Polynucleotide capture materials, and methods of using same
View Patent ↗Disclosed are methods for isolating polynucleotides (e.g., RNA and DNA) from a cell-containing sample. The disclosed methods employ polyamidoamine (PAMAM) dendrimer molecules bound to the surface of binding particles to capture and isolate the polynucleotides from the samples. The polynucleotides are released from the binding particles by contacting the binding particles with a release solution having a basic pH.
1. A method for isolating polynucleotides from a cell-containing sample in a process tube, the method comprising:
lysing the cells in the sample by contacting the sample with a lysis solution having a pH between 4 and 8, thereby releasing polynucleotides from the cells;
retaining the polynucleotides in the sample as compared to any amplification inhibitors by contacting the sample with magnetic binding particles having PAMAM(Generation 0) molecules covalently bound to the surface thereof, the polynucleotides becoming reversibly retained by the PAMAM(Generation 0) molecules on the binding particles;
compacting the binding particles retaining the polynucleotides in the process tube by positioning a magnet in communication with the binding particles;
removing the lysis solution from the process tube;
washing the binding particles with a wash solution having a pH less than 9 while the polynucleotides are retained on the binding particles; and
releasing the polynucleotides from the binding particles by contacting the binding particles with a release solution having a pH between 9 and 12.
2. The method of claim 1 , wherein the step of lysing the cells further comprises heating the lysis solution in the process tube.
3. The method of claim 1 , wherein the step of releasing the polynucleotides further comprises heating binding particles in the process tube.
4. The method of claim 1 , wherein the steps of lysing, retaining, compacting, removing, washing, and releasing all take place in a single process tube.
5. The method of claim 1 , wherein the release solution has a volume of less than 3 microliters.
6. The method of claim 1 , wherein the release solution comprises Tris.
7. The method of claim 1 , wherein the release solution comprises Bis-Tris Propane.
8. The method of claim 1 , wherein the release solution has a pH between 9 and 10.
9. The method of claim 1 , wherein the release solution has a pH between 10 and 11.
10. The method of claim 1 , wherein the release solution has a pH between 11 and 12.
11. The method of claim 1 , wherein the release solution has a pH of approximately 9.
12. The method of claim 1 , wherein the release solution has a pH of approximately 10.
13. The method of claim 1 , wherein the release solution has a pH of approximately 11.
14. The method of claim 1 , wherein the release solution has a pH of approximately 12.
15. A method for isolating polynucleotides in a sample, comprising:
contacting a solution of the sample with a plurality of binding particles, the binding particles having PAMAM molecules covalently bound to carboxyl groups on the surface of the binding particles, wherein the polynucleotides become reversibly retained by the PAMAM molecules as compared to any amplification inhibitors in the solution of the sample, the amplification inhibitors remaining in the solution;
removing the solution containing the amplification inhibitors from the presence of the binding particles;
washing the binding particles while the polynucleotides are retained by the binding particles; and
releasing the polynucleotides from the binding particles with a release solution having a pH between 9 and 12.
16. The method of claim 15 , wherein the PAMAM molecules are made from an ethylene diamine monomer core.
17. The method of claim 15 , wherein one amine group on the PAMAM molecules covalently bonds with the carboxyl groups on the surface of the binding particle.
18. The method of claim 15 , wherein a plurality of amine groups on the PAMAM molecules covalently bond with the carboxyl groups on the surface of the binding particle.
19. The method of claim 15 , wherein the PAMAM molecules on the binding particles have a molecular weight in the range of 500-600 Da.
20. The method of claim 15 , wherein the PAMAM molecules on the binding particles comprise PAMAM(Generation 0) molecules.
21. The method of claim 20 , wherein the PAMAM molecules on the binding particles have a molecular weight of approximately 516 Da.
22. The method of claim 20 , wherein the PAMAM molecules on the binding particles comprise five amine groups available for protonation.
23. The method of claim 20 , wherein the PAMAM molecules on the binding particles comprise less than five amine groups available for protonation.
24. The method of claim 15 , wherein the PAMAM molecules on the binding particles have a molecular weight in the range of 500-600 Da.
25. The method of claim 15 , wherein the PAMAM molecules are made from a monomer core selected from the group consisting of 1,2-propylene diamine, 1,3-propylene diamine, 1,2-butylene diamine, 1,3-butylene diamine, and 1,4-butylenediamine.