Isolation of biological materials using magnetic particles

• US 5,508,164 A
• Filed: 10/29/1993
• Issued: 04/16/1996
• Est. Priority Date: 10/29/1990
• Status: Expired due to Fees

• Current Assignee: DEKALB Genetics Corp. (Bayer AG)
• Original Assignee: DEKALB Genetics Corp. (Bayer AG)
• Inventors: Narayanswami, Sandya, Manning, Jerry E., Hamkalo, Barbara A., Kausch, Albert P.
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): KETTER, JAMES S
• Application Number:
  US08/146,434
• Time in Patent Office: 900 Days
• Field of Search: 435/172.3, 435/820, 435/7.2, 435/6, 536/239, 536/25.4
• US Class Current: 435/6.16
• CPC Class Codes: C12N 15/1006 by means of a solid support carrier, e.g. particles, polymers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: G01N 2035/1025 Fluid level sensing
• CPC Class Codes: G01N 33/54326 Magnetic particles
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 35/0098 involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C

Abstract

A method for the isolation and sorting of biological materials has been developed. Biological material includes chromosomes, segments of chromosomes, cell organelles, or other minute cellular components. The biological material is separated from the cellular milieu, if necessary, and anchored to a support. Example of a support are glass coverslips, glass or polymer beads. The anchoring is by means of a reversible cross-linking system. The supported biological material is then labelled with compositions capable of binding to said material, and with magnetic particles. Examples of the binding material include nucleic acid probes and antibodies. An example of the antibodies would be those directed to histones. Other labels, for example, fluoresceinbiotin-avidin may be used. The material may be released from the support and sorted by a magnetic force. This method is an alternative to flow cytometry and presents numerous advantages in terms of time, resolution, purity, and preservation of the structure of the biological material during isolation and separation.

Claims

1. A method for the magnetic labelling of a chromosome or chloroplast cellular component comprising the following steps:

(a) binding said cellular component to a binding composition comprised of an antibody or nucleic acid in the case of chromosome isolation, or antibody in the case of chloroplast isolation; and

(b) labelling said binding composition with magnetic particles having an average diameter less than about 2 μ

m.

19. The method of claim 1 wherein the particles are from about 50 nm to about 0.5 microns in diameter.

2. A method for the isolation of a chromosome or chloroplast cellular component comprising the following steps:

(a) reversibly anchoring said cellular component to a support with a DTSSP or SPDP linker;

(b) binding said cellular component to a binding composition comprised of an antibody in the case of chromosome or chloroplast isolation, or nucleic acid in the case of chromosome isolation;

(c) labelling said binding composition with magnetic particles having an average diameter less than about 2 μ

m;

(d) releasing said cellular component from the support to form free, magnetized cellular components; and

(e) isolating said cellular component by applying a magnetic force to said free, magnetized cellular components.

3. The method of claim 2, wherein the cellular component is a chromosome.

4. The method of claim 2, wherein the cellular component is a chloroplast.

5. The method of claim 2, wherein the binding composition which binds to the cellular component comprises nucleic acid probes.

6. The method of claim 2, wherein the cellular component comprises polypeptides and the binding composition comprises antibodies to the polypeptides.

7. The method of claim 2, wherein the magnetic particles comprise ferric oxide particles.

8. The method of claim 2, wherein the support comprises a silane treated surface that anchors the cellular component to the support, and a cleavable cross-linking agent which crosslinks between the cellular component and the support.

9. The method of claim 8 wherein the surface is treated with the silane N-(2-aminoethyl)-3-aminopropyl trimethoxysilane.

10. The method of claim 8 wherein the cleavable cross-linking agent comprises SPDP or DTSSP.

11. The method of claim 2, wherein the support comprises a glass coverslip.

12. The method of claim 2, wherein the support comprises controlled pore glass beads (CPG).

13. The method of claim 12, wherein the controlled pore glass beads are attached to long chain alkylamine linker arms.

14. The method of claim 2, wherein the support comprises polymer beads.

15. The method of claim 14, wherein the polymer beads are treated with oligopeptides, said oligopeptides each terminating in a cleavable amino group.

16. The method of claim 2, wherein the binding composition comprises a label.

17. The method of claim 16, wherein the label comprises a biotinylated fluorescent marker.

18. The method of claim 5, wherein the probes are capable of hybridizing to unique repetitive sequences of the centromeric regions of specific chromosomes.

20. The method of claim 2, wherein the magnetic particles comprise colloidal gold.

21. A method of separating a chromosome or chloroplast cellular component comprising the following steps:

(a) binding a supported, cellular component to a binding composition comprised of an antibody in the case of chromosome or chloroplast isolation, or nucleic acid in the case of chromosome isolation;

(b) labelling said bound cellular component with a magnetic particle having an average diameter of less than about 2 μ

m; and

(c) magnetically separating the resulting labelled cellular component.

22. The method of claim 21 wherein the cellular component comprises a chromosome and the binding composition comprises a nucleic acid probe.

23. The method of claim 21 wherein the cellular component comprises a protein and the binding composition comprises an antibody directed to that protein.

24. The method of claim 21 wherein said cellular component is supported by means mounting said cellular component on a silane treated support by a disulfide-containing bifunctional reversible crosslinker.

25. The method of claim 21 wherein the particle is from about 50 nm to about 0.5 microns in diameter.

26. A method of sorting chromosomes to achieve purified suspensions thereof, said method comprising the following:

(a) anchoring the chromosomes to a support by means of a disulfide-containing bifunctional reversible linker;

(b) hybridizing the supported chromosomes with a binding nucleic acid or antibody binding composition to form a complex;

(c) labelling the complex with magnetic particles having an average diameter less than about 2 μ

m;

(d) releasing the complex from the support; and

(e) sorting the released chromosomes by means of a magnetic force.

27. The method of claim 26, wherein the binding composition comprises nucleic acid sequence probes.

28. The method of claim 26, wherein the binding composition comprises antibodies.

29. A method of sorting chromosomes to achieve purified preparations thereof, said method comprising the following steps:

(a) anchoring the chromosomes to a support by means of a reversible DTSSP or SPDP linker;

(b) hybridizing the supported chromosomes with a binding composition comprised of an antibody or nucleic acid to form a complex;

(c) labelling the complex with a magnetic label;

(d) releasing the complex from the support; and

(e) sorting the released chromosomes by means of said magnetic label.

30. The method according to claim 29 wherein said label is a magnetic particle having an average diameter from about 50 nm to about 2 microns.

31. The method according to claim 29 wherein said label further includes a fluorescent label.