Microarray based multiplex pathogen analysis and uses thereof
Inventors
Hogan, Michael Edward • May, Melissa Rose • Eggers, Frederick Henry
Assignees
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-11421224-B2
Publication Date
2022-08-23
Expiration Date
Abstract
Provided herein is a method for manufacturing a 3-dimensional lattice microarray system for DNA sequence detection and analysis. A solid support is contacted with a formulation containing a plurality of nucleic acid probes, a plurality of bifunctional polymer liners and a solvent mixture of water and a water-miscible liquid. In a first attachment reaction the bifunctional polymer linkers are attached to the solid support and the water is evaporated. In a second attachment reaction the nucleic acid probes are attached to the bifunctional polymer linker.
Core Innovation
The invention provides a method of manufacturing a 3-dimensional lattice microarray system on a glass support having a front surface with activated surface moieties. Oligodeoxythymidine linkers each consisting of 20 to 60 thymidine bases are provided in a formulation containing a solvent mixture of water and a water-miscible liquid with a boiling point above 100° C., and the linkers include an amino group at their 3′ terminus and a fluorescent label covalently linked to their 5′ terminus.
The 3′ amino group of each oligodeoxythymidine linker is attached to one of the activated surface moieties by covalent coupling, while activated moieties that are not covalently coupled create lattice width spacing between covalently coupled linkers. Water is evaporated from the solvent mixture to progressively concentrate the nucleic acid probes together with the oligodeoxythymidine linkers covalently coupled to the activated surface moieties, while maintaining separation that supports formation of a lattice structure.
Photochemical crosslinking is then performed to crosslink thymidine nucleotides in the nucleic acid probes to oligodeoxythymidine linkers attached to the glass support. Each probe attached on the glass support is separated by both a vertical space and a lattice width, such that crosslinking forms a 3-dimensional lattice on the glass support, followed by washing to manufacture the 3-dimensional lattice microarray.
The system is configured with nucleic acid probes selected from pathogenic bacteria nucleotide probes and pathogenic fungi nucleotide probes, or combinations thereof. Each pathogenic bacteria nucleotide sequence or pathogenic fungi nucleotide sequence is sandwiched between two to seven consecutive thymidine nucleotides and is attached to both the 3′ terminus and the 5′ terminus of each nucleic acid probe.
Claims Coverage
The independent claim covers a method for manufacturing a 3-dimensional lattice microarray system using activated surface moieties on a glass support, fluorescently labeled oligodeoxythymidine linkers, a water and high-boiling-point water-miscible solvent mixture, progressive concentration by evaporating water, photochemical thymidine-mediated covalent crosslinking to form a 3-dimensional lattice, and a washing step. The inventive features are five.
Activated glass support with fluorescent oligodeoxythymidine linkers and probe-loaded formulation
Contacting a glass support having activated surface moieties with a formulation comprising a solvent mixture of water and a water-miscible liquid with a boiling point above 100° C., a plurality of oligodeoxythymidine linkers having a 3′-terminus amino group and a fluorescent label covalently linked to their 5′ terminus, and a plurality of nucleic acid probes selected from pathogenic bacteria nucleotide probes and pathogenic fungi nucleotide probes or combinations, with activated surface moieties and oligodeoxythymidine linkers present in a molar ratio of at least 10.
Covalent coupling creates lattice width spacing
Attaching, by covalent coupling, the amino group at the 3′ terminus of each oligodeoxythymidine linker to one activated surface moiety so that activated surface moieties that are not covalently coupled create lattice width spacing between covalently coupled oligodeoxythymidine linkers.
Evaporative concentration during assembly
Evaporating the water in the solvent mixture to progressively concentrate the plurality of nucleic acid probes in the solvent mixture together with the plurality of oligodeoxythymidine linkers covalently coupled to the activated surface moieties.
Photochemical thymidine crosslinking forms a 3-dimensional lattice
Photochemically crosslinking thymidine nucleotides at the 3′ and/or 5′ termini of the nucleic acid probes to the oligodeoxythymidine linkers attached to the glass support, including crosslinking to one or more linkers or to two adjacent oligodeoxythymidine linkers, such that each nucleic acid probe attached to the oligodeoxythymidine linkers is separated by both a vertical space and a lattice width and crosslinking forms a 3-dimensional lattice on the glass support.
Washing to manufacture the 3-dimensional lattice microarray
Washing the glass support at least once to manufacture the 3-dimensional lattice microarray.
The claim coverage centers on assembly of a fluorescently labeled oligodeoxythymidine linker layer on an activated glass support, evaporative concentration in a water and high-boiling-point water-miscible solvent mixture, and photochemical crosslinking of thymidine termini in pathogenic bacteria and fungi nucleotide probes to form a 3-dimensional lattice, followed by washing.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
Interested in licensing this patent?