POLYNUCLEOTIDE CAPTURE MATERIALS, AND METHODS OF USING SAME

• US 20130096292A1
• Filed: 12/03/2012
• Published: 04/18/2013
• Est. Priority Date: 07/13/2007
• Status: Active Grant

• Current Assignee: HandyLab Incorporated (Becton, Dickinson & Co)
• Original Assignee: HandyLab Incorporated (Becton, Dickinson & Co)
• Inventors: Brahmasandra, Sundaresh N., Craig, Elizaeth
• Granted Patent: US 8,710,211 B2
• Time in Patent Office: Days
• US Class Current: 536/25.41
• CPC Class Codes: C07C 233/36 having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
• CPC Class Codes: C07H 1/08 from natural products
• CPC Class Codes: C08G 83/003 Dendrimers
• CPC Class Codes: C12N 15/1013 by using magnetic beads
• CPC Class Codes: C12Q 1/6813 Hybridisation assays

Abstract

Methods for processing polynucleotide-containing biological samples, and materials for capturing polynucleotide molecules such as RNA and/or DNA from such samples. The RNA and/or DNA is captured by polyamindoamine (PAMAM (Generation 0)) bound to a surface, such as the surface of magnetic particles. The methods and materials have high efficiency of binding RNA and of DNA, and of release, and thereby permit quantitative determinations.

Claims

1. A method for isolating polynucleotides from a cell-containing sample in a process tube, the method comprising:

contacting the sample in the process tube with a lysis solution having a pH between 4 and 8, thereby releasing polynucleotides from the cells in the cell-containing sample;

contacting the sample in the process tube with a plurality of magnetic binding particles coated in PAMAM, wherein the polynucleotides becomes reversibly bound to the PAMAM, thereby creating binding particles bound with polynucleotides and a solution containing residual cellular material in the process tube;

compacting the binding particles bound with the polynucleotides in the process tube by positioning a magnet in communication with the binding particles;

removing the solution containing residual cellular material from the process tube;

washing the binding particles with a solution having a pH≦

9, thereby retaining the polynucleotides bound to the binding particles; and

contacting the binding particles bound with polynucleotides with a solution having a pH≧

9 to release the polynucleotides from the PAMAM on the binding particles, thereby isolating the polynucleotides from the cell-containing sample.

2. The method of claim 1, wherein the PAMAM on the binding particles comprises PAMAM (Generation 0).

3. The method of claim 1, wherein the polynucleotides comprise a mixture of DNA and RNA molecules.

4. The method of claim 1, wherein the contacting the compacted binding particles with a release solution further comprises contacting the binding particles with a first release solution having a pH in the range 9-12, thereby releasing any bound RNA from the PAMAM on the binding particles, while retaining any bound DNA on the binding particles.

5. The method of claim 4, wherein the contacting the binding particles with a release solution further comprises contacting the binding particles bound by polynucleotides with a second release solution having a pH in the range 12-14, thereby releasing bound DNA from the PAMAM on the binding particles.

6. The method of claim 1, wherein the binding particles are a polymeric material, the polymeric material selected from the group consisting of:

polystyrene, latex polymers, polyacrylamide, and polyethylene oxide.

7. The method of claim 6, further wherein the polymeric material comprises one or more carboxylic groups to which the PAMAM is covalently linked.

8. A method for separately isolating DNA and RNA from a cell-containing sample, the method comprising:

contacting the sample with a lysis solution having a pH between 4 and 8, thereby releasing DNA and RNA from the cells in the cell-containing sample;

contacting the sample with a plurality of binding particles, wherein the binding particles retain the DNA and RNA in the cell-containing sample;

compacting the plurality of binding particles having the DNA and RNA bound thereto;

releasing the RNA from the binding particles by contacting the compacted binding particles with a first release solution;

removing the solution containing the released RNA, thereby isolating the RNA; and

releasing the DNA from the binding particles by contacting the compacted binding particles with a second release solution, thereby isolating the DNA, wherein the pH of the second release solution is greater than the pH of the first release solution.

9. The method of claim 8, wherein the binding particles are coated in PAMAM.

10. The method of claim 9, wherein the PAMAM on the binding particles comprises PAMAM (Generation 0).

11. The method of claim 8, wherein the first release solution has a pH of 9-12.

12. The method of claim 8, wherein the second release solution has a pH of 12-14.