Polynucleotide capture materials, and methods of using same
Methods for processing polynucleotide-containing biological samples, and materials for capturing polynucleotide molecules such as RNA and/or DNA from such samples are provided. 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. The binding particles can be coated with PAMAM (Generation 0) dendrimer which is amide-bonded to the binding particles, wherein at least a portion of the nucleic acids in the biological sample become reversibly bonded to the binding particles.
1. A method for nucleic acid isolation comprising:
receiving into a process chamber a biological sample containing nucleic acid;
contacting the biological sample with a plurality of binding particles in the process chamber, the binding particles being coated with PAMAM (Generation 0) dendrimer which is amide-bonded to the binding particles, wherein at least a portion of the nucleic acids in the biological sample become reversibly bonded to the binding particles;
washing the binding particles with a solution characterized by a pH less than 10 while retaining the nucleic acid on the binding particles; and
releasing a nucleic acid sample from the binding particles by contacting the binding particles with an elution solution characterized by a pH greater than 10.
2. The method of claim 1 , further comprising compacting the binding particles having nucleic acid bound thereto prior to washing the binding particles.
3. The method of claim 2 , wherein the binding particles are magnetic, and wherein the method further comprises compacting the binding particles by applying a magnetic field to the process chamber.
4. The method of claim 2 , wherein a volume of liquid around the compacted binding particles comprises a residual solution, and wherein the method further comprises removing a substantial portion of the residual solution prior to washing the binding particles.
5. The method of claim 1 , further comprising contacting the biological sample with a lysis solution and the plurality of binding particles, thereby lysing the cells in the biological sample to release the nucleic acids.
6. The method of claim 5 , wherein the lysis solution has a pH of between 4 and 8.
7. The method of claim 5 , further comprising heating the biological sample to a temperature in the range of room temperature and 60° C.
8. A method for nucleic acid isolation comprising:
receiving into a process chamber a biological sample containing nucleic acid;
contacting the biological sample with a plurality of binding particles in the process chamber, the binding particles being coated with cationic polyelectrolyte dendrimer which is amide-bonded to the binding particles, wherein at least a portion of the nucleic acids in the biological sample become reversibly bonded to the binding particles;
washing the binding particles with a solution characterized by a pH less than 10 while retaining the nucleic acid on the binding particles; and
releasing a nucleic acid sample from the binding particles by contacting the binding particles with an elution solution characterized by a pH greater than 10.
9. The method of claim 8 , wherein the cationic polyelectrolyte dendrimer molecules are made from an ethylene diamine monomer core.
10. The method of claim 8 , wherein the cationic polyelectrolyte dendrimer is PAMAM (Generation 0).
11. The method of claim 8 , wherein the cationic polyelectrolyte dendrimer comprises six amine groups.
12. The method of claim 8 , wherein the cationic polyelectrolyte dendrimer comprises five amine groups available for protonation when amide-bonded to the binding particles.
13. The method of claim 8 , further comprising compacting the binding particles having nucleic acid bound thereto prior to washing the binding particles.
14. The method of claim 13 , wherein the binding particles are magnetic, and wherein the method further comprises compacting the binding particles by applying a magnetic field to the process chamber.
15. The method of claim 13 , wherein a volume of liquid around the compacted binding particles comprises a residual solution, and wherein the method further comprises removing a substantial portion of the residual solution prior to washing the binding particles.
16. The method of claim 8 , further comprising contacting the biological sample with a lysis solution and the plurality of binding particles, thereby lysing the cells in the biological sample to release the nucleic acids.
17. The method of claim 16 , wherein the lysis solution has a pH of between 4 and 8.
18. The method of claim 16 , further comprising heating the biological sample to a temperature in the range of room temperature and 60° C.
19. A composition comprising:
carboxyl modified microparticles; and
cationic polyelectrolyte dendrimer covalently bound via one or more amine groups per molecule to one or more of the carboxylic acid groups on the microparticles.
20. The composition of claim 19 , wherein the cationic polyelectrolyte dendrimer is PAMAM (Generation 0).