Polynucleotide capture materials, and systems using same
Inventors
Brahmasandra, Sundaresh N. • Craig, Elizabeth
Assignees
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Publication Number
US-11060082-B2
Publication Date
2021-07-13
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
The invention provides a composition comprising carboxyl modified magnetic beads having polycationic polyamine dendrimers covalently bound via one or more amine groups to one or more of the carboxyl groups on the magnetic beads. The polycationic polyamine dendrimers comprise at least two dendrimer branches having primary amine terminals and at least one tertiary amine. The document further characterizes this composition using PAMAM(0) immobilized on carboxyl-modified magnetic beads or microparticles through amine/carboxyl chemistry.
The document describes polynucleotide capture and sample preparation in which liberated RNA or DNA preferentially binds to PAMAM(0) at low pH in the presence of PCR inhibitors. After binding, the polynucleotides are washed and then released under pH/temperature-dependent conditions, with RNA release described at alkaline pH and DNA release described at more alkaline pH relative to RNA.
The document describes workflow and performance features including high binding/recovery and fast workflows, including single-tube sample preparation and rapid processing. It also states that compositions and kit formats are provided, including lyophilized PAMAM(0)-coated microparticles, and that PCR performance is demonstrated with molecular examples such as EV13 RNA and detection limits reported in the document.
Claims Coverage
The partial claims provided include one independent claim. The inventive subject matter centers on a specific bead-bound polycationic polyamine dendrimer composition, with dependent claim refinements addressing dendrimer structure and identity.
Carboxyl modified magnetic beads with covalently bound polycationic polyamine dendrimers
Carboxyl modified magnetic beads; and polycationic polyamine dendrimers covalently bound via one or more amine groups per molecule to one or more of the carboxyl groups on the magnetic beads, wherein the polycationic polyamine dendrimers comprise at least two dendrimer branches having primary amine terminals and having at least one tertiary amine.
Polycationic polyamine dendrimers having at least four dendrimer branches with primary amine terminals
The polycationic polyamine dendrimers have at least four dendrimer branches with primary amine terminals prior to covalently bonding to the plurality of magnetic beads.
Polycationic polyamine dendrimers comprising at least five amine groups when covalently bonded
The polycationic polyamine dendrimers comprise at least five amine groups when covalently bonded to the magnetic beads.
Polycationic polyamine dendrimers having molecular weight less than about 600 Da
The polycationic polyamine dendrimers have a molecular weight of less than about 600 Da prior to covalently bonding to the magnetic beads.
PAMAM (Generation 0) dendrimer species
The composition includes polycationic polyamine dendrimers that are PAMAM (Generation 0).
Across the provided independent and dependent claims, the core claim requires carboxyl-modified magnetic beads with polycationic polyamine dendrimers covalently bound through amine-to-carboxyl attachment, where the dendrimers have primary amine terminals, at least one tertiary amine, and at least two dendrimer branches. Dependent claims further specify minimum dendrimer branching and amine-group counts, a molecular-weight threshold, and PAMAM (Generation 0) as the dendrimer species.
Stated Advantages
Preferential binding/recovery of liberated RNA or DNA over PCR inhibitors at low pH.
Fast workflow and single-tube sample preparation.
Provides quantitative PCR-ready output.
Demonstrates high binding/recovery and PCR performance with reported detection limits in the document.
Provides kit formats including lyophilized PAMAM(0)-coated microparticles.
Documented Applications
Sample preparation for polynucleotide capture (RNA and DNA) from samples followed by quantitative PCR.
Use in PCR performance demonstrations for EV13 RNA, with reported detection limit examples.
Kit/device formats including compositions and kit formats with lyophilized PAMAM(0)-coated microparticles.
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