target material eluted from the silica magnetic particle in step (f) is substantially free of macromolecular or metal contaminants.

22. A method of isolating a plasmid DNA material from other materials in a medium comprising the steps of:

a) providing a medium containing the plasmid DNA;

b) providing a siliceous oxide-coated magnetic particle with the capacity to reversibly bind at least 2 micrograms of biological target material per milligram of particle;

c) forming a mixture comprising the medium, the siliceous oxide-coated magnetic particle, and a chaotropic salt, wherein the chaotropic salt concentration in the mixture is sufficiently high to cause the plasmid DNA to adhere directly to the particle in the absence of an intermediary substance;

d) incubating the mixture at about room temperature until at least some of the biological target material is adhered directly to the siliceous oxide-coated magnetic particle;

e) removing the siliceous oxide-coated magnetic particle from the mixture using an external magnetic force; and

f) eluting at least 60% of the plasmid DNA adhered to the siliceous oxide-coated magnetic particle by exposing the particle to an elution solution.

23. A method of isolating a plasmid DNA material according to claim 22, wherein the chaotropic salt in the mixture formed in step (c) is a guanidinium chaotropic salt consisting of guanidine hydrochloride or guanidine thiocyanate.

24. A method of isolating a plasmid DNA material according to claim 22, wherein the concentration of chaotropic salt in the mixture formed in step (c) is between about 0.1 M and 7 M.

25. A method of isolating a plasmid DNA material according to claim 22, further comprising a step of washing the siliceous oxide-coated magnetic particle after removal from the medium, before eluting the plasmid DNA material from the particle.

26. A method of isolating a plasmid DNA material according to claim 25, wherein the washing step is done using a wash solution comprising an alcohol and a salt.

27. A method of isolating a plasmid DNA material according to claim 25, wherein the washing step is done using a wash solution comprising at least 30% alcohol by volume and a buffer.

28. A method of isolating a plasmid DNA material according to claim 22, wherein the plasmid DNA eluted from the silica magnetic particle in step (f) is substantially free of macromolecular or metal contaminants likely to interfere with further processing or analysis.

29. A method of isolating a plasmid DNA material from other materials in a medium comprising the following steps, in the order indicated, of:

a) providing a medium containing the plasmid DNA;

b) providing a siliceous oxide-coated magnetic particle with the capacity to reversibly bind at least 2 micrograms of biological target material per milligram of particle;

c) forming a mixture comprising the medium, the siliceous oxide-coated magnetic particle, and a chaotropic salt, wherein the chaotropic salt concentration in the mixture is sufficiently high to cause the plasmid DNA to adhere directly to the particle in the absence of an intermediary substance or label;

d) incubating the mixture at about room temperature until at least some of the biological target material is adhered directly to the siliceous oxide-coated magnetic particle;

e) removing the siliceous oxide-coated magnetic particle from the mixture using an external magnetic force; and

f) eluting at least 60% of the plasmid DNA adhered to the siliceous oxide-coated magnetic particle by exposing the particle to an elution solution.