Polynucleotide capture materials, and systems using same
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 system comprising:
a container comprising:
a plurality of magnetic beads coated with polycationic polyamine dendrimers, the polycationic polyamine dendrimers being covalently bonded to carboxyl groups on the plurality of magnetic beads, and
a lytic enzyme,
wherein the container is configured to receive a biological sample containing polynucleotides and allow the biological sample to come in contact with the plurality of magnetic beads, and
wherein the plurality of magnetic beads in the container are configured to reversibly bind polynucleotides of the biological sample, through ionic bonding with the polycationic polyamine dendrimers, thereby producing a set of polynucleotide-retained beads;
a release solution, wherein the set of polynucleotide-retained beads are configured to release the polynucleotides when contacted with the release solution and heated;
a multi-lane microfluidic cartridge comprising:
a plurality of separate microfluidic networks, wherein at least two of the separate microfluidic networks comprise:
an opening for receiving polynucleotides released from the set of polynucleotide-retained beads into the microfluidic network;
an amplification reaction chamber; and
one or more channels leading from the opening to the amplification reaction chamber; and
a heater located external to the multi-lane microfluidic cartridge and configured to be in thermal communication with the amplification reaction chamber.
2. The system of claim 1 , wherein the polycationic polyamine dendrimers comprise six amine groups prior to covalently bonding to carboxyl groups on the magnetic beads.
3. The system of claim 1 , wherein the polycationic polyamine dendrimers comprise at least four dendrimer branches having primary amine terminals prior to covalently bonding to carboxyl groups on the magnetic beads.
4. The system of claim 1 , wherein the polycationic polyamine dendrimers comprise at least five amine groups when covalently bonded to carboxyl groups on the magnetic beads.
5. The system of claim 1 , wherein the polycationic polyamine dendrimers comprise at least three dendrimer branches having primary amine terminals when covalently bonded to carboxyl groups on the magnetic beads.
6. The system of claim 1 , wherein the polycationic polyamine dendrimers are amide-bonded to the magnetic beads.
7. The system of claim 1 , wherein the polycationic polyamine dendrimers comprise polyamide dendrimers.
8. The system of claim 7 , wherein the polyamide dendrimers comprise PAMAM dendrimers.
9. The system of claim 1 , wherein the plurality of magnetic beads and the lytic enzyme are in solid form in the container prior to receiving the biological sample in the container.
10. The system of claim 1 , wherein the plurality of magnetic beads and the lytic enzyme are in dry form in the container prior to receiving the biological sample in the container.
11. The system of claim 1 , wherein the container further comprises sample process control polynucleotides prior to receiving the biological sample in the container.
12. The system of claim 9 , wherein the lytic enzyme comprises proteinase K.
13. The system of claim 10 , wherein the lytic enzyme comprises proteinase K.
14. The system of claim 1 , wherein the polycationic polyamine dendrimers comprise an initial monomer core of ethylene diamine.
15. A kit comprising:
a plurality of containers, at least one container comprising:
a plurality of magnetic beads coated with polycationic polyamine dendrimers, the polycationic polyamine dendrimers being covalently bonded to the plurality of magnetic beads via amide bonds and comprising at least three dendrimer branches having primary amine terminals; and
a lytic enzyme,
wherein the at least one container is configured to receive a biological sample containing polynucleotides and to allow the biological sample to come in contact with the plurality of magnetic beads,
wherein the plurality of magnetic beads and the lytic enzyme are in dry form in the at least one container prior to receiving the biological sample in the at least one container,
wherein the plurality of magnetic beads in the at least one container are configured to reversibly bind polynucleotides of the biological sample, thereby producing a set of polynucleotide-retained beads, and
wherein the set of polynucleotide-retained beads are configured to release the polynucleotides when contacted with a release solution and heated.
16. The kit of claim 15 , wherein the polycationic polyamine dendrimers comprise six amine groups prior to covalently bonding to the plurality of magnetic beads.
17. The kit of claim 15 , wherein the polycationic polyamine dendrimers have a molecular weight of less than about 600 Da prior to covalently bonding to the plurality of magnetic beads.
18. The kit of claim 15 , wherein the polycationic polyamine dendrimers comprise at least four dendrimer branches having primary amine terminals prior to covalently bonding to the plurality of magnetic beads.
19. The kit of claim 15 , wherein the polycationic polyamine dendrimers comprise at least five amine groups when covalently bonded to the plurality of magnetic beads.
20. The kit of claim 15 , wherein the at least one container further comprises sample process control polynucleotides in dry form prior to receiving the biological sample in the at least one container.
21. The kit of claim 15 , wherein the lytic enzyme comprises proteinase K.
22. The kit of claim 15 , wherein the polycationic polyamine dendrimers comprise an initial monomer core of ethylene diamine.
23. The kit of claim 15 , wherein the plurality of magnetic beads are configured to release the polynucleotides at an extraction efficiency of at least 90%.