Polynucleotide capture materials, and methods of using same
View Patent ↗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.
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 become 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.