Selective nucleic acid separation

Patent metadata

Field	Value
Publication number	US-10604751-B2
Application number	US-201515322814-A
Country	US
Kind code	B2
Filing date	Jul 2, 2015
Priority date	Jul 2, 2014
Publication date	Mar 31, 2020
Grant date	Mar 31, 2020

Abstract

Nucleic acids in non-rare cells in a sample containing non-rare cells and rare cells are selectively released from the non-rare cells. The sample is combined with an aqueous medium, and the combination is held for a period of time and at a temperature for selectively releasing nucleic acids from the non-rare cells but not from the rare cells. The sample is subjected to filtration to separate rare cells from non-rare cells. Rare cells with intact nucleic acids are separated from non-rare cells and nucleic acids from the non-rare cells. Nucleic acids from the rare cells are subjected to one or more identification techniques either with or without extraction of nucleic acids from the rare cells.

First claim

What is claimed is:

1. A method of separating rare cells with intact nucleic acids from non-rare cells in a sample comprising the rare cells and non-rare cells, the method comprising:
(a) combining the sample with an aqueous medium,
(b) holding the combination for a period of time and at a temperature for selectively releasing nucleic acids from the non-rare cells but not from the rare cells, and
(c) separating the rare cells from the sample by filtration,
wherein the rare cells are cancer cells.

2. The method according to claim 1 , wherein the combination is held for a period of about 15 minutes to about 20 days at a temperature of about 5° C. to about 30° C.

3. The method according to claim 1 , wherein the sample is treated with a fixation agent prior to combining the sample with the aqueous medium.

4. The method according to claim 1 , wherein the filtration comprises disposing the sample on a side of the porous matrix and applying pressure to the disposed sample.

5. The method according to claim 4 , wherein the pressure applied is about 1 millibar to about 30 millibar and wherein the pore size of the porous matrix is about 0.1 μm to about 100 μm.

6. The method according to claim 1 , further comprising extracting intact nucleic acids from the rare cells.

7. A method of isolating intact nucleic acids from rare cells in a sample comprising non-rare cells and circulating nucleic acids from rare cells, the method comprising:
(a) combining the sample with an aqueous medium and nucleic acid capture particles,
(b) holding the combination for a period of time and at a temperature for selectively releasing nucleic acids from the non-rare cells but not from the capture particles,
(c) subjecting the medium to filtration to separate nucleic acids on the capture particles from non-rare cells, and
(d) extracting intact nucleic acids from the capture particles.

8. The method according to claim 7 , wherein the combination is held for a period of about 15 minutes to about 20 days at a temperature of about 5° C. to about 30° C.

9. The method according to claim 7 , wherein the rare cells are cancer cells.

10. The method according to claim 7 , wherein the filtration comprises disposing the sample on a side of the porous matrix and applying pressure to the disposed sample.

11. The method according to claim 10 , wherein the pressure applied is about 1 millibar to about 30 millibar and wherein the pore size of the porous matrix is about 0.1 μm to about 100 μm.

12. A method of determining one or more nucleic acids in rare cells in a sample comprising the rare cells and non-rare cells, the method comprising:
(a) combining the sample with an aqueous medium,
(b) holding the combination for a period of time and at a temperature for selectively releasing nucleic acids from the non-rare cells but not from the rare cells,
(c) separating the rare cells from the sample by filtration, and
(d) identifying one or more of the nucleic acids of the rare cells,
wherein the rare cells are cancer cells.

13. The method according to claim 12 , wherein the combination is held for a period of about 15 minutes to about 20 days at a temperature of about 5° C. to about 30° C.

14. The method according to claim 12 , wherein the sample is treated with a fixation agent prior to combining the sample with the aqueous medium.

15. The method according to claim 12 , wherein the filtration comprises disposing the sample on a side of the porous matrix and applying pressure to the disposed sample.

16. The method according to claim 15 , wherein the pressure applied is about 1 millibar to about 30 millibar and wherein the pore size of the porous matrix is about 0.1 μm to about 100 μm.

17. The method according to claim 12 , wherein the identifying comprises contacting the nucleic acids with one or more identification agents specific for the one or more nucleic acids.

18. The method according to claim 12 , wherein the nucleic acids are extracted from the rare cells and the extracted nucleic acids are subjected to one or more amplification techniques for amplifying nucleic acids.

Assignees

• Siemens Healthcare Diagnostics Inc
• Siemens Healthcare Disgnostics Inc

Inventors

• Pugia Michael
• Philip Julia
• Marfut Karen

Classifications

• C12N15/1003

  Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor · CPC title

• C12Q1/6806

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

• C12N15/1017 Primary

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

• C12N15/1006

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

• C12Q2527/156

  Permeability · CPC title