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 kit, comprising:
a lysis buffer;
binding particles having PAMAM(Generation 0) molecules covalently bound thereto;
a wash solution; and
a release solution having a pH of approximately 9 or higher.
2. The kit of claim 1 , wherein one amine group on the PAMAM molecules covalently bonds with the carboxyl groups on the surface of the binding particle.
3. The kit of claim 1 , wherein a plurality of amine groups on the PAMAM molecules covalently bond with the carboxyl groups on the surface of the binding particle.
4. The kit of claim 1 , wherein the binding particles are magnetic.
5. The kit of claim 1 , wherein the PAMAM molecules are made from an ethylene diamine monomer core.
6. The kit of claim 1 , wherein the PAMAM molecules on the binding particles consist of five amine groups available for protonation.
7. The kit of claim 1 , wherein the release solution has a volume of less than 3 microliters.
8. The kit of claim 1 , wherein the release solution comprises Tris.
9. The kit of claim 1 , wherein the release solution comprises bis-tris-propane.
10. The kit of claim 1 , wherein the wash solution comprises Tris-EDTA and 1% Triton X 100 at pH 8.0.
11. The kit of claim 1 , wherein the lysis buffer has a pH between 4 and 8.
12. The kit of claim 1 , wherein the release solution has a pH of approximately 11 or higher.
13. The kit of claim 1 , wherein the release solution has a pH of approximately 12.
14. The kit of claim 1 , wherein the PAMAM on the binding particles have a molecular weight in the range of 500-600 Da.
15. The kit of claim 14 , wherein the PAMAM on the binding particles have a molecular weight of approximately 516 Da.
16. The kit of claim 1 , 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.
17. The kit of claim 1 , wherein the PAMAM molecules are made from an ethylene diamine monomer core.