Polynucleotide capture materials, and systems using same
Inventors
Brahmasandra, Sundaresh N. • Craig, Elizabeth
Assignees
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Publication Number
US-11254927-B2
Publication Date
2022-02-22
Expiration Date
Abstract
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.
Core Innovation
A system is described that uses a container holding a plurality of magnetic beads coated with polycationic polyamine dendrimers, where the polycationic polyamine dendrimers are covalently bonded to carboxyl groups on the magnetic beads. The magnetic beads reversibly bind polynucleotides from a biological sample through ionic bonding with the polycationic polyamine dendrimers, thereby producing a set of polynucleotide-retained beads. A lytic enzyme is included in the container, and the container is configured for the biological sample to come into contact with the beads.
The system further includes a release solution and a heated release step in which the polynucleotide-retained beads are configured to release the polynucleotides when contacted with the release solution and heated. The released polynucleotides are then directed into a multi-lane microfluidic cartridge. The microfluidic cartridge includes a plurality of separate microfluidic networks, in which at least two separate networks include an opening for receiving polynucleotides released from the beads, an amplification reaction chamber, and one or more channels leading from the opening to the amplification reaction chamber.
A heater is located external to the multi-lane microfluidic cartridge and is configured to be in thermal communication with the amplification reaction chamber. The kit embodiment provides dry magnetic beads coated with polycationic polyamine dendrimers and a lytic enzyme in the at least one container prior to receiving the biological sample. The kit embodiment specifies that the magnetic beads and the lytic enzyme are in dry form, and that the polynucleotide-retained beads release the polynucleotides when contacted with a release solution and heated.
Claims Coverage
Two independent claims are provided: a system claim and a kit claim. The inventive features combine covalently coupled polycationic polyamine dendrimers on magnetic beads for reversible ionic binding of polynucleotides with lytic enzyme present, followed by heated release into a multi-lane microfluidic cartridge for amplification with an externally located heater; the kit claim further requires dry-form components and specifies amide bonding and primary amine dendrimer branch/terminal constraints.
Polycationic polyamine dendrimers covalently bonded to magnetic beads for reversible ionic polynucleotide binding
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, 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.
Lytic enzyme in the container with heated release solution to release bound polynucleotides
A system comprising a container further including a lytic enzyme, and a release solution, wherein the set of polynucleotide-retained beads are configured to release the polynucleotides when contacted with the release solution and heated.
Multi-lane microfluidic cartridge with separate microfluidic networks and externally located heater for amplification
A system including a multi-lane microfluidic cartridge comprising a plurality of separate microfluidic networks, wherein at least two 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.
Dry-form kit with covalently bonded dendrimer-coated magnetic beads and lytic enzyme
A kit comprising at least one container including 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 allow 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, and wherein the polynucleotide-retained beads are configured to release the polynucleotides when contacted with a release solution and heated.
Across the independent claims, the core coverage combines covalently coupled polycationic polyamine dendrimers on magnetic beads to reversibly bind polynucleotides via ionic bonding, a lytic enzyme in the container, and release of polynucleotides when contacted with a release solution and heated. The system claim further requires a multi-lane microfluidic cartridge with amplification reaction chambers and an externally located heater in thermal communication with those chambers, while the kit claim requires dry-form bead and enzyme components and specifies amide bonding and primary amine dendrimer branch/terminal constraints.
Stated Advantages
Documented Applications
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